title: Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay.
abstract: Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.
authors.affiliation: Sapidyne Instruments Inc., 700 West Diamond Street, Boise, ID 83705, USA. Electronic address: tglass@sapidyne.com.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Glass
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Winzor
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D001665:Binding Sites; D007120:Immunochemistry / Q000379:methods*; D007700:Kinetics; D011336:Probability; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D000941:Antigens
keywords: Antibody bivalence; Antigen–antibody interactions; Kinetic exclusion assay