Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen.

The heterobifunctional reagent 3-(2-pyridyl-dithio)propionate (SPDP) was used to prepare defined conjugates composed of horseradish peroxidase (HRP) and goat anti-HBs IgG. The modification of HRP and IgG with SPDP was dependent on both the SPDP: protein molar ratio and the pH of the buffer. Conjugates were separated by a single affinity chromatographic step using concanavalin A-Sepharose 4B equilibrated with 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl and 1 mM EDTA. The conjugate was eluted with 10 or 100 mM alpha-methyl-D-mannoside and appropriate pools were made reflecting various HRP/IgG molar ratios. Each pool was examined for performance in an enzyme-linked immunosorbent assay for HBsAg. Conjugate composed of an HRP/IgG molar ratio of 2.5-4 yielded the greatest sensitivity.