Immunochemical analysis of fibronectin using monoclonal antibodies

Cloned hybrid cell lines secreting antibodies directed against human plasma fibronectin were prepared according to the methods of Kohler and Milstein (Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 and (1976) Eur. J. Immunol. 6, 511-519). The specificity of each monoclonal antibody for fibronectin was established from autoradiograms of radioimmunoprecipitates following SDS-polyacrylamide gel electrophoresis. The monoclonal antibodies were reactive with both native and SDS-denatured fibronectin. Ascites fluids obtained from infected isogenic mice precipitated 85-95% of the 125I-labelled fibronectin radioactivity in indirect radioimmunoprecipitation tests. Localization of specific epitopes to restricted regions of the fibronectin molecule was carried out by monitoring monoclonal antibody binding to proteolytic fragments. Of the five monoclonal antibodies analyzed in this study, three recognized determinants which resided in the terminal 35 kDa region of the fibronectin monomer. Furthermore, these epitopes were localized to fragments as small as 20 kDa. Competition studies carried out using plasma fibronectins isolated from different species revealed that three monoclonal antibodies recognized sites which were relatively conserved, while two monoclonal antibodies recognized epitopic sequences which were unique to the human protein. The corresponding anti-fibronectin serum also demonstrated discriminatory capabilities. Immunofluorescent analysis of human fibroblasts grown in vitro demonstrated that all the monoclonal antibodies tested were reactive with pericellular fibronectin.