Fundamental studies on the measurement of antibody against C-polysaccharide extracted from cell walls of group A Streptococcus by the enzyme-linked immunosorbent assay (ELISA)

Abstract

Although there are several tools for serological diagnosis of group A streptococcal infection or its sequelae, the serological reactions are related to the extracellular products of group A streptococcus. Therefore, it is considered worthy to investigate the method of determination of antibodies in human sera to the group-specific polysaccharide (C-polysaccharide, C-poly.) which is one of the somatic antigen of group A streptococcus. Recently, passive hemagglutination test has been utilized on the measurement of anti-C-poly antibody.The present paper describes the fundamental studies on the measurement of the antibody against Cpoly in the immunized rabbit or human sera using the enzyme-liked immunosorbent assay (ELISA) technique.The purified C-poly antigen was extracted from cell walls of group A streptococcus. The C-poly was coupled with poly-L-lysine (PLL) using cyanuric chloride as coupling agent. The C-poly-PLL antigen was coated to microplate wells.The results obtained were as follows:1. When 0.7μg/well of C-poly-PLL antigen reacted with 100μl of 1: 1, 000 dilution fluid of human sera, the highest titer was shown as O. D. value (410nm) on ELISA test.2. Absorption test with whole cells of group A, B, C, D, and G streptococcus and Str. pneumoniae, purified group A C-poly and haptenic sugar (GlcNAc) indicated that the antibody specific to Nacetylglucosamine residues of C-poly is detected on ELISA test.3. The inactivation (at 56°C for 30min) of serum did not affect on the measurement of antibody by ELISA test.4. There was relatively correlation between anti-C-poly antibody levels of IgM class which were measured by ELISA and anti-C-poly antibody levels which were detected by passive hemagglutination test (r=0.62), but no correlation between the antibody levels of IgG class by ELISA and the antibody levels by passive hemagglutination test.5. When the antibody titers (IgG class) against the C-poly-PLL in the sera of patients with acute post-streptococcal glomerulonephritis and rheumatic fever were compared with those in the sera of healthy individuals using ELISA test, the titers of the former sera were significantly higher than those of the latter sera (p<0.01). There were no differences, however, in the antibody titer of IgM class to the Cpoly-PLL between sera of patients and healthy individuals.The measurement of the anti-C-polysaccharide antibody by ELISA technique is considered to be a useful tool as one of the serological diagnosis of streptococcal sequelae.