Development of high sensitive fluoroimmunoassay for synthetic adrenal agents and its clinical application

本研究为建立高灵敏度的地塞米松、泼尼松龙等肾上腺合成药物的荧光偏振免疫分析体系提供了新的途径。该系统采用氩离子激光器作为IBF-129型荧光偏振荧光计的光源、进样器和微量流动池，这些免疫分析需要制备有效的抗体和FITC标记的抗原，因此需要合成适当的甾体化合物，其上带有能与蛋白质或FITC结合的取代基。为此，我们合成了4-（羧甲硫基）-地塞米松和泼尼松龙-21-半琥珀酸酯。用混合酸酐法将这些甾体衍生物与牛血清白蛋白（BSA）偶联，并在家兔体内产生了相应的抗体。用DEAE-Sephacel柱纯化类固醇抗血清的IgG组分。皮质醇与抗强的松龙-21-半琥珀酸-BSA抗血清的交叉反应率为9.6%，其特异性与某些研究者报道的相似。然后加入抗类固醇抗血清IgG级分。将混合物在20-25 ℃的恒温下孵育60分钟，并再次测量极化值（B）。通过从偏振值（B）中减去偏振值（A），进行校正以使血清引起的干扰最小化。

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).