JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p. 2786-2791 0095-1137/90/122786-06$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 28, No. 12

Antigen Capture Assay for Detection of a 43-Kilodalton Mycobacterium tuberculosis Antigen A. A. WADEE,* L. BOTING, AND S. G. REDDY Department of Immunology of the South African Institute for Medical Research and School of Pathology, University of the Witwatersrand, Johannesburg, Republic of South Africa

Received 8 June 1990/Accepted 13 September 1990 This study describes the development of an enzyme-linked immunosorbent assay (ELISA) to detect Mycobacterium tuberculosis antigens in body fluids. A double-antibody sandwich procedure that used human and rabbit anti-M. tuberculosis immunoglobulin G antibodies was followed. The ELISA was able to detect as little as 0.8 ,ig of protein of M. tuberculosis sonic extract. Of 253 cerebrospinal fluid specimens submitted for analysis, Il (4.3%) false-positive results were recorded. Analysis of 317 pleural and ascitic fluid specimens resulted in 6 (1.9%) false-positive recordings. No false-negative results were recorded for any of the body fluids tested. This technique is rapid (5.5 h) and sensitive, may be developed and used in many laboratories with limited resources, and may prove useful in the diagnosis of extrapulmonary and pulmonary tuberculoses. Analysis of these body fluids by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that the antibody used in the ELISA detects a mycobacterial antigen of 43 kDa. Such antigens were not detected in body fluids of nontuberculous patients.

The disease caused by Mycobacterium tuberculosis is often unrecognized in its early stages because of the frequent absence of classical clinical symptoms (25) and an increasing proportion of patients with extrapulmonary involvement (9, 16, 26, 32). The diagnosis of pleural and peritoneal tuberculosis still remains problematic, as evidenced by reports indicating the absence of acid-fast bacilli on microscopic examination in more than 60% of patients (2, 19, 25, 33). Histological examination of pleural biopsy specimens can be unhelpful for diagnosis for over 20% of patients with pleural tuberculosis (33, 40), whereas peritoneal biopsy specimens are diagnostic for less than 40% of patients (2, 19, 30). Direct examination of ascitic fluid reveals acid-fast bacilli only when large volumes of fluid are concentrated (34). In addition, many patients have negative results on skin tests for tuberculosis, and chest X rays are often normal (22). Cultures of cerebrospinal fluid (CSF) specimens are not always helpful in the diagnosis of tuberculous meningitis. Cultures may show no growth or take up to 6 weeks to become positive for M. tuberculosis (12, 30). The search for reliable, reproducible, and specific serological tests for the diagnosis of tuberculosis has been an active area of research for many years. Enzyme-linked immunosorbent assays (ELISAs) are sensitive and reproducible and do not require sophisticated equipment. Several investigators have indicated that the demonstration of mycobacterial antigens in body fluids may be a clinically useful approach (1, 3, 11, 38). In particular, sandwich ELISAs have been developed for the detection of mycobacterial antigens in sputum (40) and urine (21, 29) specimens. Such assays have used anti-Mycobacterium bovis BCG antibodies to identify M. tuberculosis (8, 20, 40). Using similar techniques, other investigators have described the use of ELISAs for detecting tuberculous antigens in CSF and pleural and ascitic fluids (1, 2, 30). Circulating immune complexes have also been described in patients with tuberculosis (5, 7). However, the antigens in these complexes have not been well character*

ized and their application in diagnosis has not been established. The present report describes the development of an ELISA to detect M. tuberculosis antigens by a sandwich assay with two purified M. tuberculosis-specific antibodies. This assay was used for detecting M. tuberculosis antigens in CSF and pleural and ascitic fluids of diagnosed tuberculous patients and others with a variety of nonmycobacterial diseases. The results indicate that the assay is highly sensitive and specific in the detection of M. tuberculosis antigens. Analysis of such antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting revealed a 43-kDa protein present in these fluids that was recognized by anti-M. tuberculosis antibodies. MATERIALS AND METHODS For the establishment of the sandwich ELISA, initial experiments used 39 pleural and 24 ascitic fluid specimens obtained from patients with active pulmonary or abdominal tuberculosis. Control specimens were obtained from 49 individuals with a variety of nontuberculous diseases. CSF specimens were obtained from 63 patients with tuberculous meningitis and 109 CSF specimens were obtained from individuals with various central nervous system disorders other than tuberculous meningitis. These included patients suffering from pyogenic meningitis, febrile convulsions, and encephalitis. For the assessment of the ELISA, 200 pleural fluid, 117 ascitic fluid, and 253 CSF specimens were used without the diagnosis being known at the time of the assay. The results of the ELISA were compared with clinical and histological data obtained after the ELISA was performed. Patients were divided into two groups based on the criterion of probable or definite tuberculosis. In the cases of suspected pulmonary tuberculosis, abdominal tuberculosis, or both, specimens were submitted for histology and culture. Histological evidence from biopsy specimens and the appearance of acid-fast bacilli in culture resulted in a definite diagnosis of tuberculosis. The criteria used for the diagnosis suggestive of tuberculosis were those described by Maritz (26) and Bell and

Corresponding author. 2786

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ELISA FOR M. TUBERCULOSIS ANTIGENS IN BODY FLUIDS

Andrews (4). These included presentation with exudative effusions together with alymphocytosis, absence of neoplastic cells, less than 1% mesothelial cells, an adenosine deaminase level of more than 45 U/liter, and response to antituberculous therapy and clinical appearance suggestive of tuberculosis. A highly suggestive (probable) diagnosis of tuberculous meningitis was based on clinical presentation and on the following changes in CSF specimens: an increase in protein of >0.4 g/liter; a decrease in sugar to 6 U/liter and an absence of neoplastic cells; and negative viral, syphilitic, bacterial, or fungal serology. Mycobacterial antigen preparation. M. tuberculosis organisms were isolated as described previously (37). Organisms at 10 x 106/ml were sonicated at 4°C in an MSE Soniprep 150 sonicator (18 ,um from peak to peak) and centrifuged at 5,000 x g for 20 min. The resultant supernatant was then collected and plated (25 ,ug/ml) onto plastic petri dishes that were precoated with 25 ,ug of normal human immunoglobulin per ml. Adsorption was allowed to continue overnight at 4°C in a humidified chamber. The mycobacterial sonic extract that did not adhere to the plate was collected, pooled, and used as an antigen in the ELISA. In experiments that determined the amount of mycobacterial antigen that could be detected by our ELISA, mycobacterial extracts were diluted serially in phosphate-buffered saline (PBS)-0.05% Tween 20 (PBS-Tween) to concentrations ranging from 1 to 100 ,ug/ml, and 100 ,ul of these solutions was added as the test antigen for the assay. Antisera to mycobacterial antigens. Rabbits were given weekly intramuscular immunizations of 0.5 mg of mycobacterial sonic extracts per ml over a period of 2 months. Booster injections were administered every 14 days thereafter. Sera were collected by bleeding the ear veins of these rabbits. Human blood was obtained by venipuncture from 50 patients with proven tuberculosis; the patients had previously given informed consent. Purification of anti-M. tuberculosis antibodies. Rabbit and human immunoglobulin G (IgG) were isolated from the appropriate sera by precipitation with ammonium sulfate by the method of Campbell (6). An equal volume of saturated ammonium sulfate solution was added to serum samples while the mixture was stirred gently. The mixture was incubated for 1 h at room temperature followed by centrifugation at 5,000 x g for 5 min. The pellet so obtained was then resuspended in distilled water and dialyzed against PBS overnight. IgG was purified by passaging the mixture through Sephadex G 200 columns (Pharmacia, Uppsala, Sweden) by a modification of the technique described by Clarke and Adams (9). Immunoglobulin-rich suspensions (2 ml) were passed through preequilibrated Sephadex G 200 columns (10 by 1 cm), and 1-ml fractions were collected. IgG was present in the first 8 ml that was eluted. Enzymatic labeling of immunoglobulin. Purified human anti-M. tuberculosis antibody was enzymatically labeled with horseradish peroxidase (Sigma, St. Louis, Mo.) in the presence of sodium metaperiodate (Merck, Darmstadt, Federal Republic of Germany) by the technique described by Hudson and Hay (18). One milliliter of horseradish peroxidase (4 mg/ml) was added to 200 ,ul of 0.1 M sodium metaperiodate, and the mixture was stirred gently for 20 min at room temperature. The mixture was dialyzed against 0.001 M sodium acetate buffer overnight at 4°C. Twenty microliters of 0.1 M sodium carbonate buffer was then added to the dialyzed mixture, followed by the addition of 1 ml of

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the human anti-M. tuberculosis IgG to be conjugated. Any free enzyme was reduced by the addition of 0.1 ml of 4 mg of sodium borohydride (SaARchem, Muldersdrift, Republic of South Africa) per ml. The mixture was then dialyzed against 0.1 M borate buffer (pH 7.4) overnight at 4°C. Double-sandwich ELISA. A double-antibody sandwich ELISA was performed in flat-bottom 96-well microtiter plates (Nunc International, Roskilde Denmark). Wells were coated with 100 pi of 20 p.g of rabbit anti-M. tuberculosis IgG per ml in sodium carbonate buffer (pH 9.6) and incubated for 1 h at room temperature, followed by washing four times with PBS-Tween. Unbound sites were blocked with 100 ,ul of 0.5% bovine serum albumin for 1 h at room temperature. After a second wash with PBS-Tween, 100 ,l1 of various dilutions of pleural fluid, ascitic fluid, and CSF (in PBS-Tween) and various dilutions of mycobacterial extracts were added to duplicate wells. Plates were incubated for 2 h at room temperature. Following another four washes with PBS-Tween, 100 ,ul of enzyme-labeled antibody diluted 1:100 in PBS-Tween was added to the wells and incubated at room temperature for 1 h. After a final wash with PBS-Tween, 100 ,u1 of peroxidase substrate buffer (citrate phosphate buffer, H202, o-phenylenediamine) was added, and the reaction was terminated after 5 min with 50 pil of 2.5 M sulfuric acid. Optical densities at 492 nm (OD492s) were measured in a Dynatech Multiscan ELISA reader. All results reported here represent means of the four wells for each test antigen or specimen. Optimal concentrations of reagents (rabbit antiserum and peroxidase-labeled human antiserum) and incubation times were determined by checkerboard titrations by using 10 ,ug of M. tuberculosis extract per ml. Western blotting. Proteins in CSF and ascitic and pleural fluids were resolved by SDS-PAGE by using 10% acrylamide gels by the method of Laemmli (23). CSF and ascitic and pleural fluids were diluted 1:1, 1:50, and 1:50, respectively, in a sample buffer. Electroblotting was performed essentially as described by Towbin et al. (36). Once electrophoresis was complete, the gels were equilibrated in cold (4°C) electroblotting transfer buffer. Upon completion of electroblotting, the nitrocellulose strips were rinsed in Tris-buffered saline, (5 min for each wash), and unbound sites on the strips were blocked with bovine serum albumin (3 g/100 ml) for 1 h at room temperature. The blots were washed four times and then incubated with a 1:500 dilution of peroxidase-labeled human anti-M. tuberculosis antiserum for 2 h at room temperature with gentle agitation. The blots were washed a final four times and then incubated in an enzyme-substrate solution (methanol4-chloro-1-naphthol, H202) at room temperature for 15 to 20 minutes. After the appearance of bands, the reaction was stopped by the addition of distilled water. The blots were then compared with their corresponding stained gels to determine the sizes of the bands to which the antisera had bound. RESULTS Antigen capture immunoassay. In dose-response assays, the ELISA was capable of detecting as little as 0.8 ,ug of mycobacterial sonic extract (Fig. 1). The ODs of these values were at least two standard deviations (SDs) above the mean of the control. To determine the optimal conditions for the detection of antigen in pleural and ascitic fluids and CSF, these specimens were used undiluted and at dilutions of 1:10, 1:20, 1:30, 1:50, 1:75, and 1:100. Our results (Fig. 2) dem-

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