Monoclonal Antibodies to the Collagen Binding Domain of Human Plasma Fibronectin

Abstract

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells.

“…As shown in Fig. 4, a and b, this antibody blocked the adhesion of both Friend and WEHI-3B cells to fibronectin, while the control antibody MAB 52, which recognizes the gelatin binding domain of fibronectin (37), had no effect. Inhibition with the monoclo-nal antibody f33 was dose dependent and occurred up to a dilution of 10 ttg/ml.…”

“…Serumspreading factor preparations consisted of fetal calf serum (Flow Laboratories, Inc., McLean, VA) adsorbed on Sepharose-coupled gelatin to remove flbronectin and on Affi-gel Blue (Bio-Rad Laboratories, Richmond, CA) to remove albumin (14). These preparations were shown to be devoid of fibronectin by radioimmunoassay (37) and effectively promoted attachment of murine fibroblasts in a dose-dependent manner also in the presence of flbronectin antibodies. Laminin was purchased from Bethesda Research Laboratories (Gaithersburg, MD), bovine dermal collagen (95 % type I + 5% type ill) was from Flow Laboratories (Vitrogen 100), and human placenta basement membrane collagen (type IV) and gelatin (swine skin denatured collagen) were from Sigma Chemical Co. (St. Louis, MO).…”

“…The monoclonal antibody f 33, directed toward the cell binding site of fibronectin (33), was kindly provided by V. Miggiano (Hoffmann-LaRoche, Basel) and S. Barlati (Universit~ di Brescia, Italy). The monocioual antibody MAB 52, reacting with the gelatin binding region of fibronectin, was isolated in our laboratory (37).…”