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Title: Development and validation of competition binding assays for affinity to the extracellular matrix receptors, αvβ3 and αIIbβ3 integrin
Authors: Szabo, A
Howell, NR
Pellegrini, PA
Greguric, I
Katsifis, A
Keywords: Receptors
Tumor cells
Issue Date: 1-Apr-2012
Publisher: Elsevier B.V.
Citation: Szabo, A. M., Howell, N. R., Pellegrini, P., Greguric, I., & Katsifis, A. (2012). Development and validation of competition binding assays for affinity to the extracellular matrix receptors, αvβ3 and αIIbβ3 integrin. Analytical Biochemistry, 423(1), 70-77. doi:10.1016/j.ab.2011.12.046
Abstract: The RGD (Arg-Gly-Asp) binding integrins αvβ3 and αIIbβ3 are integral components of various pathological and physiological processes, including tumor angiogenesis, osteoclast function, and thrombus formation. Because of this, there is interest in identifying novel compounds and proteins binding to these receptors as well as investigating the mechanism of these interactions. In this article, we describe the development and validation of competition binding assays for determining the affinity of test compounds to αvβ3 and αIIbβ3 integrin. Assays were successfully developed for each receptor, and the affinity of known compounds was comparable to published results. However, the inability of binding between αIIbβ3 integrin and the labeled echistatin protein ligand to reach equilibrium resulted in an assay that did not meet the assumptions of the competition binding model. Nevertheless, there was good agreement between this assay and known literature values, and intra- and interassay variability was acceptable. Binding by conformation-specific antibodies provided evidence that solid-phase bound αIIbβ3 receptor was in an activated conformation. This study also demonstrated that current models and methods for determining receptor affinity are simplistic and fail to account for common receptor–ligand interactions such as nondissociable interactions and varying receptor activation states. © 2012 by Elsevier Inc.
Gov't Doc #: 8880
ISSN: 0003-2697
Appears in Collections:Journal Articles

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