1989 Fiscal Year Final Research Report Summary
Attempt to make Tumor Specific Human Monoclonal Antibody
| Project/Area Number |
62470142
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Niigata University |
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Principal Investigator |
YAKATA Minoru Niigata Univ. school of Medicine, department of Laboratory Medicine, Professor, 医学部, 教授 (20018352)
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| Co-Investigator(Kenkyū-buntansha) |
SUGITA Osamu Niigata Univ. Hospital, Lecturer, 医学部附属病院, 講師 (10092701)
YAMADA Toshiyuki Niigata Univ. Hospital, Assistant, 医学部附属病院, 助手 (50211636)
UCHIYAMA Kazuaki Niigata Univ. Hospital, Medical Staff, 医学部附属病院, 医員
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| Project Period (FY) |
1987 – 1989
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| Keywords | Monoclonal antibody / Tumor associated antigen / Human-mouse heterohybridoma / Carcinoembryonic antigen / Nonspecific cross-reacting antigen |
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| Research Abstract |
1. Human monoclonal antibody
(1) Making human monoclonal antibody For making human monoclonal antibody, human-mouse heterohybridoma making method was used. Human peripheral blood lymphocytes and lymphocytes in lymph nodes were fused with P3X63Ag8U.1 and P3X63-Ag8.653 mouse myeloma cell lines using polyethylene glycol.
(2) Stability of human-mouse heterohybridomas Stability of human kappa and lambda type light chain, and heavy chain expression of human-mouse heterohybridomas were researched using cytoplasmic staining. Human kappa light chain expression was more unstable than lambda type light chain or heavy chain, and it was gradually clear that it was difficult to do cloning of human kappa light chain expressing heterohybridoma when using limiting dilution. Same result was obtained at genetic level using southern blot analysis.
(3) Antigen specificity of human monoclonal antibody Tumor specific human monoclonal antibodies were not obtained. Seven clones reacted with several adenocarcinoma
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s. But after limiting dilution, two clones stopped secreting human monoclonal antibody. Finally five clones remained, and all of them reacted with albumin. So these clones considered to be not tumor related, but autoantibody or nonspecific binding.
2. Mouse monoclonal antibody
(1) Making mouse monoclonal antibody against cancer In order to investigate tumor antigen, we made mouse monoclonal antibodies. BALB/C mice were immunized with BxPC-3 human pancreatic cancer cells. Then spleen cells of the mice and PAI, mouse myeloma cell lines, were used for fusion using polyethylene glycol.
(2) Electrophoretical analysis of a carcinoembryonic antigen(CEA) related antigen A monoclonal antibody which reacted with CEA and CEA cross-reacting antigen, was used for this investigation. NCA, one of the CEA related antigens, recognized by this monoclonal antibody have apparent mol. wts of 55,000(NCA-55) in a pancreatic cancer cell line and 90,000(NCA-90) in granulocytes using SDS-PAGE. In a pancreatic cancer cell line(BxPC-3), not NCA-90 but NCA-55 is well produced with CEA, NCA-55 from the pancreatic cancer cell line has chromatographically polymorphism. NCA-55 consists of nearby four isoelectric points(ph 4.8, 5.3, 5.6, 5.8) mol. wts of 55,000 proteins. Less
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