performed with 21 patients treated for active TB, we found that the immunoglobulin G (IgG) antibody response to this

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1988, p Vol. 26, No /88/ $02.00/0 Copyright 1988, American Society for Microbiology Humoral Immune Response in Human Tuberculosis: Immunoglobulins G, A, and M Directed against the Purified P32 Protein Antigen of Mycobacterium bovis Bacillus Calmette-Guérin MIREILLE TURNEER,1* JEAN-PAUL VAN VOOREN,2 JACQUELINE DE BRUYN,' ELISABETH SERRUYS,2 PAUL DIERCKX,3 AND JEAN-CLAUDE YERNAULT2 Institut Pasteur du Brabant' and Département de Pneumologie et Service de Bactériologie, Hôpital Erasme, Université Libre de Bruxelles,2 Brussels, and Institut Georges Brugmann, Alsemberg,3 Belgium Received 29 March 1988/Accepted 14 June 1988 The P32 protein antigen of Mycobacterium bovis BCG, identified as antigen 85A in the BCG reference system, was used to investigate the humoral immune response in human tuberculosis (TB). Immunoglobulin G (IgG), IgA, and IgM directed against P32 were measured by an enzyme-linked immunosorbent assay. Mean IgG and IgA antibody levels differed significantly (P < 0.001) between active-tb patients (50 untreated and 52 treated) and healthy control subjects (111 unvaccinated tuberculin negative, 38 unvaccinated tuberculin positive, and 72 recently BCG vaccinated). Mean IgG antibody levels, but not mean IgA antibody levels, were higher (P < 0.05) in patients with positive microscopic examination for acid-fast bacilli than in patients with negative microscopic examination. A positive relation was found between mean levels and the extent of disease. There was no difference in mean IgM antibody levels between patients and controls. By setting the upper normal limit at the 95th percentile of the 221 healthy subjects, the sensitivities were 46% in untreated and 63% in treated patients for IgG and 30 and 50%, respectively, for IgA. Of the untreated patients, 56% were positive for either IgG or IgA antibodies. Among the untreated patients with negative direct smear, 35% were positive for IgG and 24% were positive for IgA. When both immunoglobulin classes were combined, the serological test was positive in 47% of those patients. Neither naturally acquired tuberculin hypersensitivity nor BCG vaccination affected positivity frequencies in healthy subjects. Only active TB seemed to induce significant anti-p32 antibody levels and to be associated with positivity. A serological test with P32 as the antigen might therefore be helpful for the rapid diagnosis of TB. Serological studies provide information concerning events of humoral immunity following mycobacterial infections and also offer more pragmatic perspectives, i.e., the possible early and rapid diagnosis of tuberculosis (TB). The first attempts at serodiagnosis, reported by Arloing in 1898, showed that sera from TB patients could agglutinate tubercle bacilli (1). Since then, many studies have been carried out by using various techniques and, in most cases, by using quite complex antigens, such as whole bacteria, culture filtrates, bacterial extracts, and tuberculins or their purified protein derivatives (PPD) (for a review, see references 18 and 25). These investigations were disappointing after the preliminary studies because of lack of sensitivity or specificity. Sensitive and reproducible assays like the enzyme-linked immunosorbent assay (ELISA) (17) allowed some technical problems to be overcome but did not improve specificity (12). Therefore, recent studies emphasized the need for more specific, and thus more purified, antigens. The difficulties in obtaining them have been repeatedly asserted (8, 11). Although novel techniques, such as expression in other vectors and purification by immunoadsorption with monoclonal antibodies, have been developed, only a few purified mycobacterial antigens are available at present. The following have been tested in serological trials: glycolipids from Mycobacterium bovis BCG by Reggiardo and colleagues (28, 29), antigen 6 from Mycobacterium tuberculosis by Stroebel et al. (31), antigen 5 from M. tuberculosis by Daniel and collaborators (2-4, 13, 26), and the 64-kilodalton (kda) protein of M. bovis BCG, expressed in Escherichia coli, by * Corresponding author Thole et al. (32). We recently purified a 32-kDa protein antigen called P32 from zinc-deficient culture filtrates of M. bovis BCG (16). This antigen was identified as the 85A component from the BCG 85 complex (9, 16). In a study performed with 21 patients treated for active TB, we found that the immunoglobulin G (IgG) antibody response to this antigen was inversely correlated to the cellular immune response measured by specific lymphoproliferation and gamma interferon production, with 77% sensitivity and 100% specificity (22). In order to evaluate the serodiagnostic potential of this purified antigen, the serological study was extended to 102 TB patients, half of whom were newly diagnosed. IgG, IgA, and IgM antibody levels were compared with those of 221 healthy subjects comprising skin test-negative and -positive unvaccinated individuals, together with BCG-vaccinated persons. MATERIALS AND METHODS Study population. (i) TB patients. The sera of 50 patients with active TB were taken before any specific treatment. Of the 50 patients, 46 had pulmonary TB, 2 had pleural TB, and 2 had urinary tract TB. The diagnosis required at least one culture of sputum, bronchial lavage, gastric aspirate, pleural fluid, or urine positive for M. tuberculosis; direct smears for acid-fast bacilli were positive in 33 patients (66%). On the basis of clinical and radiographic criteria (10), the population could be subdivided into 25 minimal, 21 moderately advanced, and 4 very advanced cases of TB. The patients (70% men) ranged in age from 19 to 88 years (mean, 48; standard deviation, 19). A second serum sample was obtained from 11

2 VOL. 26, 1988 IgG, IgA, AND IgM TO P32 ANTIGEN IN TUBERCULOSIS 1715 patients at about 2 months after the institution of antituberculous therapy. Fifty-two patients (78% men) with active TB who had been treated for at least 2 weeks at the time of blood sampling were included in the study. Of these 52 patients, 49 had pulmonary TB, 2 had pulmonary TB associated with bone or gastrointestinal TB, and 1 had pleural TB. The diagnosis was assessed by culture in all cases; 32 (62%) of the patients were sputum positive by direct smear at the time of diagnosis. Sixteen patients had mininal, 25 had moderate, and 11 had very advanced disease. They ranged in age from 22 to 83 years (mean, 48; standard deviation, 16). (ài) Control group. The 221 healthy subjects were divided into the following three subgroups. (i) One hundred and eleven unvaccinated subjects (38% men) were negative (induration diameter, <6 mm) when skin tested with 2 tuberculin units of purified protein derivative (PPD 2 UT, Institut Pasteur du Brabant). They ranged in age from 16 to 60 years (mean, 29; standard deviation, 7). (ii) Thirty-eight subjects (47% men) without any history of BCG vaccination or active TB were tuberculin positive (induration diameter, >10 mm) at the time of blood sampling. They have to be considered as having had previous TB infection. They ranged in age from 26 to 55 years (mean, 36; standard deviation, 7). (iii) Seventy-two subjects (32% men) had received bacillus Calmette-Guérin vaccine (BCG; Institut Pasteur du Brabant) less than 3 months before their blood was sampled. Sixty-three (88%) of them were skin test positive (induration diameter, >6 mm) 3 months after vaccination. They ranged in age from 21 to 49 years (mean, 28; standard deviation, 5). Antigen. P32 protein antigen was prepared as described previously (16) from unheated culture filtrates of M. bovis BCG (substrain 1173P2) grown on zinc-deficient Sauton medium for 14 days. It was purified to homogeneity as assessed by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis in denaturing conditions, NH2- terminal amino acid sequencing, and crossed immunoelectrophoresis. P32 was identified as BCG 85A antigen (16) in the reference system defined by Closs et al. (9). ELISA procedure. The ELISA for antibody determination was based on the method of Engvall and Perlmann (17). Immulon Microelisa plates (Dynatech, Kloten, Switzerland) were coated by adding to each well 1,ug of P32 in 100 Fl of Tris hydrochloride buffer (ph 8.2). After incubation for 2 h at 27 C in a moist chamber, the plates were kept overnight at 4 C. They were washed four times with 0.01 M phosphatebuffered saline (ph 7.2) containing 0.05% Tween 20 by using a Titertek microplate washer (Flow Laboratories, Brussels, Belgium). Blocking was done with 0.5% gelatin in 0.06 M carbonate buffer (ph 9.6) for 1 h. Wells were then washed as before, and 100 ul1 of serum diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin was added. According to the results obtained in preliminary experiments, the working dilutions were set at 1:200 for IgG, 1:20 for IgA, and 1:80 for IgM determinations. Each dilution was run in duplicate. After 2 h of incubation and after the wells were washed, they were filled with 100,ul of peroxidase-conjugated rabbit immunoglobulin directed against human IgG, IgA, or IgM (Dakopatts, Copenhagen, Denmark), diluted 1:400, 1:400, and 1:1,200, respectively, in phosphate-buffered saline containing 0.05% Tween 20 and 0.5% gelatin and incubated for 90 min. After the wash, the amount of peroxidase bound to the wells was quantified by using a freshly prepared solution of o-phenylenediamine (10 mg/100 ml) and hydrogen peroxide (8,ul of 30% H202 per 100 TABLE 1. Group (no. of subjects) Mean anti-p32 IgG, IgA, and IgM antibody levels in TB patients and healthy control Mean optical density (SD)l IgG IgA IgM Controls 0.14 (0.07) 0.12 (0.09) 0.23 (0.12) Skin test negative (111) 0.15 (0.07) 0.11 (0.08) 0.22 (0.10) Skin test positive (38) 0.12 (0.06) 0.12 (0.07) 0.25 (0.14) BCG (72) 0.14 (0.06) 0.13 (0.11) 0.25 (0.13) TB patients' Untreated (50) 0.38 (0.38) 0.24 (0.20) 0.25 (0.23) Direct smear negative 0.23 (0.14)c 0.21 (0.19)c 0.24 (0.26) (17) Direct smear positive 0.46 (0.44) 0.26 (0.21) 0.25 (0.22) (33) Treated (52) 0.52 (0.53) 0.32 (0.25) 0.28 (0.28) Direct smear negative 0.35 (0.40) 0.29 (0.31) 0.26 (0.37) (20) Direct smear positive 0.62 (0.58) 0.33 (0.21) 0.30 (0.20) (32) a Sera were diluted 1:200 for IgG, 1:20 for IgA, and 1:80 for IgM determinations. The Mann-Whitney U test for significance of differences (in comparison with all controls) yielded P values of <0.001 for IgG and IgA results obtained for all patient groups, except as indicated. b All TB cases were proven by culture. '' P <0.01. ml) in 0.15 M citrate buffer (ph 5.0) as a substrate. The enzymatic reaction was stopped with 8 N H2SO4 after 15 min of incubation. The optical density was read at 492 nm with a Titertek Multiskan photometer (Flow Laboratories). Wells without sera were used as controls for the conjugates. Each experiment was done by including on each plate one negative and two positive reference sera with medium and low antibody levels to correct for plate-to-plate and day-to-day variations. The antibody concentrations were expressed as the optical density values obtained after correction of the readings according to the mean variations of the reference sera. The mean interassay variation coefficients for those sera were 7.9, 7.8, and 5.3% for IgG, IgA, and IgM determinations, respectively. Statistical analysis. The significance of the difference between means was calculated by the Mann-Whitney U test, except for paired sera, for which the Wilcoxon test was used. The x2 test was used to evaluate the significance of differences in positivity frequencies between groups. RESULTS Mean antibody levels. Mean IgG, IgA, and IgM antibody levels are presented in Table 1. A significant difference (P < 0.001) in IgG and IgA levels was found between healthy subjects and TB patients irrespective of treatment. Mean levels were significantly higher (P < 0.05) in treated patients than in untreated patients. Untreated patients with positive microscopic examination for acid-fast bacilli at the time of diagnosis had a higher (P < 0.05) mean level of IgG, but not of IgA, than did those patients with a negative result. Regarding the extent of TB, severely diseased subjects presented the highest antibody levels before as well as during the course of chemotherapy (Table 2). By grouping untreated and treated patients, a positive relation (P c 0.02) was found between mean IgG and IgA antibody levels and the grade of disease. No difference was observed in mean IgM levels between patients and controls. Eleven subjects were tested before and after initiation of the treatment. The mean IgG antibody level increased from

3 1716 TURNEER ET AL. J. CLIN. MICROBIOL. TABLE 2. Mean anti-p32 IgG, IgA, and IgM antibody levels in patients matched according to the grade of TB Mean optical density ± SD (no. of cases)' Grade of TB Untreated TB Treated TB IgG IgA IgM IgG IgA IgM Minimal 0.26 ± 0.31 (25) 0.19 ± ± (16) 0.22 ± ± 0.28 Moderate 0.45 ± 0.36 (21) 0.26 ± ± ± 0.48 (25) 0.33 ± ± 0.32 Advanced 0.72 ± 0.69 (4) 0.53 ± ± ± 0.66 (11) 0.44 ± ± 0.14 a Sera were diluted 1:200 for IgG, 1:20 for IgA, and 1:80 for IgM determinations (standard deviation, 0.46) to 0.64 (standard deviation, 0.54). The therapy thus led to a significant (P < 0.01) increase in IgG levels in these individuals but not in IgA or IgM levels. Individual antibody levels. Individual anti-p32 IgG, IgA, and IgM antibody levels are shown in Fig. 1, 2, and 3, respectively. No differences were observed between the subgroups in control subjects. Therefore, the optical density value corresponding to the 95th percentile of all healthy subjects was taken as the normal upper limit in each immu- 2.0 Il 2- _ _~ 0 *4f ST- ST+ BCG Umtr Tb Tt Tb FIG. 1. Anti-P32 IgG antibody levels determined by ELISA. Sera, diluted 1:200, were tested in duplicate; each point represents one subject. The normal upper limit (--- ) corresponding to the 95th percentile of all healthy subjects and the means (-- -) are indicated. Control healthy subjects were subdivided into 111 tuberculin skin test-negative (ST-), 38 skin test-positive (ST+), and 72 BCG-vaccinated (BCG) subjects. TB patients were subdivided into 50 untreated newly diagnosed cases (Untr Tb) and 52 cases having received chemotherapy for at least 2 weeks (Tr Tb). All cases were proven by culture; negative (*) results for microscopic examination for acid-fast bacilli at the time of diagnosis are indicated. *_i:~ 614 *4l * 44 noglobulin class. Using this definition, we obtained a specificity of 95% and a sensitivity of 55 and 40% in IgG and IgA classes, respectively (Table 3). TB patients were significantly (P < 0.001) more frequently positive than the controls in both immunoglobulin classes. More treated than untreated patients were positive, although the differences were not significant. In the subgroups of untreated patients, more patients with positive direct smears at the time of diagnosis were ELISA positive compared with those with negative direct smears (Table 3). Nevertheless, 35% of the latter patients were IgG positive and 24% were IgA positive. By combining the positivities of IgG and IgA, 47% of the untreated patients with negative direct smears were positive by ELISA, but false-positives reached 10% because different control subjects were positive in the two immunoglobulin classes. As for mean antibody levels, the IgG and IgA positivity frequencies increased with the severity of the disease in untreated as well as in treated patients. The frequencies of IgM positivity were not different between groups * L3~~ ~ 4 L;t~~~ n l S T- S T + ICa Umtr Tb Tr Tb FIG. 2. Anti-P32 IgA antibody levels determined by ELISA. Sera were diluted 1:20. Symbols are described in the legend to Fig. 1.

4 VOL. 26, 1988 IgG, IgA, AND lgm TO P32 ANTIGEN IN TUBERCULOSIS ~~~~~-- r _ ST- St 6 Unr Yb Tr Tb FIG. 3. Anti-P32 IgM antibody levels determined by ELISA. Sera were diluted 1:80. Symbols are described in the legend to Fig. 1. DISCUSSION The presence of IgG, IgA, and IgM serum antibodies to P32 protein, one of the quantitatively important constituents of the cell and the major antigen secreted into the culture fluid during active growth of M. bovis BCG (16), was analyzed in TB patients and healthy subjects. Antibodies were found in skin test-negative healthy controls. These antibody levels, however, were lower than TABLE 3. Frequency of positivity of anti-p32 IgG, IgA, and lgm in TB patients and healthy controls Group (no. of subjects) No. of positive subjects (%)l IgG IgA IgM Controls 11 (5) 11 (5) 11 (5) Skin test negative (111) 9 (8) 6 (5) 3 (3) Skin test positive (38) 1 (3) 2 (5) 3 (8) BCG (72) 1 (1) 3 (4) 5 (7) TB patients Untreated (50) 23 (46) 15 (30) 4 (8) Direct smear negative (17) 6 (35) 4 (24)b 1 (6) Direct smear positive (33) 17 (52) 11 (33) 3 (9) Treated (52) 33 (63) 26 (50) 8 (15) Direct smear negative (20) 12 (60) 8 (40) 2 (10) Direct smear positive (32) 21 (66) 18 (56) 6 (19) a The upper normal limits were set at optical density values corresponding to the 95th percentiles of all healthy subjects, with sera diluted 1:200 for IgG, 1:20 for IgA, and 1:80 for IgM determinations. The x2 test for significance of differences (in comparison with 221 controls) yielded P values of <0.001 for IgG and IgA results obtained for all patient groups, except as indicated. b P <0.05. those measured in the same control sera against PPD (unpublished data), suggesting that P32 is a more specific antigen. Actually, P32 has been identified as the 85A component of the heterogeneous 85 complex in the BCG reference system (9). This complex was shown to react in crossed immunoelectrophoresis with antisera against various mycobacterial species (M. tuberculosis, Mycobacterium avium, Mycobacterium duvalii, Mycobacterium smegmatis, Mycobacterium nonchromogenicum, and Mycobacterium phlei) but to give no reaction with antisera against Nocardia asteroides, Corynebacterium pyogenes, and Listeria monocytogenes (20). P32 antigen seems to be restricted to mycobacterial species, but more refined studies, using monoclonal antibodies, are required to assess its real specificity. In addition, such studies would make it possible to identify and isolate species-specific epitopes. Neither recent BCG vaccination nor previous history of infection led to the enhancement of anti-p32 antibodies. On the contrary, higher (P < 0.001) mean IgG and IgA antibody levels were found in TB subjects than in healthy subjects, suggesting that only active disease gave rise to significant antibody levels. Although IgA was less discriminatory than IgG, its frequency of positivity was different (P < 0.001) between patients and controls. IgM did not differentiate between the groups. These results are in agreement with the findings of previous studies using complex antigens (19, 27, 35). Our study was conducted in a population of about 100 patients, half of whom were treated for TB. Among the untreated patients, 92% suffered from minimal or moderate disease. By using P32 antigen, a global sensitivity of 55% was obtained for IgG determination, with 46% sensitivity in untreated patients and 63% in treated ones and a specificity of 95%. The weakest responses were observed in patients with minimal TB who were tested before any specific treatment; the highest responses were observed in the most severely ill patients who had received chemotherapy. Mean antibody levels and frequencies of positivity were positively correlated with the extent of disease. Purified antigens from M. tuberculosis and M. bovis BCG have been previously tested in the humoral IgG response in human TB. Some proved to be unsuitable for serological purpose by being either too specific, like MPB70, a protein peculiar to BCG (21), or not specific enough, like the recombinant 64-kDa protein of BCG (32). This latter antigen, however, is known to share epitopes with proteins in many other bacterial species (37). Other purified antigens, such as a 70-kDa protein from M. bovis (7) and a 38-kDa protein from M. tuberculosis (36), have been used in preliminary studies but have not been studied extensively in serological trials, unlike glycolipids (28, 29), antigen 6 (4, 31), and principally antigen 5 (2-4, 13, 26). In 1980, Reggiardo et al. (28) developed an assay using three purified serologically active glycolipids from BCG: 88% of the TB patients (78% of the newly diagnosed cases) and only 4% of the PPD-negative or -positive healthy subjects were positive (29). Antigen 6 gave 94% sensitivity in patients treated for active bone and joint TB. The specificity was 100% (31). However, in a comparative study, Benjamin et al. (4) found antigen 6 to be less satisfactory than either antigen 5 or PPD for serological diagnosis in patients with active pulmonary TB. Antigen 5 was studied in several international trials. In a first report, Benjamin and Daniel (3) showed a sensitivity of 84% in Bolivia and 68% in the United States, with 92% specificity. Balestrino and collaborators (2) then studied antigen 5 in Argentina, where they obtained a specificity of 100% and a sensitivity of 64% (patients with

5 1718 TURNEER ET AL. active pulmonary TB, all under treatment). Later, in a prospective study in Cleveland (13), 41 patients with active TB, confirmed bacteriologically, were studied. Among them, 4 had minimal TB, 16 had moderately advanced TB, 17 had far-advanced T»; 2 had disseminated TB, 1 had extrapulmonary TB, and 1 had silicotuberculosis. They entered the study at different times after the initiation of specific therapy. The sensitivity was 49%. The specificity measured in 59 control subjects was 98%. No statistically significant relation could be found between mean titers and the extent of disease. In China, 89% sensitivity was reported by Ma et al. (26) for treated patients. In this study, a significant difference (P < 0.01) was evidenced in geometric mean titers between noncavitary and cavitary TB patients, but the frequencies of positive results did not differ significantly between the two groups. The specificity was 94%. In most of these studies, the TB status was not fully described. Comparisons are therefore difficult because of the various parameters likely to affect antibody levels and hence sensitivity (15, 24; our study). Since treated patients showed higher responses than newly diagnosed ones, the incidence of specific chemotherapy on antibody levels was tested by controlling 11 patients before and at about 2 months after the initiation of treatment. We found a significant increase (P < 0.01) of IgG anti-p32 antibody levels. In a 16-month follow-up of the response against purified antigen 5, mean IgG levels remained essentially constant; some of the patients, however, were not sampled before the initiation of specific therapy (13). It is not clear why higher IgG antibody levels were obtained in treated patients, but the higher levels may have been due to increasing amounts of antigen released as mycobacteria are destroyed by antituberculous drugs (23). In our study, the diagnosis oftb was always confirmed by culture. Among the 50 untreated patients, 33 were direct smear positive at the time of diagnosis and 17 were negative. The specificity of this bacteriological test proved to be 99% when performed in our institution. Interestingly, positive IgG levels were found in 35% of untreated patients with negative direct smears and in 52% with positive microscopic examinations. Moreover, the determination of IgA gave higher sensitivity to the assay, since the responses in IgG and IgA classes were not parallel; of the new patients with negative direct smears, 35% were positive for IgG, 24% were positive for IgA, and 47% were positive for either IgG or IgA. In untreated patients, the sensitivity of microscopic examination (66% sensitivity) combined with ELISA (56% sensitivity) attained 82%. These results are in good agreement with those previously obtained with less-purified antigens. Zeiss et al. (38), combining the first smear examination (57% sensitivity) and an ELISA using PPD as the antigen (67% sensitivity and 79% specificity), found a sensitivity of 86% in untreated patients. Daniel et al. (14), performing a study in Bolivia using an ELISA and a preparation containing, besides antigen 5, several mycobacterial proteins for IgG determination, found 69% sensitivity and 88% specificity. Combining these serological results with those of sputum smears, they found 92% sensitivity. Most of the patients had advanced cavitary disease. The real problem in discriminating between TB patients and healthy subjects might be explained by the absence of increased antibody levels in some patients rather than by the presence of antibodies in control subjects. Indeed, antibodies may be present in sera from patients but may remain undetected because of being entrapped in circulating immune complexes (6, 30). Another factor must be taken into J. CLIN. MICROBIOL. account, i.e., a possible genetic control (5) of the humoral response, as has been described for responses to multiple skin tests with new tuberculins (33) and for proliferative T-lymphocyte responses to PPD (34). Also, bacterial load in patients who recently developed limited TB may be not important enough to promote a sufficient rise in antibody levels. In conclusion, an anti-p32 humoral response in IgG and IgA classes was demonstrated specifically in active TB. The ELISA using this purified protein antigen from M. bovis BCG could be a valuable complement to bacteriological methods of diagnosis. These promising results have to be confirmed in prospective studies. ACKNOWLEDGMENTS The skillfull technical assistance of E. Van Nerom is gratefully acknowledged. This work was supported in part by grant from the Fonds de la Recherche Scientifique Médicale. J.-P.V.V. was supported by the Fondation Erasme. LITERATURE CITED 1. Arloing, S Agglutination du bacille de la tuberculose vraie. C.R. Acad. Sci. 126: Balestrino, E. A., T. M. Daniel, M. D. S. de Latini, O. A. Latini, Y. Ma, and J. B. Scocozza Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative. Bull. W.H.O. 62: Benjamin, R. G., and T. M. Daniel Serodiagnosis of tuberculosis using the enzyme-linked immunosorbent assay (ELISA) of antibody to Mycobacterium tuberculosis antigen 5. Am. Rev. Respir. Dis. 126: Benjamin, R. G., S. M. Debanne, Y. Ma, and T. M. Daniel Evaluation of mycobacterial antigens in an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of tuberculosis. J. Med. Microbiol. 18: Berzofsky, J. A., L. K. Richman, and D. J. Killion Distinct H-2-linked Ir genes control both antibody and T cell responses to different determinants on the same antigen, myoglobin. Proc. Natl. Acad. Sci. 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