Production and characterization of a novel monoclonal antibody against human sortilin

Sortilin, as a member of Vps10p-domain sorting receptor family, is overexpressed in a number of malignancies, including ovarian carcinoma. Antibodies against sortilin may contribute to further clarification of sortilin functional activities in signal transduction, intracellular sorting of proteins, and endocytosis. The aim of this study was to produce a monoclonal antibody against a synthetic peptide derived from extracellular N-terminal region of sortilin to be used as a tool for investigating sortilin characteristics in ovarian carcinoma. A synthetic peptide derived from the last 50 amino acids of extracellular domain of sortilin protein was selected and conjugated to keyhole limpet hemocyanin and used to immunize mice. The anti-sortilin monoclonal antibody (MAb), clone 2D8, was purified from supernatant of final hybridoma clone using peptide-affinity chromatography column. Reactivity of antibody with the immunizing peptide was assessed in ELISA. Furthermore, flow cytometry and Western blot analyses were used to investigate the reactivity of antibody with its target in a panel of ovarian carcinoma cell lines or tissues. MAb 2D8 was able to recognize the coated immunizing peptide in ELISA and detect its protein target, sortilin, in flow cytometry and Western blot analyses. The achieved data suggest that the developed monoclonal antibody may be applicable as a research tool for detection of sortilin protein in Western blot as well as flow cytometry tests. © Mary Ann Liebert, Inc. 2015