1989 Fiscal Year Final Research Report Summary
Development of high sensitive fluoroimmunoassay for synthetic adrenal agents and its clinical application
| Project/Area Number |
63571116
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Kobe women's College of Pharmacy |
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Principal Investigator |
WATANABE Fukuko Kobe Women's College of Pharmacy, Faculty of Pharmacy, Professor, 薬学部, 教授 (10068322)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIGUCHI Yoshino Kobe Women's College of Pharmacy, Faculty of Pharmacy, Assistant, 薬学部, 助手 (60208171)
KIGUCHI Toshiko Kobe Women's College of Pharmacy, Faculty of Pharmacy, Lecturer, 薬学部, 講師 (70068344)
KOBAYASHI Yoshiharu Kobe Women's College of Pharmacy, Faculty of Pharmacy, Lecturer, 薬学部, 講師 (20133647)
NAITOH Takeaki Kobe Women's College of Pharmacy, Faculty of Pharmacy, Professor, 薬学部, 教授 (00068339)
NINOMIYA Ichiya Kobe Women's College of Pharmacy, Faculty of Pharmacy, Professor, 薬学部, 教授 (00068321)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Fluorescence polarization immunoassay / Dexamethasone / Prednisolone / FITC-labled steroid |
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| Research Abstract |
In this study, the new approach to establishment of the high sensitive fluorescence polarization immunoassay system of the synthetic adrenal agents such as dexamethasone and prednisolone are achieved. Argon-ion laser as a light source of a model IBF-129 fluorescence polarization fluorimeter, sample changer, and micro flow-cell were used in this semi-automatic system.
These immunoassays require the preparation of effective antibody and FITC-labeled antigen, thus necessitating the synthesis of appropriate of steroids, carying substituent capable of binding with protein or FITC. For this purpose, we were synthesized 4-(carboxymethylthio)-dexamethasone and prednisolone-21-hemisuccinate. These steroid derivatives werd conjugated with bovine serum albumin (BSA) by mixed anhydride reaction, and the antibody to the conjugate were raised in rabbits. The purified IgG fraction of steroid antiserum was prepared by applying to DEAE-Sephacel column. Cross- reactivity of cortisol to anti-prednisolone-21-hemisuccinate-BSA antiserum was 9.6%, specificity of this antibody was quite similar to those of the previous reports of some investigators.
FITC-labeled antigen are prepared by conjugation the derivative steroids with the fluorescein isothiocyanate.
Procedure: Serum sample was mixed with FITC-labeled steroid and polarization value (A) was measured. Then anti-steroid antiserum IgG fraction was added. The mixture was incubated at constant temperature of 20-25 ﾟC for 60 min, and the polarization value (B) was measured again. A correction was made for minimizing interference due to serum by subtracting the polarization value(A) from the value (B).
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