Measuring antibody affinities as well as the active concentration of antigens present on a cell surface

Measuring the affinity of a therapeutic antibody to its antigen, expressed in its native form on a cell surface, is an important aspect to understanding its in vivo potency. Measured affinities can also help in selecting the best antibody for therapy. The on-cell binding affinity of antibodies was determined in the past by labelling the antibody using radioactive, fluorescent, or other probes. Labelling the antibody could potentially modify the structure of the antibody and hence could alter its original affinity. Here, we describe a label-free method to measure the affinity of antibodies to their target antigens that are expressed on the surface of cells and the number of active antigen molecules present on a given cell. In addition, this method can also be used to measure the affinity of a ligand to its receptor on a cell.