Serological Analysis of Pulmonary and Extrapulmonary Tuberculosis with Enzyme-Linked Immunosorbent Assays for Anti-A60 Immunoglobulins

Transcription

1 1084 Serological Analysis of Pulmonary and Extrapulmonary Tuberculosis with Enzyme-Linked Immunosorbent Assays for Anti-A60 Immunoglobulins Y. L. Zou, J. D. Zhang, M. H. Chen, G. Q. Shi, J. Prignot, and C. Cocito From the Microbiology and Genetics Unit. University of Louvain, Brussels. and the Pneumology Division. Louvain University Clinics. Mont Godinne. Belgium; and the Second Hospital. Da Qing, and the Tuberculosis Hospital. Chao Yang. China IgA, IgG, and IgM antibodies to mycobacterial antigen A60 were measured by ELISA in blood, pleural fluid, and cerebrospinal fluid from 560 patients with pulmonary and/orextrapulmonary who were being treated at hospitals in northern China and from 734 uninfected controls. Among 529 healthy persons (most of whom had been vaccinated with bacille Calmette-Guerin [BCG] and 287 of whom were tuberculin-positive), the rate of false-positive results was negligible; this observation ruled out interference of remote BCG vaccination with A60 assays at the chosen cutoff level. Rates of positivity for IgM and IgG, respectively, were 80% and 36% among patients with active primary pulmonary, 31% and 88.5% among patients with active postprimary pulmonary, 0 and 41% among patients with inactive pulmonary, and 30%-61% and 69%-86% among patients with extrapulmonary. Paired samples of blood and pleural fluid from patientswith pleurisy contained IgA antibody to A60 at equal titers; in contrast, most patients with tuberculous meningitis (100% of whom had a positive ELISA result) had higher levels of IgG antibody to A60 in cerebrospinal fluid than in blood-proof of intrathecal synthesis. The development of mycobacterioses entails a strong interaction ofcomponents ofpathogenic mycobacteria and the immune system of the host [I]. These immunologically active mycobacterial components are valuable as reagents in diagnostic immunoassays for mycobacterial diseases [2-6]. Some of the components are free antigenic determinants in the bacterial cytoplasm. while others are high-molecularweight complexes [7, 8]. Some proteins bound to the highmolecular-weight polymers (peptidoglycan, arabinogalactan, and lipoarabinomannan) that constitute the bacterial cell wall have proven strongly immunogenic. Indeed, these complexes play a key role in mycobacterioses and exert a strong influence on various cells of the host's immune system. These features have been conclusively documented, for example, for the cell wall-associated proteins ofm'ycobacterium lepraeand Mycobacterium. Thermostable macromolecular antigens (TMAs) are immunologically active complexes present in all mycobacteria [9]. The best-known TMAs are antigen 60 (A60)ofMycobacterium bovis and M. [10-12], antigen 7 (A7) of M. leprae [13], and antigen 36 (A36) ofmycobacterium para [14]. TMA complexes are immunodominant in mycobacterioses and are strongly recognized by infected patients' sera [9, 15, 16]. Moreover, the injectionoftma prep- arations in laboratory animals induces humoral and cellular immune reactions [17-19]. The immunodominance of TMA complexes has prompted their use as reagents in serological assays and cutaneous tests. An A60-based ELISA for and an A36-based ELISA for para have been developed and have received various applications in both human [20 26] and veterinary [14] medicine. Likewise, cutaneous tests with A60 [9, 27] and A36 [28] are exploited for diagnostic purposes. Indeed, these antigen complexes are the main active components oftuberculins and sensitins-the mycobacterial extracts widely used for cutaneous testing in medical practice [28]. The aim ofthe present work was to apply three A60-based ELISAs to the evaluation ofdifferent forms of in Chinese patients. While previous work had involved mostly patientswith pulmonary disease and had been limited to IgG antibodies [23], the current study was concerned with both pulmonary and extrapulmonary and included analyses for IgA, IgG, and IgM antibodies. In addition, antibody to A60 was evaluated in CSF and pleural fluid from patients with extrapulmonary. The goal was to clarify the immune status of patients during the course of different forms of and after chemotherapy was completed. Received 8 November 1993; revised 27 April Reprints or correspondence: Dr. C. Cocito, UCL-Vesale Avenue Mounier 52. B-1200 Brussels. Belgium. Clinical Infectious Diseases 1994;19: by The University of Chicago. All rights reserved /94/ $02.00 Materials and Methods Subjects. A total of1,294 Chinese persons were included in our survey: 734 controls (including 529 healthy persons and 205 patients with nonmycobacterial diseases) and 560

2 CID 1994; 19 (December) ELISAs for Tuberculosis Serology 1085 Table 1. Classification ofsubjects included in a serological survey of pulmonary and extrapulmonary in northern China. Category, group no. and description Controls without I: Healthy juveniles (8-15 y) BCG-nonvaccinated BeG-vaccinated 2: Healthy adults (18-35 y), BCG-vaccinated 3: Patients with nontuberculous diseases Total Patients with Pulmonary Primary 4: Active latent 5: Inactive 6: Active patent Postprimary 7: Active 8: Inactive 9: Miliary Extrapulmonary 10: Active tuberculous pleurisy II: Tuberculosis of joints, bone, lymph nodes, thoracic wall, and peritoneum 12: Tuberculous meningitis Mixed 13: Miliary and tuberculous meningitis 14: Mixed pulmonary and extrapulmonary (active) Total Grand total NOTE. BCG = bacille Calmette-Guerin. No. of subjects patients with who were being treated at hospitals in Da Quing and Chao Yang in northern China. Overall, 414 cases ofpulmonary (84 primary and 330 postprimary) and 146 cases ofextrapulmonary and mixed were analyzed (table I). The clinical categories of were defined as follows. Active latent primary was defined as the recent «2 years previously) conversion ofthe patient's tuberculin test to positivity without clinical or radiological abnormalities; active patent primary as the recent conversion of the patient's tuberculin test to positivity with clinical and/or radiological signs (hilar adenopathies); inactive primary as radiological scars resulting from primary infection (calcified primary lung lesions and calcified hilar adenopathies); active postprimary as adult-type pulmonary with clinical, radiological, and possibly bacteriologic signs ofactivity; and inactive postprimary as radiological scars of adult-type pulmonary without signs of active disease. Preparation of A60. A60 was prepared from the cytoplasm ofm. bovis strain BCG (bacille Calmette-Guerin; Pas ,294 teur Institute, Paris). The procedure used for purification by exclusion gel chromatography on Sepharose 6B (Pharmacia, Uppsala, Sweden) has been detailed elsewhere [10]. A60 was identified by crossed immunoelectrophoresis, with seconddimension migration in gels containing antiserum to M. bovis BCG [29]. A60 was found to contain three moieties-offree lipid, lipopolysaccharide, and lipopeptide nature, respectively [10, II ]-and was quantified by spectrophotometric determination ofits protein and sugar components. The virtual identity of the antigenic patterns of strain BCG and of virulent strains ofm. bovis and M. justifies the use ofa60 for the diagnosis of in humans. Serological assays for human. The ELISA used in these studies was conducted as follows [17, 23]. Each well of multiwell microtitration plates was coated with a solution of 1.5 f.lg ofa60/100 f.ll of0.05 M sodium carbonate buffer (ph 9.6). Plates were air-dried, stored at -20 C, and saturated with bovine serum albumin immediately before use. Samples of human serum diluted I:200 in the above buffer were added to the wells and incubated for I hour at 37 C. After repeated washings with buffer, horseradish peroxidase-labeled rabbit immunoglobulins to human proteins (Dako, Copenhagen; diluted I,OOO-foid immediately before use) were added. After incubation for 30 minutes at 37 C, plates were washed and peroxidase reagent was added; after incubation for an additional IS minutes at 37 C in darkness, the reaction was stopped by the addition of 100 f.ll of 2 M H 2S04, The A 450 was measured with a colorimetric plate reader (Titertech Multiskan Plus; Flow Laboratories, Brussels). For serum dilutions and plate washings, PBST buffer (0.15 M NaCl, 2 mm K 2HP04 ; ph 7.2) containing 0.005% Tween 80 was used. The serological data presented herein were obtained with three diagnostic kits (Anda Biologicals, Strasbourg, France) containing peroxidase-labeled chicken immunoglobulins to human IgA, IgG, and IgM, respectively. The peroxidase reagent used in these studies was tetramethylbenzidine. Reading of ELISA results. For the IgA and IgG assays, optical density (OD) readings were transformed into ELISA units (EU) by interpolation on a calibration curve, which was prepared for each series oftitrations with three standard human sera yielding A 4so readings of0.3, 0.5, and 1.5 (corresponding to 1.0, 2.0, and 16 EU), respectively. Since almost all controls had readings of < 104 EU, this level was considered the cutoffvalue for both IgA and IgG. Values in the 104 to 2.0-EU range were considered borderline. The IgM serological assays were considered negative when the OD was <0.5 and positive when it was ~0.5. The chosen cutoff values were 2 SDs from the mean values for healthy persons. IgG concentrationsin paired samples ofcsfand serum from patients with tuberculous meningitis were determined by irnmunonephelometry, as were IgG and IgA concentrations in paired samples ofpleural fluid and serum from patients with tuberculous pleurisy.

3 1086 Zou et al. CID 1994; 19 (December) Results c Z 0"'... 0\ N - Analysis ofcontrol subjects by A60 ELISA. The cutoff point is a basic parameter in serological analyses. Studies have shown that the cutoffvalue varies according to environmental factors and therefore must be established for each epidemiological investigation. The recommended procedure for the establishment of a cutoff point is a survey of a large number of persons with no history ofthe disease in question and no obvious contact with patients who do have that disease. Moreover, the sample surveyed should be representative ofthe region from which patients in the study have been recruited. In our survey, 734 individuals from two regions of northern China were analyzed. Among the 370 healthy juveniles and 159 healthy adults were both BCG-nonvaccinated and BCG-vaccinated subjects. In addition to these healthy controls, 205 patients with various nontuberculous diseases were surveyed (table I). Results ofthe A60 ELISAs for the three immunoglobulins in the control group are shown in table 2. Of 529 healthy individuals (nonvaccinated and vaccinated), only one had a positive result in the IgG A60 ELISA and one had a borderline IgG value. None ofthese persons had positive results in tests for IgA and IgM. Among the 205 patients with nontuberculous diseases (nontu berculous pneumopathies in 151 cases and extrapulmonary diseases in 54 cases; table 3), 13 had positive results for IgG, 3 for IgM, and 3 for IgA (these results were fully positive for only three of six patients) (table 2). In conclusion, the rate of false-positive results in the ELISA for IgG was 0.38% among healthy individuals and 6.3% among patients with nontuberculous diseases. Among healthy persons, almost no false-positivity for IgM or IgA was detected. Analysis of cases by A60 ELISA. Sera from 560 patients with different forms of were submitted for serological analysis. Included were 414 samples from patients with pulmonary, 92 samples from patients with extrapulmonary, and 54 samples from patients with both pulmonary and extrapulmonary disease. Serological results are listed by form of in table 4. The following conclusions can be drawn from these results. (I) Primary forms were characterized by high IgM and low IgG positivity rates. (2) Postprimary forms were characterized by high IgG and low IgM positivity rates. (3) Healing of postprimary was accompanied by substantial lowering ofthe IgG positivity rate and suppression of IgM positivity. (4) Most forms of extrapulmonary exhibited immunoglobulin patterns similar to those documented for postprimary. (5) The IgM-to-IgG ratio in miliary fell between the ratios found in primary and postprimary pulmonary. o...; A\ "': V c Z V'> o A\ V'> o v o...; A\ 0\ M I o M "': V Ci Z c Z 0 =' ~ g S -0\0\ 0 '<too V'> NV'> 0 N :::: o'<t N

4 CID 1994; 19 (December) ELiSAs for Tuberculosis Serology 1087 Table 3. Results of A60 ELISAs among controls with nontuberculous diseases, by clinical diagnosis. No. with indicated A60 ELISA result IgG (EU)* IgM (OD)t IgA (EU)* No. of Diagnosis patients < ~3.0 <0.5 ~0.5 NO < ~3.0 NO capo and asthma complicated by infection Acute bronchitis II Bacterial pneumonia I I 0 8 I 3 6 Viral pneumonia Pulmonary tumors Cancerous pleurisy I Pneumoconiosis complicated by infection I Other pulmonary disease Extrapulmonary disease j t 0 4 Total NOTE. EU = ELISA units: 00 = optical density: COPD = chronic obstructive pulmonary disease. * Cutoff point is <1.4 EU. t Cutoff point is an 00 of <0.5. t Acute disseminated encephalitis. Analysis ofcases oftuberculous meningitis. Serum samples from 44 cases oftuberculous meningitis (18 ofwhich were associated with miliary ) were submitted for A60 ELISA. As is shown in table 4 (group 12 and 13), 33 patients were positive for IgG (12 with borderline values), 23 were positive for IgM, and 27 were positive for IgA (9 with borderline values). In 12 cases the level of anti-a60 IgG was measured in simultaneously collected paired samples of CSF and serum. Tuberculosis was confirmed by a positive CSF culture in 5 cases and by a positive sputum culture in 5. Clinical and laboratory data for these 12 cases are displayed in table 5. All 12 patients had serum positive for IgG (1 with a borderline value). Moreover, the majority of patients had distinctly higher titers of antimycobacterial immunoglobulins in CSF (mean OD, 6.6) than in serum (mean OD, 4.9). The serological patterns for three of these cases are displayed in figure I. One patient (patient 6 in table 5) and a control had similar titers of IgG in CSF and serum ("mirror pattern"), whereas two other patients (patients I and 9) had higher titers in CSF than in serum. Analysis of pleural fluid from cases of tuberculous pleurisy. Previous reports from otherlaboratories suggested the concentration ofspecific immunoglobulins was higher in the pleural fluid than in the serum of patients with tuberculous pleurisy. This phenomenon was explored by comparative analysis of anti-a60 IgA and IgG in paired samples of pleural fluid and serum from 24 cases oftuberculous pleurisy and 27 cases of nontuberculous pleurisy (controls). The results are summarized in table 6. The A60 ELISA was positive for IgG in 22 of24 cases oftuberculous pleurisy and for IgA in 21 of24 cases. In these 24 cases the mean IgG values were similar for pleural fluid and serum. The same was true for mean IgA values. The correlation coefficient between IgA values for serum and pleural fluid was very high (r = 0.93 in tuberculous pleurisy and r = 0.87 in nontuberculous pleurisy). Discussion A cutoff point, which is essential for the interpretation of serological data, is based on large surveys ofcontrol subjects (healthy persons with no history of a given disease and patients with other diseases) and varies according to environmental conditions. The baseline OD value for anti-a60 IgG was found to be 0.2 in Belgium [23], 0.3 in France [24, 25] and Italy [26, 30], and higher in countries farther south. In the present study a cutoff OD value of 0.38 (corresponding to 1.4 EU) was established for China. These differences are presumably due to the unequal densities of the microbial populations present in each environment and to the crossreactions ofa60 from M. boviswith TMAs from other mycobacterial species. Negative serological results were found equally often in BCG-nonvaccinated and BCG-vaccinated persons. This equal distribution excluded a contribution of remote BeG vaccination to the rate of false-positivity in the A60 ELISA (table 2). Serum IgM titers documented by A60 ELISA were higher in primary than in postprimary pulmonary (table 4). Moreover, the rate of IgM positivity was higher in active than in inactive post primary (group 7 vs.

5 Table 4. Results of serological analyses for patients with different forms of. No. with indicated No. with indicated A60 ELISA result No. with indicated mycobacterial tuberculin test result* culture result IgG(EU)t IgM (OO)t IgA (EU)t Group Clinical No. of no. classification patients Positive Negative NO Positive Negative < ;;;.3.0 <0.5 ;;;.0.5 NO < ;;;.3.0 NO 4 Active latent primary I I 0 12 pulmonary Inactive primary pulmonary 6 Active patent primary I 3 pulmonary Active postprimary pulmonary Inactive postprimary II N pulmonary 0 e 9 Miliary II Active tuberculous II 0 pleurisy II Tuberculosis ofjoints II II bones. lymph nodes. thoracic wall. and peritoneum (active) 12 Tuberculous II meningitis 13 Miliary II 3 I and meningitis 14 Mixed pulmonary and II II extrapulmonary (active) n NOTE. NO = not done; EU = ELISA units; 00 = optical density. a * Positivity limit was set at a 10-mm diameter of induration. ~ \0 t Cutoff point is < 1.4 EU. f7 t Cutoff point is an 00 of <0.5. Of the seven positive results. four were for CSF and three for sputum. '0l'l> ('l l'l> 3 0-3, ~ Qj ;- \0

6 CID 1994; 19 (December) ELISAs for Tuberculosis Serology 1089 Table 5. Clinical and laboratory data for 12 patients with tuberculous meningitis whose anti-a60 IgG concentrations were measured in paired samples ofcsf and serum. Results of CSF analysis Anti-A60 IgG Time* Microbiological Pulmonary ELISA result (d) Protein Glucose culture result (OD/IO}II Patient Age of sample level level (radiological no. (y}/sex collection (gll)t (rnmol/l)! Cells/rnrrr' CSF Sputum analysis) Outcome CSF Serum I 26/F (78) + SS /F (80) CR /M (60) + MS# /M (83) + + CR /M (73) CR /F (80) + + CR /M (60) + + SS /M (66) + CR /M (88) + + SS /M (80) + + CR II 12/F (92) + CR /F (39) MS NOTE. 00 = optical density; + = positive; - = negative; SS = slight sequelae; CR = complete recovery; MS = major sequelae. * After onset ofclinical symptoms. t Normal level, <0.5 gil. t Normal level. <2.2 mmoljl. Normal count, ~8/mm3. Percentage ofall cells that were lymphocytes is shown in parentheses. II Paired samples of serum and CSF were tested at the same IgG concentration. # Major sequelae included hydrocephalus, bradypsychia, spastic paraparesis. and tardive syringomyelia. group 8, table 4). Thus, high serum levels of anti-a60 IgM are suggestive of recent activation of the infectious process. Comparison ofdata for groups 5 and 6 in table 4 (inactive and active primary ) and for groups 7 and 8 (active and inactive postprimary ) revealed a lowering of A60 ELISA values to baseline in most primary cases that resolved but in only a fraction of postprimary cases that resolved. Positive serological values in inactive may correspond to permanent sequelae. The diagnosis of human has been facilitated by serological analysis with whole mycobacterial homogenates and crude extracts such as purified protein derivative and sensitins [31-33], but these methods are rendered less specific by extensive cross-reactions among mycobacteria. In an effort to enhance specificity, several mycobacterial antigens have been purified and used as reagents for immunologic assays [3, 34-38], but biochemical fractionation has yielded purified products in amounts insufficient for clinical use. Larger amounts of recombinant proteins have recently been obtained from cloned mycobacterial genes [39-41], but most ofthe available assays have lacked the desired sensitivity and/or specificity. Performance indexes of the ELISA based on antigen 5 [3, 42-44] indicated a sensitivity of49% 89% and a specificity of 92%-1 00%. The sensitivity, specificity, and positive predictive value of the A60 ELISA for IgG were found in a previous study to be 78%, 86%, and 49%, respectively [23]. The same test applied to several immunoglobulins in French patients [24] yielded index values of 48%,71 %, and 50%for IgG; 76%, 98%.and 95%for IgM; and 68%, 100%, and 100%for IgG plus IgM [24]. On the basis of the data reported herein, index values for the specificity and sensitivity of the A60 ELISA for IgG were 83.8% and 85.4%, respectively. These performance indexes take into account ~ e :10 1:15 1:20 Serum and CSF Dilutions Figure 1. Antimycobacterial IgG levels in paired samples of serum (dashed lines) and CSF (solid lines) in three representative cases of tuberculous meningitis and one control case of purulent nontuberculous meningitis (0). Titers of anti-a60 IgG were measured by A60 ELISA. The 00 readings of increasing dilutions of CSF and serum show overlap ("mirror pattern") for two sets of paired samples (one set from patient 6 in table 5 [*]and the other set from the control) and a lack of overlap (intrathecal IgG synthesis) for two sets (from patients I [X] and 9 [0]). 1:25

7 1090 Zou et at. CID 1994; 19 (December) Table6. A60 ELISA results for paired samples of pleural fluid and serum from patients with tuberculous and nontuberculous pleurisy. Results of A60 ELISA Mean ± SO value (00) No. of No. (%) No. (%) Group patients positive Serum Pleural fluid positive IgG IgA Mean ± SO value (00) Serum Pleural fluid Tuberculous pleurisy (91.7) 1.13 ± ± (87.5) Nontuberculous pleurisy Carcinoma 15 2*(13.3) 0.30 ± ± *(6.7) Infection 12 I (8.3) 0.31 ± ± 0.13 I (8.3) Total 27 3(11.1) 0.30 ± ± (7.4) 1.08 ± ± ± ± ± ± ± ± 0.13 NOTE. 00 = optical density. * Carcinoma complicated by infection. only patients with pulmonary disease (tuberculous and nontuberculous pneumopathies). The positive predictive value ofthe test-with an assumed prevalence of0.15 for the group of individuals studied-was 48.1 %. Sensitivity values of 52%-91%and specificity values of 97%-100% have been claimed for glycolipid-based ELISAs [3]. The immunologic significance and clinical value of these indexes have been discussed repeatedly [46, 47]. The current interest in the A60 ELISA is due to the fact that the A60 complex represents an important fraction oftotal mycobacterial antigens and that it is immunodominant. Indeed, the majority of anti mycobacterial immunoglobulins in the blood [IS] and CSF [I6] of patients with are directed against A60. Tuberculous meningitis is invariably lethal unless rapidly diagnosed and treated. Several serological assays have been used to diagnose this condition [31, 48, 49]. We have recently reported the use ofan A60-basedimmunoblottingtest for the early diagnosis of this disease [16], a finding that is confirmed by the present work. Application of the A60 based ELISA to paired samples of serum and CSF revealed intrathecal synthesis of anti-a60 IgG in many cases (figure I). On the contrary, when this type ofanalysis was applied to cases oftuberculous pleurisy, titers ofanti-a60 IgA and IgG in pleural fluid matched those in serum (table 6); therefore, the accumulation of these immunoglobulins in the pleural cavity was excluded. Since no single assay allows the identification ofall forms of, the diagnosis-and thus the outcome-of this disease relies on a combination of clinical, radiological, microbiological, and immunologic data. The A60 ELISA may prove a valuable tool in the analysis of. A60 serological assays are particularly interesting in cases of extrapulmonary, which are difficult to diagnose by the usual clinical and laboratory tests. In addition, these tests can profitably be used for analysis of CSF and pleural fluid. The complementary value ofthe A60 ELISAs for different immunoglobulins is illustrated by our data, which indicate that IgM antibodies to this antigen are mainly involved in early phases ofactive tuberculous processes and IgG antibodies in later phases. References I. Chaparas SO. The immunology ofmycobacterial infections. CRC Crit Rev Microbiol 1982; 9: Coates ARM. Serology of. Eur J Respir Dis 1980;61: Daniel TM, Debanne SM. The serodiagnosis of and other mycobacterial diseases by enzyme-linked immunosorbent assay. Am Rev Respir Dis 1987; 135: Grange JM. The humoral immune response in : its nature. biological role and diagnostic usefulness. Advances in Tuberculosis Research 1984; 21: Van Vooren JP. Farber CoM. Motte S. De Bruyn J. Legros F, Yernault.IC, Assay ofspecific antibody response to mycobacterial antigen for the diagnosis of a pleural effusion in a patient with AIDS. Tubercle 1988; 69: Krambovitis E. Serodiagnosis of in perspective. Serodiagnosis and Immunotherapy 1987; 1: Daniel TM. Janicki BW. Mycobacterial antigens: a review oftheir isolation. chemistry. and immunological properties. Microbiol Rev 1978;42: Wright GL Jr. Affronti LF. Reich M. Characterization and comparison of mycobacterial antigens by two-dimensional polyacrylamide gel electrophoresis. Infect Immun 1972;5: Cocito CG. Properties ofthe mycobacterial antigen complex A60 and its applications to the diagnosis and prognosis of. Chest 1991; 100: Cocito C. Vanlinden F. Preparation and properties of antigen 60 from Mycobacterium boris BCG. Clin Exp ImmunoI1986;66: II. Fabre I. L'Hornme 0, Bruneteau M. Michel G. Cocito C. Chemical composition ofantigen 60 from Mycobacterium boris BCG. Scand J ImmunoI1986;24: Harboe M, Closs O. Bjorvatn B, Bjune G. Antibodies against BCG antigen 60 in mycobacterial infection. BMJ 1977;2: Abou-Zeid C. Harboe M. Sundsten B. Cocito C. Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria. J Infect Dis 1985; 151: De Kesel M. Gilot P. Coene M. Cocito C. Composition and immunolog-

8 GO 1994; 19 (December) ELISAs for Tuberculosis Serology 1091 ical properties ofthe protein fraction ofa36, a major antigen complex of Mycobacterium para. Scand J Immunol 1992; 36: CoetsierC, Baelden M-e. Coene M, Cocito C Immunological analysis ofcomponents ofthe antigen complex A60 ofmycobacterium bovis BCG. Clinical and Diagnostic Laboratory Immunology 1994; I: Sindic CJM, Boucquey 0, Van Antwerpen MP, Baelden Me. Laterre e. Cocito C Intrathecal synthesis ofanti-mycobacterial antibodies in patients with tuberculous meningitis: an immunoblotting study. J Neurol Neurosurg Psychiatry 1990; 53: Cocito e. Baelden MC, Benoit C Immunological properties ofantigen 60 of BCG: induction of humoral and cellular immune reactions. Scand J Immunol 1987;25: Harboe M. Antigens ofppd, old tuberculin, and autoclaved Mycobacterium bovis BCG studied by crossed immunoelectrophoresis. Am Rev Respir Dis 1981; 124: Carlucci S, Beschin A, Tuosto L, et ai. Mycobacterial antigen complex A60-specific T-cell repertoire during the course ofpulmonary. Infect Immun 1993;61: Casal M, Linares MJ. Diagnostico serologico de la. Revista Espanola de Microbiologia Clinica 1988; 3: Mattar S, Broquetas J, Sauleda J, Carceller A, Gea J, Torres JM. Detection de anticuerpos IgG con el rnetodo ELISA en pacientes tuberculosos utilizando el antigeno 60. Revista Espanola de Microbiologia Clinica 1989;4: Maes R, Homasson J-P, Kubin M, Bayer M. Development of an enzyme-immunoassay for the serodiagnostic of and mycobacterioses. Med Microbiollmmunol 1989; 178: Baelden M-e. Vanderelst B, Dieng M, Prignot J, Cocito C Serological analysis ofhuman by an ELISA with mycobacterial antigen 60. Scand J Infect Dis 1990; 22: Charpin 0, Herbault H, Gevaudan MJ, et ai. Value of ELISA using A60 antigen in the diagnosis ofactive pulmonary. Am Rev Respir Dis 1990; 142: Gevaudan MJ, Bollet C, Charpin 0, Mallet MN, De Micco P. Serological response of patients to antigen 60 of BCG. Eur J EpidemioI1992;8: Fadda G, Grillo R. Ginesu F, Santoru L, Zanetti S, Dettori G. Serodiagnosis and follow up of patients with pulmonary by enzyme-linked immunosorbent assay. Eur J EpidemioI1992;8: Benoit C, Beschin A, Desmecht M, Dekeyser P. Cocito C Delayed hypersensitivity reactions by the mycobacterial antigen A60, and cutaneous testing in. Med Microbiol Immunol 1989; 178: Gilot P, Cocito C Comparative analysis ofthree sensitins used in cutaneous testing for and para in cattle. FEMS Microbiol Lett 1993; 110: Closs 0, Harboe M, Axelsen NH, Bunch-Christensen K, Magnusson M. The antigens of Mycobacterium bovis. strain BCG, studied by crossed immunoelectrophoresis: a reference system. Scand J Immunol 1980; 12: Gulletta E. Del Pezza M, Sanduzzi A, Bariffi F, Covelli I. Serodiagnosis ~urvey of by a new ELISA method. Eur J Epidemiol 1988;4: Kalish SB, Radin RC, Levitz 0, Zeiss CR, Phair JP. The enzyme-linked immunosorbent assay method for IgG antibody to purified protein derivative in cerebrospinal fluid of patients with using purified tuberculin antigen. Tubercle 1980;61: Tandon A, Saxena RP, Saxena KC Diagnostic potentialities of enzyme-linked immunosorbent assay in using purified tuberculin antigen. Tubercle 1980;61: Viljanen MK. Eskola J, Tala E. Enzyme-linked immunosorbent assay (ELISA) for antibodies to purified protein derivative of tuberculin (PPD): IgM-, IgA- and IgG- anti-ppd antibodies in active pulmonary. Eur J Respir Dis 1982;63: Lau JHK, Leong JCY, Stroebel AB. A longitudinal study of antibody titres to antigen 6 in patients with bone and joint. Int Orthop 1983; 7: Daniel TM, Debanne SM, van der Kuyp F. Enzyme-linked irnrnunosorbent assay using Mycobacterium antigen 5 and PPD for the serodiagnosis of. Chest 1985;88: Huygen K, Van Vooren J-P, Turneer M. Bosmans R, Dierckx P, De Bruyn J. Specific Iyrnphoproliferation, gamma interferon production, and serum immunoglobulin G directed against a purified 32 kda mycobacterial protein antigen (P32) in patients with active. Scand J Immunol 1988;27: Stroebel AB, Daniel TM, Lau JHK, Leong JCY, Richardson H. Serologic diagnosis ofbone and joint by an enzyme-linked immunosorbent assay. J Infect Dis 1982; 146: Turneer M, Van Vooren J-P, De Bruyn J. Serruys E. Dierckx P. YernauIt J-C Humoral immune response in human : immunoglobulins G, A and M directed against the purified P32 protein antigen ofmycobacterium bovis bacillus Calmetre-Guerin. J Clin MicrobioI1988;26: Thole JER, Dauwerse HG. Das PK. Groothuis DG. Schouls LM. Van Embden JDA. Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli. Infect Immun 1985; 50: Young RA, Bloom BR, Grosskinsky CM. Ivanyi J. Thomas D. Davis RW. Dissection ofmycobacterium antigens using recombinant DNA. Proc Natl Acad Sci USA 1985;82: Gilot P, De Kesel M. Machtelinckx L. Coene M. Cocito C Isolation and sequencing of the gene coding for an antigenic 34-kilodaIton protein of Mycobacterium para. J Bacteriol 1993; 175: Benjamin RG. Daniel TM. Serodiagnosis of using the enzyme-linked immunoabsorbent assay (ELISA) of antibody to Mvcobacterium antigen 5. Am Rev Respir Dis 1982; 126: Balestrino EA, Daniel TM. De Latini MDS, Latini OA, Ma Y. Scocozza 18. Serodiagnosis of pulmonary in Argentina by enzyme-linked immunosorbent assay (ELISA) ofigg antibody to Mycobacterium antigen 5 and tuberculin purified protein derivative. Bull World Health Organ 1984; 62: Ma Y, Wang YoM, Daniel TM. Enzyme-linked immunosorbent assay using Mvcobacterium antigen 5 for the diagnosis of pulmonary in China. Am Rev Respir Dis 1986; 134: Grange JM. Gibson J. Nassau E. Kardjito T. Enzyme-linked immunosorbent assay (ELISA): a study ofantibodies to Mycobacterium in the IgG. IgA and IgM classes in. sarcoidosis and Crohn's disease. Tubercle 1980;61: Galen RS. Application of the predictive value model in the analysis of test effectiveness. Clin Lab Med 1982;2: Toman K. Sensibilite, specificite et valeur predictive des tests diagnostiques. Bulletin of the International Union for Tuberculosis 1981;56: Hernandez R, Munoz 0, Guiscafre H. Sensitive enzyme immunoassay for early diagnosis of tuberculous meningitis. J Clin Microbial 1984;20: Kadival GV, Mazarelo TBMS. Chaparas SO. Sensitivity and specificity ofenzyme-linked immunosorbent assay in the detection ofantigen in tuberculous meningitis cerebrospinal fluids. J Clin Microbiol 1986;23:901-4.