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.
Wang
Yu-Cai
YC
Department of Orthopaedic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, China.
Zheng
Lian-He
LH
Ma
Bao-An
BA
Zhou
Yong
Y
Fan
Qing-Yu
QY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005353:Fibronectins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D019063:Tenascin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D005353:Fibronectins; D010446:Peptide Fragments; D019063:Tenascin; C114731:tenascin fibronectin type III domain (1-92)