Binding Kinetics of Monoclonal Antibody Using Antigen-β-Galactosidase Hybrid Protein: Application to Measurement of Peptide Antigenicity

Abstract

A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the beta-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (KD), and the association (Kass) and dissociation (Kdiss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (KA) of the peptide can be estimated from the KD value of the antigen-beta-galactosidase-antibody interaction, and from the IC50 value of the peptide.

Epitope mapping of preS2 of the hepatitis B virus surface antigen against a conformation‐dependent monoclonal antibody using synthetic peptides

“…The N-terminal sequence 120/123-129 was identified to affect the B cell epitope conformation against H8 mAb. The affinity constants of the long peptides against H8 mAb range from 7.13 x 107 to 9.05 x 107 M "1 according to peptide sizes and HBV subtypes, whereas those of the short peptides with the sequence 130-145 range from 2.48 x 10 ~ to 4.05 x 105 M "1 according to HBV subtypes (12).…”

“…BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL N1, T1 and S 1 having high antigenicity against H8 mAb (11,12) were used as the template peptides for substitution. As shown in Fig.…”