The impact of different human IgG capture molecules on the kinetics analysis of antibody-antigen interaction.

ABSTRACT

Surface plasmon resonance (SPR) is a well-established method to characterize biomolecular interactions and is widely used in drug discovery and development. Here, we demonstrate that capture surfaces profoundly impact the binding kinetics parameters that are measured for antibody-antigen interactions. Six unique antibody-antigen interactions were characterized using eight different anti-human IgG capture surfaces. The antigen binding affinities for six different human monoclonal antibodies (hmAbs) captured using three different goat anti-human Fc (AHC) polyclonal antibody (pAb) surfaces were in reasonable agreement (3-7-fold weaker) with those measured by kinetic exclusion assay (KinExA). In contrast, up to 81, 32, 489, 2826, and 219-fold weaker antigen binding affinities were measured using mouse AHC mAb, Protein G, Protein A, Protein A/G, and Protein L surfaces, respectively. Protein A, Protein A/G and Protein G interacted with the Fab of hmAbs, possibly affecting antigen binding to hmAbs captured over these surfaces. Additional studies revealed that mouse AHC mAb binds hmAbs with a weak affinity (5.5-36.3 nM) and t½ values of 1.4-3.3min, compared to the sub-nanomolar affinities of the goat AHC pAbs. These results emphasize the value of measuring binding kinetics of the capture molecule before immobilizing them onto the sensor surface to perform capture kinetics assays on label-free biosensors.