PMID: 8585604Sep 1, 1995Paper

Scintillation proximity assay to measure binding of soluble fibronectin to antibody-captured alpha 5 beta 1 integrin

Analytical Biochemistry
J A PachterR Mayer-Ezell

Abstract

A scintillation proximity assay (SPA) has been developed to measure binding of alpha 5 beta 1 integrin, a heterodimeric cell-surface adhesion receptor, to fibronectin. This assay utilizes an anti-beta 1 integrin monoclonal antibody to simultaneously capture alpha 5 beta 1 from a cellular lysate and couple the integrin to anti-mouse IgG antibody-coated SPA beads for detection of 125I-fibronectin binding. The assay does not require prior purification of alpha 5 beta 1 nor physical separation of bound and free 125I-fibronectin. Chinese hamster ovary cells that stably overexpress human alpha 5 integrin (CHO#7 cells) were used as a source of alpha 5 beta 1 fibronectin receptor. Using the anti-hamster beta 1 monoclonal antibody 7E2 to capture alpha 5 beta 1 from a CHO#7 cell lysate, this SPA assay allowed measurement of specific 125I-fibronectin binding as defined by displacement by the Arg-Gly-Asp containing peptide GRGDSP or the anti-human alpha 5 antibody P1D6. IC50 values for displacement of 125I-fibronectin binding by GRGDSP and the novel cyclic peptides cRGDGF, cRGEGF, and cRRETAWA were 2.6, 0.045, 3.2, and 37 microM, respectively. Specific 125I-fibronectin binding to alpha 5 beta 1 from C8161 human melanoma cells was also meas...Continue Reading

Citations

Jan 6, 2006·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Shaogui Wu, Bailing Liu
Apr 23, 2008·Current Protocols in Neuroscience·Steven D Kahl, Christian C Felder
Apr 23, 2008·Current Protocols in Protein Science·Neil CookKelvin Hughes
Feb 4, 1998·Analytical Biochemistry·C M HoodC J Britten
Jul 19, 2002·Analytical Biochemistry·Paul H WeinrebSerene Josiah

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