Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody

Abstract

Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.