NONSPECIFIC AMINE IMMOBILIZATION OF LIGAND CAN BE A POTENTIAL SOURCE OF ERROR IN BIACORE BINDING EXPERIMENTS AND MAY REDUCE BINDING AFFINITIES

The interaction of monovalent forms of NC41, an anti-viral neuraminida se antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problem s resulting from the nonspecific amine coupling procedure. To avoid co mplications due to antibody bivalency, monovalent Fab fragments and mo nomeric recombinant scFvs were used. When immobilized by amine couplin g, the 11-1G10 anti-idiotype fragments were found to have an artificia lly reduced affinity for NC41 compared to the results obtained using s ite-directed immobilization via C-terminal thiol residue and from solu tion equilibrium measurements. The NC41 antibody fragments, on the oth er hand, were able to retain their 11-1G10 binding affinity when immob ilized nonspecifically through free amine groups. These data, in combi nation with the known sequences of the two antibodies, suggested that nonspecific immobilization through one or more lysine residues close t o or within the CDR2 region of the 11-1G10 V-H domain was responsible for the reduced strength of the interaction with NC41. These results e mphasize the need to use site-specific immobilization strategies when accurate kinetic measurements are required. (C) 1997 Academic Press.