Serum Concentrations of Allergen-specific IgG Antibodies in Inhalant Allergy: Effect of Specific Immunotherapy

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1 Serum Concentratons of Allergen-specfc IgG Antbodes n Inhalant Allergy: Effect of Specfc Immunotherapy MAJID ALI, M.D., M. P. RAMANARAYANAN, PH.D., DONALD J. NALEBUFF, M.D., RICHARD G. FADAL, M.D., AND JAMES W. WILLOUGHBY, M.D. The occurrence of antbodes belongng to IgG class of mmunoglobulns wth specfcty for short ragweed and Bermuda grass n the sera of nonatopc subjects' 5 and patents wth nhalant allergy (at the tme of ntal dagnoss, 15 followng specfc mmunotherapy 23 ) was nvestgated wth an enzyme mmunoassay. The ntra-assay and nter-assay coeffcents of varaton for ths assay ranged from 1.74%-4.62% and 3.18%- 9.12%, respectvely. IgG antbodes were found n the sera of the majorty of nonatopc and all of atopc subjects. The dfferences n the concentraton of these antbodes between these three groups were statstcally sgnfcant (P 0.001). Specfc mmunotherapy resulted n a rse n the serum levels of allergen-specfc IgG antbodes and, followng an ntal perod of modest ncrease, a decrease n the level of allergen-specfc IgE antbodes. Specfc IgG response correlated wth both the cumulatve antgen dose and the clncal beneft that accrued from specfc mmunotherapy (P 0.001). The ncrease n the serum concentraton of specfc IgG was most pronounced n patents wth hgh RAST scores at the tme of ntal dagnoss (P 0.001). The concentratons of short-ragweed specfc IgG antbodes assayed wth multple samples n three patents wth ragweed hay fever appeared not to be affected by the shortragweed season to any sgnfcant degree. We conclude that drect enzyme mmunoassay for allergen-specfc IgE and IgG antbodes are useful n vtro montors of mmunologc responses to specfc mmunotherapy for nhalant allergy. (Key words: Inhalant allergy; Allergen-specfc IgG; Enzyme mmunoassay; Specfc mmunotherapy; Immunologc responses) Am J Cln Pathol 1983; 80: THE CONCEPT of "blockng antbodes" was frst ntroduced by Cooke n 1935 as "serologc evdence of mmunty wth coexstng senstzaton n a type of frst human allergy". 5 Cooke employed the Prausntz-Kstner neutralzaton technc for ths purpose. Durng the fve decades that followed, many attempts were made to replace the cumbersome n vvo method wth an accurate and precse n vtro procedure. These efforts addressed both ndrect and drect methods. The ndrect methods nclude nhbton of the n vtro release of hstamne from allergen-challenged leukocytes, 1317 hemagglutnaton wth allergen-coated erythrocytes, 1819 subtracton of IgE antbody bndng from the total antbody bndng of rado- Receved November 1, 1982; receved revsed manuscrpt and accepted for publcaton January 28, Address reprnt requests to Dr. Al: Dvson of Pathology, Immunology and Laboratores, Holy Name Hosptal, Teaneck, New Jersey Dvson of Pathology, Immunology, and Laboratores and Department of Otolaryngology, Holy Name Hosptal, Teaneck, New Jersey; Dvson of Otorhnolaryngology, Unversty of Alabama; Allergy and Immunology Center, Waco, Texas; Department of Medcne, Unversty of Mssour School of Medcne, Kansas Cty, Mssour and Department of Pathology, College of Physcans and Surgeons of Columba Unversty, New York, New York labeled antgen, 25 radommunoprecptaton, 16 and precptaton of IgG antbodes wth staphylococcal proten A. 9 The drect methods nclude radommunoassay, 11,20 defne antgen substrate sphere (DASS), 3,6 and enzyme mmunoassays. 814 The data obtaned wth these drect mmunoassays have been lmted and largely confned to the nsect venom systems. The present study was undertaken to develop and evaluate drect sold-phase enzyme mmunoassays for n vtro montorng of the mmunologc response to specfc mmunotherapy n nhalant allergy. Patents, Materals, and Methods The patents ncluded n the study were those consultng one of the clncans n the nvestgatve team for nhalant allergy. The dagnoss of nhalant allergy was establshed by clncal evaluaton, skn tests, and the presence of allergen-specfc IgE antbodes n the serum, as determned by the Modfed RAST test. 15 The sera for the study of naturally occurrng IgG antbodes, wth specfcty for varous allergens n nonatopc subjects, were obtaned from patents hosptalzed for varous dsorders but who suffered from no known allergc dsorders; for untreated atopc state, the sera were obtaned from patents wth nhalant allergy at the tme of ntal dagnoss. Sequental samples of serum obtaned and frozen at 20 C at ntervals durng mmunotherapy over a perod of months were avalable for three short ragweed senstve patents. The clncal and laboratory data for these three short-ragweed senstve patents are gven n Table 1. All patents n the specfc IgG study were evaluated for clncal response to specfc mmunotherapy at 6-8 week ntervals. The clncan treatng the patents assgned the patents to the categores of poor, modest, good, and excellent clncal responses, wthout any knowledge of the /83/0900/0290 $01.30 Amercan Socety of Clncal Pathologsts 290 Downloaded from

2 vol. 80-No. 3 SPECIFIC IgG IN INHALANT ALLERGY Table 1. Clncal and Laboratory Data for Three Short-ragweed Senstve Patents* 291 Patent Short-Ragweed Modfed Skn RAST End-pont Before Immunotherapy Drug Useage Durng Immunotherapy Immunotherapy Dose Intal Mantenance Clncal Responset Sterod, antbotcs, anthstamnes, acetamnophen Frequent decongestant Sterods, frequent decongestant Acetamnophen Occasonal decongestant Occasonal decongestant 0.1 ml 1: ml 1: ml 1: w/v 0.3 ml 1: ml 1: ml 1:62500 w/v Excellent Excellent Excellent t Scale: poor, modest, good, and excellent. Other antgens ncluded n the therapy: Patent I: ragweed, kocha. marsh elder. Patent 2: ragweed, pgweed, kocha. chenopod, hemp, grass, marsh elder, tree, elm, Cottonwood, oak, AP dust, toe, altemara, hormodendrum, cephalosporum. helmenthosporum, pullulara. gran and grass smuts, slk moth. Patent 3: ragweed, pgweed, kocha, chenopod, hemp, grass, marsh elder, tree, elm, Cottonwood, oak. AP dust, altemara. hormodendrum, cephalosporum, helmenthosporum, pullulara, gran smut, grass smut, slk mold. changes n the serum concentratons of allergen-specfc IgG antbodes. Allergen Extracts Allergenc extracts were purchased from Pharmaca Dagnostcs (Pscataway, NJ), Hollster-Ster (Spokane, WA), and Antgen Laboratores (Lberty, MO). The extracts were dalyzed aganst 200 volumes of PBS contanng 0.01 mm mertholate, wth two changes over a perod of 16 hours at 4 C, and stored at 4 C. Rabbt Ant-Human IgG and IgE Antsera Rabbt ant-human IgG and IgE antsera (gamma globuln fracton, specfc for gamma chans), prepared by Dako-Immunoglobulns (a/s Denmark), were purchased from Accurate Chemcals (Westbury, NY). These antsera had been purfed by the manufacturer to remove antbodes aganst the other known classes of mmunoglobulns. For use n the assay for allergen-specfc IgG antbodes n serum, the antserum was affnty-purfed by sold-phase absorpton on a column of human IgG covalently mmoblzed on agarose, followed by eluton of the bound human IgG-specfc antbody from the column by employng acd 12 or base 4 or a stepwse applcaton of both. From the ant-human IgE antserum and the affnty purfed ant-human IgG, the IgG fracton was purfed by sodum sulfate fractonaton followed by on-exchange chromatography on DEAE-cellulose and repeat sodum sulfate fractonaton. The resultng IgG fractons are referred to as rabbt ant-human IgG and IgE antbodes. Immunosorbant Polystyrene mcrotter plates were purchased from Dynatec Laboratores, Inc. (Alexandra, VA). Indvdual wells of the plates were coated wth dalyzed allergenc extracts by ncubatng n each well 200 f\ of a soluton contanng 10 fg of the extract per ml coatng buffer, at 4 C for hours. Followng a washng step wth wash buffer, the coated plates were stored at 4 C for subsequent use. Enzyme-Antbody Conjugates Rabbt ant-human IgG and rabbt ant-human IgE antbodes were conjugated to horseradsh peroxdase usng a prevously descrbed method. 1-2 Horseradsh peroxdase (Sgma type VI, RZ 3.1) was conjugated to the IgG fracton by frst blockng the free amno groups on the peroxdase molecule wth phenyl sothocyanate and then oxdzng ts carbohydrate moety wth perodate to yeld the peroxdase aldehyde. After dalyss, the peroxdase aldehyde was lnked to the amno group of the IgG molecule by formaton of Schff 's base; reducton wth sodum borohydrde was employed to stablze the SchfFs base. After purfcaton by ammonum sulfate precptaton, the conjugate had an RZ value of Buffers The buffers used n the enzyme mmunoassays have been descrbed prevously. 1-2 Standard for the Immunoperoxdase Assays for Allergen-specfc IgE and IgG Antbodes Sera from patents wth a hgh level of senstvty to June-grass antgens was pooled and calbrated, by sutable dluton steps, aganst 12.5 U/mL of WHO standard IgE (usng an IP assay for total IgE) to produce standard 1 for the IP assay for allergen-specfc IgE antbodes. Standards 2, 3, and 4 were prepared wth 1:4, 1:16, and 1:32 dlutons of standard 1. The standards for the IP assay for allergen-specfc IgG antbodes were prepared Downloaded from

3 292 ABSORBANCE HUMAN IgG ANTIBODIES IN ng/ml FIG. 1. Standard curve for IP assay for IgG antbodes prepared wth dluton of an IgG standard contanng 40 ng/ml of IgG. wth pooled sera from June-grass senstve patents who were gven specfc mmunotherapy for over two years. Ths serum was calbrated, by sutable dluton steps, aganst 30 ng/ml of purfed human IgG preparaton (usng an IP assay for total IgG) to prepare standard 1 (1:100 dluton of the pooled sera). Standards 2, 3, and 4 were prepared wth 1:4, 1:16, and 1:64 dlutons of standard 1. Immunoperoxdase (IP) Assays for Allergen-specfc IgG and IgE Antbodes All assays were performed n duplcate. The detals of the IP assay for allergen-specfc IgE antbodes have been publshed before.',2 For ths study, ndvdual allergen blanks were used for all assays n order to elm ALI ET AL. A.J.C.P. September r nate the varatons n the test results ntroduced by the dfferences among the background sgnals obtaned wth dfferent allergens. Brefly, serum sample n 1:5 dluton and ant-ige-peroxdase conjugate were sequentally ncubated overnght at 4 C n polystyrene mcrotter plate wells prevously coated wth the allergens. The bound enzyme actvty was assayed wth Dynatech mcro-elsa plate reader MR 580, usng o-phenylene damne as the substrate. For assay of allergen-specfc IgG levels, serum n 1:100 dluton and ant-igg-peroxdase conjugate were used. Serum dluton factor was chosen n lght of ttraton studes. In other detals, ths assay was dentcal to the assay for specfc E antbodes. All assays for specfc C and G antbodes on sequental samples of the three ragweed-senstve patents were performed durng one assay run. Results Standard Curve for Allergen-Specfc IgG Antbodes Fgure 1 shows standard curve for the IP assay for total IgG antbodes prepared wth dlutons of a commercal IgG standard (Calbochem-Behrng, San Dego, CA) dluted to contan 40 ng/ml. Ths curve was used to calbrate the standards for IP assay for allergen-specfc IgG antbodes. Fgure 2 shows a standard curve for the IP assay for allergen-specfc IgG antbodes constructed wth four standards prepared from pooled sera from June-grass senstve patents gven specfc mmunotherapy for over two years. The data ponts on the curve that fall between the ponts for the four standards represent 1:2 dlutons of standards 1, 2, and 3, respectvely STANDARDS Pooled Sera From June Grass-Senstve Patents Gven Specfc Immunotherapy For Over Two Years (DILUTION: Standard 1. 1:00: Standard 4. 1:6400) FIG. 2. Standard curve for IP assay for allergen-specfc IgG antbodes prepared wth pooled sera from June grass senstve patents gven specfc mmunotherapy for over two years. Standard 1 represents an approxmately 1:100 dluton of the pooled serum and s calbrated aganst 30 ng/ml of IgG n an IP assay for total IgG (Fg. 1). Standards 2, 3, and 4 are 1:4, 1:16, and 1:64 dlutons of standard 1, respectvely. Assay Specfcty The specfcty of the IP assay for allergen-specfc IgG antbodes was establshed by three sets of experments: absorpton experments usng homologous and heterologous allergenc extracts; nhbton of reacton wth staphylococcal proten A; and the use of ant-igm-peroxdase and ant-iga-peroxdase conjugates. Fgure 3 shows a dluton curve obtaned wth the IP assay for allergen-specfc IgG antbodes wth the serum of a patent wth Bermuda grass senstvty who had receved specfc mmunotherapy for 16 months. Preabsorpton of the serum wth Bermuda grass allergenc extract (124 ng) was able to nhbt the reacton by approxmately 80%. Preabsorpton of the same serum wth heterologous allergenc extracts (Alternara at 120 ng and short ragweed at 124 /tg) faled to nhbt the reacton ndcatng the specfcty of the assay for the allergen nsolublzed on the polystyrene sold phase. Fgure 4 shows a smlar Downloaded from

4 Vol. 80 No. 3 ABSORBANCE IN MILLI UNITS SPECIFIC IgG IN INHALANT ALLERGY ABSORBANCE IN MILLI UNITS 293._. Absorpton Control - -" Absorbed wth Bermuda Crass E ract *- >-* Absorbed wth Short Ragweed Ex racl»- - Absorbed wln Alternana Etract soo 1200 J w-o Absorpton Control *- Absorbed wth Bermuda Grass Extract -»-«Absorbed wrtlh Short Ragweed Extract -* Absorbed wrln Alternana Extract I SERUM DILUTION FIG. 3. The specfcty of the IP assay for IgG antbodes aganst Bermuda grass antgens s shown by an absorpton experment. Preabsorpton of the serum wth Bermuda grass allergenc extracts results n nhbton of the reacton by approxmately 80%. By contrast, preabsorpton of the same serum wth short-ragweed and Alternana extracts fals to nhbt the reacton. T 100 -r 200 T 1 r SERUM DILUTION FIG. 4. The specfcty of the mmunoperoxdase assay aganst shortragweed specfc IgG antbodes s shown by an absorpton experment. The reacton s nhbted by approxmately 90% by pre-absorpton of the serum wth short-ragweed allergenc extract but not when serum s pre-absorbed wth Bermuda grass and Alternana extracts. dluton curve obtaned wth the serum of a short-ragweed senstve patent treated wth mmunotherapy for 20 months. Ths fgure confrms the specfcty of the assay for short-ragweed specfc IgG antbodes. Preabsorpton of the serum wth short-ragweed allergen extract (120 ng) was able to nhbt the reacton by 90%, whle preabsorpton wth heterologous antgen extracts (Bermuda grass and Alternara) faled to do so. The specfcty of ths assay for antbodes belongng to IgG class of mmunoglobulns was further confrmed by absorpton of the serum sample on staphylococcal proten A-Sepharose. Sold-phase absorpton of the serum sample on proten A-Sepharose elmnated the reacton by about 85% (Fg. 5). Fnally, the lack of nterference n the assay by IgM and IgA antbodes s shown n Fgure 6. At the sample dluton of 1:100 used n ths assay, IgM and IgA antbodes do not cause nonspecfc reacton. Assay Senstvty The assay senstvty was defned by calbratng the results obtaned wth the IP assay for allergen-specfc IgG antbodes, wth an IP assay for total IgG antbodes. A comparatve study of Fgures 1 and 2 shows that the concentraton of specfc IgG antbodes n a serum sample, gvng a result of 1,000 mu of absorbance n the IP assay for specfc IgG, has equvalence to the value gven by 11 ng/ml IgG antbodes n the assay for total IgG. These data show that the senstvty of the IP assay for allergen-specfc IgG antbodes used n ths study s n the range of pg/ml. Intra-assay and Interassay Reproducblty The coeffcents of varaton for the ntra-assay reproducblty of the IP assay for serum level of IgG antbodes wth specfcty for June-grass antgens ranged from % (Table 2): the correspondng range for the ntra-assay reproducblty extended from % (Table 3). Serum Concentratons of Allergen Specfc IgG Antbodes Fgure 7 shows the serum concentratons of IgG antbodes wth specfcty for short-ragweed antgens n nonatopc subjects, 15 untreated atopc patents, 15 and atopc patents treated wth specfc mmunotherapy IMMUNOPEROXIDASE ASSAY FOR IgG ANTIBODIES WITH SPECIFICITY FOR JUNE GRASS ANTIGENS 1.7 J" Serum Dluton Wth Proten A coo Wthout Proten A FIG. 5. The specfcty of the mmunoperoxdase assay for antbodes belongng to IgG class of mmunoglobulns s shown by absorpton of serum wth staphylococcal proten A. Proten A. by bndng IgG antbodes, s able to nhbt the reacton by about 85%. Downloaded from

5 294 ALI ET AL. A.J.C.P. September 1983 IP ASSAY FOR ALLERGEN-SPECIFIC IGG ANTI IgG CONJUGATE o ANTI I9H CONJUGATE A ANTI IgA CONJUGATE FIG. 6. The specfcty of the IP assay for IgG antbodes s shown by comparng dluton curves obtaned wth peroxdase conjugates of rabbt ant-human IgG, rabbt ant-human IgA, and rabbt anthuman IgM. The serum for ths study was obtaned from a patent wth hgh level of senstvty to June grass (Modfed RAST score class 5) who had been gven specfc mmunotherapy for 40 months SERUM DILUTION as expressed n mllunts of absorbance. The dfferences between these three groups, determned by the unpared /-test, were statstcally sgnfcant (P values: A and B 0.001; B and C 0.001, and A and C 0.001). The mean absorbance values for these three groups were 49, 205, and 680 mllunts of absorbance. By calbraton of the results of specfc G assay wth those of total IgG assay (Fgs. 1 and 2), these values represent approxmately 410 pg/ml, 1,250 pg/ml, and 5,800 pg/ml, respectvely. Fgure 8 shows smlar data for serum concentraton of IgG antbodes wth specfcty for Bermuda grass antgens. The dfferences n the absorbance values obtaned wth ths antgen system also are sgnfcant statstcally (P values: A and B 0.001; B and C 0.001; and A and C.001). The mean absorbance values for these three groups were 31, 193, and 589 mllunts of absorbance, respectvely. Correlaton of Specfc IgG and Clncal Responses to Immunotherapy All patents n ths study were treated for multple allergens, and the clncal response represented the sum total of responses to ndvdual allergens ncluded n the therapy. Nonetheless, the allergen-specfc IgG response to specfc mmunotherapy for nhalant allergy to Bermuda grass and short-ragweed antgens and the clncal beneft derved from such therapy correlated well (Fg. 9; correlaton coeffcent 0.852; P 0.001). The specfc IgG response to both Bermuda grass and short-ragweed Table 2. Intra-assay Reproducblty of the Immunoperoxdase Assay for Serum Levels of IgG Antbodes wth Specfcty for June Grass Antgens Table 3. Interassay Reproducblty of the Immunoperoxdase Assay for Serum Levels of IgG Antbodes wth Specfcty for June Grass Antgens Date Sample IP Unts (Mllunts of Absorbance) Mean* S.D. Coeffcent of Varaton (%) Experment Number Sample IP Unts (Mllunts Absorbance) Mean* S.D. Coeffcent of Varaton (%) 12/3/81 12/4/81 12/8/81 12/9/81 12/22/81 12/30/ I II III IV V VI * Mean of 6 determnatons. * Mean of 6 determnatons. Downloaded from

6 Vol. 80 No. 3 ABSORBANCE n 2l?rJ ( SPECIFIC IgG IN INHALANT ALLERGY over 1500' * Non-Atopc Atopc Atopc Untreated Treated FIG. 7. Serum concentraton of IgG antbodes wth specfcty for short-ragweed antgens n the sera of nonatopc subjects, atopc patents before and one year after the ntaton of specfc mmunotherapy are shown. The dfferences among these three groups are statstcally sgnfcant as determned by the unpared t-test (P values: A and B 0.001; B and C 0.001; and A and C 0.001). extract njectons was poor n three patents who dd not respond to such therapy. By contrast, the specfc IgG levels were markedly rased n most patents n whom clncal response was assessed as excellent. Seral Changes n the Serum Concentraton of Allergen-specfc IgE and IgG Antbodes durng Specfc Immunotherapy Fgures 10, 11, and 12 show the changes n the serum concentratons of IgE and IgG antbodes wth specfcty for short-ragweed antgens n three patents wth hgh levels of senstvty to short-ragweed (patents of J WW). Progressve ncrease n the serum concentraton of ragweed-specfc IgG antbodes were found n these patents over the entre duraton of therapy (404, 427, and 507 days). However, the pattern of ncrease n each patent was dfferent and appeared to be related to the dosage schedule of specfc mmunotherapy. Patents and 3 tolerated only small doses ntally (1:62,500 w/v for the frst sx months of therapy), and the ntal IgG response n both patents was modest (Fgs. 10 and 12). In both patents, the IgG response dsplayed a brsk ncrease durng the second half of the frst year of treatment as the mmunotherapy dose was advanced. Further, the serum levels of specfc IgG antbodes contnued to rse untl the end of the study perod, at a low rate n patent 1 but more brskly n patent 3. Ths relatonshp between the antgen dose and the IgG response also was observed n patent 2. Ths patent tolerated a larger ntal mmunotherapy dose (1:12,500 w/ v wthn three months of treatment). He exhbted a strong IgG response durng the frst sx months of therapy, whch was followed by a plateau (Fg. 11). It s a pont of some nterest that the IgG response to specfc mmunotherapy n patents 1 and 3 was not nfluenced by the ragweed season. In patent 2, brsk IgG response concded wth the later half of the ragweed season, and ABSORBANCE * > > Non-Atopc : I > Atopc Untreated : > Atopc Treated FIG. 8. Serum concentraton of IgG antbodes wth specfcty for Bermuda grass antgens n the sera of nonatopc subjects, Bermuda grass senstve patents before ntaton of specfc mmunotherapy, and such patents after one year of specfc mmunotherapy are gven. The dfferences between these three groups are statstcally sgnfcant as determned by the unpared Mest (P values: A and B 0.001; B and C 0.001; and A and C 0.001) Downloaded from

7 / : : - : : : 296 ALI ET AL. A.J.C.P. September 1983 A SPECIFIC IgG a low level of senstvty and correspondngly low RAST scores Dscusson ' ' 0 BERMUDA GRASS Immunotherapy wth njecton of allergen extract n SHORT RAGWEED 1300 ( 1 nhalant allergy causes a fall n the level of allergen-specfc IgE antbodes and a rse n the tter of antbodes 1 ' 1100 belongng to IgG class wth specfcty for the ncrmnated allergens. 7,2 ' 23,25 The IgG response evoked by specfc mmunotherapy relates to the amount of antgen 900- o admnstered The data concernng the correlaton : of IgG response wth clncal beneft s dsparate. 21, Lmted studes correlatng n vvo blockng antbodes ( 0' wth the n vtro assay of IgG 4 have shown good correlaton for patents wth a low level of senstvty but an 500» 0' apparent lack of correlaton for patents wth a hgh level ( oo of senstvty. 25 In one report, the IgG antbody response to specfc mmunotherapy n allergc patents was I o. 0 l 0 f ^ \ hgher than that n the nonatopc volunteers treated the 100 O' same way.'' The data obtaned n ths study wth drect enzyme mmunoassays are n basc algnment wth the results POOR MODEST GOOD EXCELLENT acheved wth the prevous, ndrect n vvo and n vtro methods. Clncal Response to Immunotherapy 13l8,2a22 ' 23 ' 25 Allergen-specfc IgG antbodes were detectable n many nonatopc ndvduals; the FIG. 9. The clncal response to specfc mmunotherapy for Bermuda grass and short-ragweed antgens s correlated wth specfc IgG response to the treatment n 20 Bermuda grass-senstve and 23 shortragweed senstve patents (correlaton coeffcent, 0.68, P 0.001). t seems lkely that the seasonal exposure dd not materally affect the IgG response n ths patent as well. The sequental changes n the serum concentraton of ragweed-specfc IgE antbodes durng specfc mmunotherapy n these three patents showed no clear relatonshp wth the dose schedule. There was an ntal ncrease n the concentraton of these antbodes n all three patents. In patents 2 and 3, ths was followed by a gradual fall n the level of serum IgE antbodes. Patent 1 also showed a decrease after the ntal rse, but the level never fell down to the ntal base value and, ndeed, showed an ncrease n the last three months of therapy. Correlaton of specfc IgG Response wth the Level of Senstvty The serum concentraton of allergen-specfc IgG antbodes showed an overall correlaton wth the level of senstvty pror to startng specfc mmunotherapy (Fg. 13, correlaton coeffcent, 0.755; P 0.001). Patents wth hgh RAST scores at the tme of dagnoss generally mounted a stronger specfc IG response than those wth ~ '200 A ;! ; ; :!. : J... j /f- *% TT mum 0. :! ^> -" I... '! IP ASSAY FOR ALLERGEN-SPECIFIC IGG ANIIBOD ES ' {S. RAGWEED) - J^f^*' ft *-**~*~"^, A/ t^9 - / ;...;. J^ ^...:..',.m...;..... : '. ;. :.. ).. j.» ; ; ; 4~U!. 1. DURATION OF [WUHOTHERAPY - DAYS! L; I I! FIG. 10. Changes n the concentratons of IgE and IgG antbodes wth specfcty for short-ragweed antgens occurrng n the serum of a patent wth short-ragweed senstvty durng specfc mmunotherapy are shown. The IgG antbodes show a progressve ncrease n ther concentraton; IgE levels, by contrast, show an ntal rse, followed by a sustaned fall. It s noteworthy that the level obtaned at the end of the study perod (507 days) s stll hgher than the level observed before startng mmunotherapy. Downloaded from

8 Vol. 80 No. 3 SPECIFIC IgG IN INHALANT ALLERGY 297 IP ASSAY FOR ALLERGEN-SPECIFIC IGG ANTIBODIES (s. RAGWEED) FIG. 11. The changes n the serum concentratons of IgE and IgG antbodes wth specfcty for short-ragweed antgens occurrng n 427 days of specfc mmunotherapy n a shortragweed senstve patent are shown. The concentraton of allergen specfc IgG antbodes show a progressve rse durng thefrstfour months of therapy, followed by a plateau. By contrast, the level of allergen specfc IgE antbodes shows an ntal rse and a sustaned fall durng the perod of treatment so that the level at the end of the study perod s substantally lower than the level before therapy was begun. 4/9/81 DURATION OF IMMUNOTHERAPY - DAYS serum levels of these antbodes were hgher n atopc patents and stll hgher followng specfc mmunotherapy. These dfferences were statstcally sgnfcant (P 0.001). The IgG response nduced by specfc mmunotherapy correlated wth the clncal beneft (Fg. 9) and the antgen dose admnstered. Further, mmu notherapy caused an ntal rse and a subsequent fall n the serum concentraton of allergen-specfc IgE antbodes n two of our three patents followed wth multple samples. Specfc mmunotherapy caused marked ncrease n the serum concentraton of allergen-specfc IgG ant- IP ASSAY FOR ALLERGEN-SPECIFIC IGG ANTIBODIES (S. RAGWEED) FIG. 12. The changes n serum concentraton of IgE and IgG antbodes wth specfcty for short-ragweed antgens seen n a short-ragweed senstve patent durng specfc mmunotherapy are shown. By contrast to the patent shown n Fgure 9, the IgG antbody response progresses unmpared for the duraton of treatment n ths patent. DURATION OF IMMUNOTHERAPY - DAYS r 3/3/81: Downloaded from

9 298 ALI ET AL. A.J.C.P. September 1983 A :. SPECIFIC IgG 0 BERMUDA GRASS SHORT RAGWEED r = 0.7SS. P =.001 o, MODIFIED RAST CLASSES FIG. 13. Serum concentratons of IgG antbodes wth specfcty for short-ragweed and Bermuda grass antgens are correlated wth those of allergen-specfc IgE antbodes. Specfc IgE levels gven are those obtaned wth the Modfed RAST test 15 at the tme of ntal dagnoss of nhalant allergy. The levels of specfc IgG were determned after two years of specfc mmunotherapy. bodes n all three patents wth a hgh level of senstvty to short-ragweed (modfed RAST Class 4) studed wth sequental samples (Fgs ). The pattern of seral changes, however, was dfferent n each case and was related to the dosage schedule of therapy. In patent 1, who could tolerate only small dose (1:62,500 w/v) durng the frst sx months, there was a moderate ncrease n the concentraton of specfc G antbodes n the frst seven months of treatment, followed by a sharp rse n the succeedng two months, and a sustaned but gradual rse n the last seven months (Fg. 10). In patent 2, for whom the dose could be rapdly advanced from 1:1,562,500 to 1:12,500 w/v wthn three months, the level of specfc G antbodes rose moderately n the frst two months, very sharply n the succeedng two months, and then leveled off, showng no sgnfcant ncrease n the last ten months of therapy (Fg. 11). The pattern of specfc G changes n patent 3 contrasted sharply wth that of patent 2. The antgen dose n ths patent could not be advanced due to local reactons beyond 1:62,500 w/v n 15 months. The seral samples revealed a moderate ncrease ntally, a plateau effect from the fourth ( ' to the eghth month, and fnally a sharp ncrease n the last fve months (Fg. 12). It s noteworthy that both patents 1 and 3 showed the sharpest ncreases n the serum levels of short-ragweed specfc IgG antbodes out of the ragweed season. In patent 2, the perod of rapd ncrease concded partally wth the ragweed season. In some other areas, however, the results of ths study are not n full accord wth those reported n some prevous studes. Zavazal and Stajner studed IgG response to specfc mmunotherapy for nhalant allergy to grass pollen. 25 They observed good correlaton between the results of an n vtro assay and the n vvo blockng antbodes for patents wth a low level of senstvty but not for hghly senstve ndvduals. By contrast, the IgG response to specfc mmunotherapy n all three hghly senstve patents n ths study (Fgs ) was strong. The same held for the majorty of patents wth hgh level of senstvty to Bermuda grass and short-ragweed antgens shown n Fgures 7 and 8. In ths study, we further observed a correlaton between the specfc IgG response to specfc mmunotherapy and the serum levels of specfc IgE antbodes (RAST scores) obtaned at the tme of ntal dagnoss (Fg. 13). Ths observaton s of consderable nterest. It seems to suggest that an atopc ndvdual's ablty to mount an IgG response to specfc mmunotherapy parallels hs ablty to produce IgE antbodes n response to the antgenc challenge n the envronments,.e., hgh IgE responders are also hgh IgG responders. Ths concluson s also supported by the observed dfferences n the serum levels of allergen-specfc IgG antbodes among the nonatopc subjects, untreated atopc patents, and treated atopc patents (Fgs. 7 and 8). However, we regard ths data prelmnary and ths concluson tentatve. The changes n the serum levels of allergen-specfc IgE antbodes durng the course of specfc mmunotherapy requre further comment. In all three short-ragweed senstve patents (Fgs. 8, 9, and 10), the levels of specfc E antbodes ntally rose and were followed by a gradual declne. However, n patents 1 and 2, the fall n the level of specfc IgE was followed later by a slght ncrease. It s noteworthy that n patent 1, the level of specfc E antbodes never reached the pre-treatment level. The clncal response to specfc mmunotherapy n patents 1 and 2 was consdered excellent. Ths fndng calls nto queston the belef that the clncal beneft of specfc mmunotherapy accrues from a fall n the concentraton of such antbodes. Our prelmnary data pont to the ncrease n the concentraton of allergenspecfc IgG antbodes as the prncpal mechansm producng clncal beneft. The majorty of atopc patents show senstvty to multple allergens. The mmunologc responses to var- Downloaded from

10 Vol. 80 No. 3 SPECIFIC IgG IN INHALANT ALLERGY 299 ous allergens ncluded n the therapy and admnstered at varous dosage levels are lkely to be varable. It s probable that consderable dfferences exst n the mmune responsveness of dfferent ndvduals and to dfferent antgens. Thus, the clncal response to specfc mmunotherapy represents the sum total of responses to ndvdual allergens n the treatment schedule as well as dfferences n mmune responsveness among ndvdual patents. The results of some studes have shown lack of correlaton between the clncal beneft and the IgG response to specfc mmunotherapy. In these studes, patents frequently had senstvty to multple allergens but were gven specfc mmunotherapy for only a sngle allergen under study. 10 The IgG response was generally good, yet the clncal mprovement was lackng. It seems probable that the observed lack of clncal correlaton resulted from the falure to treat these patents for all ther senstvtes. In concluson, the mmunoperoxdase assays for allergen-specfc IgE and IgG antbodes appear to be useful n the study of mmunologc responses to specfc mmunotherapy n nhalant allergy and warrant further evaluaton. The data presented suggest that a falure to demonstrate a good IgG response to one or more allergens ncluded n the therapy would ndcate ether a poor mmune responsveness of the patent or nadequacy of the antgen dose admnstered. As to the latter, t seems lkely that the serum specfc IgG levels can be used as a gude n the selecton of the optmum mantenance dose for specfc mmunotherapy. References 1. Al M, Ramanarayanan M, Connell JT, Nalebuff DJ, Fayem AO, Mesa-Tejada R: An mmunoperoxdase assay for serum ragweed-specfc IgE. Ann Allergy 1979; 42: Al M, Nalebuff DJ, Ramanarayanan M, Fayem AO, Mesa-Tejada R: Assay of IgE antbodes aganst June and Tmothy grass by an mmunoperoxdase technque. Cln Allergy 1980; 10: Capel PJA: A quanttatve mmunofluorescence method based on the covalent couplng of proten to Sepharose beads. J Immunol Methods 1974; 5: Chdlow JW, Bourne AJ, Baley AJ: FEBS Letters. 1974; 41: Cooke RA, Barnard JH, Helbald S, Stull A: Serologcal evdence of mmunty wth coexstng senstzaton n a type of human allergy (hayfever). J Exp Med 1935; 62: Deelder AM, Ploem JS: An mmunofluorescence reacton for Schstosoma manson usng the defned antgen substrate spheres (DASS) system. J Immunol Methods 1974; 4: Fadal RG: Immunoglobulns E and G and the allergc patent. Trans Am Soc Opthalmol Otolaryngol Allergy 1975; 15: Grant JA, Goldblum RM, Rahr R, Thueson DO, Farnam J, Gllaspy J: Enzyme-lnked mmunosorbent assay (ELISA) for mmunoglobuln G antbodes aganst nsect venoms. Cln Immunol 1981;68: Hamlton RG, Sobotka AK, Adknson NF: Sold phase radommunoassay for quanttaton of antgen-specfc IgG n human serum wth I-proten A from Staphylococcus aureus. J Immunol 1979; 122: Hll DJ, Hoskng CS, Shelton MJ, Turner MW: Falure of hyposenstsaton n treatment of chldren wth grass-pollen asthma. Br Med J 1982; 284: Johansson SGO, Mller ACML, Overell BG, Wheeler AW: Changes n serum antbody levels durng treatment wth grass pollen-tyrosne adsorbate. Cln Allergy 1974; 4: Krstansen T: Affnty Chromatography. Edted by O Hoffman- Ostenhof. Oxford, Pergamon Press, 1978, pp Lchtensten LM, Norman PS, Wnkenwerder WL, Oser AG: In vtro studes of human ragweed allergy. Changes n cellular and humoral actvty assocated wth specfc desenstzaton. J Cln Invest 1966;45: Metzger WJ, Butler JE, Swanson P, Renders, E, Rcherson HB: Amplfcaton of the enzyme-lnked mmunosorbent assay for measurng allergen-specfc IgE and IgG antbody. Cln Allergy 1981; 11: Nalebuff DJ, Fadal RD, Al M: The study of IgE n the dagnoss of allergc dsorders n an otolaryngology practce. Otolaryngol Head Neck Surg 1979; 87: Paull BR, Jacob GL, Yungnger JW, Glech GJ: Comparson of bndng of IgE and IgG antbodes to honeybee venom phospholpase-a. J Immunol 1978; 120: Pruzansky JJ, Patterson R: Hstamne release from leucocytes of hypersenstve ndvduals. I. Use of several antgens. J Allergy 1966; 38: Resman RE, Wcher K, Arbesman CE: Immunotherapy wth antgen E.'J Allergy 1969; 44: Spegelberg HL, Fredman H, Tuft L: Immunologcal responses of pollnoss patents treated wth alumprecptated pyrdne ragweed extract. Ann Allergy 1967; 25: Sobotka AK, Valentne MD, Ishzaka K, Lchtensten LM: Measurement of IgG-blockng antbodes: Development and applcaton of a radommunoassay. J. Immunol 1976; 117: Starr MD, Wenstock M: Studes n pollen allergy. Ill: The relatonshp between blockng antbody levels and symptomatc relef followng hyposenstzaton wth Allpyral n hayfever subjects. Int Archs Allergy Appl Immu 1970; 38: van der Gessen M, Homan WL, van Kernebeek G, Aalberse RC, Deges PH: Subclass typng of IgG antbodes formed by grass pollen-allergc patents durng mmunotherapy. Int Archs Allergy Appl. Immun 1976; 50: Yungnger JW, Glech GJ: Seasonal changes n IgE antbodes and ther relatonshp to IgG antbodes durng mmunotherapy for ragweed hay fever. J Cln Invest 1973; 52: Zavazal V, Stajner A, Sndelar J: Immunologc changes durng specfc treatment of the atopc state. IV. Blockng actvty of solated IgG, IgA, and IgM wth a specfc allergen-bndng capacty. Acta Allerg 1972; 27: Zess CR, Metzger WJ, Levtz D: Quanttatve relatonshps between IgE antbody and blockng antbodes specfc for antgen E n patents gven mmunotherapy wth ragweed antgen E. Cln Exp Immunol 1977; 28: ' Downloaded from