Optimization of hapten-protein conjugation by high-performance capillary electrophoresis

HPCE has been found to be efficient for the optimization of conjugation procedures employed for coupling of the haptens p-nitrophenyl-alpha-D-galactopyranoside (PNPG) and soyasaponin I to a carrier protein, Kunitz soybean trypsin inhibitor (KSTI). The carbohydrate moieties of the haptens were oxidized with periodate followed by reaction with epsilon-amino groups of the carrier. For PNPG, the periodate oxidations (0.01-0.2 M NaIO4) were followed by HPCE and 0.1 M periodate was chosen as the optimum concentration. The coupling of PNPG to KSTI was found to proceed at a constant rate for more than 250 min. The reaction rate for the soyasaponin conjugation to KSTI declined after 80 min of incubation. The coupling of soyasaponin to KSTI was confirmed by enzyme-linked immunosorbent assay with monoclonal antibodies directed against soyasaponin I. The method was found to be particularly powerful for the investigation of conjugates with low epitope densities that are difficult to determine otherwise.