Optimization of the covalent conjugating procedure (NaIO4) of horseradish peroxidase to antibodies for use in enzyme-linked immunosorbent assay.

The procedure for covalent conjugation of horseradish peroxidase (POD) to goat anti-human IgG (GAHG) molecules was systematically optimized in terms of reactant molar ratio, time of reaction, pH, and temperature. The optimum conjugation procedure was defined by the conditions that produced an enzyme-labeled Ab with the highest specific activity in immunosorbent assays for normal human IgG (NHIgG). The best conditions are: Sodium meta-periodate (NaIO4) to Ab molar ratio during oxidation is 40:1; time of oxidation is 5 min at 37 degrees C; oxidation reaction is conducted at pH 5.0; the molar ratio of POD:GAHG is 6:1; the conjugation time is 24 h at 4 degrees C; and the optimal conjugation pH is 10.0. A conjugate constructed under these conditions is capable of generating 1.8 and 12.6 times more specific signals (delta A650nm/min) than the best and worst commercial conjugates, respectively. This conjugate is also able to detect NHIgG at a concentration of 2.25 x 10(-13) M, a sensitivity 25 times that achieved by most comparable commercial products in identical assays.