| Project/Area Number |
14572150
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | Akita University |
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Principal Investigator |
ITOH Kunihiko Akita University, School of Med., Associate Professor, 医学部, 助教授 (90221770)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Toshio Akita University, School of Med., Professor, 医学部, 教授 (20108559)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | human recombinant Fab / CD98 / tumor-associated antigen / tumor diagnosis / tumor therapy / phage display / HeLaS3 / cell adhesion molecule / ヒト型リコンビナント抗体 / ファージディスプレイ法 / HeLa細胞 / 間接蛍光抗体法 / サンドイッチELISA法 |
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| Research Abstract |
The goal of this research project is to prepare human recombinant Fab (rFab) against tumor-associated antigen, CD98 and its application to diagnosis and therapy of tumor diseases. I constructed human IgG1, kappa combinatorial library (1.3 x 10^7 cfu) from bone marrow cells of a MOLT lymphoma patient. Although five-rounds of panning was performed on this library against antibody-captured CD98 antigen, no CD98-reactive rFab was obtained. We therefore changed the antigen source from CD98 protein to live CD98-positive cells (suspension form HeLa cells : sHeLa). After five-rounds of panning, 240-fold enrichment of library was observed.. We obtained 39 positives from 40 tested clones by indirect immunofluorescence assay. Positive clones were found to be identical from their BstNI fingerprinting patterns. We next performed the molecular structural and functional characterization of cloned rFab, termed AHSA anti-HeLa surface antigen) rFab. Variable heavy-and light-chain sequences of AHSA rFab were found to be highly homologous with the derived germ line gene sequences. AHSA rFab reacted with sHeLa in a concentration dependent manner, though it did not react with adherent HeLaS3 cells, and with other cell lines of human or mouse origin. Reactivity of AHSA rFab against sHeLa was decreased in parallel with the loss of cluster formation efficiency caused by a long-term culture. Cluster formation of sHeLa was inhibited by treatment of anti-Fab-cross-linked AHSA rFab against cultured sHeLa cells in vitro. These results suggest that AHSA rFab recognizes the cluster formation-associated antigen on the surface of sHeLa cells.
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