摘要
目的建立定量检测新生儿滤纸干血斑中促甲状腺素（TSH）的一步化学发光免疫分析方法。方法应用含有表面活性剂NP40s的滤纸干血斑高效蛋白洗脱液作为TSH洗脱液,抗人h-TSH单克隆抗体（mAb）作为辣根过氧化物酶（HRP）标记抗体,抗人α-TSH的mAb作为固相包被抗体,鲁米诺作为发光底物,建立一步新生儿滤纸干血中TSH双抗体夹心化学发光免疫定量检测方法。结果所用高效蛋白洗脱液NP40s最佳工作浓度为55‰,最佳洗脱反应时间为3 h,该方法检测灵敏度为1.02 mIU/L,线性范围为1.6-160 mIU/L,批内变异系数为3.18%-8.95%,批间变异系数为5.43%-9.26%,添加回收率在87.23%-114.36%之间,稀释回收率在96.80%-111.00%之间,与TSH类似物黄体生成素h链（hLH）、促卵泡激素h链（hFSH）和人绒毛膜促性腺激素（HCG）均无明显交叉反应,与现有新生儿滤纸干血TSH检测试剂盒相关性良好,相关系数为0.969 90。结论该方法 TSH洗脱和与抗体结合反应只需一步就能实现,整个检测过程在室温下就可完成,并且具有较高的灵敏度、重复性和准确性,完全可用于新生儿滤纸干血中TSH的定量检测。
To quantitatively detect thyroid stimulating hormone（TSH） in neonatal dried blood spotted fiter paper,an one-step chemiluminescence immunoassay（CLIA） was developed based on high efficiency protein eluent with surfactant NP40s,double antibodies sandwich reaction mode and luminal-hydrogen peroxide（H2 O2） system catalyzed by horseradish peroxidase（HRP）.TSH elution and antigen-antibody binding reaction could be completed in one step at room temperature in 3 h;the optimal NP40s concentration of high efficiency protein eluent was 55‰.The detection limit of the one-step CLIA method was 1.02 mIU/L;the linear range was 1.6 -1.60 mIU/L.The intraassay coefficient of variation was between 3.18%-8.95%,and interassay coefficient of variation was between 5.43%-9.26%.The results of fortified and dilution recovery tests were between 87.23%-114.36%% and 96.80%- 111.00%,respectively.There was no obvious cross reaction with hLH,hFSH or HCG.Furthermore,the developed method showed well correlation with a commercial kit in TSH detection of 58 dried blood spotted filter paper samples（correlation coefficient was 0.969 90）.In conclusion,the developed method is sensitive,repeatable,accurate,and convenient,which can be used for quantitative detection of TSH from neonatal dried blood spotted fiter paper.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2013年第10期887-893,共7页
Immunological Journal
