Molecular characterization of monovalent and multivalent hapten–protein conjugates for analysis of the antigen–antibody interaction

We prepared a hapten-protein conjugate using (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten and hen egg lysozyme (HEL) or bovine serum albumin (BSA) and defined hapten modification sites on the former protein based on results of reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometric analyses performed after enzymatic digestion. The most reactive residue for aminoacetylation in HEL was found to be Lys33, and the second was Lys96 or Lys97. The homogeneous NP-HEL conjugates were purified by HPLC and used for examining the effect of hapten valence on the antigen-antibody interaction. We also examined the molecular nature of NP conjugates of BSA. Analysis using mass spectroscopy showed that the mass distribution of NP-BSA conjugates was limited, although it became broader with an increase in NP valence. Surface plasmon resonance biosensor measurements were employed in measuring antigen-antibody interactions. The results showed that the apparent binding avidity depends on hapten valence, hapten density, size of carrier proteins, and intrinsic binding affinity of the antibody.