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1 Key words: ELISA method, streptococcal antibody, Anti-M protein antibody, whole cell antigen
2
3 Table 2 Uptake of protein A using different anti-group sera of streptococci Figure shows optical density at a wavelength of 410nm. (net value) Fig. 1 Absorption test of sera for grouping with the bacteria of corresponding groups Table 1 Procedure for quantification of serum antibodies to surface antigen of group A streptococcus by ELISA on microtiter plate 0.05m1 of 2.5mg of standard group A streptococcus suspended in lml of HSA-PBS m1 of test serum incubation at 37 Ž for lhour washing with Tw-PBS for 3times resuspension in 0.025ml of HSA-PBS +0.1m1 of protein A-Peroxidase(1:3000) incubation at room temp. for 10min. with shaking washing with Tw-PBS for 3times +0.1m1 of ABTS(1:100) incubation at 37 Žfor 30min. measurement of absorbance(od 410) by MINIR- EADER II(DYNATECH PRODUCT)
4 Fig. 2 Uptake of protein A in relation to serum and bacterial dose Table 4 Uptake of protein A in the relation to whole-cell suspensions and anti-m serum absorbed with group A polysaccharide (Bacterial dose) Original dilution 5 mg/ml(net weight) Figure shows optical density at a wevelength of 410nm. (Bacterial dose) serum dilution 1:10n Table 3 Uptake of protein A in relation to whole-cell suspensions of group A streptococci and crude anti-m sera Figure shows optical density at a wevelength of 410nm. (net value)
5
6 Fig. 3 Uptake of protein A of whole cells and trypsinized cells with anti-m serum absorbed with group A polysaccharide Fig. 4 Distribution of absorbance in sera from patients with and without valvular disease (serum dilution 1: 10) Table 5 Titration of so-called"m factor sera" by corresponding type of strain and group A strain Figure shows optical density at a wavelength of 410nm. (net value) S: statistically significant NS: statisticlly not significant
7 1) Lancefield, R. C.: Differenciation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J. Exp. Med., 106: 525 4) Rotta, J., Krause, R. M., Lancefield, R. C., Every, W. & Lackland, H.: New approaches for the laboratory recognition of M types of group A streptococci. J. Exp. Med., 134: , ) Christensen, K. K., Christensen, P., Dahiander, K., Faxelius, G., Jacobson, B. & Svenningsen, N.: Quantitation of serum antibodies of surface antigens of group B streptococci type Ia, Ib and III: Low antibody levels in mothers of neonatally infected infants. Scand. J. Infect. Dis., 12: , ) Dillon, H. C. & Reeves, M. S. A.: Streptococcal immune responses in nephritis after skin infection. Amer. J. Med., 56: , ) Kaplan, E.L., Anthony, G.F., Chapman, S.S., Ayoub, E. M. & Wannamaker, L. W.: The influence of site of infection on the immune response to group A streptococci. J. Clin. Invest., 49: , 1970.
8 (Kimura, Y., kotani, S. & Shiokawa, Y. ed.), 38 15) Lancefield, R. C.: The antigenic complex of streptococcus haemolyticus I. Demonstration of a type-specific substance in extracts of strep- tococcus haemolyticus. J. Exp. Med., 47: 91 16) Lancefield, R. C.: Persistance of type-specific antibodies in man following infection with group A streptococci. J. Exp. Med., 110: ) Stollerman, G. H. & Ekstedt, R.: Long chain formation by strain of group A streptococci in 17) Kuttner, A. G. & Krumwiede, E.: Observation the presence of homologous antiserum: A on the effect of streptococcal upper respiratory type-specific reaction. J. Exp. Med., 106: 345 infections on rheumatic children. A three-year study. J. Clin. Invest., 20: , ) Russel, H., Facklam, R. R. & Edwards, L. R.: 18) Stollerman, G. H. & Bisno, A. L.: The relative Enzyme-Linked immunosorbent assay for streptococcal M protein antibodies. J. Clin. Miptococci. IN Proceedings of International Con- rheumatogenecity of strains of group A strecrob., 3: , ference on Rheumatic fever and Rheumatic heart disease(shiokawa, Y. and Kawakita, S. ed.), 57-63, Kawakita Printing Co. Ltd., Kyoto, ) Takizawa, K., Asai, Y., Yasuda, T. & Miyamoto, Y.: Changes and changing patterens tical observation on rheumatic fever in 19) Okuda, R., Mori, C. & Nishioka, K.: Statis- Japan of T-types of group A streptococci and their drug resistance in Japan: In Recent advances in streptococci and streptococcal disease from 1963 to nd report. Ann. Paed. Japonica, 23: , Experimental Study of Quantification of Serum Antibodies Against Streptococcal Surface Antigens by ELISA on Microtiter Plate Wakahiro NOGAMI Department of Public Health, Toho University School of Medicine, Tokyo, Japan An experimental study for quantification of serum antibodies against streptococcal surface antigens was carried out, following the ELISA method, using whole bacterial cells as antigens. Results were summarized as follows: 1) When using the streptococcus group A, B, C and G strains, specific absorbance at OD 410 nm was shown against each group of specific serum, suggesting the specificity of each reaction. 2) By using the reference strains of group A streptococcus types Ml, M4, M6 and M12, the absorbances we obtained in the immune sera reflected the antibodies against M antigens as well as the C-polysaccharide and T antibodies. 3) After the absorption with C-polysaccharide of group A streptococcus and with trypsinized antigen of the corresponding type of antigen for ELISA, only the specific M antibody remained in the human serum we tested. 4) Relatively high quantities of IgG antibodies were present in the sera of patients with valvular heart disease. These antibodies were for the types of streptococci which have been prevalent in Japan during the past 20 years. Compared with the complicated methods of titration of M antibodies pulished, our method is easy to carry out for the examination of sera. In this study the results clearly reflected the presence of M antibodies for group A streptococcus.
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