Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies.

摘要: The interaction between the humanised A33 monoclonal antibody and corresponding F(ab)'2 or Fab' fragments with colonic epithelial antigen, purified by micropreparative HPLC from membrane extracts of carcinoma cell line LIM 1215, has been studied BIAcore 2000 biosensor using surface plasmon resonance detection. orientation immobilised fragment onto was controlled alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. increase signal a concomitant sensitivity detection, which facilitates analysis low levels antigen. apparent association rate (ka) dissociation (kd) constants obtained different chemistries were determined. These analyses showed that kinetic for IgG not affected method immobilisation. NHS/EDC chemistry lower affinity. By contrast use thiol coupling gave five fold observed KA, resulting similar affinity to intact molecule.