An enzyme-linked immunosorbent assay (ELISA) for quantification of antibodies to Streptococcus mutans surface antigens

Abstract

An ELISA was developed to quantitate the level of antibodies to various cell surface antigens of the Gram positive bacterium. Streptococcus mutans. Whole cells and purified cell wall components of S. mutans, lipoteichoic acid (LTA) from Streptococcus pyogenes, and dextran T 2000 were employed as coating antigens in this study. Cell walls of S. mutans were purified by mechanical disruption of whole cells followed by differential centrifugation and proteolytic enzyme treatment. Serotype-specific carbohydrate was purified from an autoclaved, lyophilized S. mutans whole cell preparation by column chromatography. LTA was prepared by Sepharose 4B chromatography of a phenol-water extract of S. pyogenes and used for detection of anti-polyglycerophosphate (PGP) antibodies. A rabbit antiserum to S. mutans 6715 (serotype g), which precipitated with purified carbohydrate antigen (RR g), gave good reactions with purified cell walls and whole cells of S. mutans 6715, less activity with RR g and low activity to LTA and dextran when tested by ELISA. Adsorption of this antiserum with whole cells of S. pyogenes resulted in antibody activity with specificity only to the serotype carbohydrate. The specificity of the antibody for homologous coating antigen was RR g > cell wall > whole cells. An antiserum to S. mutans MT573 (serotype e) contained antibody predominantly to LTA, whereas, anti- S. mutans MT703 (serotype e) reacted with both dextran and LTA; however, the activity to LTA was removed by prior adsorption of the antiserum with S. pyogenes cells. This treatment did not alter the antibody activity to dextran. To establish the sensitivity of ELISA, a purified IgG anti-serotype carbohydrate antibody was prepared by adsorption of anti- S, mutans 6715 antiserum with a mutant of S. mutans which lacks serotype carbohydrate followed by adsorption and elution of specific antibodies from S. mutans 6715 whole cells. The minimum level of sensitivity of ELISA was 12.5 ng of IgG anti-serotype carbohydrate.