Precise epitope determination of the anti-vimentin monoclonal antibody V9

Vimentin is a type III intermediate filament protein that is typically expressed in mesenchymal cells. Overexpression of vimentin is frequently observed in several types of cancer and is often associated with epithelial‑to‑mesenchymal transition. It was recently reported that the serum vimentin leve...

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Bibliographic Details
Published in:	Molecular medicine reports Vol. 16; no. 4; pp. 3917 - 3921
Main Authors:	Tomiyama, Lucia, Kamino, Hiroki, Fukamachi, Hiroki, Urano, Takeshi
Format:	Journal Article
Language:	English
Published:	Greece Spandidos Publications 01-10-2017 Spandidos Publications UK Ltd D.A. Spandidos
Subjects:	Amino Acid Sequence Amino acids Animals Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - immunology Antibodies, Monoclonal - pharmacology Antibody Specificity Asparagine Asparagine - chemistry Cancer Cancer diagnosis Cancer screening Cell division Cell Line, Tumor Clonal deletion Cloning Colon Cytoskeletal proteins Deletion mutant Epitope Mapping - methods Epitopes Epitopes - chemistry Epitopes - genetics Epitopes - immunology Fibroblasts Genetic aspects Health aspects Humans Immunoglobulins Innovations Liver Mesenchyme Mice Molecular diagnostic techniques Monoclonal antibodies Mutation NIH 3T3 Cells Nucleotide sequence Phosphorylation Protein Binding Protein Interaction Domains and Motifs Proteins Site-directed mutagenesis Tumors Vimentin Vimentin - antagonists & inhibitors Vimentin - chemistry Vimentin - genetics Vimentin - immunology
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Summary:	Vimentin is a type III intermediate filament protein that is typically expressed in mesenchymal cells. Overexpression of vimentin is frequently observed in several types of cancer and is often associated with epithelial‑to‑mesenchymal transition. It was recently reported that the serum vimentin level is significantly elevated in colon and liver tumors. Therefore, a more sensitive vimentin detection system may be useful for cancer screening and early detection. The V9 mouse monoclonal antibody (mAb), which recognizes the human vimentin protein, is widely used in routine pathology to identify mesenchymal cells using immunohistochemical analysis. Although it has been suggested that the epitope of the V9 mAb is located within the C‑terminal region of vimentin, the precise amino acid sequence that it recognizes has not yet been identified. In the present study, we constructed several deletion mutants of the vimentin protein and examined their reactivity with the V9 mAb to accurately map its epitope. We confirmed that its epitope resides in the C‑terminal region of vimentin, between amino acids 392‑466. Additionally, cross‑species comparison of amino acid sequence alignment of vimentin, as well as site‑directed mutagenesis, revealed that one residue, the asparagine at position 417, is critical for antibody binding. Using smaller vimentin fragments ranging in length from 9 to 13 residues, each containing this critical asparagine, we determined that the minimal residues required for V9 mAb recognition of human vimentin are the thirteen amino acid residues at positions 411-423 (411ISLPLPNFSSLNL423).
ISSN:	1791-2997 1791-3004
DOI:	10.3892/mmr.2017.7102