Abstract:We aimed to establish the first monoclonal antibody (MAb) against the human EMILIN-5 protein (elastin microfibril interface located protein-5) and to investigate its distribution in normal human esophageal tissues and esophageal carcinomas. The bacterially expressed 6 His-EMILIN-5 fusion protein was induced and purified. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6x His-EMILIN-5 fusion proteins or purified 6x His-ZNRD1 fusion protein as a control. The E… Show more
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.
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.
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