Using Antigen–antibody Binding Kinetic Parameters to Understand Single-Molecule Array Immunoassay Performance

This paper provides insights into the performance of single-molecule array (Simoa) immunoassays by examining the effects of various capture and detector antibody–antigen binding kinetic parameters. Simoa is similar to other immunoassays in that the overall Simoa performance is heavily dependent on the choice of antibodies; however, little is known about how the different properties of the antibodies result in the wide variations in assay performance. Here, we focus on antibody–antigen binding kinetics and demonstrate how the association ( k on ) and dissociation ( k off ) rate constants of the capture and detection antibodies affect Simoa performance. We compared six different antibodies with over a four-log range of equilibrium dissociation constants ( K D ) and found that Simoa assay performance had an inverse relationship to the k off value of the detection antibody. The Simoa fluorescent signals were highest when the k off of the detection antibody was less than 10 –5 s –1 . The capture antibody k off did not have as significant an effect, but a k off of less than 10 –3 s –1 was preferred. We also found that the k on values of the capture and detection antibodies were not important factors for Simoa performance. Therefore, the assay optimization process could be accelerated by choosing detection antibodies with the slowest k off values.