Molecular biological studies on a hepatoma-associated target antigen for a human killer T-cell clone

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We previously identified a tumor-associated target antigen, ATM-1, for a human killer T-cell clone 5B5, and found the existence of ATM-1 in sera of patients with hepatocellular carcinoma. To clone the gene that coded ATM-1, we screened the CDNA library obtained from a human hepatocellular carcinoma cell line, SK-HEP-1, immunologically using monoclonal antibodies raised against partially purified ATM-1. However we could not succeed in the cloning of ATM-1 gene possibly due to the low specificity and low sensitivity of the monocional antibodies used. Thus we changed the strategy and tried to purify the ATM-1 and determine the N-terminal amino acid sequence of the ATM-1. We purified ATM-1 from sera of patients with hepatocellular carcinoma by gel filtration on Sepharose CL-6B followed by affinity chromatography on anti-ATM-1 -bound Sepharose beads. As a control, serum from a healthy person was processed in the same way. Purified samples were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane electrophoretically and stained. The sample from sera of patients with hepatocellular carcinoma gave a protein band with a molecular wright of approximately 55, 000 that was not observed in the sample from serum of a healthy person. On N-terminal amino acid sequence analysis, the protein band was found to contain two proteins. On homology search, one protein was found to have a sequence of immunoglobulin heavy chain hypervariable region III, and the other protein did not have any known amino acid sequence. We thought that the immunoglobulin was purified as an immune complex with ATM-1. We will raise antibodies against the synthetic peptide for the N-terminal amino acid sequence of the unknown protein, and also synthesize oligomers for the amino acid sequence. We will clone the ATM-1 gene using these antibodies and oligomers. Then we will study the biological and clinical significance of ATM-1.