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) and dissociation (k) 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) and found that Simoa assay performance had an inverse relationship to the k value of the detection antibody. The Simoa fluorescent signals were highest when the k of the detection antibody was less than 10 s. The capture antibody k did not have as significant an effect, but a k of less than 10 s was preferred. We also found that the k 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 values.