tained as the result of either continual replenishment Macromolecular interactions observed using surby flowing ligate solution across the sensor surface or face plasmon resonance technology (BIAcore, Pharthrough the use of a sufficiently high ligate concentramacia) often display kinetic behavior which deviates tion for binding to be essentially without effect on its from the pseudo-first-order time dependence that has magnitude in the liquid phase. Such use of biosensor been predicted for 1:1 interactions of ligand and ligate. technology to characterize the kinetics of interaction In the present study we review the major reasons for between a monoclonal antibody and an immobilized such deviations, and present results which suggest form of its eliciting antigen (4, 8, 9) is clearly open that the most common source of deviations from the to criticism on the grounds that results for a bivalent pseudo-first-order kinetic approximation of BIAcore immunoglobulin G are not amenable to interpretation kinetic data is likely to be heterogeneity of the immobiin terms of the pseudo-first-order kinetic expressions lized ligand sites. A simplified analysis of the adsorpused for the analysis (15,16). At this stage, however, tion stage of BIAcore data is presented in terms of the it is becoming increasingly evident that systems connet observed pseudo-first-order rate constant, k obs , forming with the assumed 1:1 stoichiometry frequently rather than in terms of the association and dissociafail to exhibit pseudo-first-order kinetics (12, 13, 17, tion rate constants, k a and k d . The analysis is then ex-18). Whereas such behavior has been taken to signify tended to the determination of the dissociation equithe need for a more intricate, sequential mechanism to librium constant for the interaction of ligand and describe the ligand-ligate interaction (12), we draw ligate in the solution phase from sensorgrams reattention to other explanations for failure of the senflecting competition between soluble and immobilized sorgams to be described adequately by a single expoforms of ligand for ligate.