Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics.
In attempting to use the BIAcore instrument for the determination of binding constants of several haptens or peptides to different antibodies by measuring on- and off-rates, we found that neither the absolute nor the relative values of the binding constants corresponded to the measurements in solution. Even at the lowest coupling densities useful for measurements, rebinding and bivalency effects offset the measurements by a factor of up to 500. We caution therefore about using on- and off-rates for the determination of absolute or even relative binding constants without controlling for rebinding and avidity effects. Instead, we show that binding constants in solution can be reproduced well by using on-rate determinations of antibody preincubated with antigen, and we derive the conditions under which such an approach is valid.
Nieba
L
L
Biochemisches Institut der Universität Zürich, Switzerland.
Krebber
A
A
Plückthun
A
A
D000915:Antibody Affinity; D001667:Binding, Competitive; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006241:Haptens / Q000276:immunology*; D007700:Kinetics; D008433:Mathematics; D015394:Molecular Structure; D010455:Peptides / Q000276:immunology*; D013816:Thermodynamics
D006241:Haptens; D010455:Peptides