Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells.
BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.
McEwan
M
M
Queensland Institute of Medical Research, Australia.
Parsons
P G
PG
Moss
D J
DJ
Burrows
S
S
Stenzel
D
D
Bishop
C J
CJ
Strutton
G M
GM
D000911:Antibodies, Monoclonal / Q000032:analysis*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D008543:Melanins / Q000032:analysis; D008545:Melanoma / Q000032:analysis / Q000276:immunology / Q000473:pathology*; D014442:Monophenol Monooxygenase / Q000378:metabolism; D009363:Neoplasm Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008543:Melanins; D009363:Neoplasm Proteins; D014442:Monophenol Monooxygenase