Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay |
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Authors: | Thomas R Glass Donald J Winzor |
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Institution: | 1. Sapidyne Instruments Inc., 700 West Diamond Street, Boise, ID 83705, USA;2. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia |
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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. |
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Keywords: | Antigen&ndash antibody interactions Antibody bivalence Kinetic exclusion assay |
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