Production and Characterization of a Novel Monoclonal Antibody Against Human Sortilin

Abstract

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.

FIG. 1.

Titration of purified 2D8 in ELISA test. 2D8 was purified by peptide affinity column and its reactivity with the immunizing peptide was evaluated by ELISA. Lack of reactivity with an 18-mer irrelevant peptide and PBS alone served as negative controls.

FIG. 2.

Investigation of sortilin expression in a number of cell lines using commercial rabbit anti-sortilin antibody in Western blot analysis. All cells expressed the main isoform (NP_002950.3, 833 aa, ∼100 kDa) of sortilin protein except the mouse embryonic fibroblast cell line, NIH/3T3, which was negative for sortilin expression in the Western blot. Presence of some low-molecular-weight bands in HEK-293 and PC3 cell lines may represent the expression of smaller sortilin protein isoforms such as NP_001192157 (694 aa, ∼80 kDa).

FIG. 3.

(A) Detection of sortilin expression in NIH/3T3-sortilin (stably sortilin-transfected NIH/3T3) using reactivity with commercial rabbit anti-sortilin antibody in Western blot analysis. Commercial antibody recognized 100 kDa band of sortilin in lysates of Caov-4 (positive control) and NIH/3T3-sortilin but not in NIH/3T3-vector (stably pCMV6-Neo-transfected NIH/3T3). (B) Reactivity of 2D8 to human sortilin molecule by Western blot analysis. 2D8 recognized human sortilin as a 100 kDa band in the lysates of NIH/3T3-sortilin, ovarian carcinoma tissues, and cell lines but not in NIH/3T3-vector. NIH/3T3-sortilin and NIH/3T3-vector provided positive and negative controls, respectively. β-actin protein was detected as internal control in all samples. OC, ovarian carcinoma.

FIG. 4.

Reactivity of 2D8 with cell surface sortilin on membranes of ovarian cancer cell lines using flow cytometry analysis. Values demonstrate the percentages of positive cells. 2D8 detected transmembrane sortilin on Caov-4, A2780S, SKOV-3, and 2008/C13.R surfaces but not on NIH/3T3- vector membrane as negative control.