Human monoclonal antibody identified an immunoreactive tetrapeptide sequence (Lys-Tyr-Gln-Ile) in M(r) 43,000 protein of human melanoma.
The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma. To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92. DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF). The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein. There is no homology between the cloned sequence and other reported DNA sequences. Western blot analysis showed that the fused protein had specific binding to HuMAb L92. An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro. HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay. Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI). These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein.
John Wayne Cancer Institute, Santa Monica, California 90404.
T
T
Oka
Y
Y
Kikumoto
K
K
Itakura
D L
DL
Morton
R F
RF
Irie
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D006801:Humans; D008545:Melanoma / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; C107095:tumor-associated antigen, gp43