British Journal of Rheumatology 1992;31:615-618
DETECTION OF MYCOBACTERIUM TUBERCULOSIS ANTIGEN IN SYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS BY J. L. LAN AND C. H. WU* Allergy, Immunology and Rheumatology Division, Department of Internal Medicine and * Department of Medical Research, Taichung Veterans' General Hospital, Taichung, Taiwan, Republic of China
KEY WORDS:
MT antigen and RA, Synovial fluid, Antimycobacterial saline extract antibody, ELISA.
RHEUMATOID arthritis is recognized as an immunological, possibly autoimmune, disease [1]. Although some microorganisms have been implicated [2-6] in RA, no inciting agents or target antigens have been unequivocally identified. Mycobacteria are able to induce not only immunopathological granuloma as seen in leprosy and tuberculosis, but also arthritis in rats [7]. Adjuvant arthritis (AA), triggered by MT in Freund's complete adjuvant after intracutaenous immunization, has remarkable similarities to the arthritic process seen in human RA. The disease features a proliferative and inflammatory reaction in synovial membranes, progressive pannus formation, destruction of cartilage, erosion of bone, and ultimately ankylosis [8]. Mounting evidence [9-17], has implied that MT may be the autoantigen in RA. Since many antigenic determinants can be found within a microorganism, and some may mimic host macromolecules by chance, the verification of MT antigen in synovium or synovial fluid is important for the pathogenesis of RA. Recently, a highly specific and sensitive double-antibody sandwich ELISA, using antimycobacterial saline extract (anti-MSE) antibody able to detect MT antigen in cerebrospinal fluid and sputum of patients with meningitis or pulmonary tuberculosis, was reported by Wu et al. [18,19]. The ELISA was able to detect as little as 6.0 ng of antigen, and maximum cross-reactivity with non-tuberculous mycobacterial antigens was less than 2%. The present study describes the detection of MT antigen in synovial fluid of RA patients by the anti-MSE antibody based ELISA.
METHODS Patients and specimens This study was carried out on 65 patients (26 females and 39 males) with RA, as defined by the 1987 revised American Rheumatism Association criteria [20], attending the immunology and rheumatology clinic at Taichung Veterans' General Hospital, Taichung, Taiwan. Fifteen patients (two females and 13 males) with gout, 14 patients (five females and nine males) with OA, 12 patients (one female and 11 males) with SA and eight patients (two females and six males) with OID (two psoriatic arthritis, two septic arthritis, two pseudogout and two pigmented villonodular synovitis) were studied as control groups. SF specimens were treated with hyaluronidase (HU, 100 (ig/ml) for 30 min at 37°C and centrifuged at 10 000 g for 15 min at 4°C. Supernatants were used for ELISA or stored at -70°C until use. Preparation of MSE antigens MSE of M. tuberculosis H^Ra (MSEMT) was prepared according to the method of Bennedsen [21] as previously described [18]. Briefly, the cells were suspended in 0.15 M NaCI and incubated at 37°C for 72 h with gentle shaking. After centrifugation at 10 000 g for 30 min, the supernatant was treated with 80% saturated ammonium sulphate to precipitate proteins. The precipitates were dissolved in 0.05 M PBS, pH 7.2 and dialysed against the same buffer overnight at 4°C. After dialysis, the solutions were filtered through 0.45 |im membrane and stored at —70°C until use. Production of anti-MSEMT antibodies Antibodies against MSEMT were raised in rabbits. Young adult New Zealand white rabbits were immunized with multiple intradermal injections of 200 ug
Submitted 3 June; revised version accepted 27 August 1991. Correspondence to Chii-Huei Wu, Department of Medical Research, Taichung Veterans' General Hospital, Taichung, Taiwan 40705, Republic of China.
© 1992 British Society for Rheumatology
0263-7103/92/090615 + 04 $08.00/0 615
Downloaded from http://rheumatology.oxfordjournals.org/ at Simon Fraser University on June 8, 2015
SUMMARY By use of antimycobacterial saline extract antibodies, Mycobacterium tuberculosis (MT) antigens have been detected in synovialfluid(SF) of patients with rheumatoid arthritis (RA) by a highly specific and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Absorbance for 15 gout, 14 osteoarthritis (OA) patients, 12 patients with spondylarthropathies (SA) and eight patients with other inflammatory disorders (OID) ranged from 0.001 to 0.025 with the mean values of 0.0086 ± 0.0078,0.0077 ± 0.0051,0.0069 ± 0.0059 and 0.0113 ± 0.0059, respectively. Among 65 SF of patients with RA examined, 34 were found to be negative for MT antigen with absorbance ranging from 0.002 to 0.024 and a mean value of 0.0114 ± 0.0070. For 31 (47.7%) MT antigen-positive specimens of RA, optical density ranged from 0.052 to 2.446 with a mean value of 0.5564 ± 0.7354. Significant statistical difference (P
DETECTION OF MYCOBACTERIUM TUBERCULOSIS ANTIGEN IN SYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS BY J. L. LAN AND C. H. WU* Allergy, Immunology and Rheumatology Division, Department of Internal Medicine and * Department of Medical Research, Taichung Veterans' General Hospital, Taichung, Taiwan, Republic of China
KEY WORDS:
MT antigen and RA, Synovial fluid, Antimycobacterial saline extract antibody, ELISA.
RHEUMATOID arthritis is recognized as an immunological, possibly autoimmune, disease [1]. Although some microorganisms have been implicated [2-6] in RA, no inciting agents or target antigens have been unequivocally identified. Mycobacteria are able to induce not only immunopathological granuloma as seen in leprosy and tuberculosis, but also arthritis in rats [7]. Adjuvant arthritis (AA), triggered by MT in Freund's complete adjuvant after intracutaenous immunization, has remarkable similarities to the arthritic process seen in human RA. The disease features a proliferative and inflammatory reaction in synovial membranes, progressive pannus formation, destruction of cartilage, erosion of bone, and ultimately ankylosis [8]. Mounting evidence [9-17], has implied that MT may be the autoantigen in RA. Since many antigenic determinants can be found within a microorganism, and some may mimic host macromolecules by chance, the verification of MT antigen in synovium or synovial fluid is important for the pathogenesis of RA. Recently, a highly specific and sensitive double-antibody sandwich ELISA, using antimycobacterial saline extract (anti-MSE) antibody able to detect MT antigen in cerebrospinal fluid and sputum of patients with meningitis or pulmonary tuberculosis, was reported by Wu et al. [18,19]. The ELISA was able to detect as little as 6.0 ng of antigen, and maximum cross-reactivity with non-tuberculous mycobacterial antigens was less than 2%. The present study describes the detection of MT antigen in synovial fluid of RA patients by the anti-MSE antibody based ELISA.
METHODS Patients and specimens This study was carried out on 65 patients (26 females and 39 males) with RA, as defined by the 1987 revised American Rheumatism Association criteria [20], attending the immunology and rheumatology clinic at Taichung Veterans' General Hospital, Taichung, Taiwan. Fifteen patients (two females and 13 males) with gout, 14 patients (five females and nine males) with OA, 12 patients (one female and 11 males) with SA and eight patients (two females and six males) with OID (two psoriatic arthritis, two septic arthritis, two pseudogout and two pigmented villonodular synovitis) were studied as control groups. SF specimens were treated with hyaluronidase (HU, 100 (ig/ml) for 30 min at 37°C and centrifuged at 10 000 g for 15 min at 4°C. Supernatants were used for ELISA or stored at -70°C until use. Preparation of MSE antigens MSE of M. tuberculosis H^Ra (MSEMT) was prepared according to the method of Bennedsen [21] as previously described [18]. Briefly, the cells were suspended in 0.15 M NaCI and incubated at 37°C for 72 h with gentle shaking. After centrifugation at 10 000 g for 30 min, the supernatant was treated with 80% saturated ammonium sulphate to precipitate proteins. The precipitates were dissolved in 0.05 M PBS, pH 7.2 and dialysed against the same buffer overnight at 4°C. After dialysis, the solutions were filtered through 0.45 |im membrane and stored at —70°C until use. Production of anti-MSEMT antibodies Antibodies against MSEMT were raised in rabbits. Young adult New Zealand white rabbits were immunized with multiple intradermal injections of 200 ug
Submitted 3 June; revised version accepted 27 August 1991. Correspondence to Chii-Huei Wu, Department of Medical Research, Taichung Veterans' General Hospital, Taichung, Taiwan 40705, Republic of China.
© 1992 British Society for Rheumatology
0263-7103/92/090615 + 04 $08.00/0 615
Downloaded from http://rheumatology.oxfordjournals.org/ at Simon Fraser University on June 8, 2015
SUMMARY By use of antimycobacterial saline extract antibodies, Mycobacterium tuberculosis (MT) antigens have been detected in synovialfluid(SF) of patients with rheumatoid arthritis (RA) by a highly specific and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Absorbance for 15 gout, 14 osteoarthritis (OA) patients, 12 patients with spondylarthropathies (SA) and eight patients with other inflammatory disorders (OID) ranged from 0.001 to 0.025 with the mean values of 0.0086 ± 0.0078,0.0077 ± 0.0051,0.0069 ± 0.0059 and 0.0113 ± 0.0059, respectively. Among 65 SF of patients with RA examined, 34 were found to be negative for MT antigen with absorbance ranging from 0.002 to 0.024 and a mean value of 0.0114 ± 0.0070. For 31 (47.7%) MT antigen-positive specimens of RA, optical density ranged from 0.052 to 2.446 with a mean value of 0.5564 ± 0.7354. Significant statistical difference (P
