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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。
Objective To prepare anti-human T3 polyclonal antibody and develop a new type of unbalanced competition technique by using fluorescein isothiocyanat (FITC) system to establish a new method that can be widely used in the clinical detection of free triiodothyronine , FT3) method. Methods New Zealand white rabbits were immunized with bovin serum albumin (BSA) as antigens to prepare anti - human T3 polyclonal antibody. HRP was purified and conjugated with horseradish peroxidase (HRP) by affinity chromatography ). T3-analog was labeled with FITC. The anti-FITC antibody was used to coat the luminescent plate. FITC-T3 analogs were combined with anti-FITC antibody to form solid-phase antigen. By the principle of non-equilibrium competition, the solid phase antigen competed with anti-T3 antibody-HRP in serum with FT3 and calculated by standard curve FT3 concentration, a methodological evaluation was established and compared to a detection system coated directly with Bovine serum gamma globulin (BGG) (non-FITC system). The method was applied to the detection of 120 clinical serum specimens and the quantitative results were compared with the quantitative results of the Echo-2010 automatic chemiluminescence immunoassay system (Elec-sys 2010). Results The antibody was proved to be highly specific to T3 by enzyme-linked immunosorbent assay. The calibration curve of non-equilibrium competitive chemiluminescence immunoassay established by FITC system showed a linearity of R> 0.99, a linear range of 0.25 ~ 50 pg / ml and a sensitivity of 0.25 pg / ml. CV and CV of inter- %, The test result is better than the non-FITC system test. For the detection of clinical serum samples, the quantitative results with the quantitative results of the German Elecsys2010 quantitative kit has a good correlation, the correlation coefficient r = 0.962 2, the two measured values were not significantly different. Conclusion The prepared anti-human T3 polyclonal antibody is highly specific. The unbalanced competitive FT3 detection chemiluminescence method based on FITC system has the advantages of good precision, high sensitivity, good stability and good accuracy, and achieves the clinical determination Demand, can replace imported chemiluminescence products for the detection of clinical samples.