Generation and Characterization of Anti-MUC4 Monoclonal Antibodies Reactive with Normal and Cancer Cells in Humans

Journal of Histochemistry and Cytochemistry - Tập 52 Số 2 - Trang 253-261 - 2004
Nicolas Moniaux1, Grish C. Varshney1,2, Subhash C. Chauhan1, M.C. Copin3, Maneesh Jain1, Uwe A. Wittel1, Mahefatiana Andrianifahanana1, Isabelle Van Seuningen3, Surinder K. Batra1
1Department of Biochemistry and Molecular Biology and Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, Nebraska
2Institute of Microbial Technology, Chandigarh, India.
3Unité INSERM 560, Lille, France

Tóm tắt

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 μm above the cell surface, in which MUC4α is an extracellular mucin-type glycoprotein subunit and MUC4β is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDT-TPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.

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Thông tin xuất bản

Journal of Histochemistry and Cytochemistry

Tập 52 Số 2

253-261

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