Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.
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 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta 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, STGDTTPLPVTDTSSV. 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.
Moniaux
Nicolas
N
Department of Biochemistry and Molecular Biology and Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, Nebraska 68198-4525, USA.
Varshney
Grish Chandra
GC
Chauhan
Subhash Chand
SC
Copin
Marie Christine
MC
Jain
Maneesh
M
Wittel
Uwe A
UA
Andrianifahanana
Mahefatiana
M
Aubert
Jean-Pierre
JP
Batra
Surinder Kumar
SK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D018613:Microscopy, Confocal; D055264:Mucin-4; D009077:Mucins / Q000276:immunology* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D020080:Tandem Repeat Sequences / Q000276:immunology
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C524658:MUC4 protein, human; C524659:Muc4 protein, mouse; D055264:Mucin-4; D009077:Mucins; D010455:Peptides