摘要
目的制备抗人三碘甲腺原氨酸(T3)多克隆抗体,应用异硫氰酸荧光素(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总三碘甲腺原氨酸(TT3)的方法。方法以T3-牛血清清蛋白(BSA)为抗原免疫新西兰大白兔,制备抗人T3多克隆抗体,亲和层析法纯化特异性的抗人T3多克隆抗体并连接辣根过氧化物酶(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,固相抗原与T3校准品或样本中的T3非均衡竞争结合T3-HRP抗体,建立标准曲线,血清中TT3通过标准曲线计算浓度。由此建立TT3化学发光检测方法进行方法学评价并与直接用T3-牛血清γ球蛋白(BGG)包被(非FITC系统)的检测系统进行比较。将该方法应用于临床100份血清标本的检测,定量结果与德国Roche2010电化学发光全自动免疫分析系统(Elecsys2010)结果进行比较。结果抗体对T3具有高度特异性,线性相关系数r>0.99,线性范围为0.1～8ng/mL,分析灵敏度为0.1ng/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定量试剂盒的结果有很好的相关性,相关系数为0.9619,临床符合率良好。结论该方法精密性好、灵敏度高、稳定性强,有良好的准确性,适用于临床标本的检测。
Objective To prepare polyclonal antibodies（mAb） against human triiodothyronine（T3） and to establish a new method of non-equilibrium competitive chemiluminescence immunoassay（CLIA） with fluorescein isothiocyanate（FITC） for detection of serum total T3（TT3） .Methods Polyclonal antibody of human T3,prepared by immunizing New Zealand rabbits with human T3-bovin serum albumin（BSA） ,was purified by affinity chromatography and was labeled with horse radish peroxidase（HRP） .FITC was conjugated to T3 analogue.Microwell was coated by anti-FITC antibody.In this assay,calibrators（or samples） ,FITC-T3 analogue and anti-T3 antibody-HRP were added to coated microwell plate.FITC-T3 analogue would combine to anti-FITC antibody to form solid phase T3 analogue on microwell.T3,if exist in samples,would combine to anti-T3 antibody-HRP compete with solid phase T3 analogue,and less T3 analogue-anti-T3 antibody-HRP complex would be formed on microwell.The amount of complex,which showed negative correlation with concentration of TT3 in samples,would be measured through CLIA reaction.Compared with the method of coating by T3-BGG（non-FITC system） ,sensitivity,precision,stability and clinic correlation for this method were evaluated.This method as well as Roche Elecsys 2010 System were used for detecting TT3 in 100 clinical serum samples.Results The prepared antibody was with high specificity to T3.The linear correlation coefficient of constructed CLIA with FITC system for the detection of human TT3 was more than 0.99,with linear range of 0.1-8 ng/mL,sensitivity of 0.1 ng/mL,intra-assay and inter-assay precision under 5%,which was better than those of non-FITC system.Compared with Elecsys 2010 system,the correlation coefficient was 0.961 9.Conclusion The quantitative diagnostic kit for TT3 by non-equilibrium competitive CLIA using FITC system could provide a rapid and accurate determination of TT3 and be suitable for application for clinical diagnosis.
出处
《国际检验医学杂志》
CAS
2012年第8期899-901,903,共4页
International Journal of Laboratory Medicine
基金
天津市科技型中小企业技术创新专项资金项目(09ZXCXSH05300)
