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1 Key words: syphilis, anti- Treponema pallidum-igm, purified antigen, serodiagnosis, TPHA
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4 Table 1 Influence of purification of Tp antigen by Hypaque and MgC12 on TPHA titer and on M/G ratio in primary syphilitic serum Fig. 1 Relationship of and-tp-igm SPHA titers with crude and purified Tp antigens Table 2 Sensitivity and specificity of fractionated TPHA, SPHA and captured Tp-IgM-RIA N.D., not determined; RF, rheumatoid factor.
5 Table 4 Amounts of rabbit immunoglobulins associated with surface of crude and purified Tp preparations Table 3 Inhibition test by purified Tp in SPHA * 1; serum from patients with untreated primary syphilis * 2; less than 1: 2 Rabbit immunoglobulins were detected by immunofluorescence test using goat anti-rabbit IgG or IgM labeled with FITC.
6 Fig. 2 Production of specific IgM and IgG in rabbits inoculated with Tp
7 1) Herbest, B.-R., Goerz, G. & Muller, F.: Diagnostischer and therapeutischer Ausagewert des IgM-FTA-ABS and IgM-FTA-19S- Tests bei der Syphilis. Aktuel. Dermatol., 5: , ) Muller, F. & Oelerich, S.: Korrelation im- munologischer Parameter zu den Stadien der apparenten and klinisch stumm en Syphilis. Dermatol. Monatsschr., 165: , 1979.
8 13) Miller, J. N., Bruijn, J. H., Bekker, J. H. & Onvlee, P. C.: The antigenic structure of Tre- 3)Miiller, F. & Oelerich, S.: Serological patterns and their correlation with clinically stration, nature and location of specific and ponema pallidum, Nichols strain. 1. The demon- apparent or latent syphilis. WHO/VDT/RES, shared antigens. J. Immunol., 96: , 79: 361, ) Weinbach, R.: Die Verwendbarkeit formol behandelter Etythrozyten als Antigen trager in der indirekten Haemagglutination. II. Schweiz. Z. Allgem. Pathol., 22: 1-11, ) Wide, L.: An immunological method for the assay of human chorionic gonadotropin. Acta Endocrinol. Suppl., 70: 1-111, ) Lowry, O. H., Rosebrough, N. J., Farr, A. L. & 7) Dobson, R. M.S., Taber, H. L. & Baughn, E. R.: Recognition of Treponema pallidum antigens by IgM and IgG antibodies in congenitally infected new borns and their mothers. J. Infect. Dis., 157: , ) Pedersen, S. N., Sheller, P. J., Ratnam, V. A. & Hira, K. S.: Enzyme-linked immunosorbent assays for detection of immunoglobulin M to nontreponemal and treponemal antigens for the diagnosis of congenital syphilis. J. Clin. Microbiol., 27: , ) Lewis, L. L., Taber, H. L. & Baughn, E. R.: Evaluation of immunoglobulin M western blot analysis in the diagnosis of congenital syphilis. J. Clin. Microbiol., 28: , ) Tanaka, S. & Suzuki, T.: Anti-Treponema pallidum IgM, IgA, and IgG subclass antibody responses after treatment in patients with syphilis at various stages. 1. Assessments by enzyme-linked immunosorbent assay. Genitourin Med., 66: , ) Sato, T., Kubo, E., Yokota, M., Kayashima, T. & Tomizawa, T.: Treponema pallidum specific IgM haemagglutination test for serodiagnosis of syphilis. Br. J. Vener. Dis., 60: 364 Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: ) Schmidt, B. L.: Solid-phase Hemadsorption: A method for rapid detection of Treponema pallidum-specific IgM. Sex Transm. Dis., 7: 53 19) Fieldseel, A. H., Cox, D. L. & Moeckli, R. A.: Cultivation of virulent Treponema pallidum in tissue culture. Infect. Immun., 32: , ) Alderete, J. & Baseman, J.: Surfaceassociated host proteins on virulent Treponema pallidum. Infect. Immun., 26: , ) Okamoto, S. & Tanabe, Y.: Studies on Treponema pallidum haemagglutination antibodies. I. TPHA antibodies in experimental syphilitic rabbits. Br. J. Vener. Dis., 47: 77-80, 1971
9 Development of Anti- Treponema pallidum-igm Antibodies Detection Methods Using Purified Antigen \Comparison with Conventional Methods \ Takashi SATO Department of Microbiology, Yamaguchi University School of Medicine Treponema pallidum subsp. pallidum Nichols (Tp) antigens were purified by centrifugation in the presence of Hypaque (sodium diatrizoate) or MgC12. When the TPHA (Treponema pallidum hemagglutination) tests with crude and purified antigens were carried out in the sera of patients with untreated primary syphilis, the purification by MgCl2 resulted in enhanced sensitivity to Tp-specific IgM as the same as the purification by Hypaque. In addition, the SPHA (solid phase hemadsorption) test with the Hypaque-purified antigen to and-tp-igm antibodies was carried out using the sera of patients with primary and secondary syphilis. Fifty-nine percent (27/46) of them showed positive for the crude antigen and 100% (46/46) for the purified antigen, indicating that sensitivity to and Tp-IgM was clearly enhanced with the antigen purification. The captured Tp-IgM-RIA test with the purified antigen was also undertaken in 16 patients with primary syphilis, 31 patients with secondary syphilis and 73 patients with latent syphilis. All the patients in the former two groups were positive, while all of the patients in the latter group were negative. To elucidate the mechanism whereby the purification improves the sensitivity to and-tp-igm antibodies, host-derived antibodies associated with crude and purified Tp cells were measured by a fluorescent antibody technique. As the results, a great amount of rabbit anti-tp-igm antibodies were found on the surface of the crude antigen, whereas only a trace amount was detected on the purified antigens. On the basis of these findings, it was considered that the purified antigen exhibits a strong reactivity to human anti-tp-igm antibodies due to the removal of rabbit anti-tp-igm antibodies from the surface by the purification procedure.
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