Generation and identification of monoclonal antibodies against FNIII domain D of human tenascin-C.
Tenascin-C (TN-C), a key component of extracellular matrix (ECM), is strongly expressed in fetal and cancer tissues. Large-molecular-weight variants of TN-C, including different combinations of its alternative spliced FNIII repeats, are specifically expressed in tissues under certain pathological conditions. Here we report the production of monoclonal antibodies (MAbs) against FNIII domain D (FNIII D) of human TN-C. Complementary DNA encoding the FNIII D region was generated by RT-PCR from human osteosarcoma (OS) cell line, and the recombinant FNIII D-GST fusion protein was expressed and purified. Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against FNIII D were obtained by routine murine hybridoma technique. The MAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). Both of them were applicable in Western blot and IHC. With our MAbs, we found TN-C was positive in OS and most of it was among the tumor stroma. To conclude, these MAbs to human FNIII D domain of TN-C may be useful for exploring OS pathogenesis and potential clinical application.