应用FITC系统建立非均衡竞争游离甲状腺素化学发光法

目的]制备抗人甲状腺素多克隆抗体并应用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)系统建立非均衡竞争FT4化学发光法.方法]以T4-牛血清白蛋白(bovin serum albumin,BSA)为抗原免疫新西兰兔,制备抗人T4多克隆抗体,亲和层析法吸附掉与3-碘-L-甲状腺原氨酸(3-iodo-L-tyrosine)、3,5-二碘-L-甲状腺原氨酸(3,5-diiodo-L-tyrosine)、D-三碘甲状腺原氨酸(D-T3)、L-三碘甲状腺原氨酸(L-T3)、反三碘甲状腺原氨酸(rT3)有交叉反应多抗,纯化特异性的抗人T4多克隆抗体并用FITC标记T4类似物,用抗FITC抗体包被微孔板.反应时,将FT4校准品或样品、FITC-T4类似物、T4抗体-HRP依次加入包被有抗FTTC抗体的化学发光板中,FITC -T4类似物与FT4校准品或样品,非均衡竞争结合T4抗体-HRP,从而在微孔板上形成免疫反应复合物T4类似物-T4抗体-HRP,通过酶促化学发光反应,就可以显示出与样品FT4浓度负相关的相对光单位(relative light unit,RLU),建立FT4化学发光检测方法.结果]抗体经酶联免疫吸附实验证明对T4具有高度特异性,与3-iodo-L-tyrosine、3,5-diiodo-L-tyrosine、L-T3、D-T3、rT3无交叉反应.该方法建立的试剂盒标准曲线的线性相关系数不小于0.9900;分析灵敏度为0.9 pmol/L;线性检测范围0.9～130 pmol/L;精密度测试批内变异系数CV＜ 10％;与进口发光免疫分析系统做线性相关比较,线性回归系数为1.0852,相关系数为0.9673,临床符合率良好；本方法建立的试剂盒经加速稳定性试验37℃放置7d与2～8℃放置7d无异.结论]综上所述,应用FITC系统建立的非均衡竞争FT4化学发光免疫分析灵敏度高、特异性好适于临床诊断应用.

Detection of human serum free thyroxine by non-equilibrium competitive chemiluminesce immunoassay using FITC system

【Objective】To prepare polyclonal antibodies（mAb） against human thyroxine and to establish a new method of non-equilibrium competitive chemiluminescence immunoassay（CLIA） with fluorescein isothiocyanate（FITC） for serum free thyroxine determination.【Methods】 Ployclonal antibody was prepared by immunizing New Zealand rabbits with human T4-BSA and purified by affinity chromatography.T4 polyclonal antibody was labeled with horse radish peroxidase（HRP） and FITC was conjugated to T4 analogue.Microwell was coated by anti-FITC antibody.In the assay,the calibrators（or samples） FITC-T4 analogue and T4 antibody-HRP were added to the microwell plate.FITC-T4 analogue would combine to anti-FITC antibody to form solid phase T4 analogue on the microwell.T4,if exist in sample,would combine to T4 antibody-HRP compete with solid phase T4 analogue,and less T4 analogue-T4 antibody-HRP complex would be formed on the microwell.The amount of the complex,which showed negative correlation to the concentration of free T4 in the samples,would be measured through enzymatic chemiluminescence immunoassay reaction.Sensitivity,precision,stability and clinical correlation for this method were evaluted.【Results】 Through ELISA showed high specificity of the prepared antibody but no cross reaction with 3-iodo-L-tyrosine,3,5-diiodo-L-tyrosine,D-T3,L-T3,rT3.The detection of human serum FT4 by chemiluminesce immunoassay with FITC system was that the linear correlation of the master curve was not lower than 0.9900;the analytical sensitivity was 0.9 pmol/L;the measuring range was 0.9~130 pmol/L,the coefficients of variability among batch were all under 10%;the linear regression coefficient was 1.0852,the linear correlation was 0.9673;the stability was established to be good through 37℃ accelerated stability test.【Conclusion】The quantitative diagnostic kit for free T4 by non-equilibrium competitive chemiluminesce immunoassay using FITC system provides a rapid and accurate determination of FT4 and is suitable for application to clinical diagnosis.