Scintillation proximity assay to measure binding of soluble fibronectin to antibody-captured alpha 5 beta 1 integrin.
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 measured using anti-human beta 1 antibodies. This method should be generally useful to measure cell-free ligand binding to receptors that are difficult to purify.
Pachter
J A
JA
Department of Molecular Pharmacology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
Zhang
R
R
Mayer-Ezell
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D016466:CHO Cells; D002474:Cell-Free System; D006224:Cricetinae; D004354:Drug Screening Assays, Antitumor; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D008863:Microspheres; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D017469:Receptors, Fibronectin / Q000096:biosynthesis / Q000737:chemistry*; D012588:Scintillation Counting; D012995:Solubility
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D010456:Peptides, Cyclic; D017469:Receptors, Fibronectin