Monoclonal antibodies to the collagen binding domain of human plasma fibronectin

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.     

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.     

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit alpha9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration.