| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Norihiro GRADUATE SCHOOL OF PHARMACEUTICAL SCIENCES, TOHOKU UNIVERSITY, RESEARCH ASSOCIATE, 大学院・薬学研究科, 助手 (90205477)
IKEGAWA Shigeo GRADUATE SCHOOL OF PHARMACEUTICAL SCIENCES, TOHOKU UNIVERSITY, ASSOCIATE PROFESSOR, 大学院・薬学研究科, 助教授 (90111301)
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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 attempt to generate novel antibody molecules, which may allow us to develop excellent analytical systems with ultrahigh sensitivity and specificity.
α-type and β-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of an anti-hapten antibody have been produced by the B-cell hybridoma technique, for developing a highly sensitive noncompetitive immunoassay system which has been difficult to establish for haptens. Ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG) and 11-deoxycortisol (11-DC) were selected as the model hapten. The combined use of the α- and β-type anti-idiotype antibodies together with the anti-hapten antibody provided a highly sensitive assay system : the detection limit was 70 fg (118 amol) for UDCA 7-NAG and 1 pg (2.9 fmol) for 11-DC. A cross-reaction study revealed that both assay systems have practical specificity. The UDCA 7-NAG noncompetitive assay system has already been well validated to be available for direct measurement (without any pretreatment) of human urine levels of this metabolite.
On the other hand, a single-chain Fv fragment (scFv) against 11-DC, which shows very high affinity (Ka 1.3 x 10ィイD110ィエD1 MィイD1-1ィエD1) and specificity (cross-reactivity with cortisol, 0.14%, cortisone 0.21%), has been clones. Cloning of a fusion protein between this scFv and green fluorescent protein, which would enable a sensitive fluoroimmunoassay system, is now ongoing in our laboratory.
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