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.