Fischer, J.; Kaufmann, J.O.; Weller, M.G. Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays. Preprints2024, 2024040996. https://doi.org/10.20944/preprints202404.0996.v1
APA Style
Fischer, J., Kaufmann, J.O., & Weller, M.G. (2024). Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays. Preprints. https://doi.org/10.20944/preprints202404.0996.v1
Chicago/Turabian Style
Fischer, J., Jan Ole Kaufmann and Michael G. Weller. 2024 "Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays" Preprints. https://doi.org/10.20944/preprints202404.0996.v1
Abstract
The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.
Keywords
ELISA; microplate; competitive immunoassay; IC50; test midpoint; point of inflection; law of mass action; thermodynamics; equilibrium constant; dissociation constant; binding strength; monoclonal antibodies; clones; surface-plasmon resonance; SPR; interaction; isothermal titration calorimetry; ITC; microscale thermophoresis; MST; Kd value
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
