Both kinetic data and epitope mapping provide clues for understanding the anti-coagulant effect of five murine monoclonal antibodies to human beta(2)-glycoprotein I

The interaction between live murine monoclonal antibodies (mAb) and beta(2) -glycoprotein I (beta(2)GPI) in the absence of phospholipids was studied us ing surface plasmon resonance-based biosensor technology. Two separate epit ope regions were confirmed for the five mAb but epitopes of two mAb were sh own to be overlapping but not identical. The characteristics of binding on both immobilized beta(2)GPI, using different chemistries of coupling to a d extran matrix and antibody surfaces prepared by two strategies of immobiliz ation, were compared. Binding was strongly influenced by the orientation of the immobilized partner, and the five mAb showed heterogeneity in their bi nding to immobilized and soluble beta(2)GPI. The observed stoichiometries o f mAb-beta(2)GPI complexes and the detailed analysis of the kinetics of the association and dissociation phases of the interactions with soluble and i mmobilized beta(2)GPI revealed differences in the dissociation rate constan ts, resulting in a 10-fold higher affinity for immobilized beta(2)GPT compa red to soluble beta(2)GPI for four out of five mAb. This suggests bivalent binding of these mAb to immobilized beta(2)GPI. In addition, the kinetic da ta helped explain the differing anti-coagulant properties of these mAb.