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

本研究实现了建立地塞米松、强的松等肾上腺合成药物高灵敏度荧光偏振免疫分析系统的新方法。该半自动系统采用氩离子激光作为光源，由IBF-129型荧光偏振仪、样品交换器和微流动池组成。这些免疫分析需要制备有效的抗体和FITC标记抗原，因此需要合成适当的类固醇，携带能够与蛋白质或FITC结合的取代基。为此，我们合成了4-（羧甲基硫）-地塞米松和21-半琥珀酸强的松。这些类固醇衍生物通过混合酸酐反应与牛血清白蛋白（BSA）偶联，并在家兔中培养了针对该偶联物的抗体。应用DEAE-Sephacel柱制备纯化的类固醇抗血清IgG。皮质醇与抗强的松龙-21-半琥珀酸- bsa抗血清的交叉反应性为9.6%，特异性与部分文献报道相当。fitc标记抗原是由衍生物类固醇与异硫氰酸荧光素偶联制备的。操作步骤：将血清样品与fitc标记的类固醇混合，测量极化值(A)。然后加入抗类固醇抗血清IgG部分。在20-25℃恒温下孵育60 min，再次测量极化值(B)。通过从值(B)中减去偏振值(A)，进行校正以使血清干扰最小化。

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).