Anti-melanoma monoclonal antibody HMB-45 on enhanced chemiluminescence-western blotting recognizes a 30-35 kDa melanosome-associated sialated glycoprotein.
HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.
Chiamenti
A M
AM
Institute of Biological Chemistry, University of Verona, Italy.
Vella
F
F
Bonetti
F
F
Pea
M
M
Ferrari
S
S
Martignoni
G
G
Benedetti
A
A
Suzuki
H
H
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D008163:Luminescent Measurements; D008544:Melanocytes / Q000737:chemistry*; D008545:Melanoma / Q000737:chemistry* / Q000276:immunology*; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000032:analysis* / Q000276:immunology*; D012794:Sialic Acids / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D012794:Sialic Acids; D012967:Sodium Dodecyl Sulfate