Serodiagnosis of Early Lyme Disease: Analysis of IgM and IgG Antibody Responses by Using an Antibody-Capture Enzyme Immunoassay

THE JOURNAL OF INFECTIOUS DISEASES VOL. 158, NO.4. OCTOBER 1988 1988 by The University of Chicago. All rights reserved. 22-1899/88/584-7$1. Serodiagnosis of Early Lyme Disease: Analysis of IgM and IgG Antibody Responses by Using an Antibody-Capture Enzyme Immunoassay Victor P. Berardi, Karen E. Weeks, and Allen C. Steere From the Department of Virology, Center for Disease Control, Massachusetts Department of Public Health, and the Department of Internal Medicine, Division of Rheumatology/Immunology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts We used an antibody-capture enzyme immunoassay (EIA) to evaluate the early antibody responses to Borrelia burgdorferi in paired sera from 3 patients with erythema chronicum migrans. During acute disease, 2 (67,7) patients had elevated specific IgM responses, and by convalescence(one to four weeks after treatment), 28 (93%) patients had increased IgM or IgG responses. In acute specimens, elevated IgM responses correlated with disseminated infection; however, by convalescence, most patients with either localized or disseminated disease had positive tests. Among 133control subjects, IgM cross-reactivity was observed in 4 of 37 patients with either Epstein-Barr virus or rickettsial infections, and false-positive IgG tests were seen in 8 of 28 patients with syphilis. With antibodycapture EIA, the diagnosis of Lyme disease can be confirmed in the majority of acutely ill patients and in almost all patients by convalescence. Borrelia burgdorferi, the etiologic agent of Lyme disease, was first isolated from ticks in 1981 [1] and later from blood, skin, or spinal fluid of patients with acute disease [2, 3]. The infection characteristically begins with an erythematous skin lesion, erythema chronicum migrans (ECM), which is often accompanied by influenza-like or meningitis-like symptoms [4, 5]. Early antibiotic therapy is effective in treating primary infection and often prevents the neurological, cardiac, and arthritic manifestations that occur later in the disease [6]. Although ECM is the distinctive clinical marker ofacute Lyme disease, it is atypical or absent in 27-47 of the patients; these patients are often thought to have viral infections [4, 5]. Because culturemethodsare a low-yield procedure [2, 3], serological tests are preferred for the laboratory confirmation of infection. In 1982, an indirect immunofluorescence assay was first used to evaluate the antibody response in Lyme disease [2]. Subsequently, ELISAs were shown to be more sensitive and specific [7-9]. Both the im- munofluorescence assay and the ELISA, however, are insensitive in patients with acute infection [7, 1]. In one prospective study using indirectelisa, only 347 ofpatients had elevated antibodytiters acutely and only 57 had them by convalescence [1]. Using immunoblotting, the earliest detectable antibodies appear to be directed primarily against a genusspecific flagellar polypeptide (41 kilodalton) of the spirochete [11-13]; however, attemptsto improve the sensitivity of ELISA with flagellin-enriched or purified flagellar antigens have produced variable results [12, 14]. More recently immunoblotting was found to be superior to indirect ELISA for diagnosing early Lyme disease [14]. In an attempt to improve serodiagnosis of early Lyme disease, we developed an antibody-capture enzyme immunoassay (EIA) for detecting IgM and IgG antibodies to B. burgdorferi. We found this method to be superior to other currently available methods (ELISA or immunoblotting) for diagnosing early Lyme disease. Received for publication 23 December 1987and in revised form 4 May 1988. This work was supported in part by grant AR-2358 from the National Institute ofarthritis and Musculoskeletal and Skin Diseases. Please address requests for reprints to Victor P. Berardi, Department of Virology, Center for Disease Control, 35 South Street, Boston, Massachusetts 213. Patients and Methods Patients. We tested acute and convalescent sera from 3 patients with ECM who had participated in a prospective study of Lyme disease in 1983 [1]. Their specimens have been previously used to compare the sensitivity of culture, indirect ELISA with various antigen preparations, and immunoblotting for diagnosing early Lyme disease [1, 14]. The 3 754

Antibody-Capture EfA in Early Lyme Disease 755 patients represented the clinical spectrum of early Lyme disease and were included in the present study on the basis of clinical criteria alone. In eight patients, evidence of infection remained localized to the skin or the regional lymph nodes. The remaining 22 patients had clinical evidence ofdisseminated infection in multiple organ systems. Sera. Acute serum samples were collected at the time ofdiagnosis and treatment (mean, 11.3 d; range, 1-31 d after onset of symptoms), and convalescent serum samples were collected at a mean of 27.2 d after disease onset (range, 7-55 d). All patients were treated successfully with oral tetracycline or penicillin. Control sera were obtained from 35 healthy medical personnel and from 24 patients with primary Epstein-Barr virus infection, 13 with Rocky Mountain spotted fever, 28 with syphilis, and 33 patients hospitalized at Yale-New Haven Hospital. Patients in the latter group had many different diagnoses, including systemic lupus erythematosus and renal allograft rejection. All case and control sera were coded, randomized, and tested without knowledge of the diagnosis. Antigen. The G39/4 strain of B. burgdorferi, isolated from ticks in Connecticut [2], was propagated in BSK medium [15] containing antibiotics (21lg of amphotericin B/mL, 2 J.Lg of neomycin sulfate/ml). The strain was cloned using serial limiting dilution. Subcultures were inoculated into 1 ml flasks of medium and were incubated for five to seven days at 33 C. The organisms were harvested by centrifugation at 15 g for 2 min at 1C, were resuspended in 4 ml of PBS (7.6 mm NaHP 4, 2.2mMNaH zp4, 15mMNaCI; ph 7.2), and were washed three times. The pellet obtained from the final wash was resuspended in 12 ml of distilled demineralized water and was sonicated in an ice bath with eight, 15-s bursts by using a Microson'" ultrasonic disruptor (Heat Systems Ultrasonics, Farmingdale, NY) at 57 output power. This sonicate was used as the immunogen for production of rabbit hyperimmune serum. For antigen in the capture assays, the sonicate was clarified by centrifugation at 4 g for 2 min at 4 C. The soluble fraction was collected, aliquoted, and stored at - 7 C. Protein concentrations were estimated using the method of Lowry et al. [16]. Immune rabbit serum. We used rabbit hyperimmune serum to detect antigen bound by patients' antibody in the capture EIA. We prepared the se- rum by injecting a New Zealand white rabbit with a total of 1 mg of the borrelial sonicate emulsified in Freund's complete adjuvant. The adjuvant-antigen emulsion was administered at four sc and two im sites. A l-mg booster dose in Freund's incomplete adjuvant was administered 32 d later, and the serum was obtained five weeks after the second dose. By immunoblotting, the serum showed an antibody response to 2 polypeptides of B. burgdorferi. Antibody-capture EIA. For the IgM capture assay, affinity-purified goat IgG, directed against human IgM (u-chain specific; Tago, Burlingame, Calif), was diluted 1:4 (1.4 ug/ml.) in carbonate/bicarbonate buffer (6.4 mmnahc 3, 5.9 mmna zc3 ; ph 9.1).The diluted antibody was then added in 1 ul.volumes to the inner 6 wells ofimmulon-z" flatbottomed polystyrene plates (Dynatech Laboratories, West Chester, Pa) and allowed to adsorb for 18h at 4 C. After washing eight times (3 ul.zwell) with PBS containing.5% Tween-2 (PBS-T buffer), by using a Titertek microplate washer (Flow Laboratories, McLean, Va), we added 25 u.l of PBS containing.5% Tween-2 and 5% nonfat dry milk (PBS-T-M buffer) to the wells, and the plates were incubated for 1 h at 22 C. The wells were washed again in PBS-T buffer, and similarly washed after the sequential addition and incubation (l h at 37 C) of5 ul, ofeachofthe reagents diluted in PBS-T-M buffer as follows: (1) patient's serum, 1:1;(2) 1.52 ug ofb. burgdorferi antigen/ml; (3) rabbit antibody to B. burgdorferi, 1:128; (4) horseradish peroxidase-labeled goat IgG Ftab'), antibody to rabbit IgG (heavy- and light-chain specific, 1:1 ; Cooper Biomedical, Malvern, Pa). We included controls on each plate as follows: (1) patients' serum incubated with buffer and without B. burgdorferi antigen (serum control); (2) buffer without serum (buffer control); (3) a positive serum sample (positive control); (4) a negative serum sample (negative control). After washing an additionaleight times with PBS (without lween-2), we added 1 ul,of the substrate 2,2' azino-di[3-ethylbenzthiazoline-6-sulfonate] (Kirkegaard and Perry Laboratories, Gaithersburg, Md) to all wells, and the plates were incubated at 22 C. The ODs were measured at 45 nm by using a Titertek Multiscan" spectrophotometer (Flow Laboratories). When the well containing the positive control reached an OD of.4 (1'\.115 min), the ODs of all the wells were recorded. The IgG-capture assay was carried out as described for IgM with modifications as follows. The

756 Berardi et al. wells were coated with affinity-purified goat IgG directed against human IgG (y-chain specific; Tago) diluted 1:8 (1.8 ug/ml.), the patient's sera were diluted 1:1, and 21 ug of B. burgdorferi antigen/ml was used. The optimal concentration ofeach reagent was determined by preliminary checkerboard titration. Reagent concentrationsyielding a maximumod of the positive control and a minimum OD ofthe negative control were considered optimum. The IgM antibody response was expressed as a ratio ofthe OD ofthe well containing the patients' serum with antigen divided by the OD of the buffer control. For IgG antibody determination, the OD ofthe wellcontaining the patients' serum with antigen was divided by the OD ofthe serum control. OD ratios were considered to be positive if they were 3 SD above the mean of normal control sera. Positive OD ratios were l.s and 2.S for the IgM and IgG tests, respectively. To quantify the IgM and IgG antibody response to B. burgdorferi, we diluted the positive control serum with the negative control serum in serial twofold dilutions (l:2 to 1:SI2); each was further diluted in PBS-T-M buffer (l :1 for IgM; 1:1for IgG) and the OD ratios were determined. A standard curve was constructed from these OD ratios (figure 1), and the relative geometric proportions of specific antibodyin samples from patients were estimated from the standard curve. Results 18 16 14 12 )...... ii) 1 8... 6 i:: & 4 2 IqM i UNOlL 2 4 8 IgG iii i i 16 32 64 128 256 512 SERUM DILUTION Figure 1. Standard curves used to quantify proportions of IgM and IgG antibodies to B. burgdorferi in sera from patients with Lyme disease. The endpoint dilutions (+) of the standard were 1:8 for IgM antibody and 1:156for IgG (OD ratios [arrows] of 1.5and 2.5, respectively). Units of specific antibody (shown in parentheses) were calculated for each patient's sample by using the formula as follows: units = (reciprocal of endpointdilutionof standard)/(dilution of serum corresponding to specimen OD ratio). Antibody response in acute and convalescent sera. Westudied the antibodyresponse in acute and convalescent sera of3 patients with early Lyme disease. Among the 22 patients seen within the first 14 d of onset of symptoms, IgM antibody to B. burgdorferi was detected in 12 acute samples and in 18 by convalescence one to four weeks after treatment (figure 2). Of the eight patients from whom acute sera were obtained 2-31 d after disease onset, all had elevated specific IgM antibody responses in both acute and convalescent sera. In contrast, IgG antibody to B. burgdorferi was not detected in any of the 22 acute samples from patients first seen within two weeks of onset and was detected in only four of their convalescent samples. Among those seen later, four ofthe eight patients had specific IgG antibodyacutely, and only five had IgG by convalescence. Collectively, 2 (67%) patients had detectable IgM antibody to B. burgdorferiat the time of diagnosis, and 28 (937) developed IgM and/or IgG specific antibodies during convalescence. Only one patient had a positive IgG test by convalescence, in the absence of detectable IgM. Of the two patients not diagnosed serologically, one, with localized infection, was seen seven days and the other, with disseminated infection, was seen 12 days after disease onset. Antibodyresponse in localized anddisseminated infection. The antibody response in patients with localized disease (ECM alone) was compared with that of patients with clinical evidence of disseminated infection (table 1). During the first two weeks ofclinical disease, the 14patients with disseminated infection had specific IgM antibody significantly more often in acute-phase samples than did the eight patients with localized disease (P <.OS, Fisher's exact test). By convalescence, 6 of8 patients with localized disease and 13 of 14 with disseminated infection had detectable IgM antibody. None of the patientsin eithergroup, had positive IgG tests acutely and only four did by convalescence. This comparison was not possible among patients first seen three

Antibody-Capture EfA in Early Lyme Disease 757 1 It 1 o, o i 1 i 2 DAYS i i 3 4 AFTER ONSET Figure 2. Results of serological tests for IgM (top) and IgG (bottom) antibodies to B. burgdorferi in sera from 3 patients with erythema chronicum migrans. Patients wereseenat 1-14d (left) and 2-31d (right);, acute sera; e, convalescent sera. IgG or IgM responses of 1 are 3 SD above the mean of normal control subjects (dotted area). 1 1 1 or four weeks after disease onset because all eight of these patients had evidence of disseminated infection. We attempted to determine if the early administration of antibiotics aborted or suppressed the production of antibody. The post-treatment convalescent IgM and IgG responses in patients with disseminated infection seen within the first two weeks of disease (mean, 22.9 d post-onset) were compared with the pre-treatment responses in the group seen three or four weeks after the onset of symptoms (mean, 22.6 d post-onset). As shown in table 1, the post-treatment convalescent IgM values in the early group were similar to the pre-treatment IgM values in the later group. IgG was detected more frequently in the pre-treatment samples of the later group, but the differences were not statistically significant (P >.5, Fisher's exact test). Specificity ofantibody-capture EfA. To assess the specificity of the antibody-capture EIA, we tested sera from 35 healthy subjects and from 24 patients with primary Epstein-Barr virus (EBV) infection, 28 with syphilis, 13 with recent Rickettsia rickettsii infection, and 33 who were hospitalized with other diagnoses (figure 3). Of the 133 control subjects, 1 of 13 patients with Rocky Mountain spotted fever and 3 of 24 with EBV infection had positive tests in the IgM assay. In the IgG assay, eightof28 patientswith syphilis had positive tests. No control subject had both false-positive IgM and IgG results. When controls were compared among themselves, patients with EBV infection had higher OD ratios in the IgM assay than did those in the other groups (mean, 1. ±.5 vs,.7 ±.24), and those with syphilis had higher IgG antibody ratios (mean, 2.2 ±.8 vs. 1.4 ±.3). Discussion In an attempt to improve serodiagnosis ofearly Lyme disease, we developed an antibody-capture EIA for detecting IgM and IgG antibodies to B. burgdorferi. Table 1. Serological results of patients with early Lyme disease who have localized or disseminated infection. Acute sera Convalescent sera No. (7) positive No. (7) positive Initial serum Days post-onset Days post-onset collection was (mean) IgM IgG (mean) IgM IgG 1-4 d post-onset Localized (n = 8) 7 2 (25)* 29 6 (75) 1 (13) Disseminated (n = 14) 7 1 (71)* 23 13 (93) 3 (21) 2-31 d post-onset Disseminated (n = 8)t 23 8 (1) 4 (5) 39 8 (1) 5 (63) NOTE. Sera were tested by using antibody-capture EIA as described in Patients and Methods. * p <.5, Fisher's exact test. t All patients seen more than two weeks after disease onset had clinical evidence of disseminated disease.

758 Berardi et 1. l5 3 25 2 15 1!S 3 25 2 ; 15 1 5.5 1. 1, 1.5 2. 2.5 3. GLr-,,..J;oI,-LfUfU.h-r..lfLr"T"'fL, 1.5 2. 2.5 3. 3.5 5. OPTICAL DENSITY RATIO Figure 3. Distribution ofod ratios observed in tests for IgM (top) and IgG (bottom) antibodies to B. burgdorferi in 133 control subjects. _, Healthy and hospitalized controls;, patients with syphilis; 1iliI, patients with Epstein Barr virus infection;, patients with Rocky Mountain spotted fever. Dottedline separatesnegative(left) and positive (right) test results. To facilitate comparison with previous data, we tested acute and convalescent sera from 3 patients who participated in an earlier study that compared methods for diagnosing early Lyme disease [1]. In that study, as in previous ones [2, 3, 1], both culturing and direct visualization of spirochetes in affected tissues were low-yield procedures; therefore, serological testing was the only practical laboratory method for diagnosing the infection. Although most investigators have found ELISA to be more sensitive and specific than immunofluorescence [7-9], insensitivity during early disease remains a problem with either method. In a recent study, using sonicated whole spirochetes as the antigen for an indirect ELISA, 4/ ofpatients had elevated antibodytiters acutely and only 6% had them by convalescence (table 2) [14]. Investigators have subsequently modified methods of serological testing in an attempt to overcome the problem of insensitivity during early disease. Coleman and Benach [12] reported significantly better results when using a flagellin-enriched antigen preparation for the ELISA instead of using sonicated whole spirochetes, but they did not achieve better results by using immunoblotting. Conversely, Grodzicki and Steere [14] had only slightly better results with a flagellin-enriched preparation on the solid phase of an ELISA compared with sonicated whole spirochetes, when using the same sera we used (table 2) [14]. They found immunoblotting, however, Table 2. Comparison of antibody-capture EIA, indirect ELISA, and immunoblotting methods for serodiagnosing early Lyme disease. No. of patients with positive IgM or IgG response to B. burgdorferi Acute sera 2-31 d 1-14 d Post-onset Post-onset Localized Disseminated Disseminated Convalescent sera Method (n = 8) (n = 14) (n = 8) (n = 3)* Antibody-capture EIA 2 1 8 28 Indirect ELISA Whole sonicated spirochetest 1 4 7 18 Flagellin-enriched preparationt 1 3 8 19 Immunoblottingt 2 7 7 25 * Compared with indirect ELISA using sonicated whole spirochetes, significantly more patients had positive responses with immunoblotting (P <.5) or antibody-capture EIA (P <.5; Fisher's exact test). t These data are found in [14].

Antibody-Capture EIA in Early Lyme Disease 759 to be superior to ELISA with either antigen preparation. Using immunoblotting, 53% ofthe patients had positive tests acutely and 837 had them by convalescence (table 2) [14]. In the current study, we obtained even better results by using antibody-capture EIA. With this method, 677 of the patients had positive antibody responses to B. burgdorferi acutely and 937 had them by convalescence (table 2). In most instances, it was only the IgM test that was positive; only one patient had a positive IgG test result by convalescence in the absence ofdetectable IgM. Diagnosing patientswith localized disease (ECM alone) early in the illness remains a problem with any of the assays, including antibody-capture EIA. With the antibody-capture EIA, however, all but one of these patients developed positive tests duringconvalescence one to four weeks after treatment. To explain the phenomenon ofinsensitivity ofantibody testing, it has been suggested that the early humoral immune response in Lyme disease may be suppressed or aborted entirely by early antibiotic therapy [1]. Our study shows that the majority of patients with early Lyme disease had IgM antibody to B. burgdorferi acutely, and in most of them, the amounts increased duringconvalescence, despite successful antibiotic therapy. As previously noted [8, 1, 11], the majority of patients did not have specific IgG responses duringthe first monthofillness, a finding that may indicate a problem in switching from IgM to IgG production. In addition, our study suggests that their IgG levels may have been dampened by antibiotic therapy. In our study, sera from 35 healthy control subjects and 33 hospitalized patients were not reactive in eitherthe IgM or IgG assays. A fewpatientswith EBV or rickettsial infection had false-positive IgM tests, and 37 of those with syphilis had positive IgG tests. B. burgdorferi, other spirochetes, and certain other bacteria [7, 8, 17] possess conserved polypeptides; therefore, false-positive tests might still be expected in patients with certain other diseases in addition to those tested here. The increased sensitivity ofantibody-captureeia for detecting specific IgM antibody, observed here with B. burgdorferi, has been found with other infectious agents, including Toxoplasma gondii [18], cytomegalovirus [19], and more recently with eastern and western equine encephalitis viruses [2]. Antibody-capture assays have been particularly useful in demonstrating IgM responses early in these illnesses [2] or in those patients with congenital infection [18]. In adult patients with recently acquired toxoplasmosis, Naot and Remington [21] observed significantly greater sensitivity in detecting specific IgM antibodies by using antibody-capture EIA compared with indirectimmunofluorescence. In a recent study of2 patients with alphavirus infections, serological confirmation of eastern equine encephalitis virus infection was possible by using IgM-capture EIA in all acute sera, even those collected as early as one day after onset [2]. There are several reasons for the increased sensitivity ofantibody-capture EIA in detecting IgM antibody. First, the IgM antibody in the patient's serum is bound to the solid phase by the capture antibody (goat antibody to human IgM), an occurrence that allows the specific antigen-antibody reaction to occur in the absence of competing antibody isotypes. Second, in our experience, solubleantigen may be added in approximately 1 times the concentration used in the standard indirect ELISA, a process thereby allowing the antigen and antibody to react in antigen excess. Finally, with antibodycapture EIA one measures the proportion of specific to total antibody of a given isotype [22] rather than the amount of specific antibody alone, and a proportional increase may be detectable before the absolute increase. In conclusion, we found antibody-capture EIA to be superior to other currently available methods for diagnosing early Lyme disease. It is considerably more sensitive and as specific as the standard indirect ELISA. We also found it to be better than immunoblotting. The relativecomplexity ofantibody-capture EIA and immunoblotting are deterrents to their use in smaller clinical laboratories; however, either method may be successfully used in larger reference or specialized laboratories. Because antibiotic therapy is curative in this disease, diagnosis remains important so that treatment can be begun without delay. With antibody-capture EIA, the clinical diagnosis can be confirmed in the majorityofacutely ill patients and in almost all by convalescence. Thus, we believe that the use of IgM-capture EIA will probably solve the previous problem ofinsensitivity in serological testing of patients with early Lyme disease. References 1. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP. Lymedisease-a tick-borne spirochetosis? Science 1982;216:1317-9

76 Berardi et al. 2. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmid GP, JohnsonE, Malawista SE. The spirochetal etiology of Lyme disease. N Engl J Med 1983;38:733-4 3. Benach JL, Bosler EM, Hanrahan JP, Coleman JL, Habicht GS, Bast TF, Cameron DJ, Ziegler JL, Barbour AG, Burgdorfer W, Edelman R, Kaslow RA. Spirochetes isolated from the blood of two patients with Lyme disease. N Engl J Med 1983;38:74-2 4. Steere AC, Malawista SE, Hardin JA, Ruddy S, Askenase PW, Andiman WA. Erythema chronicum migrans and Lyme arthritis. The enlarging clinical spectrum. Ann Intern Med 1977;86:685-98 5. SteereAC, Bartenhagen NH, Craft JE, Hutchinson GJ, Newman JH, Rahn DW, Sigal LH, Spieler PN, Stenn KS, Malawista SE. The early clinical manifestations of Lyme disease. Ann Intern Med 1983;99:76-82 6. Steere AC, Hutchinson GJ, Rahn OW, Sigal LH, Craft JE, DeSanna ET, Malawista SE. Treatment of the early manifestations of Lyme disease. Ann Intern Med 1983; 99:22-6 7. RussellH, Sampson JS, Schmid GP, Wilkinson HW, Plikaytis B. Enzyme-linked immunosorbent assay and indirect immunofluorescence assay for Lyme disease. J Infect Dis 1984;149:465-7 8. Craft JE, Grodzicki RL, Steere AC. Antibody response in Lyme disease: evaluation of diagnostic tests. J Infect Dis 1984;149:789-95 9. Magnarelli LA, Meegan JM, Anderson JF, Chappell WA. Comparison of an indirect fluorescent-antibody test with an enzyme-linked immunosorbent assay for serological studies of Lyme disease. J Clin Microbiol 1984;2:181-4 to. Shrestha M, Grodzicki RL, SteereAC. Diagnosing early Lyme disease. Am J Med 1985;78:235-4 11. Craft JE, Fischer DK, Shimamoto GT, Steere AC. Antigens of Borreliaburgdorferi recognized during Lyme disease. Appearance of a new IgM response and expansion of the IgG response late in the illness. J Clin Invest 1986;78:934-9 12. Coleman JL, Benach JL. Isolation of antigenic components from the Lyme disease spirochete: Their role in early diagnosis. J Infect Dis 1987;155:756-65 13. BarbourAG, Hayes SF, HeilandRA, SchrumpfME, Tessier SL. A Borrelia-specific monoclonal antibody binds to a flagellar epitope. Infect Immun 1986;52:549-54 14. Grodzicki RL, Steere AC. Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease. J Infect Dis 1988;157:79-9 15. Barbour AG. Isolation and cultivation of Lyme disease spirochetes. Yale J BioI Med 1984;57:521-5 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J BioI Chern 1951;193:265-75 17. Barbour AG, Burgdorfer W, Grunwaldt E, Steere AC. Antibodies of patients with Lyme disease to components of the Ixodesdamminispirochete.J Clin Invest 1983;72:54-15 18. Naot Y, Desmonts G, Remington JS. IgM enzyme-linked immunosorbent assay test for the diagnosis of congenital Toxoplasma infection. J Pediatr 1981;98:32-6 19. YolkenRH, Leister FJ. Enzyme immunoassaysfor measurement of cytomegalovirus immunoglobulin M antibody. J Clin Microbiol 1981;14:427-32 2. Calisher CH, Berardi VP, Muth DJ, Buff EE. Specificity of immunoglobulin M and G antibody responses in humans infected with eastern and western equine encephalitis viruses: Applicationto rapid serodiagnosis. J Clin Microbiol 1986;23:369-72 21. Naot Y, Remington JS. An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis. J Infect Dis 198;142:757-66 22. Siegel JP, Remington JS. Comparison of methods for quantitating antigen-specific immunoglobulin M antibody with a reverse enzyme-linked immunosorbent assay. J Clin Microbiol 1983;18:63-7