Interaction of fibronectin with antibodies and collagen in radioimmunoassay

Abstract

Human fibroblast surface and plasma protein, fibronectin, has been shown to bind to collagen, and it can be purified by affinity chromatography on gelatin (denatured collagen) coupled to Sepharose particles (Engvall, E. and Ruoslahti, E. (1977) Int. J. Cancer 20, 1–5). The same procedure was used for purification of fibronectin from mouse plasma. Radioiodination of the gelatin-purified mouse and human fibronectins gave labeled proteins from which 60–70% of the radioactivity could be bound to anti-fibronectin in antibody excess. Fibronectin preparations isolated using other methods showed lower immunoreactivity after labeling. A major part of the labeled fibronectin retained its affinity for collagen. This allowed removal of weakly immunoreactive material by fractionation of the iodinated protein on gelatin-Sepharose. The bound and eluted fraction of the labeled protein showed more than 90% binding to antibody and to collagen. The labeled fibronectin was used to study two questions of immediate interest: the effect of collagen, a component likely to be present in samples containing fibronectin, on fibronectin radioimmunoassay, and the effect of anti-fibronectin on the fibronectin-collagen interaction. Quantitation of fibronectin by radioimmunoassay was found to be unaffected by the presence of collagen in the sample, suggesting that the interaction of fibronectin with antibody was not affected by the presence of collagen. On the contrary, the binding of labeled fibronectin to collagen was inhibited when the labeled protein was mixed with purified antibodies to fibronectin. Fibronectin previously bound to a gelatin column was released when antibodies were passed through the column. That antibodies can interfere with the fibronectin-collagen binding suggests that the avidity of the antibody-fibronectin interaction is higher than that between fibronectin and collagen.