A technique for preparing defined conjugates of horseradish peroxidase and immunoglobulin

Conjugates of horseradish peroxidase (HRP) and sheep anti-human immunoglobulin (anti-Ig) were prepared by means of a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The molar ratios of the conjugates could be changed by varying the degree of chemical substitution of anti-Ig and HRP. Gel filtration and affinity chromatography were used to separate conjugated anti-Ig from free HRP and unconjugated anti-Ig. The distribution of complex sizes and the presence of free HRP and anti-Ig in the final products were monitored by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. After final purification and characterisation conjugates were studied as reagents in enzyme immunoassays (EIA) with various antigens. Conjugates prepared by SPDP bridging were shown to yield more than 60% of the anti-Ig as conjugated small defined enzyme-protein complexes. A suitably labelled conjugate reacted in a standardised way resulting in a reproducible dose-response curve when a positive serum was assayed in different dilutions.