高灵敏电化学发光法特异性测定人血清抗甲状腺球蛋白抗体

The Establishment of Electrochemical Luminescence Detection Method for Human Anti-Thyroglobulin Antibodies

目的通过竞争法一步法原理,建立一种检测人血清抗甲状腺球蛋白抗体(anti-TgAb)的电化学发光免疫分析(ELICA)方法。方法将样本、三联吡啶钌标记抗体和生物素标记甲状腺结合球蛋白(thyroglobulin,Tg)抗原三者共同孵育形成抗体-抗原复合体,再加入链霉亲和素包被的磁微粒与该复合物共同孵育形成钌标记抗体-生物素抗原-链霉亲和素磁颗粒复合物,通过磁场固定洗涤去除游离物质;然后加入三丙胺得到相对光子强度(relative light units,RLU)。检测该方法的灵敏度、线性范围、精密度、准确度及稳定性,并与罗氏公司的anti-TgAb检测试剂盒进行对比。结果anti-TgAb的最低检测限为5 IU/mL,线性范围为10~1000 IU/mL,准确度为0.900~1.100,批内变异和批间变异均小于5%。与罗氏公司anti-TgAb检测试剂盒的相关系数r=0.996(n=50),稳定性测试(37℃,8d)中,各项性能指标变异程度均符合标准[线性相关系数r=0.9998,准确度低值0.94,准确度高值0.93,批内精密度(CV)高值3.77%,低值4.34%]。结论该方法各项性能指标(灵敏度、精密度、准确度、稳定性等)均能达到临床检测要求,可供临床检测使用。

Objective To establish an electrochemiluminescence immunoassay method for detecting human serum anti-thyroglobulin antibody by the principle of one-step competition method.Methods The antibody,the antimony-labeled antibody and the biotin-labeled thyroid-binding globulin antigen were co-incubated to form an antibody-antigen complex,The complex was incubated with streptavidin-coated magnetic particles to form streptavidin magnetic particles-biotin antigen-tripyridinium labeled antibody complex.The unbound material was washed away by magnetic field washing,and then tripropylamine challenge buffer was added.Immediate detection of relative light units was then conducted.Results The method established in this study had a minimum detection limit of 5 IU/mL,a linear range of 10-1000IU/mL,and an accuracy of 0.900-1.100,within batch variation less than 8%,between batch variation less than 10%.Compared with Roche company’s anti-TgAb Assay Kit,the correlation coefficient was 0.9996(n=50).After 8 days of heat treatment at 37℃,the performance indicators were up to standard,and the test results showed not much difference.Conclusion The method from the current study has a high sensitivity,wide linear range,high accuracy and good stability,and various indexes can meet the requirements of clinical testing and can be popularized and for the clinical usage.