Preparation and characterization of a novel monoclonal antibody specific to peptide VEHVVADAGAFLRH.
EMILIN-5, consisting of 766 amino acids with a cysteine-rich EMI domain at the NH(2) terminus, is likely to play a significant role in the process of osteogenesis. Here we aimed to efficiently generate monoclonal antibody against EMILIN-5 protein. We synthesized the peptide VEHVVADAGAFLRH based on published EMILIN-5 cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-EMILIN-5 fusion protein or the peptide. One monoclonal antibody (MAb) named C14 (IgG1), effective in detecting the native EMILIN-5 protein, was characterized by ELISA and Western immunoblot analysis. By using the MAb, we found EMILIN-5 protein was expressed in human cancer tissues of stomach, liver, and testis. Taken together, the MAb C14 bound to native EMILIN-5 protein and should be useful in studies of EMILIN-5 protein functions.
Cao
Jun
J
Department of Orthodontics, Qindu Stomatology College Affiliated with the Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China. zhanganuo@126.com
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006433:Hemocyanins; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000276:immunology*; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D013274:Stomach Neoplasms / Q000276:immunology; D013736:Testicular Neoplasms / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C089826:EMILIN3 protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin