Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).