title: Using Antigen-antibody Binding Kinetic Parameters to Understand Single-Molecule Array Immunoassay Performance.
abstract: 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 (kon) and dissociation (koff) 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 (KD) and found that Simoa assay performance had an inverse relationship to the koffvalue of the detection antibody. The Simoa fluorescent signals were highest when the koffof the detection antibody was less than 10-5s-1. The capture antibody koffdid not have as significant an effect, but a koffof less than 10-3s-1was preferred. We also found that the konvalues 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 koffvalues.
authors: Department of Chemistry, Tufts University , Medford, Massachusetts 02155, United States.
authors: Trinh L
authors: TL
authors: Dinh
authors: Department of Chemistry, Tufts University , Medford, Massachusetts 02155, United States.
authors: Kevin C
authors: KC
authors: Ngan
authors: Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine , Grafton, Massachusetts 01536, United States.
authors: Charles B
authors: CB
authors: Shoemaker
authors: Department of Chemistry, Tufts University , Medford, Massachusetts 02155, United States.
authors: David R
authors: DR
authors: Walt
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D001665:Binding Sites; D005453:Fluorescence; D007118:Immunoassay; D007700:Kinetics
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex