Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin.
|Title||Development and validation of competition binding assays for affinity to the extracellular matrix receptors, α(v)β(3) and α(IIb)β(3) integrin.|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Szabo, AM, Howell NR, Pellegrini P, Greguric I, Katsifis A|
|Date Published||2012 Jan 8|
The RGD (Arg-Gly-Asp) binding integrins α(v)β(3) and α(IIb)β(3) 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 α(v)β(3) and α(IIb)β(3) 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 α(IIb)β(3) 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 α(IIb)β(3) 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.
|Short Title||Anal Biochem|