Improved presentation of antigenic sites in enzyme-linked immunosorbent assay: antigenicity of reduced and carboxymethylated riboflavin carrier protein

Abstract

Disulphide reduced and carboxymethylated riboflavin carrier protein (RCM-RCP), an unfolded derivative of chicken RCP, does not bind riboflavin and there is a drastic reduction in its ability to interact with antiserum to cRCP. Antibodies to RCM-RCP are directed against sequential epitopes(s). On radioiodination of RCM-RCP, a maximum of 30-50% binding at dilution of 1:500 was obtained with rabbit antibodies RCM-RCP [n = 5]. However, high titer antibodies was obtained when radioiodinated RCM-RCP was immobilized on ELISA microtiter plates suggesting that immobilization of RCM-RCP leads to either preservation of antigenic sites or improved presentation of the antigenic determinants. An avidin-biotin system was utilized to develop an ELISA.