An avidin-biotin based ELISA for quantitation of antibody to bacterial polysaccharides

Abstract A solid phase immunoassay utilizing avidin-biotin binding has been developed for measuring anticapsular polysaccharide antibodies. Capsular polysaccharides of Escherichia coli K1, Haemophilus influenzae type b, Staphylococcus aureus types 5 and 8, and levan from Aerobacter levanicum have been biotinylated through -OH or COOH groups with retention of antigenicity. Polysaccharides were immobilized on avidin-coated microtiter wells for use in an enzyme-linked immunosorbent assay (ELISA) to detect antibody. Two preparations of biotinylated S. aureus type 8 polysaccharide were equivalent as antigens in ELISA. Specificity was demonstrated by absorption of antisera, by competitive inhibition with purified antigens, and by reaction with specific monoclonal or myeloma antibodies. Reproducibility of the assay for H. influenzae type b and S. aureus type 8 antibody was demonstrated by replicate titrations of high and low level antisera.