1999 Fiscal Year Final Research Report Summary
Preparation of Monoclonal Antibody against Mutagenic Sugar Chain Which Specifically Occurs in Hepatoma Cells and Its Evaluation for Diagnostics
| Project/Area Number |
10556079
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KUMAGAI Hidehiko Kyoto Univ. Grad. Sch. Biostudies, Professor, 生命科学研究科, 教授 (70027192)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIHARA Hideharu Gifu Univ. Fac. Eng., Professor, 工学部, 教授 (60021599)
KOUKETSU Mamoru Gifu Univ. Fac. Eng., Research Associate, 工学部, 助手 (50178208)
YAMAMOTO Kenji Kyoto Univ. Grad. Sch. Biostudies, Professor, 生命科学研究科, 教授 (70109049)
SAWABU Norio Kanazawa Univ. Canser Inst., Professor, がん研究所, 教授 (90019969)
FUKUMI Hironobu Fukui Industrial Univ. Fac. Eng., Professor, 工学部, 教授 (90288340)
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| Project Period (FY) |
1998 – 1999
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| Keywords | hepatoma / monoclonal antibody / mutagenic sugar chain / α-fetoprotein / alkylglycoside / endoglycosidase / transglycosylation |
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| Research Abstract |
It is known that the structure of oligosaccharide existed in glycoproteins and cell membrane frequently mutate in cancer cells. It was found that the structure of oligosaccharide in glycoprotein α-fetoprotein from serum was varied in hepatoma patients. Therefore, we intended to develop a simple method to prepare the monoclonal antibody specific for the mutagenic oligosaccharide of glycoprotein. In this study, we synthesized the novel glycoconjugate (alkylglycoside) having oligosaccharide of glycoprotein as immunogen using transglycosylation activity of microbial endoglycosidase (endo-β-N-acetylglucosaminidase), and prepared the monoclonal antibody against asparagine-linked bianntenary complex-type of oligosaccharide.
1. We prepared a large amount of glycopeptide containing bianntenary complex-type of oligosaccharide from hen egg yolk as the donor of ligosaccharide for binding to alkylgucoside.
2. We synthesized alkylglycoside as the immunogen of asparagine-linked bianntenary complex-type
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of oligosaccharide using transglycosylation activity of Mucor endoglycosidase (Endo-M). We incubated egg yolk glycopeptide and n-octyl-β-D-glucoside with Endo-M, pH6.0, at 37℃ for 12hr, and isolated the transglycosylation product by HPLC. The product was confirmed as the alkylglycoside having complex-type of oligosaccharide by mass spectrometry.
3. We immunized mice by the alkylglycoside. The immunogen was injected to mice intraperitonealy four times. After last booster, spleen cells were harvested and mixed with myeloma cells, and fused. Three weeks later, hybridomas grew and the screening of hybridomas producing antibody against oligosaccharide was carried out by ELISA method using human serum tranferrin glycopeptide and egg yolk glycopetide, both of which contained bianntenary complex-type of oligosaccharide. As the result, five hybridoma cell lines were obtained as the producer of antibody. Using these hybridomas, cloning was carried out by limiting dilution method. Then, we obtained three hybridoma clones which produced antibody against bianntenary complex-type of oligosaccharide.
4. We also prepared the hybridoma producing asialo-bianntenary complex-type of oligo-saccharide according to the same method described above. Less
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