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 neuraminidase antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problems resulting from the nonspecific amine coupling procedure. To avoid complications due to antibody bivalency, monovalent Fab fragments and monomeric recombinant scFvs were used. When immobilized by amine coupling, the 11-1G10 anti-idiotype fragments were found to have an artificially reduced affinity for NC41 compared to the results obtained using site-directed immobilization via C-terminal thiol residue and from solution equilibrium measurements. The NC41 antibody fragments, on the other hand, were able to retain their 11-1G10 binding affinity when immobilized nonspecifically through free amine groups. These data, in combination with the known sequences of the two antibodies, suggested that nonspecific immobilization through one or more lysine residues close to or within the CDR2 region of the 11-1G10 VH domain was responsible for the reduced strength of the interaction with NC41. These results emphasize the need to use site-specific immobilization strategies when accurate kinetic measurements are required.