Methods of screening antigen-binding molecules by normalizing for the concentration of antigen-binding molecule

A method of screening a candidate antigen-binding molecule for affinity to an antigen, comprising:

• a. contacting (1) a control compound at a plurality of concentrations, and (2) a candidate antigen-binding molecule with a substrate, the substrate comprising (i) an antigen immobilized on the substrate, and (ii) a normalization binding molecule immobilized on the substrate;
• b. detecting a binding of the candidate antigen-binding molecule to the antigen;
• c. detecting a binding of the candidate antigen-binding molecule to the normalization binding molecule;
• d. detecting a binding of the control compound at the plurality of concentrations to the normalization binding molecule, wherein the detecting produces a plurality of detection signals;
• e. generating a standard calibration curve utilizing the plurality of detection signals;
• f. determining a first and a second concentration for each candidate antigen-binding molecule, wherein the first concentration is an apparent concentration determined from the binding of the antigen-binding molecule to the antigen of (b), and the second concentration is an actual concentration determined from the binding of the antigen-binding molecule to the normalization binding molecule of (c), wherein the actual concentration of the candidate antigen-binding molecule binding is determined using the calibration curve;
• and
• g. calculating a ratio of the first and second concentrations for candidate antigen-binding molecule, wherein the ratio is a relative affinity of the antigen-binding molecule to the antigen.