应用FITC系统建立非均衡竞争游离三碘甲腺原氨酸（FT3）化学发光法

目的制备抗人T3多克隆抗体,应用异硫氰酸荧光素(fluorescein isothiocyanat,FITC)系统开发新型非均衡竞争技术,建立一种能广泛应用于临床检测游离三碘甲腺原氨酸(free triiodothyronine,FT3)的方法。方法以T3-牛血清白蛋白(bovin serum albumin,BSA)为抗原免疫新西兰大白兔,制备抗人T3多克隆抗体,亲和层析法纯化并联接辣根过氧化物酶(horse radish peroxidase,HRP)。用FITC标记T3-类似物。采用抗FITC抗体包被发光板,FITC-T3类似物与抗FITC抗体结合形成固相抗原,通过非均衡竞争法原理,固相抗原与血清中FT3竞争结合抗T3抗体-HRP,通过标准曲线计算FT3浓度,建立方法学评价并与直接用T3-牛血清γ球蛋白(Bovine serum gamma globulin,BGG)包被(非FITC系统)的检测系统相比较。将本方法应用于临床120份血清标本的检测,定量结果与德国罗氏2010电化学发光全自动免疫分析系统(Elec-sys 2010)定量结果进行比较。结果抗体经酶联免疫吸附实验证明对T3、具有高度特异性。应用FITC系统建立的非均衡竞争化学发光免疫分析标准曲线的线性R>0.99,线性范围0.25～50 pg/ml,分析灵敏度为0.25 pg/ml,精密度测试批内、批间变异系数CV<5%,检测结果优于非FITC系统的检测。对于临床血清标本的检测,其定量结果与德国Elecsys2010定量试剂盒的定量结果具有很好的相关性,相关系数r=0.962 2,两者的测定值无显著性差异。结论制备的抗人T3多克隆抗体特异性强,以FITC系统为平台建立的非均衡竞争FT3检测化学发光法具有精密性好、灵敏度高、稳定性强,有良好的准确性,达到临床测定的需求,可代替进口化学发光产品用于临床样本的检测。

Detection of free triiodothyronine in human serum by non-equilibrium competitive chemiluminescence immunoassay using FITC system

Objective To prepare a polyclonal antibody(mAb) against human triiodothyronine(T3) and to establish a new method of non-equilibrium competitive chemiluminescence immunoassay(CLIA) with FITC system for clinical determination of serum free triiodothyronine(FT3).Methods Polyclonal antibody of T3 was prepared by immunizing New Zealand rabbits with human T3-bovine serum albumin(BSA),purified by affinity chromatography,and labeled with horse radish peroxidase(HRP).FITC was conjugated to T3-analogue,and microwell was coated by anti-FITC antibody.FITC-T3 analogue would combine to anti-FITC antibody to form solid phase T3-analogue on microwell.FT3,if existed in sample,would combine to T3 antibody-HRP and compete with solid phase T3-analogue.The amount of the complex would be measured through enzymatic chemiluminescence immunoassay reaction.Compared with those of the method coating by T3-bovine serum gamma globulin(BGG)(non-FITC system),the sensitivity,precision,stability and clinic correlation of this method were evaluated.This method as well as Roche Elecsys 2010 System was used in 120 clinical serum samples for FT3 determination.Results ELISA showed the prepared antibody had a high specificity to T3.The linear range and sensitivity of human serum FT3 detected by chemiluminescence immunoassay with FITC system were 0.25 to 50 pg/ml(R>0.99) and 0.25 pg/ml,respectively,and the coefficients of variability within and among batches were all under 5%,and these results were better than those of the non-FITC system.The results obtained from chemiluminescence immunoassay with FITC system were correlated with those of the Elecsys 2010 system,and the correlation coefficient was 0.962 2.Conclusion The antibody against human T3 with high purity and specificity is successfully prepared.The quantitative diagnostic kit for FT3 by non-equilibrium competitive chemiluminescence immunoassay using FITC system provides a rapid,sensitive and accurate determination of FT3 and is suitable for clinical diagnosis.