Surface plasmon resonance (SPR) is the gold standard for determining rate and equilibrium constants of bimolecular complexes. Accuracy of these parameters depends on the correct determination of the concentration of the injected analyte. Calibration free concentration analysis (CFCA) has been developed to overcome the limitation of measuring protein concentrations spectroscopically, which may overestimate the fraction of the protein that really binds to the immobilized ligand, i.e. the active concentration. In this work, we demonstrate that CFCA can also be implemented in a capture format for measuring active concentrations. Capture CFCA (CCFCA) was first validated by measuring the concentration of a HLA-B*44:02 antigen solution. The active concentration of this molecule determined by CCFCA was similar to that obtained by covalent CFCA. CCFCA was then used to determine the concentration of the W6/32 pan class I HLA monoclonal antibody over three different HLA molecules captured by another specific antibody. This could not have been performed by covalent CFCA because immobilized HLA molecules cannot withstand regeneration. By exploring different capture levels we also show that CCFCA gives consistent results even at low capture levels. Knowing the active concentration of W6/32, we then determined the rate and equilibrium constants of W6/32-HLA complexes on the same flow cell. CCFCA is of general use for measuring active concentrations and of great interest for analytes recognizing ligands that cannot be covalently immobilized on sensor chips. The capture mode also allows determining the kinetic constants of multiple analyte-ligand complexes on the same flow cell. This increases experiments throughput and reduces sensor chip consumption.
Keywords: Affinity; Calibration free concentration analysis; Capture; HLA antibodies; Single cycle kinetics; Surface plasmon resonance.
Copyright © 2015. Published by Elsevier B.V.