Experimental design for analysis of complex kinetics using surface plasmonresonance

Using BIAcore surface plasmon resonance technology, we found that the real- time association kinetics of Fabs specific for hen egg-white lysozyme did n ot conform to a 1:1. Langmuir association model. Heterogeneity of the compo nents is not the source of the complex kinetics. Informed by independent st ructural data suggesting conformational flexibility differences among these antibodies, we chose global mathematical analysis based on a two-phase mod el, consistent with the encounter-docking view of protein-protein associati ons. Experimental association times (T-a) from 2 to 250 min revealed that i nitial dissociation rates decreased with increasing T-a, confirming a multi phasic association. The relationship between observed dissociation rate and T-a is characteristic of each antibody-antigen complex. We define a new pa rameter, T-50, the time at which the encounter and final complexes are of e quimolar concentration. The observed T-50 is a function of analyte concentr ation and the encounter and docking rate constants. Simulations showed that when the ligand is saturated at high analyte concentrations, T-50 reaches a minimum value, T-50(MIN), which can be used to compare antigen-antibody c omplexes. For high-affinity complexes with rapid rearrangement to a stable complex, T-50(MIN) approaches T-1/2 of the rearrangement forward rate const ant, We conclude that experiments with a range of T-a are essential to asse ss the nature of the kinetics, regardless of whether a two-state or 1:1 mod el is applicable. We suggest this strategy because each T-a potentially rev eals a different distribution of molecular states; for two-step analysis, a range of T-a that brackets T-50 is optimal.