| Project/Area Number |
24710251
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Living organism molecular science
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| Research Institution | Ehime University |
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Principal Investigator |
TAKEDA Hiroyuki 愛媛大学, プロテオサイエンスセンター, 助教 (40609393)
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| Project Period (FY) |
2012-04-01 – 2014-03-31
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| Project Status |
Completed (Fiscal Year 2013)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2012: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
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| Keywords | 膜タンパク質 / 無細胞タンパク質合成 / プロテオリポソーム / GPCR / モノクローナル抗体 / スクリーニング / AlphaScreen / 抗体 / DRD1 |
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| Research Abstract |
We developed a high-throughput screening method using cell-free synthesized proteoliposome based on AlphaScreen, ELISA, Biacore, respectively. AlphaScreen and ELISA had excellent throughput, thus suitable for primary screening of monoclonal antibodies. Especially AlphaScreen was superior for conformational sensitive antibody selection. Using the methods developed in this study, we conducted screening and functional analysis of anti-GPCR monoclonal antibody. We immunized mice with cell-free synthesized and purified DRD1 proteoliposome and developed 800 hybridoma lines. Primary screening of 800 hybridoma was performed using AlphaScreen and ELISA, respectively, and 36 specific monoclonal antibodies were selected. They were high affinity and applicable for multiple applications. These results demonstrated that the technology based on cell-free synthesized GPCR proteoliposome may provide advantageous materials and assay methods for monoclonal antibody production.
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