Preparation of protein A-peroxidase monoconjugate using a heterobifunctional reagent, and its use in enzyme immunoassays

Abstract

Protein A-peroxidase monoconjugate was prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate. The yield of the monoconjugate was much higher than that obtained with current methods. An immunoassay method was developed in which protein A-peroxidase monoconjugate served as a universal tool. Protein A-peroxidase monoconjugate was taken up by IgG molecules immobilized on an excess of solid phase antigen. The ability of free antigen to inhibit the binding of antibody, measured as inhibition of conjugate up take, served as the basis for quantification in the assay. The method was applied to human chorionic gonadotropin (HCG) and human IgG. Optimal assay conditions were developed and it was found that as little as 1 ng of HCG/ml and 2 ng of IgG/ml could be detected. The method is of comparable sensitivity to other available immunoassay methods and gives accurate, reproducible results.