Cell surface vimentin detection in cancer cells by peptide-based monoclonal antibody
Cell surface vimentin detection in cancer cells by peptide-based monoclonal antibody
Niloufar Sadeghi,1Hodjattallah Rabbani,2,*Omid Zarei,3Ali-ahmad Bayat,4
1. Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 2. Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 3. Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran 4. Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Introduction: 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.
Methods: A 14-mer synthetic peptide from the N-terminal region of 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.
Results: Amid the several obtained producing anti-vimentin antibody hybridoma, 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, 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.
Conclusion: Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tools where detection or targeting of vimentin in a wide range of organisms is required.