Experimental design for analysis of complex kinetics using surface plasmon resonance.
Using BIAcore surface plasmon resonance technology, we found that the real-time association kinetics of Fabs specific for hen egg-white lysozyme did not conform to a 1:1 Langmuir association model. Heterogeneity of the components is not the source of the complex kinetics. Informed by independent structural data suggesting conformational flexibility differences among these antibodies, we chose global mathematical analysis based on a two-phase model, consistent with the encounter-docking view of protein-protein associations. Experimental association times (T(a)) from 2 to 250 min revealed that initial dissociation rates decreased with increasing T(a), confirming a multiphasic association. The relationship between observed dissociation rate and T(a) is characteristic of each antibody-antigen complex. We define a new parameter, T(50), the time at which the encounter and final complexes are of equimolar concentration. The observed T(50) is a function of analyte concentration 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 complexes. For high-affinity complexes with rapid rearrangement to a stable complex, T(50)(MIN) approaches T(1/2) of the rearrangement forward rate constant. We conclude that experiments with a range of T(a) are essential to assess the nature of the kinetics, regardless of whether a two-state or 1:1 model is applicable. We suggest this strategy because each T(a) potentially reveals a different distribution of molecular states; for two-step analysis, a range of T(a) that brackets T(50) is optimal.
Lipschultz
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Basic Research Laboratory, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Li
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Smith-Gill
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D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D006801:Humans; D007700:Kinetics; D020349:Surface Plasmon Resonance
D000936:Antigen-Antibody Complex