| Project/Area Number |
12557236
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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 |
Laboratory medicine
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| Research Institution | Tohoku University |
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Principal Investigator |
GOTO Junichi TOHOKU UNIVERSITY HOSPITAL, PROFESSOR, 医学部附属病院, 教授 (80006337)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Norihiro KOBE PHARMACEUTICAL UNIVERSITY, PROFESSOR, 教授 (90205477)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥11,100,000 (Direct Cost: ¥11,100,000)
Fiscal Year 2002: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2001: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2000: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | Antibody engineering / Anti-motatype antibody / Hyper-antibody / Single-chain Fv fragment / Bioanalytical chemistry / Noncompetitive immunoassay / Hapten / Bile acid / 免疫工学 / デオキシコール酸 / アフィニティーラベル / アシルアデニレート / アシルグルクロニド / ステロイド |
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| Research Abstract |
Antibodies are widely used as a key reagent in various immunoassays or immunoaffinity purification systems, which are essential in the biomedical analyses. However, conventional antibodies have some limitations on both affinity and specificity, resulting in insufficient sensitivity or low selectivity of these immunochemical techniques, In this study, we attempted to generate novel antibody species, anti-metatype antibodies (the antibodies recognizing antigen-antibody complex) and "hyper-antibodies" (the genetically engineered antibodies having improved affinity and specificity), which may allow us to develop excellent analytical systems for bioactive small molecules (haptens).
Anti-metatype antibodies that specifically recognize the complex between a target hapten and an anti-hapten antibody are expected to enable noncompetitive immunoassay systems for steroids and synthetic drugs, showing much higher sensitivity than conventional competitive immunoassays. First, we prepared single-chai
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n Fv fragments (scFvs) against lithocholic acid 3-sulfate and deoxycholic acid (DCA) because scFvs lack the constant regions of immunoglobulins and thus would be advantageous to induce such anti-metatype antibodies. The V_H- and V_L-genes of mouse monoclonal antibodies against these bile acids were amplified by RT-PCR using RNA extracted from the corresponding hybridoma cells, which were then assembled into the scFv sequence encoding 5'-V_H-(Gly_4-Ser)_3-V_L-3' and expressed in E. coli XLOLR strain. The resulting scFv proteins showed similar reactivity against the relevant haptens to those of the parent mouse antibodies.
Then we investigated the affinity-labeling of the auti-DCA antibody paratope with a relevant hapten, DCA, because this would be effective to inhibit the dissociation between the haptens and antibodies and increase the chance for generating anti-metatype antibodies. We found DCA-adenylate is highly reactive to primary amino groups on proteins, and works very well as the labeling agent. Immunization of mice with the affinity-labeled DCA-anti-DCA scFv complex would provide us the anti-metatype antibodies that enable sensitive noncompetitive immunoassay procedures. Less
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