Identification of the immunodominant regions of the melanoma antigen tyrosinase by anti-tyrosinase monoclonal antibodies.
Tyrosinase, the critical enzyme in melanin synthesis, is also found to be expressed in most malignant melanomas and can serve as a target for the immune response by both CD4+ and CD8+ T-cells. Therefore it could be used as a potential target for therapeutic intervention in tyrosinase-positive melanomas. In order to develop serological reagents for the immunodetection of human tyrosinase and to find the most immunogenic region of the protein, we have raised a panel of monoclonal antibodies (MAbs) against recombinant tyrosinase expressed and purified from bacteria. Epitope mapping revealed the 79 amino acid long stretch between 163 and 241 residues to be the most immunodominant region of the tyrosinase. This region could be further divided into three parts by binding different MAbs. These MAbs were very useful tools for the detection of tyrosinase expression from different constructs in tissue culture cells by immunocytochemistry and in melanocytes by immunohistochemistry. Some of the MAbs that recognized epitopes between 163 and 204 amino acids also recognized an additional distinct protein of about 70 kDa seen on Western blot analysis of transfected and non-transfected COS-7 cells. One of these, the MAb 4B1, was used in immunohistochemistry, and cross reaction with the basement membrane of the human tissue was observed. The analysis of the 4B1 MAb epitope showed that the C-terminal part of that region almost entirely overlaps with the sequence of the recently reported basement membrane protein beta-netrin.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001485:Basement Membrane / Q000473:pathology; D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D019556:COS Cells; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D008545:Melanoma / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; D004247:DNA; D014442:Monophenol Monooxygenase