Hypersensitivity. Human Reagins: Appraisal of the Properties of the Antibody of Immediate-Type

Transcription

1 BACTROLOGICAL REVIEWS, Sept. 1972, p Vol. 36, No. 3 Copyright American Society for Microbiology Printed in U.S.A. Human Reagins: Appraisal of the Properties of the Antibody of Immediate-Type Hypersensitivity JOHN A. FLICK Microbiology Department, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania INTRODUCTION Definition of Terms Relationship of Reagins to IgE Atopy and Reagins Comparison of Sensitivities of Antibody Detection Techniques RELATIONSHIP OF THE DIRECT SKIN TEST REACTION TO THE PRESENCE OF REAGIN REAGIN PRODUCTION Types of Antigens Producing Reagins Special Case of Polysaccharide Antigens Reagin Production in Nonatopics Intradermal Antigen and Reagin Production OCCURRENCE OF REAGINS IN BODY FLUIDS BIOLOGIC ACTION OF REAGINS Reagin Shock Tissues Human Skin Susceptibility to Reagin Shock Reagin Shock and Univalent Haptens Reagin Reactions and Eosinophiles PROPERTIES OF REAGINS Heat Inactivation Thiol Sensitivity Heat Conversion of Reagins to Blockers IgE and Molecular Structure Sedimentation Constants and Molecular Weight Electrophoretic Characteristics Solubility Characteristics Stability Transplacental Transmission Reagin Serology (Noncellular Assay) Protection Specificity Reagin-Antigen Union in Solution Reagin-Hapten Dissociation Latent Periods Duration of Fixation Sensitization of Laboratory Animals PARTIAL REAGINS AND QUEER REAGINS REAGIN DETECTION AND ASSAY USING VIABLE CELLS Assay in the Skin Use of Leukocytes Basophile-Mast Cell Degranulation DISCUSSION LITERATURE CITED.346 INTRODUCTION behind clouds of mystery into at least a partial daylight of recognition of its physical and The reagin type of antibody, long associated chemical attributes with a much better delinewith a variety of human allergic reactions gen- ation of its biologic properties. Whereas in the erally considered to be histamine mediated, past it was often ignored by teachers and textrecently has had a remarkable emergence from books dealing with immunology, perhaps be- 311

2 312 FLICK cause of only infrequent studies of reagins in animals and because of its close association with human clinical disease, now it has matured into its own realm of importance with strong evidence supporting its occurrence and potentiation of allergic reactions in all mammalian species that have been closely examined. Because of the extensive investigation of reagins derived from humans as compared to those of other animal species, this review will consider the properties of human reagins and will draw on information concerning laboratory animal reagins only in those occasional instances where it is believed that further background is needed. Over the years it has become evident that the antibodies of an immune response are usually quite diverse in properties either when compared from individual to individual or even within a single individual with time. Since reagins represent one type of antibody among the many, it seems reasonable that they too should be heterogeneous in various aspects. Because of this belief, an attempt has been made to collect and present here information for each property that might tend to support such an hypothesis of variability. Definition of Terms The problem of the diversity of properties leads to a related problem of the proper definition of reagin. A narrow, restrictive definition may eliminate from the classification some antibodies of like biological properties which might better be included; a broad definition, on the other hand, might undesirably include some antibodies that have no allergic potential. There is dissension concerning the best definition among the ranks of reagin immunologists. It is not intended here to settle such an issue; only future research and integration of data can produce a more solid foundation for that. However, it does seem desirable to slightly broaden the definition of reagin in order to better look at the available data, savor it, and perhaps gain insight into which way we are going. Relationship of Reagins to IgE The serum antibody associated with much human allergic disease was first detected by Prausnitz and Kustner (327) in 1921 and subsequently referred to as "reagin" by Coca and Grove (61). This unfortunate term has persisted in usage since then in spite of years of confusion engendered by the application of the BACTERIOL. REV. same term to the unrelated syphilitic antibody that reacts serologically with cardiolipin. Other names in use for this allergic antibody include atopic reagin, skin-sensitizing antibody, and more recently, homocytotropic antibody (but mostly for the reagins of laboratory animals). Within the last 5 years, the elegant work of Ishizaka especially (reviews in 165 and 168), and of others, have indicated that the examples of human reagins so far studied belong to a distinct immunoglobulin class, immunoglobulin E (IgE). It has been very tempting to substitute, in current usage and thinking, the term IgE for reagin, as being equivalent and more precise. However, such constriction of meaning seems to me as yet unwarranted in that the term IgE defines an antibody molecule through its distinctive antigenic structure and not through its biologic properties. As yet, we are uncertain if occasional reagins belong to other Ig classes and if all IgE molecules possess the biologic properties of reagins. To the physician and biologist, reagin represents a kind of antibody that has strong associations, probably etiologically related, with certain diseases of humans including hayfever, allergic asthma, anaphylaxis, urticaria, angioneurotic edema, and other ills of lesser frequency or recognition. Reagins are defined by the Prausnitz- Kiistner (PK) test of local passive transfer of the serum antibody to the skin of a normal human recipient. After a latent period of 18 hr or longer, the injection of the specific antigen causes a wheal and erythema to develop at the doubly injected site within 15 to 30 min. This whealing reaction has generally been attributed to the local release of histamine from reagin-sensitized mast cells by antigen based on three lines of evidence. (i) The intradermal injection of histamine, from the minimally reactive dose of 20 to 25 ng (64, 289) upwards, reproduces the salient features of this allergic reaction. (ii) Antihistamine therapy usually reduces or abolishes this allergic reaction. (iii) Histamine is released into the dermal lymph by the reagin-antigen interaction in humans (199). However, it is still uncertain whether or not other suggested mediators of inflammation, such as bradykinin, which can mimic the action of histamine in human skin (27, 114, 137), also play some role in the allergic mechanism. This question will be avoided here and, for brevity, the reagin-antigen allergic reaction will be considered to be mediated solely by histamine release. Thus, by definition, reagins minimally must

3 VOL. 36, 1972 possess two properties: ability to fix rather irreversibly to skin elements and ability to release histamine from an intra- to an extracellular environment upon triggering by antigen. Another property often attached to the definition of reagin is that of being heat labile as compared to other kinds of antibodies. However, I prefer not to limit the scope of the term reagin at this time by incorporating this property into its definition. PROPERTIES OF HUMAN REAGINS Atopy and Reagins The occurrence of human reagins has been so intimately entwined with the concept of atopy, possessive of many controversial facets, that the latter term needs clarification. Atopy represents a group of human, inheritable allergic diseases that are mediated by a reaginantigen interaction. The incidence of atopy in the United States population has been estimated to be in the neighborhood of 10 to 20% (117, 274, 389). But not all reagin-antigen allergic reactions occur in atopics. Those diseases universally included within the scope of the term are hayfever and asthma. There exists a strong suspicion that human anaphylaxis belongs in this category. However, atopic dermatitis is very controversial in causation, appearing to have no mechanistic relationship to reagin-antigen-histamine mediation. I consider this term to be a misnomer and believe that the sole development of atopic dermatitis by a person is insufficient reason to classify that person as atopic. Some students of allergy classify persons as atopic whose sole allergic disorder is urticaria; others do not. I will not here. Persons yielding direct wheal and erythema skin tests to common pollen, mold, or food antigens, which often are responsible for hayfever and asthma in atopics, but who show no clinical atopic disease are often classified as potential or latent atopics. It is true that some of these persons will sooner or later develop clinical atopy but others will not. This classification is open to doubt. The importance of classifying people or reagins as atopic is becoming progressively less clear as will develop later in this article. The major current value of l appending the label atopic to a person is related to its prognostic propensity; the atopic person is probably more prone toward anaphylaxis than the nonatopic person when injected with allergenic drugs, serums, or vaccines. Because of uncertainty about distinct differences, if any, in reagins derived from atopic and from nonatopic persons, the type of source, atopic or nonatopic, will be indicatod 313 as much as practical in the following account of the properties of these allergic antibodies. Comparison of Sensitivities of Antibody Detection Techniques Until recent years it was impossible to arrive at an estimate of the concentration of reagin in serum in terms of antibody nitrogen per milliliter, and therefore, to determine the sensitivity of the PK test in weight units of antibody. With the progressive purification of the serum fraction containing reagin, it has become possible to make reasonable estimates of the reagin protein needed for a minimal PK response. In Table 1 are listed values for the minimal antibody nitrogen detectable by various procedures; mostly common serological techniques above and reagin procedures below. In the reagin portion, the first listed value, 580 pg of N, was calculated indirectly from the minimal amount of purified antigen, 200 pg of N, that produced whealing at a 48-hr PK site as reported by Fink et al. (103). This value could well be much too large because the antigen specific for the reagin could have been a minor fraction of the purified horse serum albumin necessarily assumed to be the specific antigen in its entirety. The second listed value, 50 pg of reagin N, is derived from observations on the IgE-reagin fraction of serum EK of Ishizaka and Ishizaka (164). The dose producing a minimal (5-min latent period) PK reaction using excess antigen contained an amount of antibody capable of specifically binding in vitro 2.5 x 10-,ug of radiotagged antigen. In this case, an additional assumption made in addition to those needed from Table 1 is that in the fraction only reagin was present to bind the antigen. The third listed value, 16 pg of reagin N, is based on observations of serum AR by Ishizaka et al. (183), which contained 16 ug of IgE/ml of undiluted serum. A minimal 24-hr PK reaction was obtained with a dose of 0.05 ml of a 1: 8,000 dilution of this serum. The reagin estimate here assumes that all of the IgE in the serum was specific reagin; an assumption that should yield a rather large value. The fourth and fifth values for minimal reagin N are not derived from PK tests but rather represent the minimally detectable IgE already fixed in the skin of persons subsequently injected at the same site with heterologous animal-precipitating sera of anti-human IgE specificity. The specific precipitin protein content and the maximal dilution that yielded minimal whealing were determined for each serum. These values permit the calculation of

4 314 FLICK BACTERIOL. REV. the minimal amount of precipitin antibody needed to combine with the IgE-reagin and, with the assumptions in Table 1, the amount of reagin. These values, especially the 0.2 pg of reagin N, seem to be the best estimates of the minimally detectable reagin and the latter will be used in subsequent calculations with the added assumption, known to be inaccurate (380), that all human skins react to the same degree to all specimens of human reagins. Whereas, then, the PK test detects less than picogram quantities of reagin nitrogen, or concentrations of about 10 pg of N/ml, it is evident from the upper part of Table 1 that other methods of detecting antibodies do not possess this degree of sensitivity. Thus, the most sensitive in vitro test, hemagglutination, utilizing bis-diazo-benzidine coupling of the antigen, requires about 10 times the concentration of possible, for example, that the sensitivity of the PK test at 0.2 pg of N is in reality 500 times the sensitivity of the hemagglutination test at 100 pg of N rather than just 10 times. RELATIONSHIP OF THE DIRECT SKIN TEST REACTION TO THE PRESENCE OF REAGIN Although some people will wheal at the site of minimal skin trauma and some chemicals upon intradermal injection will produce local histamine release and whealing on a nonimmunologic basis in most persons so tested, these reactions can be brushed aside from our consideration by the finding in the literature of suitable controls or studies. Then the question posed is: When an antigen is injected into the skin of a person and a whealing reaction ensues at the site, is this reaction indicative of the presence of the reagin type of antibody as antibody N needed for the PK test. However, it is unclear at the moment, in the absence of definitive data, whether the minimal amount or the minimal concentration is the best ex- its sole cause? Or, as an alternative, can other types of antibodies, which might be present, cause the whealing? If the whealing of a direct pression of sensitivity for comparing tests. For skin test always signifies the presence of reagins then there are a lot more data available this reason, the volumes used in each test are indicated parenthetically in the table. It is in the literature for consideration in dealing TABLE 1. Comparison of minimal detection levels of antibody by various methodsa Technique Antibody Antigen used Method of Minimum antibody source calculation measured Year Ref. Nonreagin antibody Quantitative precipitin R-S Pneumotype 1 6,g of N (1 ml) Quantitative precipitin Hu-S Diphtheria toxin 4 sg of N (5 unit AT) Oudin-precipitin G-S Hu-IgA Reversed role 1.6 Ag of N (of IgA) Complement-fixing R-IgG Hu-RBC (A) 70 ng of N (1 ml) Complement-fixing R-IgM Hu-RBC (A) 2 ng of N (1 ml) Agglutination-bactericidal R-S Typhoid ng of N (0.5 ml) Agglutination-RBC Hu-IgG Hu-RBC (A) 10 ng of N (1 ml) Agglutination-RBC Hu-IgM Hu-RBC (A) 0.4 ng of N (1 ml) Agglutination-RBC Hu-S Ragweed (BDB)- 0.1 ng of N (1 ml) RBC Bactericidal R-S Typhoid ng of N (0.1 ml) PCA (6 hr) R-S Egg albumin 3 ng of N (0.1 ml) PCA (6 hr) Hu-S Diphtheria toxin 13 ng of N (0.1 ml) PCA (3 hr) Hu-S A-substance 100 ng of N (0.1 ml) Reagin or IgE PK (48 hr) Hu-S HoSA I 580 pg of N (0.1 ml) PK (5 min) Hu-IgE Ragweed E I 50 pg of N (0.02 ml) PK (24 hr) Hu-S Ragweed E D 16 pg of N (0.05 ml) RCA-Hu R-anti-Hu- Hu-IgE I 3 pg of N (0.02 ml) IgE RCA-Hu GP-anti- Hu-IgE I 0.2 pg of N (0.02 ml) Hu-IgE WBC - H Hu Ragweed E I 0.8 pg of N (1 ml) WBC - H Hu Ragweed E I 8,000 pg of N (1 ml) a In some instances the data from the original sources were incorporated into the calculations used for the above values with the following values and assumptions. All proteins have 16% N; antigen and reagin combine in a 1:1 molecular ratio. Molecular weights used: reagin or IgE, 200,000; HoSA, 68,000; ragweed antigen E, 37,800 (202). Abbreviations: A, blood group A; C, complement; BDB, bisdiazobenzidine coupled; D, direct calculation of antibody N; G, goat; GP, guinea pig; H, histamine; Ho, horse; Hu, human; I, indirect calculation of antibody N; N, nitrogen; PCA, passive cutaneous anaphylaxis (in guinea pig); R, rabbit; RCA, reversed cutaneous anaphylaxis; S, serum; SA, serum albumin; WBC, white blood cells; RBC, red blood cells.

5 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 315 with the biologic properties of reagins. Experience since 1921 indicates that there is a good but not perfect association between wheal and erythema production by direct skin test and the presence of serum reagins as judged by PK test, using the same antigen for both tests. This association was noted early for "atopic reagins" but has been extended to such diverse antigens or haptens as penicillin and its derivatives, foreign serum proteins, or microbial and parasitic antigens. Furthermore, there is commonly a direct relationship between the size of the wheal of the direct skin test and the reciprocal PK titer of the same subject's serum (67, 68, 144, 242, 339). The lack of perfect correlation between the results of these two tests has been readily explained on the basis that some skin cells can absorb reagins to their surface for detection by direct skin test when the circulating reagins are at levels in the plasma undetectable by PK test. As a result, the direct skin test is usually a more sensitive means of detecting reagins in a subject for a given antigen than the PK test. Evidence from other approaches fortifies the concept that the whealing skin test signifies the presence of reagin. Complexes of antigen and specific IgE antibody (reagin) cause skin whealing upon injection, whereas complexes formed between antigen and antibodies of various other classes of Ig do not produce skin whealing under similar conditions (164). Or, the injection of animal anti-human IgE serum (163, 289) by Ishizaka and associates into the skin of humans caused whealing reactions, but the injection of similar antisera specific individually for human immunoglobulins A, D, G, or M (IgA, IgD, IgG, or IgM) failed to produce whealing. These experiments also tend to indicate that most kinds of human antibodies other than those of class IgE, which contain most reagins, will not produce whealing in human skin. But for final proof that reagins alone are responsible for direct skin test whealing, we need to exclude all other antibodies by past, and unfortunately, by future experience. Past experience is tempered by a relative lack of pertinent human, experimental data. The concept, which needs data for support or refutation, is that other serum antibodies, if present in the plasma or lymph at sufficient concentration, may produce a whealing reaction upon complexing locally with injected antigen. There are now a number of immune mechanisms studied in laboratory animal systems which have the potentials for releasing histamine and which probably do not involve the reagin or IgE categories of antibodies. Although only rarely have any of these mechanisms been incriminated in humans as alternate pathways for the production of whealing skin test reactions or anaphylaxis, they must still be considered as pretenders for these roles. Perhaps the oldest of the recognized mechanisms of immune histamine release in animals involves first the formation of anaphylotoxin from either of several components of serum complement (22) by an antigen-antibody precipitate. The anaphylotoxin so produced can then induce the release of both cytoplasmic granules and histamine from rat mast cells (282). Mechanisms of histamine release involving rabbit platelets, the major carrier of blood histamine in this species, can require complement or not (150). In one mechanism, a complex formed of antibody, antigen, and complement (Cl through C6 required) will induce histamine release from and lysis of platelets present. In another, perhaps due to a different kind of antibody, but studied with particulate antigens, the antigen-antibody complex requires C1 through C3 components of complement in order to produce a nonlytic release of histamine from the platelets. The addition of neutrophiles to either of these systems will augment the release of histamine from the platelets. In these interactions, the antibody seems not to be fixed to either the platelets or the neutrophiles. On the other hand, a nonlytic release of histamine occurs from rabbit platelets in mixture with leukocytes sensitized with a fixed antibody when antigen is added. Complement is not involved. The biological nature of the fixed antibody promoting this reaction remains speculative; it could be a reagin or not. In the suitably immunized rat there appears a heat-stable antibody which gives a homologous passive cutaneous anaphylaxis reaction with a 4-hr but not with a 24-hr latent period (281). It can also sensitize rat mast cells for the release of histamine by antigen without the intervention of complement. Platelets are not involved in the rat. Because of the peculiarities of the thermal stability and of the latent period as compared to those of typical reagin, this antibody is not classed as a reagin by most investigators, but, as will be brought to the fore later, this topic is subject to debate. How many of the mechanisms, especially those involving platelets, will be found to op-

6 316 FLICK erate in humans remains to be determined. Although the human platelet contains inappreciable amounts of histamine (136), it still remains possible that certain other cells in the body might substitute for the platelet in the types of mechanisms recognized to operate on rabbit systems. So far, human antibodies resembling those described above for the rat have been uncovered only occasionally. Kuhns and Pappenheimer (218) described two such human sera, OD and Fo, with antibodies against diphtheria toxoid. These sera failed to give positive conventional PK tests but caused whealing when the latent period for the PK test was reduced to no more than an hour or so. Recently Parish (308) has studied similar human antibodies that transfer successfully to monkey skin when latent periods of 2 to 4 hr, but not of 24 hr, were employed. This type of antibody was incapable of sensitizing guinea pig skin. Another explanation for the activity of these antibodies will be considered in Partial Reagins and Queer Reagins (p. 339). I have been unable to find other examples of nonreaginic whealing antibodies for humans. Usually other antibodies seem to be specific blockers of reagin activity rather than producers of wheals. When penicillin is administered therapeutically, a high percentage of the recipients subsequently develop antibodies against penicillin as judged by hemagglutination using penicillin coupled to red cells (233). In the majority of cases recorded, blood containing these hemagglutinins (either IgM or IgG) to penicillin failed to sensitize the patient for whealing by direct skin testing with penicillin or its derivatives, or for the production of urticaria by systemically administered penicillin (99, 100, 233, 238, 240). In those cases where sensitization for immediate type allergy has occurred, usually there were penicillin-specific reagins also present or present alone. Patients rarely have been observed (43) who gave negative skin tests but whose serum collected concurrently produced a positive PK test with the same antigen. The stimulus for doing a PK test in the face of a negative skin test was usually a previously generalized urticarial reaction on the part of the patient to the antigen. In several such cases (240, 339) allergic to penicillin, the direct skin tests ultimately became positive a few days after harvesting the serum that produced the positive PK test. A number of good, alternative explanations are possible other than the assumption that a nonreaginic antibody was responsible. BACTERIOL. REV. The patient's skin may have been temporarily refractory to whealing, or blocking antibody may have been of sufficient temporary activity to prevent the direct reaction, but not the PK test; or the patient's skin may have been less sensitive to reagin-antigen interaction than the PK recipient's skin. Finally, reagin-like allergic reactions have been reported only rarely in humans where careful investigation by direct skin test and PK test seems to rule out the mediation of reagins. Schmidt et al. (367) reported observations dealing with a woman who developed anaphylaxis on two occasions upon receiving only 10 ml of human blood. Her serum contained an IgG precipitin against human serum IgA globulin, a protein undetectable in her blood. She lacked reagins to allogeneic serum antigens. Circumstantial evidence suggested that the antigen responsible for the anaphylaxis was human IgA globulin. A similar case was studied in detail by Vyas et al. (429). Although the patient reacted to the intravenous injection of human plasma containing IgA (but no reaction to IgA-free plasma) with anaphylaxis, including urticaria, the skin test with purified IgA failed to elicit a whealing reaction. Animal models of nonreaginic shock involving other antibody mechanisms (150), as outlined above, could explain this human situation, but also reagin mechanisms, as well as nonimmune mechanisms, cannot be completely eliminated. These lines of evidence lead me to believe that antibodies other than reagins rarely mediate the wheal of the direct skin test. The validity of this conclusion will still require continuing critical appraisal and will depend, in part, upon the prevailing definition of reagin. Based on these conclusions, one can interpret, with possibly a small percentage of error, that the direct whealing skin test indicates the presence of reagin. Henceforth, for ease of expression in this review, the antibody responsible for a wheal response by direct or by PK skin test will be termed reagin. For clarity, the type of skin test used will be indicated generally. REAGIN PRODUCTION Types of Antigens Producing Reagins Ever since Blackley in 1873 (39) demonstrated that he could incite an attack of hayfever by transferring pollen from a flower to the mucosa of his own nose via his pollenpicking finger, inhalant antigens have been recognized as associated commonly with atopic

7 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 317 disorders and, hence, with reagin production. Aside from inhalant pollens, fungi, and food antigens, which have been regarded as common reagin producers, many other types and sources of antigens have induced, on occasion, specific reagin formation. Foreign serum proteins commonly produce reagins as judged by direct skin and PK tests (13, 198, 285, 335, 344, 382, 421). Many drugs on occasion will immunize a person for reagin production and cause urticaria. The most notable of these is penicillin, and its derivatives formed in the body (233). Even heavy metals occasionally stimulate specific reagin formation as determined by PK tests (41). Antigenic products of infectious agents, especially those of worms, are common producers of reagins. Thus, ascaris antigens readily induce reagin production in humans as indicated by both direct and PK tests (44, 45, 80, 326). African children with verified ascariasis have an average serum IgE level 28 times that measured in Swedish children presumably free of worm infections (192). Other worm infections also produce reagin formation regularly (44, 121, 332, 411, 412). Although there is a paucity of pertinent data, other types of infectious agents can induce reagin formation. I have observed occasionally the presence of reagins by direct and PK tests against OT or PPD, or both, in persons having received BCG vaccination or a natural tuberculous infection. In cases where OT only gives a positive skin reaction, one cannot eliminate the possibility that the reagin is directed against some nonmycobacterial component of the culture medium without using suitable controls; but PPD is prepared from a synthetic medium (372, 373), making it very likely that only mycobacterial antigens are involved. Sulzberger and Kerr (407) observed patients with Trichophyton infection who developed reagins by PK test to products of the fungus. Silverman and Efron (390) described four persons with chronic bacillary dysentery who reacted upon skin testing with a vaccine of Shigella paradysenteriae with local whealing, generalized symptoms resembling those of histamine intoxication, and an exacerbation of the dysentery. Special Case of Polysaccharide Antigens Polysaccharide antigens have represented to me a special category with respect to reagin production because there has been a gradual accumulation over the years of suggestive evidence that as a group they can regularly stimulate specific reagin formation. In 1929, Tillett and Francis (416) observed that patients recovering from pneumococcal pneumonia develop a whealing reaction upon skin testing with the capsular polysaccharide of the same type specificity as the pneumococcus causing the disease. Subsequent studies (3, 4, 101, 105, 106, 115, 259, 351) confirmed that recuperating, pneumonococcal pneumonia patients and many normal persons possess type-specific reagins (by direct skin test only) to pneumococcal polysaccharides. (Because the administration of rabbit anti-pneumococcal serum to patients prior to skin testing can be responsible for whealing responses and thus cloud the interpretation of the skin test results, those references dealing solely with antiserumtreated patients have been omitted.) Since carrier states are common for the prevalent pneumococcal types, one would expect immunological reflection of this. Thus, Felton and Prather (101) observed that before and after immunization with pneumococcus type 1, about 48% and 84%, respectively, of adults gave whealing to the type 1 polysaccharide. Other polysaccharides have given similar results. Purified dextrans (196, 232, 271) and staphylococcal polysaccharide (195, 197), as well as staphylococcal taichoic acid (266, 418), produce whealing reactions by skin test in humans. Taichoic acid probably represents the immunologically active polysaccharide (405, 418) that was originally isolated and used for skin testing by Julianelle and Hartman (195). Barker et al. (24) isolated a glycoprotein from Trichophyton mentagrophytes, a cause of human ringworm, and presented evidence indicating that the active component responsible for the skin whealing of infected persons is a polysaccharide. On a similar basis, an allergen of the inhalant fungus Alternaria seemed to be a polysaccharide at least for some individuals (402). Kropp and Foley (207) isolated a polysaccharide from the tubercle bacillus. This antigen produced whealing upon skin testing in some active cases of tuberculosis and in some normal subjects who presumably had healed infections. But not all reports are favorable for the generalization that polysaccharides are reagin producers. A polysaccharide capable of precipitating with immune serum was isolated from a strain of gonococcus by Casper (50). It failed to induce skin whealing in patients with gonorrhea. Staphylococcal taichoic acid failed to release histamine from presumably reagin-sensi-

8 318 FLICK BACTERIOL. REV. tized leukocytes in vitro (267) although a staphylococcal protein antigen was capable of such activity using the same patient's leukocytes. The detection of antipolysaccharide reagins by PK test in the sera of subjects who give whealing by direct skin test has been reported upon only infrequently. Kabat and Berg (196) found only negative PK tests with such antidextran sera. Palczuk and Flick (305) observed that some immunized subjects had reagins by PK test against pneumococcal polysaccharides, but that the PK test results were nullified in many but not all recipients by the occurrence of a reagin-blocking, skin-fixing antibody in the donor's serum. The presence of this blocking, fixing antibody would then tend to lower the incidence of reagins detectable by PK test. Rogers and Wagner (351) reported that 5% of 40 sera from persons whealing upon direct injection of pneumococcal type 1 polysaccharide immunization with specific reagin production arises from the inability to estimate the role played by possible minute protein impurities in the polysaccharide prepawhealing caused by polysaccharides, like whealing by other antigens, is mediated by reagins. Perhaps the greatest difficulty for the proper interpretation of these data connecting polysaccharide immunization with specific reagin production arises from the inability to estimate the role played by possible minute protein impurities in the polysaccharide preparations. Vaughan and Kabat (428) and Finger and Kabat (102) have emphasized that minor antigenic impurities can be responsible for reagin activity. Even the type specificity shown by the pneumococcal polysaccharides in their interaction with reagins may be insufficient evidence to completely incriminate the polysaccharide as the reactive antigen, because Austrian and McLeod (23) have described pneumococcal type-specific protein antigens. The latter could be responsible for causing false interpretations. Reagin Production in Nonatopics It has been held that only atopic persons or potentially atopic persons can produce reagins. To counter this belief one need only produce reagins in normal persons, except that one cannot as yet distinguish the normal from the person who has the atopic trait but who does not express it clinically and may never express it. Another approach, the one attempted here, is to determine in the general population if the ability to produce reagins markedly exceeds the 20% estimated incidence of atopy. Evidence bearing on the incidence of reagin production can be derived from two types of situations involving iatric immunizations of humans: (i) from the maximal incidence of serum sickness developed from the prior injection of therapeutic foreign serum as the antigen, and (ii) data derived from persons experimentally immunized with a variety of special antigens, the most noteworthy one being ascaris antigen. A study of the immune responses in persons with worm infections also provides acceptable data, and finally, ancillary evidence has been derived recently from measurements on IgE production. The most common manifestations of human serum sickness are skin rash, soft tissue edema, arthralgia, fever, and lymphadenopathy. By far the most common skin rashes (probably better than 90% incidence) are urticaria or diffuse erythema (406), or both. These rashes, the edema and arthralgia, are best ascribable to histamine release from a reaginantigen union in the body. It seems highly unlikely that these particular allergic manifestations are due to specific precipitins also frequently present in these patients, and which seem responsible for the necrotizing arteritis type of serum disease studied in rabbits. The reagin mediation of the two common rashes of serum sickness is based on several lines of evidence: (i) the similarity between the morphology of the PK reaction and that of the rash; (ii) a good degree of correlation between onset of human serum sickness and the time of conversion of the direct skin test from negative to a whealing reaction using the foreign serum as antigen (254, 420, 421); and (iii) a fair correlation between the disease and early reagin appearance in the serum by PK test (74, 335, 344, 421, 422). Of 45 patients with serum sickness recorded in these five reports, 82% became PK test positive. Often the serum sickness reagins disappear rapidly from the patient's blood (422) so that proper timing of blood sampling for reagins is important in order to obtain the best correlation. Thus, for these positive reasons, we can reasonably assume that the occurrence of the disease, serum sickness, is a valid indicator of human reagin production. The incidence of serum sickness following therapeutic foreign antiserum injections varies greatly depending upon such factors as the dose of antigen (406), number of injections of antigen (135), route of administration, degree

9 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 319 of refinement of the serum globulins, perhaps, trials years earlier probably because of the use and other factors, but probably has an overall of larger doses of antigen or of adjuvants. incidence of 20%. Thus, Sturtevant (406) in Thus, by employing water-oil emulsions of antigen, Feinberg and his associates in a series of 1916 reported 17%; Gordon et al. (135) in 1929, 32%; Davis (81) in 1938, 24%; Arbesman et al. studies produced reagins as judged by direct (13) in 1960, 20%; but Longcope and skin tests in 21% (94), in 46% (28), and in 62% Rackeman (254) in 1918, reported 84%. The (392) of nonatopics and in 100% (392) of overall incidence does not seem appreciably atopics. Malley and Perlman (264) obtained an different from the incidence of atopy. Yet further examination of the data of Gordon et al. by both direct skin and PK tests in a small incidence of 75% reagin production detected (135) indicates that the incidence of serum group of normal persons. sickness increased progressively with an increase in the number of sequential doses of indicates that normal and atopic persons can Thus, the evidence from a variety of sources horse serum administered. Thus, the incidence produce reagins when given a suitable stimulus. There is suggestive evidence at hand that after one dose was 18%; after two doses, 43%; and in 556 cases receiving four doses, 74%. atopics may produce reagins faster, or to a This latter figure is probably sufficiently different from the 20% incidence of atopy and However, Adams (1) concluded that, following higher titer, than normals (214, 360, 392). seems to indicate that most humans, given a vaccination, atopics produce antityphoid agglutinins (nonreaginic) to a significantly higher sufficient challenge, can develop serum sickness, or more importantly here, can produce titer than did normal persons. Levine and Vaz reagins. (239) similarly found in eight strains of inbred An extract of Ascaris lumbricoides as antigen has been used to produce reagins in good specific reagin production occurred in mice that, upon suitable antigenic stimulation, people. The incidence of reagin production, as those mice that were also good immunologic in serum sickness, depends upon the number responders by other criteria. Conversely, mice of injections of antigen given. Davidson et al. of strains that were poor reagin responders (80) produced reagins in 60% of their subjects failed to produce specific antibodies of the with six immunizing doses and in 85% with 12 nonreagin category. Caution is needed in interpreting the latter type of correlation involving doses. The subjects were mostly nonatopic. Zohn (443) successfully stimulated reagin production with the same antigen in all of 11 non- indicates that upon active immunization, at a failure of antibody production. Experience atopic pregnant women. In these studies, the times, animals will produce a very limited sort judgment that reagins were produced was of antibody response, in that rather than producing the usual broad assortment of kinds of based on a whealing direct skin test in all and a confirmatory positive PK test in some. antibodies, only a single kind will appear. Kuhns and McCarty (216) used diphtheria Such a limited response, although in itself toxoid to immunize 104 rheumatics and 91 perhaps a good one, may not be uncovered by nonrheumatics, neither group being selected the assay technique chosen. It is evident that with any regard for atopy. The incidence of further studies of a critical sort are needed to direct skin test whealing with the antigen after clarify the difference, if any, between atopics immunization was 78% and 88%, respectively, and normals with respect to reagin production in the two groups, an approximate twofold increase in positive skin tests as compared to the Persons selected not for atopy but for pos- or antibody production in general. preimmunization status. sessing natural worm infections usually develop reagins against the worm antigens. Thus, Bovine deoxyribonuclease, as an alum vaccine, was used by Salveggio and Leskowitz 82% of 23 persons with proven Wuchereria (359) for human immunization. Reagin production, mostly by skin test but also by PK test in with proven Schistosoma rnansoni infection bancrofti infection (411) and 87% of 46 persons some, occurred in 66% of nonatopic and in 75% (412) gave direct skin test whealing reactions of atopic subjects; a difference probably not to suitable worm extracts. From both groups, significant. 46% of 15 persons with positive skin tests furnished sera that yielded positive PK tests. In Pollen antigens have been used successfully now for reagin production both in normal persons and in atopics who lack reagins specific tive transfer tests for reagins when obtained one recent study (90) 81% of 16 sera gave posi- for the antigen employed as a vaccine. The from persons with proven schistosomiasis; in rate of success has increased as compared to another (151), 80% of five proven cases of larval

10 320 FLICK BACTERIOL. REV. migrans had reagins by passive transfer. Recent data concerning the presence of IgE tend to fortify the foregoing information about reagins. The skin testing of humans with foreign anti-human IgE serum (6, 163, 289) indicates that most people, normal or atopic, possess some IgE, the major reagin-bearing Ig class. Quantitative serum IgE measurements indicate (34, 188, 189, 191, 192) that atopics have a higher average serum concentration of this class of globulin than normals, although there is considerable overlap in the ranges. Intradermal Antigen and Reagin Production There is suggestive evidence that immunization is more apt to produce specific reagin if the antigen is injected intradermally rather than by other routes in humans. When Kuhns (213) immunized 16 persons with 10 intradermal doses of diphtheria toxoid, 100% developed reagins, whereas when 17 other persons were given the same total dose of toxoid, but as a single subcutaneous injection, only 53% produced reagins as judged by a whealing direct skin test subsequently. Groups of eight to thirteen subjects in the Rostenberg and Welch study (354) were given the same total dose of penicillin, but by different routes and with either 2 or 5 days between each of three series of injections. In the group receiving intradermal injections with a 5-day interval between injection series, 100% subsequently developed whealing skin tests indicating reagin formation. The incidence of reagin formation in all other groups was zero, including the group that received intradermal injections with a 2-day interval between series. Since no alternative-route group reported was strictly comparable in design to the one developing 100% results, the interpretation is open to question. Similarly, the following recorded observations tend to add some reinforcement to the belief that the skin has an adjuvant effect in producing reagins, but they lack the power of well controlled experiments. Recipients used for PK testing are given the challenging injection of antigen by intradermal injection and are selected for the property of being nonreactive to the antigen used in the PK tests. Tuft (391) provoked reagin production in 29% of 17 such recipients each of whom received a total dose of only to ml of horse serum as antigen. No alternative-route group is available for comparison, but the incidence of serum sickness from much larger injections of horse serum given by the intramuscular or intravenous routes is probably not significantly different from this figure. Richter et al. (349) and Connell (65) also noted that some of their PK recipients developed reagins rather easily to the challenging antigen. In these three reports the interpretation that reagins were produced is based on direct skin testing only. Malley and Perlman (264) stimulated reagin formation, as indicated by both skin tests and PK tests in 75% of eight normal subjects given four intradermal doses of purified timothy antigen D. Mote and Jones (285) injected 79 rheumatic children intradermally with rabbit serum; 54% developed evidence of reagin formation by subsequent direct skin testing. The emphasis on reagin production by the intradermal route should not imply that this route will induce only reagin formation. There is much evidence, here ignored, that it is an excellent route for production of other kinds of antibodies and of delayed hypersensitivity. OCCURRENCE OF REAGINS IN BODY FLUIDS In addition to the occurrence of reagins in the serum, a number of other body fluids have been examined for the presence of reagins by PK test or by tests which detect the presence of IgE. These fluids have been derived from atopic subjects possessing detectable reagins for specific antigens in their sera or from normal and allergic subjects for IgE assay. Nasal secretions (86, 180, 290, 345, 365) and nasal polyp fluid (31) can contain reagins or concentrations of IgE that are often considerably higher than the values for IgE in the atopic patient's serum, suggesting local production to a degree (87). Saliva or sputum (86, 157, 180, 290) as well as tears (374) can contain reagins or detectable levels of IgE, especially if derived from atopics. Skin blister lymph can contain reagins (31, 311, 391) but spinal fluid, even when the serum reagin titer is high, so far has lacked detectable reagins (252). Urine contains detectable quantities of IgE, which Turner et al. (423) indicate is probably produced locally rather than being cleared from the plasma. IgE-containing plasma cells (probably representing the synthesis of IgE) were found scattered among respiratory and intestinal epithelial cells of humans (410). Such IgE plasma cells also were prominently present in the adenoids and tonsils.

11 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 321 BIOLOGIC ACTION OF REAGINS Reagin Shock Tissues Tissues shocked by the reagin-antigen interaction must be capable of responding to the histamine and other physiologically active substances that are released by this union. Because histamine is readily diffusible and can be rapidly transported to distant target cells in the body, it is difficult at times to be sure if a local tissue allergic response is due to locally fixed reagin or to distantly fixed reagin uniting with antigen. But under conditions of relatively low concentrations of antigen applied locally with evidence of lack of spread of the ensuing allergic reaction to distant structures, one can pinpoint the tissue involved in the fixation of reagin and undergoing the local shock reaction. The best and oldest example of this is the PK test itself, which indicates both sensitization and shock of dermal elements. Samsfe-Jensen and Kristensen (364) and later Parish (307) found that human skin pieces absorb reagin from serum in vitro whereas certain other tissues prepared similarly do not. Loveless (256) transfused reagin-rich serum into normal persons and at intervals tested tissues for local allergic reactivity to antigen as an indication of reagin fixation. The skin became reactive to antigen at about 1.25 hr after the intravenous injection of reagin, the conjunctiva at 2.75 hr, and the nasal mucosa in 24 hr. Because of the timing, the small local dose of antigen used, and the nonspreading character of the reactions, this information pinpoints reagin fixation to the reactive tissues. Freeman (118) discovered that application of atopic serum and antigen in succession to one subject's nasal mucosa caused a typical local hayfever type of reaction. In atopics the lungs can respond to the reagin-antigen union with clinical asthma, a result of bronchiolar constriction and thick mucous production, which cause a reduced vital capacity. Asthma has been considered to be due, at least in part, to histamine release and it is of interest that the intravenous injection of histamine in adequate amounts will reproduce the asthma syndrome in persons who have asthmatic attacks (77, 88, 352) or in persons with certain other types of pulmonary disease (434) but generally not in persons who lack pulmonary disease (78, 88, 435). Pieces of viable, asthmatic (atopic), human lung upon stimulation in vitro with the specific antigen will release histamine into the fluid phase and also show constriction of the bronchiolar smooth muscle (366). Human lung pieces can be successfully sensitized passively in vitro as determined by either loss of reagin from the absorbed serum (307) or by subsequent histamine release by addition of antigen (273, 306, 375). However, Sheard et al. (375) recorded that 9% of lungs tested failed to shock by this technique using known active sera. The human intestinal tract clinically shows evidence of reacting (usually excessive contractions) in an allergic manner, and in vitro experiments support this contention. Discarded human appendices can be passively sensitized in vitro (59, 204) with reaginic sera for shock with antigen. Pieces of human ileum and gravid uterus also can be sensitized similarly by reagins (417) with about 50% success. All of the diverse tissues susceptible to reagin sensitization and shock have in common the presence of scattered mast cells. The current evidence incriminates the mast cell as the cell type that fixes reagins and releases histamine upon antigen addition. The evidence derived from subprimate animals (21) fortifies that obtained in humans and monkeys. The fluorescent-antibody studies of Rappaport (337, 338) attempted to identify the cells of the human skin that fixed reagin. Unfortunately his technique could not differentiate cell-fixed reagin in union with antigen from other kinds of antibodies that might be fixed to cells and also be in union with the antigen. By using a somewhat different fluorescent-antibody technique, Hubscher et al. (155) were able to identify the cell in monkey skin that fixed human IgE as the mast cell. However, it would be difficult to exclude some degree of IgE fixation to epidermal or glandular cells because of the stray background fluorescence of these cells in their photomicrographs. Parish (306), using pieces of human lung sensitized passively with reaginic sera, positively correlated three effects of the addition of antigen; the release of histamine, of SRS-A (slow reactive substance of anaphylaxis that causes a slow contraction of smooth muscle), and of the basophilic granules of mast cells. Heating the sensitizing serum at 56 C for 0.5 hr prevented the release of all of these substances. Monkey lung, which presumably reacts like that of humans, also seems to release both histamine and SRS-A when the shock is mediated by IgE but not by other classes of human Ig (184, 297). However, in rats the SRS-A seems to be derived from neutrophiles (298). Also, in human tissue some uncertainty exists that all the SRS-A released is due to reagin in the human sera

12 322 FLICK (375). There is a lack of evidence to indicate that the mast cell (or its blood counterpart, the basophile) is the only cell type that can fix reagin, or even that it is the only cell type which can be sensitized by reagin for antigen release of active products like histamine in the human. The ability of the blood basophile to liberate histamine when activated by antibody, especially reagin, and antigen will be considered in detail in Use of Leukocytes (p. 342) and Basophile-Mast Cell Degranulation (p. 344). Human Skin Susceptibility to Reagin Shock Human skin varies in its responsiveness to sensitization by reagin in the PK test. Coca and Grove (61) noted that 11% of their normal PK recipients failed to react. Subsequent studies have indicated that such PK nonreactors do exist but that the incidence varies from 0% of 65 subjects of Walzer (430) to 2% of 400 subjects of Lippard and Schmidt (251) and to 4% of 25 subjects of Pedersen-Bjergaard (317) with an overall incidence of 2% of 490 subjects. In some cases a recipient fails to react with a reagin of one specificity but can react by skin test to an antigen of another (430). But an occasional individual fails to react in the PK test with all of several specificities tried (317). It is of interest that, in the case of some monkeys, failure to respond to PK (or to passive cutaneous anaphylaxis [PCA]) testing is associated with an inability to react to histamine in the skin (317). The responsiveness of individual humans as PK recipients is not limited to the all-or-none categories; variation in degree also occurs (380), due certainly to a variety of factors. Reagin Shock and Univalent Haptens Patients with clinical evidence of reaginmediated allergy due to a drug or chemical, upon being skin tested with the offensive hapten, fail to wheal at the site in the majority of cases. Two explanations have emerged to account for this common inability to detect drug-specific reagins by skin testing: (i) the hapten used for testing is of the wrong specificity for the responsible reagin; a by-product formed from the hapten in the body actually corresponds in specificity to the reagin; and (ii) the proper hapten must be in a multivalent form in order to effect a release of histamine from the reagin-sensitized cell. However, in some cases of penicillin allergy, direct skin tests with penicillin itself or with one of its simple derivatives do produce whealing. This is usually interpreted to be the result of rapid BACTERIOL. REV. complexing between the univalent hapten and proteins in the skin to form multivalent derivatives. This concept, the classical theory, rests on many observations commencing with those of Landsteiner (220, 221) and of Tillet et al. (304), that univalent haptens incapable of coupling to proteins fail to produce anaphylaxis in sensitized animals and, indeed, will silently, specifically desensitize such animals for subsequent challenge with a multivalent form of the hapten. A similar situation commonly but not always exists in relation to human anti-drug reagins. For example, in PK tests, univalent phenyl mercuric cysteine, used as allergen, failed to shock the sensitized site, and at once desensitized the site for subsequent challenge with the multivalent phenyl mercuric protein (270). Similarly, benzyl-penicilloyl- E-amino caproate (BPO-EACA) in suitably sensitized animals (85, 234) acts entirely as theory predicts for a univalent hapten, and also behaves in an analogous manner in human direct skin testing. Selected penicillin-allergic subjects wheal by direct skin test with multivalent benzyl-penicilloyl-polylysine, fail to wheal with univalent BPO-EACA, and show depressed whealing with mixtures of the uni- and multivalent forms (104, 234, 236, 309). Dinitrobenzene in its uni- and multivalent forms acted similarly in whealing skin tests of an allergic author (92). Thus, for many human reaginic situations, the above concept expressing a requirement for the multivalence of a hapten in order to cause shock seems to fit the observations, but not entirely. A minor fraction of penicillin-allergic persons will wheal rapidly when skin tested with benzyl-penicillin, a substance that does not form covalent bond complexes detectably at the ph of body fluids according to the work of Josephson et al. (193). Furthermore, a small number of these persons have whealed when skin tested with univalent BPO-EACA (104, 236, 309, 387), a penicilloyl derivative generally thought to be blocked from further complexing with proteins. Can the classical theory still explain these results with seemingly univalent haptens? It could if there existed allergically active, multivalent contaminants in the penicillin derivatives used for skin testing. Batchelor et al. (26) have shown that some batches of commercial penicillin contain penicilloyl-protein complexes and even polymerized penicillin derivatives, although the latter were found to be immunologically inactive in their studies. Yet their report includes the study of two exceptional human reaginic sera with which protein-free benzyl-penicillin caused whealing of PK sites,

13 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 323 whereas the purified, isolated penicilloyl-protein did not, indicating that in some cases the contaminating protein derivative was inactive allergically. A few more subjects, included in other studies (18, 203), yielded similar skin test results when protein-free benzyl-penicillin was used. Unfortunately, reports have not as yet appeared on the successful use of BPO- EACA preparations proven free of multivalent contaminants that might be active in skin tests. Thus, for the exceptional situation, one has a choice of two theories to explain the observed skin test results. If the classical theory holds, faith must be placed upon the invariant existence of a multivalent contaminant in a given drug solution as the allergically active ingredient. The alternate theory that some samples of reaginic antibodies have the capacity to set off histamine release upon union with a univalent hapten seems somewhat more plausible. Further work is needed to add support for or against the latter concept. As a start, Raffel and his co-workers (5, 120, 138) have provided convincing evidence that univalent haptens can elicit heterologous (nonreaginic) PCA reactions with some animal antisera. Based on the results of their work with uniand multivalent forms of penicilloyl derivatives, Levine and Fellner (235) suggested that the mediation of immune histamine release required a linking or a bridging by a multivalent antigen of two cell-fixed antibody molecules. Subsequently, Stanworth (396) proposed that the union of antigen with fixed antibody during bridging caused a change in the steric folding of the peptide chains of the antibody molecules, and that the latter event initiated the cell membrane changes leading to histamine release. Such allosteric alterations do occur in human and rabbit precipitins when immunoglobulin is caused to aggregate either by union with specific antigen or by chemical or thermal treatment (148, 149), as judged by the appearance, upon aggregation, of a new antigenic determinant on the immunoglobulin molecule. Heating likewise produces a new determinant on human IgE molecules (187). A univalent antigen, theoretically incapable of combining with two antibody molecules simultaneously, could not effect bridging nor the release of histamine. Ishizaka et al. (164, 167) added evidence in support of the bridging theory. In several in vitro cellular systems, bivalent anti-ige, or its bivalent fragment F(ab')2 produced histamine release upon uniting with cell-fixed IgE, whereas the corresponding univalent fragment Fab', proven to possess antibody activity, failed to release histamine under similar circumstances. Further support can be found in the observations (164) that specific IgE-antigen complexes formed in solution and probably having molecular ratios of AgAbl or Ag2Ab3 based on measured sedimentation constants cause whealing upon intradermal injection, whereas antigen-excess complexes of ratio Ag2Ab do not. The former complexes can be involved in bridging upon attachment to cell surfaces, the latter complex predictably cannot. Thus, the evidence favors the conceptual requirement of bivalence minimally for antigen in order to have histamine release. But it is still possible that certain aberrant reagin molecules, because of structural peculiarities, might effect histamine release when triggered by a univalent form of antigen. Reagin Reactions and Eosinophiles Clinically one often finds an association between a reagin-antigen reaction and the local or systemic increase in eosinophilic leukocytes. During hayfever attacks, eosinophiles become numerous in the nasal secretions. Worm infections, especially trichinosis, often induce an eosinophilia, probably associated with a continuing reagin-antigen (of the worm) interaction in the tissues (44). On the other hand, eosinophilia is not a common finding according to Longcope (253) in human serum sickness, in which the most prominent symptoms are attributable to reagin-antigen reactivity. Eidinger et al. (91) used the skin window technique of Rebuck to measure the leukocyte response to applied antigen in atopic persons. Superimposed upon the expected allergic response of the skin was a gradual increase in eosinophiles in the area of the reaction continuing even after subsidence of the gross allergic reaction. They believed that histamine, but not bradykinin nor serotonin, would also cause an early, transitory eosinophile infiltration in atopics only. Feinberg et al. (95) confirmed these findings for both antigen and histamine and also observed that shocking a PK test site in normal recipients similarly led to a local eosinophile infiltrate. But Felarca and Lowell (98) disagreed that histamine causes a greater eosinophile response than saline in atopics, based on a technique involving a coding of the test and control sites used for cell counts. All agree, however, that antigenreagin, allergic, local reactions attract eosinophiles.

14 324 FLICK Zolov and Levine (444) gave doses of penicillin to persons with antipenicillin antibodies and studied them for an eosinophilia, allergic reactions, and the type of antibody present. Eleven of twelve persons who developed eosinophilia had detectable reagins against penicillin derivatives (by direct skin test, some confirmed by PK test). There was a lack of association between the presence of antipenicillin of IgG or IgM class and eosinophilia. It is presumed that other, nonimmunologic mechanisms can produce an eosinophile response in humans. PROPERTIES OF REAGINS Heat Inactivation The work of Coca and Grove (61), early in reagin history, laid the groundwork for differentiating reagins from other antibodies on the basis of sensitivity to heat. They used a temperature-time relationship known to inactivate complement, 56 C for 0.5 hr, and observed that these conditions incompletely inactivated reagins in a few samples of serum. Gradually the problem emerged to find the proper minimal conditions which would inactivate and, thereby, characterize all reagins derived from atopic persons. It became evident that the conditions 56 C for 4 hr or less would produce 100% inactivation of reagins in most atopic sera (9, 131, 176, 243, 255, 348, 353, 390). A somewhat less commonly used set of conditions quite comparable in results is 60 C for 1 hr or less (42, 255, 257). But much variation in heat lability occurs. At one end of the scale is a single, atopic, reaginic serum reported by Flick and Feinberg (113) that was inactivated completely at 37 C for 2 hr. At the other end are occasional sera from atopics requiring greater temperatures or longer periods of treatment for complete inactivation. Thus, Loveless recorded occasional sera requiring 5 hr at 56 C (255) or 2 hr at 60 C (257) for complete inactivation. Schmidt and Lippard (368) found one serum requiring 7 hr at 56 C, and Richter et al. (348) found three sera needing more than 8 hr at 56 C for complete inactivation. Within these extreme conditions needed for atopic reagin inactivation there is still much variation in heat lability as indicated by studies of the amount of inactivation with increments of time (9, 255, 353). Although variability in thermal inactivation suggests the existence of some sort of responsible variability in the structure of reagin molecules, an alternate viewpoint is that the former may be related to variation in the com- BACTERIOL. REV. position of the serum samples used. The study of Richter et al. (348) would tend to negate this influence. Three atopic sera, each containing reagins of two different specificities, were examined for heat sensitivity. In each case, reagin of one specificity was inactivated completely at 56 C for 2 hr, whereas the other reagin showed residual partial activity even after 8 hr at 56 C. The influence of associated serum constituents on the heat lability itself appears to be nil, based on studies of partially purified reagins. Ishizaka et al. (176) were able to inactivate by 56 C for 4 hr the specific reagin contained in a nearly pure sample of IgE. Goodfriend and Perelmutter (131) fractionated two atopic sera by chromatography. In each case the reagin contained in the early fraction was inactivated at 56 C for 0.5 hr, whereas the reagin in a late fraction required 56 C for 1 hr. These observations, then, tend to incriminate the molecular structure of the reagin rather than the associated serum constituents as the primary cause of thermal lability or its variability. At one time it was thought that heat lability could be used to differentiate atopic from nonatopic reagins or, to use other equivalent terms of questionable value in common use, to differentiate natural or spontaneous reagins from induced or immune reagins, respectively. Reagins occurring in the serum of persons having experienced serum sickness from the therapeutic injection of horse immune serum were judged to be heat stable. Largely for this reason such antibodies were and are now considered not to be reagins by many, even though they were detected by PK test. Thus, human serum sickness reagins (i.e., PK antibodies) in early reports were found to be resistant to 56 C for 1 hr (335), for 1.5 hr (198), and for 4 hr (73) in one case of partial inactivation. The total number of sera studied in these reports is small. Variation in heat lability was uncovered by Terr and Bentz (414) who fractionated one of three sera from serum sickness cases in which the reagins were considered heat stable when measured in the whole serum. The reagin which separated with the -y2-globulin was resistant to 56 C for 4 hr whereas the reagin separating with the -y M- globulin was completely inactivated by these same conditions. Other investigators using 56 C for 4 hr have usually found that artificially induced or immune reagins are heat labile. Thus, Sherman et al. (382) using seven bleedings from three serum sickness cases, Fisher and Cooke (111) with four serum sickness sera

15 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 325 and one serum containing antipneumococcal S-3 reagins, Kuhns and Pappenheimer (218) with serum Hu containing antidiphtheria toxin reagins, and Siegel and Levine (388) with two sera from cases of serum sickness due to penicillin found only heat-labile reagins present. The data indicate to me that reagins, whether atopic or nonatopic, natural or immune in origin, are usually heat labile; however, some more than others, and an occasional example may be relatively resistant to heat. The mechanism of the inactivating effect of heat upon reagin remains largely unknown although the evidence presented by Ishizaka et al. (176) indicates that adequate heating somehow prevents IgE (reagin) from fixing to the skin by inducing changes in the structure of the Fc fragment. For reagins to be classified distinctively as heat labile, other antibodies should then be heat stable. This has usually been the case with human antibodies, but not always so, depending in part upon the type of serological technique that is used to measure the antibody activity. Reagin-blocking antibodies are usually heat stable. Human (63, 209, 393) and laboratory animal (63, 111, 125, 272, 328) immune sera which, when unheated, are capable of precipitating antigen, can lose the precipitating ability following heating at 56 C for 0.5 to 5 hr. The loss of precipitating ability may be partial or complete. However, such heated sera usually do not show a concomitant loss of PCA activity (111, 272, 328) for guinea pigs, a significant decrease in hemagglutinating activity (393), nor decrease in toxin neutralizing ability (63, 209). Human or animal precipitin that has been separated from serum albumin is no longer inactivated by heat (63) but, if the albumin is restored, the antibody again becomes susceptible to heat inactivation for precipitin. These observations have prompted the conclusion that heating of immune serum causes a complexing of the precipitating antibody with albumin or some constituent therein. Thus, in mechanism, the heat inactivation" of precipitating ability, when it occurs, seems to be on a different basis than the corresponding inactivation of reagins, and does not seem to disturb the biological activities of such antibodies. It is evident that, although heat inactivation of biologic activity is mostly a unique property of reagin, there exists considerable variability among different reagins in their responsiveness to heat. Because of this variability, I prefer not to incorporate this characteristic into the definition of reagin although the majority of workers in the field do. Also, from the available data, heat lability does not seem to be a property that is useful in differentiating atopic from nonatopic reagin. Thiol Sensitivity Just as reagins show variable susceptibility to heat so do they to reductive inactivation by thiols. But this property is not unique for reagins because, following the initial observations of Deutsch and Morton (84) that 0.1 M mercaptoethanol split 19S globulin into 7S subunits, the technique was used mainly to differentiate 19S antibody, which the treatment inactivated, from 7S antibody, which survived such treatment (55, 122). A further complication was introduced by the observations of Rockey and Kunkel (350) that some samples of IgA antibody (probably polymerized) were inactivated by this concentration of mercaptoethanol. With this background, a series of reports (127, 131, 140, 166, 350, 436) indicated that 0.1 M thiol would inactivate the reagins in somewhat more than a dozen atopic sera so studied. Although the concentration of thiol used in these studies was uniform, the time and temperature of treatment were variable factors that are important (131, 342) in deciding if an antibody is inactivated or not by thiols. Leddy et al. (231) observed that certain reaginic sera (atopic) were completely inactivated by 0.1 M thiol when tested on one PK recipient but, when the same treated serum was tested in another recipient, partial reaginic activity remained. These results are consistent with partial inactivation of the reagin with different degrees of sensitivity on the part of the recipients for the detection of the residual reagin. They also observed that some atopic sera containing reagins of two different specificities upon treatment with 0.1 M thiol had the reagin of one specificity completely inactivated but the other only partially so. Other investigators have had similar experiences which indicate that reagins show a degree of variable resistance at times to 0.1 M thiol (119, 126, 342). The reagin separating out with the y2-globulin from a serum of a patient with serum sickness was both thiol- and heatresistant (414), whereas a reagin separating with the -y M-globulin of the same serum was completely susceptible to thiol and heat. No reagin has yet been reported to be resistant to 0.2 M thiol when so tested. Heat Conversion of Reagins to Blockers It had been assumed that the heating of reagins destroyed all activity-the fixing capac-

16 326 FLICK TABLE 2. Calculations of antibody content of serum FEL (258)a ity, the ability to unite with antigen, and the sensitizing capacity for allergic shock-until Kuhns and Pappenheimer (218) suggested that such treatment might inactivate only the capacity to sensitize. This suggestion was based upon a study of their antidiphtheria toxoid serum, Hu, which when heated had lost the ability to sensitize skin sites for PK testing, but had gained a new, specific blocking activity. This was detected when a mixture of heated serum and antigen was injected into skin sites sensitized with unheated Hu serum. Furthermore, this heat-produced, blocking antibody in experiments of a different design did not diffuse away like the usual blocking antibody but fixed to the site for long periods of time. In contrast, Loveless (255, 258), Delorme et al. (83), and Sherman et al. (378, 379, 382) could not detect the production of new blocking antibody by heating reaginic sera. In retrospect it seems likely that the technique used with its large error was incapable of detecting any possible conversion of reagin to blocker by heat because of the frequent presence of natural, specific, blocking antibodies in many atopic sera even from untreated persons, and because reagin generally occurs in minute amounts. Table 2 provides the results of retrospective calculations based on data for atopic serum FEL reported by Loveless (258) in order to illustrate the problem. Serum FEL bound antigen (by antigen neutralization) to the same extent, heated or unheated, but heating inactivated the reagin. The reagin content turns out to be about 0.1% of the total measurable specific antibody. The variation in the neutralization titers given in Loveless' tables appears to be of the order of 50 to 100%. Even if the assumed values used for these calculations introduced 100-fold errors in the an- Se- Antibody rum Vol Value (ml) Reagin, PK titer :2,560 Neutralizing (of antigen) ,200 ng of antigen N Calculated reagin content ng of N Calculated neutralizing antibody ,800 ng of N aassumptions: 0.2 pg of N of reagin minimum detectable (from Table 1); 10% of total timothy antigen is antigen B (active); molar combining ratio of antigen/antibody, 1. Molecular weights: IgG, 150,000 (blocker); IgE, 200,000 (reagin); timothy antigen B, 10,000 (261). BACTERIOL. REV. tibody estimates, the technique used probably could not have detected the hypothesized conversion of reagin to blocking antibody. This still leaves the blocking, fixing antibody of Kuhns and Pappenheimer in need of an explanation. It will be offered in Partial Reagins and Queer Reagins. Ishizaka et al. (176) have restudied the problem of the effect of heat upon purified IgE with specific reagin activity from one atopic serum. Heating caused a loss of whealing function, a loss of ability to fix to the skin and only about a 35% loss of antigen binding capacity. In this one case much more sensitive techniques indicate that most of the reagin retains its ability to bind antigen, but because of probable heat damage to the F, portion of the molecule, the antibody was converted to a blocking, nonfixing antibody. Currently this appears to be the only valid fact concerning the fate of heated reagin. IgE and Molecular Structure Vigorous attempts have been made in recent years to identify the immunoglobulin class of reagins, assuming hopefully that they belong to a single class. In this area, all sustained investigations have so far dealt with atopic reagins rather than those of other origins. In 1966, Ishizaka and Ishizaka (160, 161) realized that the reagins in their two sera did not belong to any of the then known Ig classes. A specific antiserum was developed by them (171) that defined a new immunoglobulin class, IgE, with which reagin activity was associated. In collateral work, Johansson and Bennich in 1967 (190) described a novel, human meyloma protein, ND (or IgND), which was antigenically different from other known serum proteins. The immune serum prepared against it was used to investigate panels of normal and atopic sera for the presence of normal protein with the same antigenic specificity. Normal persons (164) possessed a specifically reactive serum protein of very low concentration and atopics usually had greater concentrations of the same protein. The purified ND myeloma protein also had the ability to fix to the skin and to block reagin fixation (396). The two groups of investigators finally reported in 1969 that anti-ignd was identical in specificity to anti-ige (30) and that IgND as an abnormal protein corresponded in properties to IgE, a normal serum globulin class. Most subsequent studies have found that reagins belong to the IgE class (75, 119, 162, 172, 176, 179, 183, 401, 436). The assignment of reagins to the IgE class is

17 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 327 based on two major lines of evidence: (i) In serum fractionation studies by several different techniques, including the use of specific precipitating sera for each of the Ig classes, reagin is associated with the IgE, not with the other Ig classes; and (ii) IgE of all the Ig classes of globulins alone (with rare possible exceptions) possesses biological and physical properties closely akin to those of reagins. Thus, rabbit anti-ige serum specifically precipitated both IgE and reagin activity from solution (30, 162, 172, 185) whereas specific antisera against the other four known Ig classes did not appreciably do so (160, 161, 162, 172, 185, 333). Or as reagin is separated from serum and purified by various chemical or physical means, it correlates in titer with the concentration of IgE (75, 171, 172, 401, 436). IgE protein alone of the Ig classes can fix to human skin like reagin (171, 174, 179, 397) and competitively blocks the fixation of reagin. When IgE is suitably heated, specific reagin activity therein disappears and the IgE loses both the ability to unite with anti-ige (75, 176, 187) and to block the fixation of reagins in the PK test (176). Rabbit and guinea pig anti-ige serum at high dilution upon injection into the skin of most persons will produce whealing in a reversed-role anaphylactic reaction, whereas similar antisera against other classes of Ig will not produce a local shock reaction (163, 183). Artificially aggregated, but soluble IgE or IgE myeloma protein in minute amounts will cause whealing upon injection into human skin (174, 182). Similar aggregates prepared from some other purified Ig proteins do not do this except that much larger amounts of aggregated IgG did produce skin whealing. This result could have been due to an undetectable amount of contaminant IgE according to the investigators. Although the available evidence very strongly indicates that reagins usually belong to the IgE class of globulins in humans, the apparent heterogeneity of reagin properties in particular and of all antibodies in general necessitate looking for reagin activity that may be associated with other Ig classes. At the moment, only a few possible deviations from the reagin-ige relationship seem to have been uncovered. Reagins in the chromatographic fraction I of the two atopic sera studied by Reid (341) were not precipitated with anti-ige but were precipitated with an anti-igg, suggesting to him that these reagins belonged to IgG or some unknown minor class of Ig buried in the IgG. Radermecker et al. (333) absorbed reaginic sera with anti-igg, a procedure which removed better than 90% of the IgG present and which removed 3 to 6% of the reagin activity. They could not distinguish between the possibilities that the reagin loss was due to error of assay or that a small amount of reagin belonged to the IgG class. Similarly, the already quoted work of Ishizaka et al. (182), that aggregated IgG could cause whealing, could be interpreted as error, or that sometimes reagin can belong to IgG. In the hands of Paul and Weir (316), an anti-igg serum added to slices of human lung caused the release of 15 to 28% of the available histamine, values within the range of release of 23 to 54% effected by an anti-ige serum and well above the maximal control value of 12%. They interpreted their result, too, as suggestive that some IgG could be responsible for histamine release, or that the anti-igg serum was slightly contaminated with anti-ige. IgE, hence most reagin, appears to have a structure similar to IgG molecules. IgE has L- chains similar to those of IgG (160, 172) with kappa- or lambda-specific determinants, or both (162, 179, 183). The class determinant (epsilon) is localized to the F, portion (30) of normal or myeloma IgE as is the substructure responsible for fixation to cells (174, 398). Gel diffusion studies (174) with the F, fragments derived from two IgE myeloma proteins probably indicate the occurrence of IgE subclasses comparable to those of other classes of immunoglobulins. The H-chains possess carbohydrate (190). The sedimentation constant of IgE is 8.0 to 8.3S (161); of IgE myeloma proteins, 7.9S, with a molecular weight of 196,000 (174, 190). Sedimentation Constants and Molecular Weight Reagin has been found to sediment in association usually with the 7 to 8S globulins but not usually with the 19S globulins (7, 126, 147, 161, 172, 353, 394, 413). However, reagin rarely has been reported to be associated with the 19 to 22S proteins with no light-weight reagins concurrently present (141) in the serum, or to be associated in three antipenicillin reaginic sera with both the light and heavy molecules well separated in a sucrose density gradient (126). In one of these three sera studies by Girard, the light and heavy reagins were also well separated by passage through a column of Sephadex G200.

18 328 FLICK Electrophoretic Characteristics In electrophoresis with a variety of supporting media, reagin migrates usually with the 'y-globulins (48, 49, 172, 210, 371, 395) whether from atopic or nonatopic sources. But some sera contain reagins that migrate with the y2-globulins (371). The IgE myeloma protein (191) also migrates with the -y-globulin very close to the IgA and IgD peaks. Solubility Characteristics The separation of serum proteins by ammonium sulfate precipitation still has usefulness as a starting procedure. Many reagins precipitate at 50% saturated ammonium sulfate or less (20, 127), but on occasion 55% saturation is needed to cause 75% or more of the reagin to go into the globulin precipitate (20, 72). Unfortunately, some investigators who utilize ammonium sulfate for fractionating serum proteins fail to specify the temperature at which the solution became saturated. Since the solubility of ammonium sulfate changes 0.33 g per 100 g of water per degree over the range of 0 to 20 C, the temperature will markedly influence the salt concentration used for salting-out proteins. When reaginic sera or fractions are dialyzed against to 0.02 M phosphate buffer in preparation for chromatography, the reagin often remains with the water-soluble pseudoglobulin (20, 72, 156, 158). But sometimes most of the reagin precipitates with the euglobulin instead (103, 340), or may be distributed over both fractions (20, 103). Stability Reagins have earned the reputation for being unstable. However, for storage in serum they are usually quite stable. They can be stored frozen at -20 C (325) or at -10 C for 6 months (41) without appreciable loss of activity in the serum. Even refrigerator storage over many months (146, 371) or even up to 4 years (69) produces little or no loss. But in these studies relatively few reaginic sera were used. Some untested samples may not hold up as well as others. When one attempts to separate reagins from the extraneous serum proteins, their activity may be easily jeopardized. Partially purified reagins are less stable upon storage (371) than when kept in whole serum. Ammonium sulfate precipitation of reagins can cause a 75 to 90% loss of activity (103, 340). Passage of reaginic serum through a starch column or through a filter paper column caused a 50 to BACTERIOL. REV. 90% loss (72) of activity. Diethylaminoethyl (DEAE) chromatography of such sera can produce a loss of 50 to 70% of the activity (319, 394), and even ultracentrifugation can produce a marked loss, although the same serum subjected to sucrose density gradient ultracentrifuging suffered no loss (394). Zone electrophoresis can give variable losses as with other procedures. Terr and Bentz (414) observed such reagin loss with one serum and Sehon et al. (371) found that two of eleven atopic sera had complete reagin inactivation by starch electrophoresis. Many reports fail to indicate the amount of reagin loss when sera are subjected to a variety of mild procedures for fractionation. It is possible that the present state of our technology permits the characterization mainly of robust reagins which have selectively survived fractionation of the serum. The effect of divergent ph values upon the activity of reagins in serum is influenced by the time and temperature of exposure (72). At ph 4.0 to 4.5 and below or at ph 10 to 12 and above for 24 hr at 7 C, most of the reagin activity was lost (72, 127) in a few samples. When several reaginic sera were studied at ph 3.0 for 2 hr, variable results occurred, with the majority showing little loss but some marked loss showing of reagin (142). Transplacental Transmission The ability of reagin to cross the placenta in humans is of some interest in relation to the passive acquisition of maternal sensitivity in utero by the fetus and its ability to respond allergically after birth. In five unrelated studies, a total of 65 mother-newborn pairs, in which the mother had circulating reagin, have been investigated for reagins occurring in the cord blood collected at parturition (2, 29, 66, 383, 443). Of the cord bloods, 97% lacked reagins by PK tests. Connell et al. (66) found one cord blood that contained reagin at about 'b5 of the maternal titer and Allensmith and Buell (2) also found a single cord blood that contained reagins. In all of these studies but that of Zohn (443), reagins of presumably atopic mothers were assayed. Zohn studied reagins produced in normal mothers who were immunized with ascaris antigen during the last trimester. The results were the same as in atopics. Since it is possible that reagins reaching fetal tissues transplacentally might be fixed to fetal cells with sufficient rapidity to reduce their concentration in the fetal plasma to undetectable levels, Sherman et al. (383) also skin tested 12 newborn infants with

19 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS antigens corresponding in specificity to the maternal reagins. None of the infants gave evidence of skin-fixed reagins. At least some newborn infants can respond allergically by PK test (2). Approaching the problem from a different point of view, Johansson (189) assayed the content of IgE in cord blood of 37 infants, and found the mean value to be 36 ng of protein/ml with a range of 16 to 97 ng/ml of serum. As age increased toward adulthood, the mean concentration of IgE in the serum increased progressively (33, 189). The progressive increase with age suggests that the IgE of cord blood was of fetal rather than maternal origin. It also complicates the interpretation of the origin of the specific reagins found in the two cord bloods cited above. In one of these two cases (2), the reagin activity in the cord blood had disappeared from the infant's serum by PK test and direct skin test by 30 hr after parturition suggesting that the reagin was of maternal origin. However, in adults and perhaps in infants, reagin production and appearance can be very transitory indeed. It seems likely that on rare occasions, reagins can pass the placenta with difficulty. Whether a placental lesion is needed to permit direct passage of maternal whole blood to the fetus is uncertain. The passage of fetal red cells into the maternal circulation is fairly common, and easy to detect when there is an Rh incompatibility. A high titer of maternal reagin alone seems not to be important in establishing transfer to the fetus because at least one mother of the pairs studied (383) had a titer of 1: 1,000 without reagins being detectable in the cord blood. Reagin Serology (Noncellular Assay) Attempts to detect complement fixation by sera containing reagins mixed with specific antigen have been without success (16, 61, 211), and often it has been assumed that reagins are incapable of fixing complement. However, with the recently acquired knowledge that reagins often occur in human sera at concentrations well below the minimal concentrations of IgG or IgM antibodies that can detectably fix complement (see Table 1), one can reasonably ask whether the failure is due to peculiarities of reaginic molecular structure or to inadequate concentrations of this antibody. Currently, the best answer is derived from the study by Ishizaka et al. (175) of the ability of aggregated purified serum globulins to fix complement or some of its components in the 329 absence of antigen. Whereas aggregated IgG can fix complement, aggregated IgE (myeloma protein) does not fix complement at concentrations comparable to those used successfully for the former. Thus, it seems likely that reagins of IgE class usually do not fix complement. Similarly, reaginic sera have failed to give antigen precipitation but again this lack of detectability may be due to the low concentration of reagin in the serum. Structural knowledge would suggest that, like IgG-antibodies, reagin should have two combining sites for antigen. Miller and Campbell (279) as well as Kuhns and Pappenheimer (218) presented quantitative evidence that reagin co-precipitated with a matching antigen-antibody precipitate. Unfortunately, in neither case was the supernatant of the precipitate examined for the loss of reagin activity as proof of co-precipitation. The increment of nitrogen in the precipitate could have been due to now recognized, associated antibodies of the same specificity, or even to complement in the Miller and Campbell study. In general, it seems unlikely that one can detect co-precipitation of human reagins by the conventional quantitative precipitin techniques without using radioisotope tracers. Attempts have been made to measure reagins by hemagglutination techniques, usually by coupling the soluble antigen to red cells by the use of tannic acid or with bisdiazotized benzidine. The latter procedure seems to carry greater sensitivity for the detection of antibody than the former but it may also be measuring something other than specific antibody in human sera (14). By hemagglutination it is possible to detect specific, heat-stable antibodies against allergens in atopic sera before hyposensitization therapy (96, 299) and often to a higher titer after such therapy. If one used unheated sera in order to attempt detection of reagins, there is a lack of correlation between the hemagglutination and the PK titers. This is brought out best by finding sera with low or negative hemagglutination titers with good PK titers (12, 83, 96), and especially with PK titers of 1: 1,000 or more (10, 83). Other methods of correlation also fail. Thus, the chemical inactivation rates of reagin and hemagglutination are different (140), or when sera containing reagin and hemagglutinin of the same specificity are absorbed with a particulate antigen, the reagin is removed less rapidly than the hemagglutinin (320). In all of these experi-

20 330 FLICK ments, the ability of reagins to produce hemagglutination has not been excluded; reagins at the concentrations available merely did not seem to give the effect. In contrast, Ishizaka and Ishizaka (165), by using purified, concentrated IgE with its associated specific reagin, did succeed in obtaining hemagglutination. The agglutination was augmented markedly by an anti-ige serum but not by an anti-iga nor an anti-igg serum. Coombs et al. (75) have devised a hemagglutination test which measures specific IgE in unfractionated serum. They employ an anti-ige serum to bring about visible hemagglutination after the specific IgE antibody has united with the antigen coupled to the red cell. Hemagglutination here did correlate with the reagin titer in the three positive sera studied. Another similar technique, the radioallergosorbent test (RAST), for detecting IgE antibodies specific for a particular antigen on a semiquantitative basis has been devised and reported upon more extensively. Wide et al. (438) coupled antigens to batches of Sephadex G25 particles or, in a later modification by Berg et al. (32), to cellulose particles, through the action of cyanogen bromide. The resulting insoluble antigen-sorbent was used to remove all classes of antibodies present in an atopic serum specific for the antigen-sorbent. After washing away uncombined proteins from the particles, the specific IgE antibodies absorbed from the serum were detected on the insoluble sorbent by adding a standard amount of purified 125I-tagged anti-human IgE. The latter, after washing away uncombined, tagged antibody from the complex, was measured in a counter. The amount of radiotag adsorbed to the insoluble antigen when buffer or normal serum was substituted for atopic serum was a small and rather constant correction for the unknown serum. An atopic serum was considered positive if the 125I-anti-IgE adsorbed was 1.5 (32), 2.0 (401, 438), or more times the control background values. These low levels are equivalent to about 0.2 pg of anti-ige nitrogen (401). Therefore, the minimal specific IgE detected by RAST corresponds to the same order of sensitivity of the PK test at its best, and is of greater sensitivity than can be attained by any form of the hemagglutination test (see Table 1 for comparison). So far the test has been considered only semiquantitative, in that the amount of radioactivity adsorbed to the insoluble antigen-antibody complex is graded 1+ to 5+ rather than expressing indirectly in quantitative terms the amount of IgE antibody adsorbed. The latter, desirable procedure BACTERIOL. REV. would probably be difficult because of the variable ratio in atopic sera of specific IgE antibodies to other classes of antibodies of the same specificity with their variable association constants and competitive interactions with the antigen. So far the use of serum dilutions, a procedure which would magnify the number of tests required, has not been described or needed. The specificity of RAST has been studied and found adequate (438). Studies also have been made of its clinical usefulness to determine those antigens which cause clinical disease, among the many against which an atopic person has reagins. The percentage of agreement between the results of RAST and of skin tests, in the case of atopics for the multiple inhalant antigens used, was 68% (32, 438) and 73% (401). When the results of RAST were compared to those of clinical provocative tests (i.e., inhalation or instillation of progressively greater concentrations of antigen to bring about an attack of asthma or hayfever, respectively), agreement was found in 74% (32) and 96% (438). It is evident that there are sizable groups of atopics in which RAST fails to predict the clinically offensive antigens. A technically more difficult procedure was used by Ishizaka et al. (173) to measure quantitatively the amount of specific IgE in each of 10 atopic sera and to correlate the concentration with the PK titers against the same antigen. The reagin serum was mixed with a rabbit anti-human IgE serum and with 131I_ tagged antigen. The anti-ige specifically precipitated the IgE antibodies; the tagged antigen combined with its specific IgE antibodies in the precipitate in proportion to the amount of the latter. Washing freed the precipitate of unwanted contaminant. The amount of radiotag in the precipitate correlated well with the PK titers of the sera. In this technique for quantitative precipitation of the IgE, it is important to have the ratio of IgE to anti-ige at equivalence or in slight excess anti-ige, necessitating a testing of supernatants for excess reactants. Ishizaka et al. also found that a good excess of tagged antigen should be present to maintain maximal binding to the precipitate. Often other classes of antibodies to the same antigen are present, as they demonstrated, and will take up the antigen competitively. Their undesirable precipitation by the antigen, not, of course, by the anti-ige, will be prevented if the antigen is in sufficient concentration to form soluble complexes. Considerable centrifuging is required to capture and wash the small amount of precipitate

21 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 331 formed between anti-ige and IgE when the latter is present in serum in its normal, relatively low concentration. A qualitative, radiotag method of detecting antibodies specific for a labeled antigen in the immunoelectrophoresis procedure was developed by Yagi et al. (442) and used for IgE antibodies by Ishizaka et al. (171, 172) as well as by Ito et al. (186). Due to the use of the radioactive label and of subsequent radioautographs, great sensitivity was attained in detecting immunoelectrophoretic lines of precipitate. The serum was first fractionated by gel electrophoresis. Then anti-ige serum was added to the trough in the gel to develop the bands of precipitate, either visible, or invisible if of insufficient density. The radiotagged, purified antigen either could be mixed with the anti-ige in the trough or, in a separate step, could be diffused into the gel from the surface. In either case, the labeled antigen combined with its specific antibody complexed with anti- IgE in the gel. This procedure emphasizes the insolubility of antigen-antibody precipitates even at such low concentrations of reactants that no visible precipitate can be detected by ordinary means. In response to the desire to measure the total IgE concentration in serum and body fluids, Johansson et al. (191) modified for this purpose the radioimmunosorbent test (RIST) described by Wide and Porath (439). Myeloma protein of IgE specificity and tagged with 125I was employed to determine, in the unknown sample, the concentration of untagged IgE, which is put in competition with the former for anti-human IgE presented in an insoluble form. In practice, purified anti-ige is coupled chemically to Sephadex particles. The ultimate specificity of RIST for IgE depends upon rendering this particulate antibody monospecific for the epsilon determinant. In the original procedure (439), the amount of insoluble immunosorbent used in a test was that which would bind 50 to 60% of 0.1 ng of tagged antigen added. With these amounts of reactants held constant, a standard curve was constructed based on the amount of inhibition of binding of the tagged antigen when the system was mixed with falling dilutions of the same antigen untagged. Unknowns could then be assayed in the same system when substituted for the purified untagged antigen of the standard. The minimal amount of IgE protein detectable was about 5 ng of protein/ml (191), and the error is represented by standard deviations of -23% or less (189). The sensitivity of the procedure can be markedly increased to detect as little as 1 pg of protein/ml by two modifications of the standard technique (439): preincubation of the particulate-antibody and labeled antigen before addition of the unlabeled antigen in place of mixing all reactants simultaneously, and reduction of the amounts of insoluble antibody and labeled antigen used. For the same purpose, Rowe (355) and also Ishizaka and Newcomb (180) have modified the single radial diffusion technique developed by Mancini et al. (265) for assaying in the clinical laboratory the more plentiful of the classes of serum Ig. The modifications made use of radioactive tracers for detecting the ring of antigen-antibody precipitate formed during gel diffusion of the reactants. A two-stage process was used. In the first stage, as in the Mancini technique, the antigen, here IgE, deposited in serial dilutions in wells in the gel slab is allowed to diffuse outward against a suitable concentration of anti-ige initially distributed uniformly throughout the gel. Either the area or the square of the diameter of the ring of precipitate, which forms about each well, at maximum size is proportional to the initial concentration of the antigen in the well. Because of the usually low concentration of IgE in body fluids, the precipitate is invisible unless the second stage of radiotagging is brought into play, that is, the overlaying of the gel with 13II-labeled antiserum against the immunoglobulins of the animal species which was used to prepare the anti-ige of the first stage. A thorough washing away of soluble proteins from the gel should occur between stages 1 and 2, as well as after stage 2, in order to increase the contrast of the radiolabeled precipitate bands from the surrounding gel in the subsequent radioautographs. The time required for this complete procedure, 8 to 9 days, about the same as in the Mancini technique, is greatly in excess of the time needed for RAST. The sensitivity of the test as estimated by Rowe for IgE was about 40 ng of protein/ml or about 60 times more sensitive than the Mancini procedure. Arbesman and Ito (11) recently observed that the second stage of the Rowe procedure could be eliminated with a saving of time and materials by simply tagging the anti-ige with 1311 directly without loss of sensitivity. Centifanto and Kaufman (54) have modified the second stage of the Rowe technique by using attached fluorescein in place of the radiotag as a means of detecting the otherwise invisible precipitate. In this case the tag exhibits itself by fluorescence when exposed to blue light. This procedure probably has intermediate sen-

22 332 FLICK sitivity between the Mancini and Rowe methods. Protection Do reagins offer protection against harmful agents to the person possessing them? It is a well known fact that some persons who experience hayfever would find it very difficult to believe that reagins have any value whatsoever. Nevertheless, there is a little data indicating a protective role for reagins in infections. In 1929, Neill and Fleming (288) and much later Kuhns (211) described sera possessing both reagin activity for diphtheria toxin constituents and also toxin neutralizing ability at PK sites in normal, Schick-test positive persons. The evidence that the reagin present was, itself, neutralizing the toxin is based on the observation that the antitoxin activity remained fixed to the PK site as did the reagin for at least 2 to 4 days. An alternate, less likely explanation might involve the simultaneous occurrence in each of the sera of a reagin specific for a contaminant antigen in the toxin and of a skin-fixing, nonwhealing, antitoxin. It is also possible that a small but sufficient amount of a nonfixing, nonwhealing antitoxin remained at the skin site and that the reaginic whealing was specific for a nontoxic contaminant. This hypothesis seems very unlikely when viewed with some of Kuhn's quantitative data in mind. The half-life of antitoxin was approximately 10 hr at human skin sites (212), a value conforming to that determined by other investigators. Using the usual value of the half-life of antitoxin at a 4-day PK site, approximately /5oo of the original nonfixing antitoxin should have remained. Yet Kuhns found that, with reaginic serum Gr (211) at a 4-day PK site, the loss of neutralizing power was probably less than 50%. Thus, it seems very likely that a tissue-fixing type of antitoxin was involved. I have observed a human serum that both neutralized streptococcal erythrogenic toxin as antigen and gave whealing at a 24-hr PK site in the skin of a Dick-test positive individual. Again, the most likely explanation is that the reagin present neutralized this toxin. There are few other data available for humans on the subject, but this meager information can be supported a bit by some laboratory animal observations. In rats, Ogilvie et al. (295, 296) have found protective value of reagins developed against worm antigens. The assignment of protection to the reagin rather than to other antibodies present in the serum is based on BACTERIOL. REV. both the specific immunity activity and the usual specific reagin sensitizing activity remaining at the injected local skin site for 3 days. The challenge for detecting these two activities locally was the injection of live worm larvae. Similar studies in monkeys, however, failed to indicate that monkey reagins were protective against worm larvae (90, 296). Sabin, in 1935 (357, 358) in a provocative report, indicated that certain rabbit antivaccinia virus sera would not neutralize virus nor fix complement when mixed with the virus in vitro but would neutralize virus when living rabbit tissue was present, either in vivo or in tissue culture. A local injection of immune serum into rabbit skin followed by virus at the same site 3.5 days later led to specific protection, suggesting that a tissue-fixing antibody, though not necessarily a reagin, was offering the protection. Thus, there is just a bare shred of evidence that reagins may be beneficial to the person or animal making them. Specificity Limited data have been presented to support the view that reagins in general have a special property, that of low grade specificity as compared to other antibodies. Thus, in studies conducted by Sherman and Stull (385) and by Cooke (70), antigens of diverse phylogenetic origins cross-reacted at PK sites with the reagins in a single serum. One such serum possessed a reagin which cross-reacted with both horse dander and ragweed antigens with unidirectional, complete neutralization of the reagin. But an alternative explanation might be that a common antigenic determinant existed in the unrelated sources; heterophile antigens are moderately frequent. More recent evidence furnished from studies of reagins to penicillin and its derivatives strongly indicate that reagins can possess a specificity as narrow or, on occasion, as broad as the variable specificity demonstrated by precipitins. Some antipenicillin reagins can distinguish various side chain substituents on the penicillin nucleus as judged by direct skin test results on the penicillin-allergic patients of Parker and Thiel (310) or of Shibata et al. (387). But in the majority of cases the side chain was not important in determining reactivity of penicillins. Mathews and Pan (269) and also Batchelor and Dewdney (25), by using PK tests, found some reaginic sera which reacted with benzyl-penicillin but not with the derivative, benzyl-penicilloyl-polylysine. Ped-

23 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS ersen-bjergaard (317) used benzyl-penicillin, benzyl-penicilloate (BPO), and benzyl-penicilloly-polylysine (BPO-PL) as allergens in PK tests. There were reaginic sera which would react only with benzyl-penicillin, only with BPO-PL, with any two of the allergens but not the third, and with all three allergens. Thus, some sera showed a high degree of specificity; others possessed either reagins with broader specificity or multiple reagins of different specificities as recorded for two of a group of sera studied by Levine and Redmond (237). It is unclear why some reagins can make a readout of the structure of univalent BPO but not of BPO-PL, the multivalent form. That a high degree of specificity of reagins is not limited to those directed toward haptens is indicated by the observations of Bernton et al. (36), that single atopic sera contain reagins of several noncross-reacting specificities toward purified antigens of cottonseed. Reagin-Antigen Union in Solution A concept was developed that reagin and antigen fail to unite in the absence of cells. Of course, once reagin fixes to cells there would be no problem of union as evidenced by the results of the PK tests. This interpretation was based, in part, upon observations of Levine and Coca (241) that the injection of a suitable mixture of reagin and antigen produces a wheal and the reagin becomes neutralized for future challenge with antigen. It also depends upon later observations of Swineford and Mason (268, 408), who injected high-titered reaginic serum back into sites of the donor's own skin and later injected antigen at a distance subcutaneously. The result in some but not all such trials was a whealing at the prepared site only, evidence for the circulation of antigen in the plasma side by side with reagin to the site of greater sensitization where antigen escaped the plasma in sufficient amount to produce shock. Subsequently, reports appeared of a variety of experiments developed to throw more light on the problem. The early ones (291, 403) produced no evidence of reaginantigen union in solution, helped to strengthen the concept of nonunion, but in reality were inadequate in design to give any meaningful answer. More recent experiments in some cases also have had design flaws. Experience indicates that certain principles must be followed for such experiments to offer valid evidence of either union or nonunion. When reaginic serum is absorbed with a particulate 333 antigen, in a test of union or nonunion, not only should the supernatant be examined for loss of specific reagin, but an attempt should be made to elute the reagin from the antigen, a process that can be difficult (142), in order to prove union. In the absence of elution data, the apparent loss of reagin from the supernatant could be due to the release of soluble antigen from the particles with neutralization of the reagin. An erroneous interpretation of the status of the reagin could easily occur because a suitable mixture of reagin and antigen upon intracutaneous injection can produce an early missed, or misinterpreted whealing. Later challenge of the site with antigen to detect the reagin will result in a negative response suggesting the absence of reagin rather than its neutralization. That the antigen coupled by various procedures to particles and washed can somehow elute at times into the suspending medium is indicated in the case of diazopolystyrene particles by the work of Yagi et al. (441); in the case of tannic acid coupling to some batches of red cells by the work of Freinberg and Flick (97), and with bisdiazotized benzidine coupling to red cell ghosts (water hemolyzed) by that of Royal and Flick (356). In the latter situation, as antigen "elutes," there appear in the supernatant of the ghosts minute particles visible by darkfield microscopy. Antigen elution may not be through a rupture of the coupling bond but rather that some batches of red cells can release membrane components carrying attached antigen by fragmentation, a process described by Furchgott (123). In the case that an absorption experiment indicates nonunion between reagin and antigen, then one must prove that (i) antigen of the same specificity as that of the reagin was actually coupled to the particulate carrier, and (ii) the specific particulate antigen was present in sufficient amount to effect reagin removal. Only those reports that seem to fulfill these basic requirements for properly interpreting experiments on antigen-reagin union will be considered. Malley et al. (263) undertook the absorption of specific reagin in one atopic serum with timothy antigen coupled to red cells. No loss of reagin occurred from the supernatant. That the particulate antigen was adequate for the job at hand was indicated by the ability of samples of the antigen to absorb blocking antibody in adequate amounts and of the same specificity as the reagin, the blocking antibody and the reagin being measured in PK tests modified to handle detection of the

24 334 FLICK blocker. This experiment is the only adequate one yet found in the literature that reports a failure of union between reagin in solution and antigen. Perelmutter et al. (320) absorbed 90% or more of the reagin contained in each of five atopic sera using antigen coupled to red cells. Proof of reagin-specific union to the particulate antigen was based on the successful elution of reagin from the antigen by mixing the washed complex with a hemagglutinating antibody of the same apparent specificity. This latter antibody replaced the reagin on the antigen. Richter et al. (346) detected no release of ragweed antigen from washed complexes of antigen-polyaminostyrene. This complex was able to absorb specific reagin from sera (177, 346) as judged by the loss of PK activity from the supernatant. Goldstein et al. (128) were successful in eluting some reagin, after specific absorption to an insoluble form of the antigen, by the use of either 2 M NaCl or 2 M KI solutions. Malley et al. (260), using a different insoluble specific immunoadsorbent, also eluted with 6 M urea solution the reagins of ten antitimothy grass pollen sera. Recovery was excellent; the amount of reagin in the eluate was usually 10- to 20-fold greater than that in the original serum. Whether this marked increase in recovered reagin represents titrational error or the removal of serum inhibitors by the associated purification process remains to be ascertained. Ishizaka and Ishizaka (158) used a different, adequate procedure for detecting union between reagin and its antigen. A mixture of purified reagin and excess antigen was subjected to passage through a column of Sephadex G200. An early fraction, with a passage mobility roughly equivalent to that of reagin alone, upon injection into the skin caused whealing directly but not when challenged with antigen 24 hr later, indicating the presence of both reagin and antigen in the peak fraction. A later peak contained only antigen, indicating that uncombined antigen had a much slower column mobility. Thus, a reagin-antigen complex was indicated without using a particulate form of antigen. If only antibody other than reagin had united with the antigen and carried it along beside uncombined reagin, the skin test results should have been different. Similarly, electrophoresis of a reagin-antigen mixture caused separation of a reagin-antigen complex from antigen alone, and here also, from reagin alone. Apparently these conditions permitted some of the complex to dissociate into free reagin and free antigen. Subsequently, they (164) subjected purified IgE, carrying specific reagin activity, to BACTERIOL. REV. gradient ultracentrifugation following mixing with excess ragweed antigen E, which has a molecular weight 19% of that of reagin. A skinwhealing complex of 13.1S and a nonwhealing complex of 9.7S were separated, indicating again that union of reagin and antigen could occur in solution. The occurrence of union between specific IgE and antigen is indicated also by the results of certain types of radioimmunodiffusion experiments (86, 166). A precipitate was formed in a gel between IgE containing some specific antibody and an anti-ige serum. Then the radiotagged specific antigen was diffused into the reaction area and successfully combined with the specific IgE antibodies in the preformed precipitate. However, it can be argued whether or not these conditions of union reflect the ability of free IgE antibody to unite with antigen. Perhaps the prior attachment of the anti-ige to the F, portion of the IgE antibody is comparable in action to the prior attachment of the mast cell receptor to the same general area of the latter antibody, thus providing the hypothesized augmentation of bonding between IgE (reagin) and antigen. It seems quite evident that most reagins can readily unite in solution with antigen. More work will be needed to determine if occasional ones act differently in this property. It seems likely that reagins, like other types of antibodies, will have variation in their association constants for antigen. Whether low values for the association constant in some cases can account completely for failure of union as in the experiment of Malley et al. (263) or whether one needs to postulate that cell-fixed reagin as compared to free reagin, due to special flexions of the molecule, can have a greater affinity for its antigen must be decided by further investigations. The knowledge that the reagin-antigen complex, if not too loaded with antigen (164), can cause shock reactions gives a sufficient and satisfactory explanation for the early observations of Levine and Coca (241). Reagin-Hapten Dissociation Attention should be addressed to one example of a reagin that has the ability to cause shock in skin in collaboration with its antigen but then seemingly releases the antigen, thereby preparing itself for producing subsequent shocks when fresh antigen is offered. A reaginic serum described by Sherman and Cooke (381) acts in this fashion. This serum had a 48-hr PK titer of 1: 10,000 with sodium sulfadiazine as the allergen, or by using data

25 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 335 and assumptions of Table 1, 0.05 ml of serum contained 6 x 10-8 Mmoles of reagin. For the purposes of neutralization, this amount of reagin was mixed with an equal volume of either 1.8 x 101 Amoles (10% solution) or 1.8 x 10-1,gmoles (0.1%) of sodium sulfadiazine and the mixtures were injected into separate normal skin sites. Both mixtures produced the same degree of whealing, although a control serum plus saline mixture caused no reaction. This result certainly suggests that the hapten was in excess over reagin in the mixture containing 10% drug and either at equivalence or in excess in the one containing 0.1% drug. Each test site was then reinjected with 1.8 umoles (1.0% solution) of sodium sulfadiazine at 48 hr and again at 72 hr. Each injection at these two sites produced the same size wheal, suggesting that for each challenge the reagin present had to combine maximally with fresh hapten in order to trigger equal, maximal reactions. One hypothesis, that a minor impurity in the hapten solution caused the reaction, leads to a contradiction in that the same amount of residual reagin seems to be present at the 0.1% and the 10% hapten sites as indicated by the equal reactions obtained by repeat challenges. Also the initial, equal reactions at the two sites indicate not a 100-fold difference in concentrations of the shocking hapten, but rather a reagin-limited reaction with excess hapten. An alternate hypothesis that fits the observations is that hapten, probably given in excess each time, dissociated from the fixed reagin and diffused away during the interval between injections, leaving the reagin fixed and free to combine with fresh hapten later. It is unknown how the body normally handles the reagin-antigen complex fixed to cells. That the removal must occur at a greater rate usually than the rate of dissociation of the reagin-antigen complex is indicated by many observations that, in PK tests, reagin can be neutralized by excess antigen as judged by a lack of reactivity of the site upon subsequent challenge. However, in this interesting study of sulfadiazine-specific reagins, the rate of allergen dissociation appeared to be much faster than the rate of complex removal. Other examples of probable hapten dissociation from fixed antibodies (not necessarily reagins) in vivo can be found in the studies of Tillett et al. (415) and of Ovary and Karush (304) in both of which rabbit antisera passively sensitized guinea pigs for systemic, or local cutaneous anaphylaxis. In the former study, a univalent glucoside hapten and, in the latter, lactose as a univalent hapten were used, yielding similar results. Injection of the univalent hapten into the sensitized guinea pigs did not produce anaphylaxis and, in fact, did desensitize the animals temporarily for challenge with the hapten-conjugate. But if sufficient hours intervened between hapten desehisitization and hapten-conjugate challenge, full sensitivity for anaphylaxis had returned. In the case of the PCA tests of Ovary and Karush, the data were interpreted to indicate in vivo dissociation of the hapten from the fixed antibody. This apparent ease of dissociation in vivo of haptens from fixed antibody is not necessarily due to the hapten entirely, because many such systems do not exhibit this peculiar behavior (325). At least in part, this dissociative phenomenon seems to be a function of the antibody. Farr (93), for example, found that antibody produced early in the immunization of some rabbits dissociated more readily from its protein antigen (bovine serum albumin [BSA]) than antibody produced later. But univalent hapten, being unable to combine with more than one fixed antibody molecule as compared to multivalent haptens or antigens, may find it easier to dissociate than the corresponding multivalent antigen under conditions of high density of fixed antibody. This phenomenon of in vivo dissociation of hapten or antigen from fixed reagin may account for the inability to desensitize clinically some allergic persons by using repeated injections of allergen over a relatively brief period of time, whereas other individuals have been successfully desensitized. In the former situation, possibly due to the rapid dissociative process, fixed antibody is never exhausted. Latent Periods In the human PK test, the latent period has been the subject of controversy. Some contend that a minimum latent period is necessary for elicitation of the shock reaction by antigen; others hold that no such minimum period is needed. Or, although the allergenicity of injected reagin can develop immediately, quantitatively it requires time to reach a maximum. All of these viewpoints have factual foundations in human reagin studies. Two well-recognized factors, the production of skin whealing by some sera alone (56, 60, 430) and the presence of blocking antibodies (255) in some reaginic sera, have tended to complicate the interpretation of data in these studies, particularly when mixtures of reaginic serum and antigen are injected. The whealing ability of human sera is serum dependent, in-

26 336 FLICK creasing with in vitro age and heating, as well as recipient dependent (371). Moderate whealing can be produced in about 7% of humans (206) just by injecting saline intradermally. Aggregated IgE (174, 178, 182), prepared by heating or other means, produces skin whealing upon injection. Therefore, spontaneously aggregated IgE in some sera may account for much of the irritant properties of these sera. Blocking antibodies in reaginic sera whether from untreated or hyposensitized atopics by combining with antigen preferentially can block out the antigen-reagin union in mixtures. Only if antigen is added to such sera in excess can shock reactions be elicited upon injection of such mixtures. Coca and Grove (61) first tried studying a zero latent period of the PK test by injecting mixtures of reaginic serum and antigen. They could not properly interpret their results, a whealing reaction within 0.5 hr, because one of the two sera used also produced whealing without antigen at a control site. But subsequently many other investigators recorded that mixtures of reagin and antigen upon injection caused specific whealing within 15 to 60 min of injection (35, 42, 62, 71, 124, 145, 158, 211, 223, 255, 370, 386). It is also evident that such mixtures need not be incubated any length of time prior to injection in order to produce whealing (145). Alternately, the latent period for elicitation of a reaction can be 5 min (60) or 1 hr (223). Ishizaka and Ishizaka (158, 164) have produced skin whealing complexes of antigen and purified specific reagin or IgE. Their findings are of interest in the case of reagin from one serum in that complexes produced in vitro with the ratio of two antigen molecules per antibody molecule upon injection would not produce shock whereas complexes with less antigen did shock. This observation offers a possible explanation for the nonreactivity in skin of some reagin-antigen mixtures prepared in antigen excess (35, 347). All of the above studies used atopic sera. The occurrence of a time-dependent maximal reactivity in the PK test has been reported for some atopic sera. The serum of Richter et al. (347) failed to yield whealing with a 5- to 20-min latent period and produced progressively larger whealing with latent periods of 1 hr to a maximum at 4 hr, using constant doses of reagin. When the dose of reagin was varied the minimum latent period for reactivity was inversely related. Antigen challenge of all sites 24 hr later indicated that the antigen used to terminate each latent period had BACTERIOL. REV. been sufficient to completely neutralize the reagin. A single serum of Clark and Gallagher (60) produced a smaller reaction with a latent period of 5 min than with a latency of 24 hr. Reed (341), using two semipurified reaginic fractions separated by chromatography from a single serum, studied their latent periods for maximal reactivity by titrating the sera to a 2+ wheal end point. Chromatographic peak I reagins reached maximal reactivity at about 12 hr whereas peak II reagins reached a maximum in a latent period of between 12 and 24 hr. Both reagin fractions possessed some reactivity with the shortest latent period tested. Using purified IgE reagin from one atopic serum, Ishizaka and Ishizaka (166) showed that the amount of IgE required for the sensitization to produce minimal whealing by antigen at 2 hr was four times the amount needed for the same degree of whealing at 24 hr. Finally, the quantitative assays of Stanworth and Kuhns (399) on a single batch of reaginic serum could offer explanations for at least some of these effects. When a single dilution of the serum was used to sensitize sites in three recipients for determining the latent period of maximal reactivity, the effect depended upon the recipient, not the serum. In recipient G.W., antigen injection of PK sites at 53 hr produced whealing significantly greater in area than at 2 hr. In the other two recipients there was no significant change in wheal size with length of latent period from 1.25 to 53 hr. There was considerable irregular variation in wheal size at sites tested within this large time interval, but this variation seemed to be due to differences in reactivity of skin sites on a recipient's back. Inspection of their data indicates that, when replicate-sensitized sites in one recipient are injected with antigen simultaneously, the majority of sites yield good wheals but a small proportion of sites exhibit zero whealing. This sort of quantitative data tends to foul the interpretation of the preceding studies, which seem to indicate that a latent period for maximal reactivity expresses a property of the reagin, of the serum with its mixture of antibodies and enzymes, or of the involved cell. The opinion has been expressed that the need for a latent period of some hours in order to obtain optimal reaginic whealing is due to a slow fixation of reagins to tissues. However, this view has little factual data to support it; rather it is an interpretation based on the optimum in the latent period. In the situation where reagin does seem to require time to develon its full reactivity, if fixation to the cells

27 VOL. 36, 1972 were retarded one would expect the unfixed reagin to diffuse away just as common blocking antibody does-rather rapidly, within a few hours. A more likely hypothesis to explain the slow build-up of reactive capacity is that after a rapid fixation to cells some reagins require an interaction with the cell surface, a ripening of the antibody or the cell, in order to release histamine maximally upon union with antigen. Studies of reagins or homocytotropic antibodies of the guinea pig (58), the rat (38), or the rabbit (445) likewise indicate considerable variation in the latent period for maximal reactivity of PCA and also for the early part of the latent period when no reactivity can be expressed. PROPERTIES OF HUMAN REAGINS 337 Duration of Fixation The maximal latent period in PK tests for reagin reactivity, or even better, the half-life of fixed reagin represents measures of the avidity of attachment of reagin to cell receptors. That some reagin samples can fix to skin sites for long periods has been indicated by nonquantitative observations of reactivity persisting after latent periods of 4 weeks or more (61, 124, 218, 255) in normal recipients. The six atopic sera of Goodfriend and Perelmutter (131) gave variable results in that some showed diminution in whealing ability over 18 days whereas others showed no reduction in reactivity. These PK assays were done in each of two recipients so that the differences between sera in the apparent loss of reagin activity with time are probably not due to recipient factors primarily but to differences in the reagins themselves. Fireman et al. (107, 108) described an atopic serum that provided a PK titer of 1: 10 at 24 hr, but was unreactive with longer latent periods. Some studies have attempted to determine the half-life of reagin at PK sites by quantitative means. Kuhns (212) determined and compared the half-life in skin sites of a number of antitoxic, nonatopic sera, some possessing reagins, others only nonreaginic antitoxin. The use of antitoxin and toxin in the skin of persons with no antidiphtheria immunity enabled him to measure the persistence of antibody not through its whealing ability in the case of reaginic sera but by its residual antitoxic activity at injection sites. The half-life of the antitoxin was about the same for all sera tested, 7 to 14 hr, regardless of the type of antitoxin present, reaginic or nonreaginic. This value seems remarkably small for reagins but then in retrospect Kuhn's data indicate that he probably was not measuring the skin site half-life of reagin but of associated nonreaginic antitoxin. For each serum he determined the antitoxin concentration at two times, at zero time in the skin of rabbits and separately after a latent period of several days in human skin. Technical reasons prevented assaying these sera at both times in humans only. The two values were plotted in order to derive the above half-life values, a procedure valid only if the skins of the two species used are quantitatively identical in their expression of toxin neutralization and if the antitoxin antibodies in each serum are reasonably homogeneous. However, published data (218) indicate that the difference in antitoxin values of a serum as determined in the skin of a rabbit and of a guinea pig is greater than the assay error of the technique in rabbits alone (217). So perhaps, human and rabbit skin are not nearly identical in this same property. Kuhns also assumed, based on available information, that at least one reaginic antitoxic serum, Hu, contained only reagins as the dominant antitoxin. This particular serum (211, 212, 218, 301) contained antitoxoid reagins titering 1: 500 in 48-hr PK tests, 20 units of antitoxin/ml, no precipitins, but co-precipitated 128 gg of anti-toxoid N/ml. Using 0.2 pg of N as the value of minimally detectable reagin from Table 1, the reagin content of serum Hu is of the order of 0.001% of the total co-precipitating antibody. The stated error (217) in the toxin neutralization test was about 10,000 times larger than the percentage content of reagin. Therefore, certainly the first and perhaps the second plot points used for the half-life determination represent values for nonreaginic antibody in serum Hu. Fireman et al. (109) used serial dilutions of atopic sera to sensitize skin sites and determine the highest dilution that produced reactions after various latent periods up to 6 to 7 weeks. Their data indicated heterogeneity of reagins with respect to half-life. The disappearance rate of some reagins was greater during the first 2 weeks of the latent period than during the subsequent 2 weeks, indicating within a single serum the presence of reagins of differing half-lives. The average half-life of these reagins varied between 2.4 and 4.5 days; values not strikingly different from values obtained for radiotagged IgG (1.8 days), IgA (2.4 days), or IgM (1.7 to 3.3 days) similarly injected into normal skin sites. These comparisons led the authors to doubt the firm binding of reagins to human skin. Cass and Anderson (51), by using the same quantitative techniques employed by Fireman et al., calculated

28 338 FLICK for atopic reagins a half-life of 11 to 15 days. The reagins of one serum persisted 12 weeks or more at a PK site. The two reaginic fractions from another atopic serum studied quantitatively by Reid (341) had half-lives of 9 to 10 days. A "cold" reagin (see Partial Reagins and Queer Reagins) triggered to produce whealing at PK sites by the application of ice gave titers for Houser et al. (152) of 1:64 with latent periods of 1, 7, and 14 days, and of 1:8 at 28 days. Thus, the evidence indicates that reagins are probably quite diverse in their ability to fix to skin cells independent of the concentration of the reagin in the serum. One must also consider that the avidity of fixation depends not only upon the structure of the reagin but also upon the structure of the receptors of recipient cells. Such surface membrane structures could be under genetic control similar to the control of transplantation antigens. That reagins do fix to skin cells is further confirmed by the competitive, nonspecific blocking action of normal serum constituents upon the fixation of specific reagin. Such serum constituents can be either IgE globulin (179) or material with a sedimentation constant of 1 to 3S (133). The blocking protein is effective when in sufficient quantity in a mixture with the specific reagin (19, 170) used in PK testing, or when injected 24 to 48 hr (132, 133, 134, 159, 343) or even 6 days (170) before the injection of the specific reagin at the same skin site. The blocking protein is not active if injected into a reagin-sensitized site 46 hr after the reagin and 2 hr before the injection of antigen (170). Sensitization of Laboratory Animals It would be desirable to be able to sensitize laboratory animals for shock with human reagins as a substitute for the hazardous PK test in humans with its attendant risk of transmission of viral hepatitis. It has usually not been possible to produce shock in guinea pigs with human reaginic sera (10, 19, 47, 53, 57, 61, 82, , 169, 204, 301, 327, 336, 344), but exceptions have been reported among the sera tested. The evidence presented by Franklin and Ovary (116) as well as by others (19, 47, 168, 169, 303) indicates that the exceptions are due not to reagins but to associated IgG antibodies. However, probably not all subclasses of IgG antibodies can sensitize guinea pigs because Minden et al. (280) reported that 11 of 14 human anti-bsa sera tested by guinea pig BACTERIOL. REV. PCA failed to produce reactions, yet all negative sera contained anti-bsa of IgG class and some in sufficient amount to precipitate the antigen. Rabbits (53, 224), cats (224), and the dogs (215, 224) also seem insusceptible to sensitization with human reagins. Actually it is unknown whether human reagins are incapable of fixing to the cells of these animals or if reaction failure is due to an inability of the reagin-antigen complex to effect release of inflammatory substances from the cells. In the case of the rabbit, there is a further complication in that its skin does not wheal or regularly produce redness in response to histamine (79, 89). Various species of monkeys have been used successfully to detect human reagins. Grove (139) and Caulfeild (52) sensitized one monkey each with atopic sera in PK tests. Of 17 monkeys used by Straus (404), 16 gave positive PK tests with human atopic sera. The antigen could be injected either locally into the sensitized site as in humans or by the intravenous route to shock the skin sites. Layton and associates in a series of studies (224, ) found that most of the primate species tested reacted adequately at either PK or 24-hr PCA sites which had been sensitized with human reaginic sera. The active antibody when tested was always heat-labile. However, the PK test in monkeys gave rather variable results with respect to the appearance of the wheal and often with no visible erythema developing. The 24- to 48-hr PCA test with its intravenous injection of a mixture of antigen and a blue dye, the latter to enhance the visibility of the reaction of the sensitized skin site, proved to be an excellent substitute. Others have confirmed these findings that human reaginic sera produce monkey PCA (19, 46, 313, 317, 353). There is a good correlation between the results of human PK tests and monkey PCA tests with human sera. Most of these investigators have found that quantitative human PK tests are 10 to 100 times more sensitive than corresponding monkey PCA tests, although this is not always the case (353) and certainly must depend in part upon the reactivity of the human recipient used for making the comparison as well as the monkey. Human precipitin sera fail to yield positive 24-hr PCA tests in monkeys (19). Ishizaka et al. (169) were able to produce monkey PCA reactions only with IgE specific reagins but not with antibodies of IgA or IgG class. I believe that, at the present time, sufficient correlation data exist to indicate that monkey PCA measures the same an-

29 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS tibody in human sera that the human PK test does. Small pieces of viable monkey skin can be used to assay human reagins (130) by passive sensitization in vitro with measurement of the histamine released upon addition of antigen. The antibody involved was heat labile and test results correlated well with those of human PK tests performed on the same sera. Monkeys can be sensitized for anaphlyaxis when injected with sufficient human reaginic sera but not with human precipitating sera (46, 47, 139, 225, 313, 314). Monkey lung and smooth muscle structures have been used successfully in vitro for detecting human reagins. In the case of pieces of monkey lung, passive sensitization has been tried with a relatively small number of human sera so far. The criterion of antigenic shock is the release of histamine into the bath fluid (129, 262, 273). Results parallel those of human PK tests and the antibody activity for both tests is heat-labile. Two nonreaginic sera containing high-titered hemagglutinins, included in the report of Goodfriend et al. (129), were incapable of sensitizing monkey lung for histamine release. So far the data are consistent with the hypothesis that only reagins in human sera will sensitize monkey lung in vitro. Monkey ileum strips may be sensitized with human reaginic sera or their reagin-rich IgE fractions in vitro (10, 204, 219, 273, 436, 437) for antigen-induced contractions in a Schultz- Dale technique. One disadvantage of this test is that such strips are unpredictable in their responsiveness (10, 273). Usually the antibody measured in this test is heat labile and there has been a good correlation between the results of it and those of the human PK test. Another possible disadvantage is that the test may measure antibodies other than reagins as suggested by both Kunz et al. (219) and Wicher (436) after observing some reaginic sera that were active in this test before and after heating for 4 hr at 56 C. The reagins in these particular sera after heating were inactive as determined by human PK test. It remains to be decided whether monkey ileum is detecting antibodies other than reagin or if the heating incompletely inactivated the reagins in the samples and the human recipients possessed low sensitivity for their detection. There is evidence developing that human reagins can sensitize rat peritoneal mast cells for an antigen-induced shock reaction. This in vitro interaction will be considered in detail as a possible assay technique in Reagin Detection 339 and Assay Using Viable Cells (p. 341). PARTIAL REAGINS AND QUEER REAGINS Partial reagins, for the purpose of this discussion, are those that resemble reagins in conforming to either one but not both basic criteria that set reagins apart-prolonged fixation to tissues and sensitization for the release of histamine. Clear-cut descriptions of human antibodies with properties consistent with this concept are not common. Kuhns and Pappenheimer (218) and Parish (308) have described human antibodies that transferred successfully to recipient skin sites only when brief latent periods were used, and failed to provoke whealing when antigen was injected 24 hr or more after the serum. Two of these sera sensitized skin sites with 40 min, but not with 100-min latent periods; the others for a variable number of hours. The former authors suggested that this antibody formed reactive complexes with antigen without fixation, although brief reversible fixation was not ruled out. If it is assumed that some fixation of short duration occurred, then should this antibody be classed as a reagin? Usage of the term rigidly requires that the antibody should fix for at least 18 to 24 hr to be so classified. However, future experience may indicate a need for broadening the term. I prefer to believe that this antibody represents one end of a spectrum of reagins with respect to the ability to fix to tissues. This view is certainly at variance with current thought. Kuhns and Pappenheimer (218) also found that the antidiphtheria toxoid serum, Hu, contained a reagin that, upon heating, disappeared. But in its place they detected an antibody that blocked antitoxoid reagin and, furthermore, fixed to injected skin sites for long periods. Thus, this antibody resembled reagin in the ability to fix to skin but failed to sensitize. It was their belief that the heating converted the reagin present to a blocking, fixing antibody. An alternative viewpoint is that this fixing blocker occurred in the unheated serum as one of several types of antitoxoid present and that assay difficulties or error prevented its detection until the reagin was inactivated. A similar, fixing, blocking antibody was detected in unheated, human, antipneumococcal type 3 sera by Palczuk and Flick (305). This antibody was heat stable, blocked antipneumococcal type 3 reagins specifically, and was able to fix for prolonged periods to skin sites of most but not all recipients tested. When this

30 340 FLICK BACTERIOL. REV. fixing, blocking antibody was present in a serum along with reagin specific for type 3 polysaccharide, it was difficult to detect the presence of the reagin by PK test except in the skin of unusual recipients who, perhaps, did not fix the blocker. The data indicate a competition between reagin and the fixing blocker for the common antigen, and that when present together the relative concentrations of each would decide if the serum in a suitable recipient would give a reagin-antigen reaction or not Ȯther studies, done in vitro, indicate that a variety of antibodies, not yet identified fully as to biologic properties, can attach to homologous or nearly homologous cells. Homologous monocytes and neutrophiles possess surface receptors for human class G immunoglobulin (153, 154, 181, 275) and especially for proteins of subclasses IgG1 and IgG3 (153, 154, 275). For comparison under similar experimental conditions, human or monkey basophiles and mast cells have receptors for human IgE (155, 181). The baboon antibody of Kay et al. (201) that fixed to homologous monocytes fractionated from the serum along with the IgG but could not be identified with any of the known immunoglobulin classes with monospecific antisera. In addition, Hubscher et al. (155) presented evidence that their chromatographic fraction I of one human serum contained antibody, associated with IgG, possessing the ability to fix to monkey mast cells. This fixation ability was heat labile. That reagin was not involved in the fixation was indicated by the failure of fraction I to give a positive PK test. However, the presence was not disproven of a peculiar, hypothetical IgE antibody that had the ability to fix to cells but not the ability to mediate histamine release. It is unclear from these in vitro experiments if the involved immunoglobulins can fix to cells for prolonged periods comparable to the fixation of the average reagin, or if the fixation is a rapidly reversible one. The temperature used for washing the cells to remove loosely absorbed proteins, often not specified in the above reports, also could markedly influence the ability of some of these antibodies to fix to cells as indicated by the work of Boyden and Sorkin (40) on the fixation of rabbit or guinea pig antibodies to homologous cells. In their experiment, antibodies that fixed to splenic cells at room temperature rapidly eluted at 37 C. A peculiar reagin has been found in the sera of some patients with acquired cold urticaria, a disease brought on either in a localized or generalized form by chilling. If such a serum is injected intradermally into a normal recipient, the application of ice to the site after a latent period of 24 hr will induce a wheal and erythema reaction. Chilling thus substitutes for specific antigen injection of the PK test. A brief period of warming of the site following chilling is required (194, 384) to elicit the whealing. The reagin involved is peculiar in two respects: the specificity of the "cold reagin" or of its antigen (if any) is unknown and the triggering of the cold reagin-cell complex for shock requires chilling. Otherwise, the properties of cold reagins correspond to those of the usual reagins. Thus, cold reagins are heat labile (152, 384) and fix to skin sites for variable, long periods of time; at least 28 days for some sera and lesser times for others (152). Local shock can be produced by ice after latent periods of 1 to 4 hr (194, 384), the shortest periods tested. The sera from two reported patients (152, 384) contained usual reagins to known antigens as well as cold reagins. PK sites sensitized with these sera upon shock and desensitization with injections of the known antigens were still fully reactive upon exposure to ice and vice versa, indicating a specificity for cold reagins. Cold reagins in two of the sera described by Houser et al. (152) were identified as IgE globulins; the activity being absorbed from the sera by anti-ige only and not by antibodies specific for other known human Ig classes. Also in this study, a normal IgE fraction of sufficient concentration blocked the fixation of cold reagins to normal skin just as it does for the usual reagin. The application of ice to skin sites passively sensitized with cold reagins not only causes shock but also desensitizes the sites with varying degrees of difficulty, possibly depending upon the titer of cold reagins present. The serum described by Sherman and Seebohm (384) sensitized sites that became desensitized by a single application of ice. But one serum reported upon by Houser et al. (152) and another by Samsoe- Jensen (362) produced whealing at sensitized sites with each of three successive applications of ice about 24 hr apart. One of these serum sites (152) when tested with ice a fourth time was found to be desensitized. Cold reagins have certain characteristics remarkably similar to those possessed by human cold hemagglutinins suggesting that the latter might well serve as a model of sorts for the former. Cold hemagglutinins occur in association with a variety of disease states but

31 PROPERTIES OF HUMAN REAGINS VOL. 36, especially with infections by Mycoplasma pneumoniae. The immunogens responsible for their production are unknown except for M. pneumoniae (76). Cold hemagglutinins seemingly cross-react with an antigen present on most human red cells, including those of the person producing the antibody. The union between the cold hemagglutinin and the erythrocyte is a weak one, as indicated by the rapid dissociation of the antibody as the temperature increases toward 37 C from the optimal temperature of about 4 C for union and agglutination (400). The data dealing with cold reagins suggest that the antigen uniting with this fixed antibody to produce whealing upon chilling is a cross-reacting normal constituent of the plasma or lymph of the donor and also of the PK recipient. That such a hypothetical union occurs only with chilling suggests that the bond is a weak or cross-reacting one and that, therefore, the original immunogen for cold reagin production does not coincide with the serum antigen. This hypothesis possibly could be tested in monkey skin because, unlike the usual reagin, one sample of cold reagin (152) failed to sensitize for an ice-induced response in monkeys. Such a serum under the same conditions might produce a reaction provided the sensitized skin site in the monkey received an injection of normal human serum just prior to the application of ice. One area where cold reagins seem not to react like cold hemagglutinins involves the reversible dissociation of antigen upon warming. So far there is no evidence definitely indicating that the warming of a shocked cold reagin PK site caused renewal of the ability of the cold reagin to produce future shocks, although some available data (152, 334, 362) could be so interpreted. Various body conditions and functions might interfere with this process, however. The serum from some patients with solar urticaria also contains an antibody which acts like a reagin but again the antigen remains unidentified. This reagin triggers a typical whealing reaction either in the serum donor or in a passively sensitized recipient upon exposure of skin sites to photons in the ultraviolet and short visible wavelength regions (143). The reagin is heat labile and fixes for long periods to the skin (143, 362). With some sera (143), the necessary exposure of the skin site to light can be accomplished either before or after the injection of the serum and will still lead to whealing. These observations suggest that the hypothetical antigen involved is a skin constituent, modified and released over a period of time by the light energy absorbed momentarily. That such a similar mechanism, release of skin cell antigen by chilling, might apply also for cold reagins, seems unlikely from the trials along these lines of Sherman and Seebohm (384) and by the negative results obtained in the experiment of Rajka and Asboth (334) designed to test this hypothesis. REAGIN DETECTION AND ASSAY USING VIABLE CELLS Assay in the Skin The concentration of serum reagin can be estimated by transfer to the skin of recipients by three techniques. The simplest, but relatively inaccurate method is to inject serum, either undiluted or as a single dilution, into a PK site and measure the wheal size developed upon injecting an excess of antigen. The reagin concentrations of several sera being compared are then roughly related to the wheal sizes. A more quantitative method, probably the best of the three, involves injecting serial dilutions of the reaginic serum into PK sites to determine the minimal amount of serum that yields a detectable reaction (71). Finally, reagin assay by the neutralization technique is based on observations of Coca and Grove (61) that when a mixture of reagin and antigen, the latter in excess, is injected intradermally the reagin becomes neutralized as determined by a lack of reactivity when the same site is later challenged with more antigen. A control mixture of reagin and saline injected into a nearby skin site would readily wheal upon challenge with antigen in a parallel procedure. From this, the technique that developed to measure reagin concentration (251, 255, 312, 431, 440) involved injecting into separate skin sites a series of mixtures of serum in constant amount, and antigen serially diluted. At some convenient time, 6 hr to several days later, the sites were challenged with excess antigen to determine the presence or absence of residual reagin at each site. The reagin concentration, expressed in terms of antigen equivalents per milliliter, was indicated by the mixture containing the least amount of antigen that just neutralized the reagin. Arbesman and Eagle (8) found the minimum error, based on replicate determinations, to be 50 to 100% for this technique. However, this procedure of neutralization not only measures reagins but also other antibodies of the same specificity in the serum

32 342 FLICK specimen, a fact realized gradually as data accumulated to indicate that most sera containing reagins also possess other antibodies of the same specificity. Therefore, in most situations, the serial dilution technique of obtaining a reagin titer is the most accurate method yet devised of expressing reagin concentration by skin testing. The usual technique of the PK test has been modified in a variety of ways for special purposes at times. It seems very likely that the modifications described below still permit the resulting test to measure the same antibody as measured by the original PK procedure. No proof of this belief has been offered; rather it is based on intuitive reasoning. Thus, if antigen is pricked rather than injected into the sensitized skin site (399), the measurements are essentially the same. Antigen can be injected at a distance from the sensitized site by the intravenous route (430), or by the intramuscular route (251, 440) with comparable results, although the doses required are larger than those needed for local injection. Evans blue dye given intravenously to human PK recipients at the time of antigen injection into the sensitized site (315) will produce a bluing of the whealing reaction as it unfolds, comparable to the PCA reaction produced by human reagins in monkeys. The antigen for PK tests can be administered by the oral (15, 127, 245, 430) or rectal (430) routes with successful shock of the sensitized sites resulting in some cases. When these latter, more bizarre routes of antigen administration are employed, the onset of whealing is delayed, requiring 0.5 to 2.5 hr. Use of Leukocytes Instead of using serum as a source of reagin to sensitize PK sites, Walzer and associates (432, 433) have successfully used washed leukocytes from some atopics. However, the intradermal injection of leukocytes causes a local, slowly subsiding, indurated reaction, necessitating latent periods of 7 to 11 days before injecting antigen. Whealing reactions were usually small, possibly a result of the long latent periods and the small amount of reagin transferred by leukocytes. The white cells of one patient produced a positive PK test whereas his serum did not, indicating a distinct usefulness for leukocytes when exploring for the presence of reagins occurring in low concentration. BACTERIOL. REV. The employment of leukocytes for detecting reagins in vitro had its origins in the observations of Katz (200) that blood cells from atopics would release histamine to the plasma phase upon addition of antigen. Over a period of years studies (200, 248, 292, 294, 426) have indicated that histamine release by antigen correlated positively with the use of atopic, but negatively with nonatopic bloods. From groups of atopic persons, evidence also developed that a rough relationship existed between the amount of histamine released in vitro from each blood under standard conditions and the size of the wheal developed upon skin testing of the donor of the same blood (293, 329, 426). Finally it became evident that leukocytes from some normal blood samples could be sensitized passively in vitro by reaginic sera for histamine release by the proper antigen (183, 243, 244, 247, , 427). In passive sensitization experiments, the amount of histamine released by antigen or its substitute, anti-ige, was directly proportional to the concentration of reagin added (244, 246). The antibody in those sera that successfully sensitized leukocytes for this shock reaction was heat labile to the same extent as reagin (183, 243, 244, 426, 427). Sera lacking reagin detectable by PK test failed to passively sensitize normal white cells for histamine release (294). IgE, but not IgA, IgD, and IgM (183), passively sensitized leukocytes for shock produced by the addition of the respective specific Ig class heterologous antiserum. In this study, IgG at high concentration also sensitized cells for shock by anti-igg addition, but Ishizaka et al. indicated that the result could well have been due to a small amount of IgE contamination in the IgG preparation with a small amount of anti-ige as contaminant in the anti-igg serum. A subsequent restudy (246) indicated that the antibody specificity involved in the leukocyte shock by anti-igg serum was probably not due to traces of anti- IgE. The addition of an excess of IgE protein to the antiserum failed to reduce the ability of the serum to release histamine. When known reaginic bloods were used in this in vitro leukocyte shock reaction, the presence of serum complement seemed to play no role (246, 249, 277). Conceivably, complement attached to the surface of leukocytes might be involved in the shock reaction, but Middleton and Sherman (277) were unsuccessful in attempts to extract hemolytic complement from leukocytes known to be active in the histamine release reaction. It thus seems very likely that complement is not involved in reagin-mediated leukocyte shock. Furthermore, from the evidence cited above, it is evident that the reagin is the anti-

33 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 343 body responsible for the human leukocyte Conditions for the histamine release phase of shock reaction and that under the conditions the reaction are rather exacting, especially in of experimentation usually no other type of the absence of serum, with respect to temperature of incubation and cation needs (243, 248, antibody of humans produces this effect. In fact, some other antibodies specific for the allergen and associated with the reagin in sera mine) of: (i) specific antigen (17, 244, 318, 321, 278, 330, 426). The maximal release of hista- from untreated and especially from hyposensitized atopic patients seem to play a protective centrations release lesser amounts of hista- centration (in that greater than optimal con- role by successfully competing with the reagin mine) of, (i) specific antigen (17, 244, 318, 321, for the antigen in these in vitro systems (250, 426), (ii) antigen's equivalent, anti-ige (321), 425). or (iii) aggregated IgE myeloma protein, nonspecifically (174). Interestingly, an antigen One step, usually employed in the passive sensitization of leukocytes, could play a crucial concentration optimum also occurs for maximal whealing when complexes of antigen and role in limiting the mediation of the shock reaction to reagins alone. This procedure is the reagin in a series of ratios are injected into the washing of the sensitized leukocytes before skin, but not when antigen alone is injected addition of antigen; a process which could into presensitized sites (164). remove reversibly adsorbable antibodies, especially when completed at 37 C, that otherwise that release histamine upon reagin-antigen The type and concentration of leukocytes might be involved in complement-dependent shock could be important factors in deciding or complement-independent histamine release whether or not a given sample of blood is capable of detecting reagin. The involved cell of the types studied in laboratory animals (300). When Sampson and Archer (361) mixed type(s) must contain histamine. The standard human group A red cells with human anti-a work on the histamine content of human blood agglutinating serum and added homologous leukocytes, that of Graham et al. (136), indicates that the basophile, which constitutes leukocytes, histamine was released in a reaction probably complement dependent. That usually less than 2% of the total leukocytes, complement was involved was indicated by carried 51% of the total blood histamine. The the loss of reactivity of the serum following histamine present in tissue and in blood or any each of three separate treatments: (i) heating of its compartments is that which can be extracted for subsequent assay with acid or by at 60 C for 3 min, (ii) two absorptions with zymosan, or (iii) treatment of the serum with boiling (18, 244). Lymphocytes, about 35% of cobra venom. Although these three methods of the total leukocytes, carried about 1%, and eosinophiles, about 3% of the leukocytes, carried inactivating components of complement strongly implicate the role of complement in 29% of the total histamine. After subtracting the reaction, yet even here we cannot exclude small amounts detected in platelets, serum, the inactivation of a possible reagin of anti-a and erythrocytes, the remaining histamine of specificity. The heating conditions were sufficient to inactivate some reagins probably and remaining cell types, i.e., 0.5% to monocytes, the total was assigned by Graham et al. to the the effects of the other two inactivators of which were assumed to be like lymphocytes in complement upon reagins are unknown. histamine concentration, and 14.5% to neutrophiles. Thus, three cell types were believed to The use of human blood leukocytes for the in vitro detection of reagins can hardly be considered as a routine test at this time. Many involved in reagin-mediated shock. Lichten- contain most of the histamine and could be batches of blood fail to release histamine in stein and Norman (247) state that in their best the presence of reagin and antigen. Only 20%o sensitized leukocyte preparations 80 to 100% of of the blood samples tested by Levy and Osler the total histamine was released by reagin-antigen shock. In such a situation, all cellular (243) yielded satisfactory levels of histamine release. Middleton (276) also noted that only sources of histamine must have been involved. certain bloods could be sensitized and shocked However, more recent analyses for the source and that some reaginic sera gave negative results when used to sensitize satisfactory blood are capable of separating to a degree various of histamine employing newer techniques that cells. It should be expected that some sera leukocyte types, indicate that the human basophile is probably the sole possessor of hista- with low titered reagins by PK test will fail to promote in vitro leukocyte shock because the mine. Sampson and Archer (361) could detect PK test is more sensitive than the leukocyte no histamine in preparations of mixed neutrophiles and eosinophiles, but free of shock test according to Levy and Osler (244). basophiles.

34 344 FLICK Similarly, the analyses of Pruzansky and Patterson (331) place 78 to 94% of the total histamine in the blood basophile and little if any in neutrophiles. It is becoming more evident that the basophile is the only cell in human blood that experiences reagin shock, i.e. histamine release, and as a result perhaps the conclusion is valid that one reason for the failure of a blood to yield reagin-antigen shock is its low basophile content. Another reason is that basophiles from some persons suitably sensitized with IgE fail to release histamine upon addition of anti-ige (181) suggesting a defective enzyme system in the donor's cells, perhaps a very desirable defect to possess. Basophile-Mast Cell Degranulation Shelley and Juhlin (377) found that the basophiles of blood taken from humans who had experienced anaphylaxis or urticaria, upon brief exposure in vitro to the alleged allergen, often showed depletion of their basophile granules. The granules of shocked cells either lie abnormally at the periphery of the cytoplasm or have been mostly expelled from the cell. In the case of rat basophiles or mast cells this process of degranulation seems to be a prerequisite for the release of histamine from the granules under certain conditions of antigenantibody shock (283, 284, 424). The results of the human (direct) basophile degranulation test, from various studies, had a degree of association with past allergic reactions, especially to drugs like penicillin, but were not investigationally related to the presence of reagins in the blood as detected by the PK test. Because human blood often contained too low a concentration of basophiles to permit a significant evaluation of the test results, Shelley (376) tried using rabbit blood as a richer source of basophiles to be passively sensitized by "allergic" human serum in an indirect test. As a result of studies in some laboratories, this test has been discredited as being clinically useful for prognostication. Schwartz et al. (369) tried rat peritoneal mast cells in a similar indirect test and reported that four human "allergic" sera successfully caused degranulation in the presence of the alleged antigen. In addition, the direct, basophile degranulation test, involving the blood of each patient, was positive with the same antigen. Perelmutter and associates (321, 323, 324) have studied the rat mast cell degranulation test in more detail, especially with respect to the type of human antibody involved in its induction. In these reports, a BACTERIOL. REV. total of 14 human sera considered to contain reagins were tested and found positive in the rat mast cell test. The evidence supporting the hypothesis that the test detects human reagins is multifaceted. The activity in the sera so tested was heat labile at 56 C for 0.5 hr (conditions which also inactivate complement). Rat mast cells sensitized with human serum were shocked into degranulation by anti-ige serum, but not by anti-human Ig sera of class specificity A, D, G, or M. The sensitizing antibody in the few sera so tested remained fixed to the mast cells at least through one washing but did not fix strongly enough to withstand two washings. The percentage of cells degranulating correlated with the PK titer in rank-order tests involving eight reaginic sera. Hemogglutination titers of these sera did not correlate with the results of the degranulation tests. The serum activity involved in the mast cell test was sensitive to 0.1 M mercaptoethanol as was the PK activity. One serum fractionated by sucrose density gradient ultracentrifugation and separately by DEAE-Sephadex chromatography had PK activity and mast cell degranulation activity coincide in the fractions in both cases. Finally, a purified human myeloma protein (ND) of IgE specificity sensitized rat mast cells for degranulation by anti-human IgE serum. Korotzer et al. (205) also have reported positive results for this test with all of eleven human "allergic" sera (no PK test results reported). Here also anti-ige serum, but not anti-ig sera specific for the other four human classes of Ig, caused degranulation of "allergic" serum-sensitized rat mast cells. They too, found the activity in the "allergic" sera to be heat labile. Thus, the early evidence seems good that human reagin of IgE class can sensitize rat mast cells for degranulation. However, there are some suggestions that perhaps antibodies other than IgE reagins can prepare rat mast cells for this shock reaction. Schwartz et al. (369) noted that one human serum studied in some detail seemed to require complement for the degranulation reaction, in that "allergic" serum inactivated at 56 C for 0.5 hr had its degranulating potential restored by the addition of fresh, normal serum. Perelmutter and Liakopoulou (323) likewise found similar evidence for some sera that.a heat-labile cofactor might be involved. Although the review of the evidence presented earlier indicates that human reagins probably do not involve complement in their allergic interactions with human cells and antigen, no data are available as to such requirements for their involvement with rat

35 VOL. 36, 1972 PROPERTIES OF HUMAN REAGINS 345 mast cells. However, the "burden of proof' would seem to require that a human antibody utilizing complement in the rat mast cell test would be considered to be nonreaginic in properties until proven otherwise. In its quantitative aspects, the rat mast cell degranulation test at present depends upon the percentage of mast cells showing definite morphological changes as an expression of the concentration of the reagin in the serum under test, at best a semiquantitative expression. Normal serum under the revised conditions of the test described by Perelmutter and Millard (324) still produces a low incidence of degranulation (2 to 8% in reference 323) which must be subtracted from the higher percentage produced by the positive serum under test. Normal human serum is toxic for rat mast cells, but this effect can be reduced by the additional presence of normal rat serum. The sensitivity of the rat mast cell test is probably somewhat less than that of the human PK test. The former test could detect a minimum of about 2 ng of N/ml of purified IgE myeloma protein used to sensitize the cells subsequently shocked with anti-ige (323). If this estimate of sensitivity extends to human reagins in serum, then by using the minimal, detectable concentration derived from the best sensitivity value of Table 1 for the PK test, the comparison indicates that the latter is about 200 times more sensitive. On the other hand, one of the sera used by Perelmutter and Liakopoulou (323) had a PK titer of 1:20 and yielded 15% degranulation in the rat mast cell test. Future work must unravel this discrepancy, if significant, in comparative sensitivity. Degranulation tests are relatively simple and quick in operational procedure as compared to the more quantitative and laborious histaminerelease assay techniques. DISCUSSION The evidence that has accumulated concerning the nature of human reagins indicates that this biological entity represents a spectrum of antibodies, different in degrees in their properties one from another but all possessing a few properties in common. Within this spectrum, the usual or common human reagin belongs to class IgE, is heat labile and thiol sensitive, rapidly sensitizes the skin of humans to mediate shock after little or no latent period, fixes for a long half-life, and sensitizes monkeys in addition to humans. Triggered by antigen, it causes histamine to be released from the cytoplasmic granules of mast cells and basophiles. It is probably produced to a varying degree by all persons except rarely those whose defects, genetic or acquired, interfere with its synthesis. Production of reagin is not limited to atopic persons. Reagin can sensitize for various immediate-type (i.e. histamine-release type) allergic conditions in almost all humans including atopics. But in the latter group, perhaps because of the irregular development of a special, mysterious potential for reactivity of certain tissues, reagins can also sensitize for diseases like hayfever and asthma. Because of the difficulties associated with any investigation of reagins, it is common to encounter reports that deal with the reagin found in single or a very few samples of serum. The question must then be asked if the reagin is typical in its properties of the common type of reagin, or if it is peculiar and uncommon. Only further study, of course, of the property in many specimens of reagin can delineate its spectral position in relation to others. It is the task of immunochemistry to assign the biologic functions of antibodies to local structural areas of the molecule. In the case of reagins, there are at least three properties that need to be so assigned: (i) the antibody function, (ii) the ability to fix to certain cell types, and (iii) the ability to sensitize or mediate for shock of the cell. The antibody functional groups of reagin or IgE, based partly on enzyme dissection directly, and indirectly more on analogy to the structure of IgG, are relegated to the Fab portion of the molecule, are two in number, and involve portions of both the H and L chains. Although no data yet exist for reagins, it seems likely from a comparison with other types of antibodies that some reagins can have one of the two antibody sites nonfunctional. This could occur as a result of a masking of one site by an external agent, or perhaps, through an uncommon type of folding of the chains, i.e., steric masking, yielding a univalent-acting reagin. Of course, by the same process, both sites might be inactivated for complete failure of antibody function. The cell fixation of reagin in the few cases studied can be assigned to an area on the Fc portion of the molecule. This function seems largely independent of the antibody areas. The fact that reagin or IgE fix to basophiles and that IgG fixes to neutrophiles and monocytes suggests that structural differences exist between these two classes of antibodies at the cell-fixation areas. Within the group of reagins, variation in ability to fix to cells, as evidenced

36 346 FLICK by duration of fixation, also indicates some variation in the structure of the fixation area. The third or sensitizing ability of reagin might belong to a separate area of the molecule, an area also assignable to the F, portion, because both anti-ige uniting with cell-fixed Fc fragment (of IgE) and aggregated F, fragment of IgE (174) cause histamine release. It seems evident that before sensitization can occur, fixation must occur. It has been generally assumed that sensitization is a sole function of fixation, but the cited work of Hubscher et al. (155) suggests that for some antibodies fixable to mast cells the two properties of fixation and sensitization are indeed separate functions, dependent upon distinct structural areas. Further suggestive evidence of separate functions can be found in the cited bridging investigations of Ishizaka et al. in that bivalent but not univalent anti-ige, added to cell-fixed IgE, caused cell shock. Bridging perhaps brings a structural area of the IgE molecule into apposition with cell surface components, thereby activated, which in turn are responsible for histamine release. It can be argued that since other properties of antibodies show much variation, this sensitizing property will likewise show variation, as will the hypothetical structural area responsible for it. The assumption is also made that the variation in structure of this area is independent for the most part of the other functional areas under consideration. Then, in general, IgE molecules that show extreme variation from the average in the direction of (i) lacking all antibody function, or (ii) lacking the ability to fix to histamine-containing cells, or (iii) lacking the ability to sensitize, cannot be classed as reagins or be expected to give rise to human immediate-type hypersensitivity. On the other hand, an antibody that contains the necessary structure on its F, portion to conform to a class other than IgE might also possess structural areas resembling those of IgE responsible for the properties of fixation to mast cells and sensitization for histamine release. Then this hypothetical antibody would be classed as a reagin. BACTERIOL. REV. LITERATURE CITED 1. Adams, D. D Antibody formation in allergic and in normal people. Lancet 2: Allansmith, M., and D. Buell The relationship of yla-globulin and reagin in cord sera. J. Allergy 35: Alston, J. M., G. R. Galbraith, and D. Stewart The soluble specific substance of pneumococcus type I: antibactericidal action: skin reactions. J. Pathol. Bacteriol. 33: Alston, J. M., and A. S. R. Lowdon Studies of the skin reactions to the specific soluble substances of the penumococcus types I and II. Brit. J. Exp. Pathol. 14: Amkraut, A. A., L. T. Rosenberg, and S. Raffel Elicitation of PCA by univalent and divalent haptens. J. Immunol. 91: Ammann, A. J., W. A. Cain, K. Ishizaka, R. Hong, and R. A. Good Immunoglobulin E deficiency in ataxia-telangiectasia. N. Engl. J. Med. 281: Andersen, B. R., and W. E. Vannier The sedimentation properties of the skin-sensitizing antibodies of ragweed-sensitive patients. J. Exp. Med. 120: Arbesman, C. E., and H. Eagle Assay of ragweed pollen extracts. J. Allergy 10: Arbesman, C. E., and H. Eagle Thermolability of ragweed pollen extract and its corresponding reagin. J. Allergy 11: Arbesman, C. E., P. Girard, and N. R. Rose Demonstration of human reagin in the monkey. II. In vitro passive sensitization of monkey ileum with sera of untreated atopic patients. J. Allergy 35: Arbesman, C. E., and K. Ito A new method for measuring IgE. J. Allergy 47: Arbesman, C. E., S. Z. Kantor, D. Rapp, and N. R. Rose Immunologic studies of ragweed-sensitive patients. III. Clinical aspects: the relationship of reagin and hemagglutinating antibody titers to results of hyposensitization therapy. J. Allergy 31: Arbesman, C. E., S. Z. Kantor, N. R. Rose, and E. Witebsky Serum sickness; serologic studies following prophylactic tetanus antitoxin. J. Allergy 31: Arbesman, C. E., N. R. Rose, S. Z. Kantor, and R. B. Beede Immunologic studies of ragweed-sensitive patients. I. Specificity and sensitivity of hemagglutination reactions. J. Allergy 31: Arkins, J. A., P. P. Ruetz, and T. L. Squier Mephenesin (Tolserol) sensitivity with demonstrable skin-sensitizing antibody in the serum. J. Allergy 28: Armstrong, C A study of ragweed pollen extracts for use in the treatment of ragweed pollen hypersensitiveness. Pub. Health Rep. 39: Assem, E. S. K., and M. K. McAllen Serum reagins and leucocyte response in patients with house-dust mite allergy. Brit. Med. J. 2: Assem, E. S. K., and H. 0. Schild Detection of allergy to penicillin and other antigens by in vitro passive sensitization and histamine release from human and monkey lung. Brit. Med. J. 3: Augustin, R., R. C. Connolly, and G. M. Lloyd.

37 VOL. 36, 1972 PROPERTIES OF IHIUMAN REAGINS Atopic reagins as a prototype of cytophilic antibodies. Protides Biol. Fluids Proc. Colloq. 11: Augustin, R., and B. J. Hayward Human reagins to grass pollens and moulds: their purification and physicochemical characterization. Immunology 3: Austen, K. F., and E. L. Becker Mechanisms of immunologic injury of rat peritoneal mast cells. II. Complement requirement and phosphonate ester inhibition of release of histamine by rabbit anti-rat gamma globulin. J. Exp. Med. 124: Austen, K. F., et al Nomenclature of complement. Int. Arch. Allergy 37: Austrian, R., and C. M. MacLeod A type specific protein from pneumococcus. J. Exp. Med. 89: Barker, S. A., C. N. D. Cruikshank, J. H. Morris, and S. R. Wood The isolation of trichophytin glycopeptide and its structure in relation to the immediate and delayed reactions. Immunology 5: Batchelor, F. R., and J. M. Dewdney Some aspects of penicillin allergy. Proc. Roy. Soc. Med. 61: Batchelor, F. R., J. Dewdney, J. G. Feinberg, and R. D. Weston A penicilloylated protein impurity as a source of allergy to benzyl penicillin and 6-aminopenicillanic acid. Lancet, 1: Becker, E. L., I. Mota, and D. Wong Inhibition by antihistamines of the vascular permeability increase induced by bradykinin. Brit. J. Pharmacol. Chemother. 34: Becker, R. J., D. B. Sparks, S. M. Feinberg, R. Patterson, J. J. Pruzansky, and A. R. Feinberg Delayed and immediate skin reactivity in man after the injection of antigen in emulsion. J. Allergy 32: Bell, S. D., and Z. Eriksson Studies in the transmission of sensitization from mother to child in human beings. I. Transfer of skin sensitizing antibodies. J. Immunol. 20: Bennich, H., K. Ishizaka, T. Ishizaka, and S. G. 0. Johansson A comparative antigenic study of 'YE-globulin and myeloma IgND. J. Immunol. 102: Berdal, P Serologic investigations on the edema fluid from nasal polyps. J. Allergy 23: Berg, T., H. Bennich, and S. G. 0. Johansson In vitro diagnosis of atopic allergy. I. A comparison between provocative tests and the radioallergosorbent test. Int. Arch. Allergy 40: Berg, T., and S. G. 0. Johansson Immunoglobulin levels during childhood, with special regard to IgE. Acta Paediat. Scand. 58: Berg, T., and S. G. 0. Johansson IgE concentration in children with atopic diseases: a clinical study. Int. Arch. Allergy 36: Berquist, G Studies on signs of antigenantibody reaction in blood and serum from allergic subjects: a preliminary report. Acta Allergol. 15: Bernton, H. S., J. R. Spies, and H. Stevens Evidence of the multiplicity of allergens and reagins in cottonseed sensitiveness. J. Allergy 13: Biberstein, H Beitrage zur passiven Uebertragung der Ueberempfindlichkeit gegan chemischebekannte Stoffe. Z. Immunitaetsforsch. Exp. Ther. 48: Binaghi, R. A., and B. Benacerraf The production of anaphylactic antibody in the rat. J. Immunol. 92: Blackley, C. H Experimental researches on the causes and nature of catarrhus aestivus (hayfever or hay-asthma). Bailliere, Tindall, and Cox, London. 40. Boyden, S. V. and E. Sorkin The adsorption of antibody and antigen by spleen cells in vitro: some further experiments. Immunology 4: Brattsten, I., H. Colldahl, and A. H. F. Laurell The distribution of reagins in the serum protein fractions obtained by continuous zone electrophoresis. Acta Allergol. 8: Brown, E. A., E. M. Holden, and C. Nobili Skin test blocking antibody response to oral pollen therapy and criteria for its use. Ann. Allergy 5: Brown, H., and H. S. Bernton Studies on the hymenoptera. IV. Correlation of passive transfer in hymenoptera allergy with culprit insect and direct skin tests. J. Allergy 44: Brunner, M Immunological studies in human parasitic infestations. I. Intradermal testing with parasitic extracts as an aid in the diagnosis of parasitic infestation. J. Immunol. 15: Brunner, M Active sensitization in human beings. J. Allergy 5: Buckley, R. H., and R. S. Metzgar The use of nonhuman primates for studies of reagin. J. Allergy 36: Buckley, R. H., and R. S. Metzgar Reactivity of human IgG antibodies in primate and guinea pig passive anaphylaxis. Int. Arch. Allergy 29: Cann, J. R., and M. H. Loveless Distribution of sensitizing antibody in human serum proteins fractionated by electrophoreses-convection. J. Immunol. 72: Cann, J. R., and M. H. Loveless Biophysical characterization of reaginic and blocking sera. J. Allergy 28: Casper, W The preparation of the typespecific carbohydrates of gonococci. J. Immunol. 32: Cass, R. M., and B. R. Andersen The disappearance rate of skin-sensitizing antibody activity after intradermal administration. J. Allergy 42: Caulfeild, A., M. Brown, and E. Waters

38 348 FLICK Suitability of the monkey (Macacus rhesus) as a recipient for the Prausnitz-Kiistner reaction. Proc. Soc. Exp. Biol. Med. 35: Caulfeild, A., C. Cohen, and G. Eadie The antigenic properties of pollen fractions. J. Immunol. 12: Centifanto, Y. M., and H. E. Kaufman A simplified method for measuring human IgE. J. Immunol. 107: Chan, P. C. Y., and H. F. Deutsch Immunochemical studies of human serum Rh agglutinins. J. Immunol. 85: Chant, E. H., and L. N. Gay Skin reaction to human sera. Bull. Johns Hopkins Hosp. 40: Chase, M. W Production of local skin reactivity by passive transfer of anti-protein sera. Proc. Soc. Exp. Biol. Med. 52: Chase, M. W Studies on the sensitization of animals with simple chemical compounds. X. Antibodies inducing immediate-type skin reactions. J. Exp. Med. 86: Chopra, S. L., B. A. Kovacs, and B. Rose Detection of human reagins by the Schultz- Dale technic using human appendix. Int. Arch. Allergy 29: Clark, J. A., and M. G. Gallagher Neutralization of atopic reagins in vivo. J. Immunol. 12: Coca, A. F., and E. F. Grove Studies in hypersensitiveness. XIII. A study of the atopic reagins. J. Immunol. 10: Cohen, M. B., and T. Nelson A sheep antibody which blocks the Prausnitz-Kiistner reaction. J. Immunol. 34: Cohn, M., and A. M. Pappenheimer A quantitative study of the diphtheria toxinantitoxin reaction in the sera of various species including man. J. Immunol. 63: Collins, J., E. Dundas, H. Edgar, and J. H. Toogood Graded response of experimental skin wheal and its suppression by chlorpromazine and antihistamine. J. Allergy 31: Connell, J. T Sensitization of human subjects after multiple intracutaneous injections of aqueous ragweed extract. J. Allergy 40: Connell, J. T., E. B. Connell, and D. Lidd Studies on the placental transfer of skin-sensitizing antibody, specific binding of a ragweed fraction, and immunoglobulins. J. Allergy 39: Connell, J. T., and W. B. Sherman Skinsensitizing antibody. I. Relationship of the skin sensitizing antibody titer to the occurrence of symptoms in untreated persons with a positive ragweed skin test. J. Allergy 34: Connell, J. T., and W. B. Sherman Skinsensitizing antibody titer. III. Relationship of the skin-sensitizing antibody titer to the intracutaneous skin test to the tolerance of injections of antigens, and to the effects of BACTERIOL. REV. prolonged treatment with antigen. J. Allergy 35: Connell, J. T., and W. B. Sherman Changes in skin-sensitizing antibody titer after injections of aqueous pollen extract. J. Allergy 43: Cooke, R. A Consideration of some allergic problems. II. Serologic studies of skin reacting allergies (hay fever type). J. Allergy 15: Cooke, R. A., J. H. Barnard, S. Hebald, and A. Stull Serological evidence of immunity with coexisting sensitization in a type of human allergy (hay fever). J. Exp. Med. 62: Cooke, R. A., A. E. 0. Menzel, and P. A. Myers The antibody mechanism of ragweed allergy. Electrophoretic and chemical studies. II. The skin sensitizing factor. Int. Arch. Allergy. 17: Cooke, R. A., A. Menzel, P. Myers, S. J. Skaggs, and H. Zeman Spontaneous and induced allergies of the immediate type in man. J. Allergy 27: Cooke, R. A., and W. C. Spain Studies in hypersensitiveness. XXVI. A comparative study of antibodies occurring in anaphylaxis, serum disease and the naturally sensitive man. J. Immunol. 17: Coombs, R. R. A., A. Hunter, W. E. Jonas, H. Bennich, S. G. 0. Johansson, and R. Pansani Detection of IgE (IgND) specific antibody (probably reagin) to castor-bean allergen by the red-cell linked antigen antiglobulin reaction. Lancet 1: Costea, N., V. J. Yakulis, and P. Heller The mechanism of induction of cold agglutinins by mycoplasma pneumoniae. J. Immunol. 106: Curry, J. J The effect of antihistamine substances and other drugs on histamine bronchoconstriction in asthmatic subjects. J. Clin. Invest. 25: Curry, J. J Comparative action of acetylbeta-methyl choline and histamine on the respiratory tract in normals, patients with hayfever and subjects with bronchial asthma. J. Clin. Invest. 26: Darsie, M. L., S. M. Perry, D. Rosenfeld, and J. A. Zaro Size of histamine wheal in relation to dose and animal species. Proc. Soc. Exp. Biol. Med. 59: Davidson, A. G., B. Baron, and M. Walzer Factors influencing reagin formation in experimental human sensitization to ascaris lumbricoides antigen. I. Influence of chronic infection (tuberculosis) on the rate of sensitization. J. Allergy 18: Davis, H. M Horse serum skin tests. J. Hyg. 38: De Besche, A Studies on the reactions of asthmatics and on passive transference of hypersusceptibility. Amer. J. Med. Sci. 166:

39 VOL. 36, 1972 PROPERTIES OF H[UMAN REAGINS Delorme, P. J., M. Richter, S. Grant, H. Blumer, A. Leznoff, and B. Rose Immunologic studies of ragweed sensitive patients treated by a single repository antigen injection. J. Allergy 32: Deutsch, H. F., and J. I. Morton Human serum macroglobulins and dissociation units. I. Physicochemical properties. J. Biol. Chem. 231: De Weck, A. L., and C. H. Schneider Some observations on the molecular mechanisms of immediate-type allergic reactions. Int. Arch. Allergy 36: Dolovich, J., T. B. Tomamsi, and C. E. Arbesman Antibodies of nasal and parotid secretions of ragweed allergic subjects. J. Allergy 45: Donovan, R., J. F. Soothill, S. G. 0. Johansson, and H. Bennich.,1970. Immunoglobulins in nasal polyp fluid. Int. Arch. Allergy 37: Dowell, R. C., J. W. Kerr, and V. A. Park The metabolism of C14 histamine in subjects with bronchial asthma. J. Allergy 38: Dreisbach, R. H Failure of benadryl and pyribenzamine in experimental skin sensitization to penicillin and horse serum. J. Allergy 18: Edwards, A. J., V. E. Jones, S. R. Smithers, and R. J. Terry The occurrence and properties of reagins in Rhesus monkeys infected with Schistosoma mansoni. Ann. Trop. Med. Parasitol. 61: Eidinger, D., R. Wilkinson, and B. Rose A study of cellular responses in immune reactions utilizing the skin window technique. J. Allergy 35: Farah, F. S., M. Kern, and H. N. Eisen Specific inhibition of wheal and erythema responses with univalent haptens and univalent antibody fragments. J. Exp. Med. 112: Farr, R. S A quantitative immunochemical measure of the primary interaction between I*BSA and antibody. J. Infect. Dis. 103: Feinberg, A. R., R. J. Becker, R. G. Slavin, and S. M. Feinberg Induction of immediate and delayed skin reactivity with emulsion of purified ragweed antigen. J. Allergy 38: Feinberg, A. R., S. M. Feinberg, and F. Lee Leukocytes and hypersensitivity reactions. I. Eosinophile response in skin window to ragweed extract, histamine, and compound 48/80 in atopic and nonatopic individuals. J. Allergy 40: Feinberg, R. J., J. D. Davison, and J. A. Flick The detection of antibodies in hayfever sera by means of hemagglutinins. J. Immunol. 77: Feinberg, R. J., and J. A. Flick Elution of pollen antigens from tannic acid treated erythrocytes. Proc. Soc. Exp. Biol. Med. 96: Felarca, A. B., and F. C. Lowell Failure to elicit histamine eosinophilotaxis in the skin of atopic man, description of an improved technique. J. Allergy 41: Fellner, M. J., E. Van Hecke, M. Rozan, and R. L. Baer Studies on penicilloyl-specific IgG antibodies in man. J. Invest. Dermatol. 54: Fellner, M. J., E. Van Hecke, M. Rozan, and R. L. Baer Mechanisms of clinical desensitization in urticarial hypersensitivity to penicillin. J. Allergy 45: Felton, L. D., and P. F. Prather Studies in immunizing substances in pneumococci. IX. Cutaneous tests in non-immunized and immunized individuals in relationship to serum antibody content. Pub. Health Rep. 54: Finger, I., and E. A. Kabat A comparison of human antisera to purified diphtheria toxoid with antisera to other purified antigens by quantitative precipitin and gel diffusion techniques. J. Exp. Med. 108: Fink, J. N., R. Patterson, and J. J. Pruzansky Human antibody to heterologous serum protein. J. Allergy 38: Finke, S. R., M. H. Grieco, J. T. Connell, E. C. Smith, and W. B. Sherman Results of comparative skin tests with penicilloyl-polylysine and penicillin in patients with penicillin allergy. Amer. J. Med. 38: Finland, M., and H. F. Dowling Cutaneous reactions and antibody response to intracutaneous injection of pneumococcus polysaccharides. J. Immunol. 29: Finland, M., and W. D. Sutliff Specific cutaneous reactions and circulating antibodies in the course of lobar pneumonia. I. Cases receiving no serum therapy. J. Exp. Med. 54: Fireman, P., M. Boesman, and D. Gitlin Association of a skin-sensitizing antibody with 7S y2-globulins. J. Allergy 36: Fireman, P., M. Boesman, and D. Gitlin Heterogeneity of skin-sensitizing antibodies. J. Allergy 40: Fireman, P., M. Boesman, and D. Gitlin Disappearance of intradermally administered plasma immunoglobulins and skin-sensitizing antibodies. J. Allergy 40: Fisher, J. P., and J. T. Connell Passive cutaneous anaphylaxis in the guinea pig with serum of allergic patients treated with ragweed extract emulsions. J. Allergy 33: Fisher, J. P., and R. A. Cooke Passive cutaneous anaphylaxis (PCA) in the guinea pig. J. Allergy 28: Fisher, J. P., E. Middleton, and A. E. 0. Menzel Studies on passive cutaneous anaphylaxis with serum of allergic patients. J. Allergy 27: Flick, J. A., and R. J. Feinberg The activity of human reagins exposed to the disinfectant, beta-propiolactone. J. Allergy 26:

40 350 FLICK 114. Fox, R. H., R. Goldsmith, D. J. Kidd, and G. P. Lewis Bradykinin as vasodilator in man. J. Physiol. 157: Francis, T., and W. S. Tillett Cutaneous reactions in pneumonia. The development of antibodies following the intradermal injection of type-specific polysaccharide. J. Exp. Med. 52: Franklin, E. C., and Z. Ovary On the sensitizing properties of some normal and pathologic human immune globulins and fragments obtained by papain or pepsin digestion. Immunology 6: Freeman, G. L., and S. Johnson Progression of allergies in adolescents. Amer. J. Dis. Child. 118: Freeman, J Discussion on paroxysmal rhinorrhoea. Proc. Roy. Soc. Med. 18: Frick, 0. L., and K. Ishizaka Association of IgE with reaginic activity in sera from grass pollen- and horse dander-sensitive individuals. J. Allergy 45: Frick, 0. L., W. Nye, and S. Raffel Anaphylactic reactions to univalent haptens. Immunology 14: Frisch, A. W., C. B. Whims, and J. M. Oppenheim Intradermal reactions in trichinosis. Amer. J. Clin. Pathol. 17: Fudenberg, H. H., and H. G. Kunkel Physical properties of the red cell agglutinins in acquired hemolytic anemia. J. Exp. Med. 106: Furchgott, R. F Observations on the structure of red cell ghosts. Cold Spring Harbor Symp. Quant. Biol. 8: Gay, L. W., and E. Chant The passive transfer of hypersensitivity (local and contralateral passive transfer experiments). Bull. Johns Hopkins Hosp. 40: Gell, P. G. H., C. R. Harington, and R. P. Rivers The antigenic function of simple chemical compounds: production of precipitins in rabbits. Brit. J. Exp. Pathol. 27: Girard, J. P Antibody synthesis in vitro by human peripheral lymphocytes. Int. Arch. Allergy 32: Goldstein, G. B., D. C. Heiner, and B. Rose Studies of reagins to a-gliadin in a patient with wheat hypersensitivity. J. Allergy 44: Goldstein, G. B., D. C. Heiner, B. Rose, L. Goodfriend, and B. J. Underdown Isolation of antibodies to gliadin and ragweed antigen E with the use of immunosorbents. Int. Arch. Allergy 36: Goodfriend, L., B. A. Kovacs, and B. Rose In vitro sensitization of monkey lung fragments with human ragweed atopic serum. Int. Arch. Allergy 30: Goodfriend, L., and I. Luhovyj In vitro detection of reagins in human atopic sera by monkey skin suspension technique. Int. Arch. Allergy 33: BACTERIOL. REV Goodfriend, L., and L. Perelmutter Properties of two chromatographically distinct reagins in the sera of ragweed atopic individuals. Int. Arch. Allergy 33: Goodfriend, L., L. Perelmutter, J. A. Phills, and B. Rose Relationship of normal human serum immunoglobulins to blocking of the Prausnitz-Kiistner sensitization. Int. Arch. Allergy 30: Goodfriend, L., J. A. Phills, and L. Perelmutter Low molecular weight factors in normal human serum which block passive sensitization by reagins. Int. Arch. Allergy 35: Goodfriend, L., J. A. Phills, and B. Rose Characterization of factors in normal human serum which block P-K sensitization with reagins. J. Allergy 39: Gordon, J. E., and S. M. Creswell To what extent do toxin-antitoxin mixtures sensitize to therapeutic serum? J. Prev. Med. 3: Graham, H. T., 0. H. Lowry, F. Wheelwright, M. A. Lenz, and H. H. Parish Distribution of histamine among leukocytes and platelets. Blood 10: Greaves, M., and S. Shuster Responses of skin blood vessels to bradykinin, histamine, and 5-hydroxytryptamine. J. Physiol. 193: Green, J. F., K. G. Thielke, and S. Raffel Univalent antigen as elicitor of anaphylactic reactions. J. Immunol. 104: Grove, E. F Studies in specific hypersensitiveness. XXXI. On passive transfer of atopic hypersensitiveness to monkeys. J. Immunol. 15: Gyenes, L., S. 0. Freedman, A. H. Sehon, and Z. Ovary The properties of fragments of skin sensitizing and blocking antibodies as revealed by the Prausnitz-Kiistner passive cutaneous anaphylaxis and hemagglutination reactions. Int. Arch. Allergy 24: Gyenes, L., J. Gordon, and A. H. Sehon Ultracentrifugal characterization of antibodies in sera of ragweed-sensitive individuals. Immunology 4: Gyenes, L., and A. H. Sehon The use of polystyrene-allergen conjugates for the removal of antibodies from sera of allergic individuals. Can. J. Biochem. Physiol. 38: Harber, L. C., R. M. Holloway, V. R. Wheatley, and R. L. Baer Immunologic and biophysical studies in solar urticaria. J. Invest. Dermatol. 41: Harley, D Hayfever: the mechanism of specific desensitization. Lancet 2: Harley, D Hayfever. I. A study of reaginallergen mixtures. Brit. J. Exp. Pathol. 14: Harter, J. G The effect of storage on human skin-sensitizing antibody (reagin). Fed. Proc. 20:259.

41 VOL. 36, Heimlich, E. M., W. E. Vannier, and D. H. Campbell Sedimentation studies of skin-sensitizing antibody. J. Allergy 31: Henney, C. S., and K. Ishizaka Antigenic determinants specific for aggregated y-g globulins. J. Immunol. 100: Henney, C. S., and D. R. Stanworth Effect of antigen on the structural configuration of homologous antibody following antigenantibody combination. Nature (London) 210: Henson, P. M Role of complement and leukocytes in immunologic release of vasoactive amines from platelets. Fed. Proc. 28: Hogarth-Scott, R. S., S. G. 0. Johansson, and H. Bennich Antibodies to toxocara in the sera of visceral larva migrans patients: the significance of raised levels of IgE. Clin. Exp. Immunol. 5: Houser, D. D., C. E. Arbesman, K. Ito, and K. Wicher Cold urticaria; immunologic studies. Amer. J. Med. 49: Huber, J., and H. H. Fudenberg Receptor sites of human monocytes for IgG. Int. Arch. Allergy 34: Huber, J., S. D. Douglas, and H. H. Fudenberg The IgG receptor: an immunological marker for the characterization of mononuclear cells. Immunology 17: Hubscher, T., J. I. Watson, and L. Goodfriend Target cells of human ragweed-binding antibodies in monkey skin. II. Immunoglobulin nature of ragweed-binding antibodies with affinity for monkey skin mast cells. J. Immunol. 104: Humphrey, J. H., and R. R. Porter Reagin content of chromatographic fractions of human gamma-globulin. Lancet 1: Ishizaka, K., E. G. Dennis, and M. Hornbrook Presence of reagin and 'ya-globulin in saliva. J. Allergy 35: Ishizaka, K., and T. Ishizaka Biologic activities of soluble antigen-antibodies. X. Skin-reactive properties of allergen-reagin complexes. J. Allergy 36: Ishizaka, K., and T. Ishizaka Physicochemical properties of reaginic antibody. III. Further studies on the reaginic antibody in YA-globulin preparations. J. Allergy 38: Ishizaka, K., and T. Ishizaka Physicochemical properties of reagin antibody. I. Association of reaginic activity with an immunoglobulin other than gamma A or gamma G globulin. J. Allergy 37: Ishizaka, K., T. Ishizaka, and E. H. Lee Physicochemical properties of reaginic antibody. II. Characteristic properties of reaginic antibody different from human gamma A- isohemagglutinin and gamma D-globulin. J. Allergy 37: Ishizaka, K., and T. Ishizaka Identifica- PROPERTIES OF 1HIUMAN REAGINS 351 tion of -ye-antibodies as a carrier of reagin activity. J. Immunol. 99: Ishizaka, K., and T. Ishizaka Reversed type allergic skin reactions by anti-ye-globulin antibodies in humans and monkeys. J. Immunol. 100: Ishizaka, K., and T. Ishizaka Induction of erythema-wheal reactions by soluble antigen 'YE antibody complexes in humans. J. Immunol. 101: Ishizaka, K., and T. Ishizaka Human reaginic antibodies and immunoglobulin E. J. Allergy 42: Ishizaka, K., and T. Ishizaka Physicochemical properties of human reaginic antibody. VIII. Effect of reduction and alkylation on -y antibodies. J. Immunol. 102: Ishizaka, K., and T. Ishizaka Immune mechanisms of reversed type reaginic hypersensitivty. J. Immunol. 103: Ishizaka, K., and T. Ishizaka Biological function of ye antibodies and mechanisms of reaginic hypersensitivity. Clin. Exp. Immunol. 6: Ishizaka, K., T. Ishizaka, and C. E. Arbesman Induction of passive cutaneous anaphylaxis in monkeys by human ye antibody. J. Allergy 39: Ishizaka, K., T. Ishizaka, and M. Hornbrook Blocking of Prausnitz-Kiistner sensitization with reagin by normal human globulin. J. Allergy 34: Ishizaka, K., T. Ishizaka, and M. M. Hornbrook Physicochemical properties of human reaginic antibody. IV. Presence of a unique immunoglobulin as a carrier of reaginic activity. J. Immunol. 97: Ishizaka, K., T. Ishizaka, and M. M. Hornbrook Physicochemical properties of reaginic antibody. V. Correlation of reaginic activity with -YE-globulin antibody. J. Immunol. 97: Ishizaka, K., T. Ishizaka, and M. M. Hornbrook Allergen-binding activity of ye, yg and ya antibodies in sera from atopic patients. In vitro measurements of reaginic antibody. J. Immunol. 98: Ishizaka, K., T. Ishizaka, and E. H. Lee Biologic function of the F, fragments of E myeloma protein. Immunochemistry 7: Ishizaka, K., T. Ishizaka, E. H. Lee and H. Fudenberg Immunochemical properties of human ya isohemagglutinin. I. Comparisons with -yg- and ym-globulin antibodies. J. Immunol. 95: Ishizaka, K., T. Ishizaka, and A. E. 0. Menzel Physicochemical properties of reaginic antibody. VI. Effect of heat on ye-, Y-y, and ya-antibodies in the sera of ragweed sensitive patients. J. Immunol. 99: Ishizaka, K., T. Ishizaka, and M. Richter Effect of reduction and alkylation on allergen-combining properties of reaginic anti-

42 352 FLICK body. J. Allergy 37: Ishizaka, K., T. Ishizaka, and T. Sugahara Biological activity of aggregated y- globulin. III. Production of Arthus-like reactions. J. Immunol. 86: Ishizaka, K., T. Ishizaka, and W. D. Terry Antigenic structure of ye-globulin and reaginic antibody. J. Immunol. 99: Ishizaka, K., and R. W. Newcomb Presence of 'ye in nasal washings and sputum from asthmatic patients. J. Allergy 46: Ishizaka, K., H. Tomioka, and T. Ishizaka Mechanisms of passive sensitization. I. Presence of IgE and IgG molecules on human leukocytes. J. Immunol. 105: Ishizaka, T., K. Ishizaka, H. Bennich, and S. G. 0. Johansson Biologic activities of aggregated immunoglobulin E. J. Immunol. 104: Ishizaka, T., K. Ishizaka, S. G. 0. Johansson, and H. Bennich Histamine release from human leukocytes by anti-ye antibodies. J. Immunol. 102: Ishizaka, T., K. Ishizaka, R. P. Orange, and K. F. Austen The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J. Immunol. 104: Ito, K., K. Wicher, and C. E. Arbesman Insoluble immunoadsorbents containing anti- IgE; removal of reaginic activity and subsequent elution. J. Immunol. 103: Ito, K., K. Wicher, and C. E. Arbesman Comparison of two myeloma IgE by monkey antiserum to human IgE. Int. Arch. Allergy 39: Ito, K., K. Wicher. and C. E. Arbesman Antibodies to denatured IgE and new antigenicity of the IgE acquired during heating. J. Immunol. 106: Johansson, S. G Raised levels of a new immunoglobulin class (IgND) in asthma. Lancet 2: Johansson, S. G Serum IgND levels in healthy children and adults. Int. Arch. Allergy 34: Johansson, S. G. 0. and H. Bennich Immunological studies of an atypical (myeloma) immunoglobulin. Immunology 13: Johansson, S. G. O., H. Bennich, and L. Wide A new class of immunoglobulin in human serum. Immunology 14: Johansson, S. G. O., T. Mellbin, and B. Vahlquist Immunoglobulin levels in Ethiopian preschool children with special reference to high concentrations of immunoglobulin E (IgND) Lancet 1: Josephson, A. S., E. C. Franklin, and Z. Ovary The characterization of antibodies to penicillin. J. Clin. Invest. 41: Juhlin. L., and W. B. Shelley Role of BACTERIOL. REV. mast cell and basophile in cold urticaria with associated systemic reactions. J. Amer. Med. Ass. 177: Julianelle, L. A., and A. F. Hartmann The immunological specificity of staphylococci. IV. Cutaneous reactions to the typespecific carbohydrates. J. Exp. Med. 64: Kabat, E. A., and D. Berg Dextran. An antigen in man. J. Immunol. 70: Kahn, M., H. S. Baldwin, B. R. Zeitlin, and M. Smart Cutaneous reactions to staphylococcus polysaccharide, protein, and an unfractioned extract in hypersensitive and normal individuals. J. Allergy 22: Karelitz, S., and A. Glorig Studies on the specific mechanism of serum sickness. III. Passive sensitization with antibody contained in serum hickness convalescent serum. J. Immunol. 47: Katz, G Histamine release in the allergic skin reaction. Proc. Soc. Exp. Biol. Med. 49: Katz, G., and S. Cohen Experimental evidence for histamine release in allergy. J. Amer. Med. Ass. 117: Kay, A. B., B. N. Gurner, and R. R. A. Coombs Passive sensitization of tissue cells. III. A primate-macrophage-cytophilic antibody. Int. Arch. Allergy 37: King, T. P., P. S. Norman, and J. T. Connell Isolation and characterization of allergens from ragweed pollen. Biochemistry 3: Knudsen, E. T., 0. P. W. Robinson, E. A. P. Croyden, and E. C. Tees Cutaneous sensitivity to purified benzylpenicillin. Lancet 1: Kobayashi, S., J. P. Girard, and C. E. Arbesman Demonstration of human reagins in monkey tissue. Ill. In vitro passive sensitization of monkey ileum with sera of atopic patients. Physiologic and enhancing experiments. J. Allergy 40: Korotzer, 0. L., Z. H. Haddad, and A. F. Lopapa Detection of human IgE antibody by a modified rat mast cell degranulation technique. Immunology 20: Kremen, A. J., H. L. Taylor, and H. Hall Skin sensitivity of man to bovine plasma and its albumin and globulin fractions. Proc. Soc. Exp. Biol. Med. 43: Kropp, G. V., and J. A. Foley Studies on polysaccharide from tubercle bacillus. J. Lab. Clin. Med. 29: Krueger, R. C., and M. Heidelberger Microestimation of antibodies with one milliliter samples of antisera of low antibody content. J. Lab. Clin. Med. 38: Kuhns, W. J Immunochemical studies of antitoxin produced in normal and allergic individuals hyperimmunized with diphtheria toxoid. HI. Studies of the passive Arthus reaction in guinea pigs using human precipi-

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