Evaluation of a method for affinity measurement using solution equilibrium titration with magnetic beads

Some of the most well-characterized interactions between biomolecules occur between antibodies and their respective antigens; these interactions are the backbone of the modern pharmaceutical and diagnostic industries. Solution equilibrium titration (SET) is the gold standard method for measuring affinities between biomolecules, where a fixed concentration of analyte is titrated with a variable concentration of titrant. However, SET measurements require innate or external signal differences to distinguish between free and bound analyte. Here, we evaluated a solution-phase reaction, solid-phase detection (SRSD) method that probes the fraction of free analyte in an equilibrium titration with titrant-coated magnetic beads to estimate the apparent binding affinity. We comprehensively compared SRSD against traditional methods using two different antibody-antigen pairs and relevant controls. We show that SRSD produces affinity measurements of the same order of magnitude as those measured by well-established label-free kinetic (SPR) and solution-phase equilibrium (SET) approaches. We anticipate that the SRSD method may be of general interest for affinity estimation, as it can be performed with unpurified analytes in complex mixtures. However, this method cannot be assumed to be generally valid or interchangeable across all systems, as the study illustrates system-specific, situational utility when appropriate validation controls are implemented.