| Project/Area Number |
09672349
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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 |
Laboratory medicine
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| Research Institution | Tohoku University |
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Principal Investigator |
KOBAYASHI Norihiro Faculty of Pharmaceutical Sciences, Tohoku University, Assistant Professor, 薬学部, 助手 (90205477)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | noncompetitive immunoassay / anti-idiotype antibody / monoclonal antibody / hapten / bile acid metabolite / ll-deoxycortisol |
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| Research Abstract |
Conventional immunoassays for small molecules (haptens) such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (analyte) and labeled-antigen against a limited amount of antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity (attomole-zeptomole order) and a wider working range, this principle has not been suitable for measuring haptens. Thus, we attempted to develop a noncompetitive immunoassay system using anti-idiotype antibodies as the key reagent ; ursodeoxycholic acid7-N-acetylglucosaminide (UDCA 7-NAG ; a bile acid metabolite) and I 1-deoxycortisol (11-DC) were selected as the model hapten. BALB/c or A/J mice were immunized with amonoclonal antibody against the hapten which had been conjugated with keyhole limpethemocyanin. The fusion between the immune spleen cells and P3INS l/l-Ag4-1 myeloma cells afforded a-type and beta-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of the anti-hapten antibody. The combined use of selected alpha-type and beta-type antibodies together with the anti-hapten antibody provided a sensitive noncompetitive assay system of UDCA 7-NAG, whose measurable range was approximately0.2- 500 pg, and the minimum detectable amount was 130 fg (= 220 amol).
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