摘要
目的 通过双位点夹心化学发光免疫分析原理,建立一种高灵敏度测定人血清促甲状腺激素(thyroid stimulating hormone,TSH)的化学发光免疫分析方法.方法 将样本与预包被有抗促甲状腺激素(TSH)抗体的超顺磁珠,以及抗TSH-碱性磷酸结合物添加到反应管中共孵育,待测样本中的TSH通过夹心法与磁珠和抗TSH-碱性磷酸结合物与形成双位点复合物.与磁珠结合的复合物吸附到磁场上,用缓冲液洗去未结合的物质.加入发光底物3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐[3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane,AMPPD],测定相对光子强度(RLU),通过标准曲线计算待测样本中TSH的浓度.结果 本研究建立的TSH检测方法,最低检测限为0.003μIU/mL,批内CV小于1％,总CV小于3％,正确度样本实测值与靶值之间相对偏差均在±12.5％范围内,线性范围为0.005～100μIU/mL且相关系数(r)高于0.9900.开封稳定性试验的相对偏差<10％.结论 该方法检测血清TSH浓度具有灵敏度高、准确性好、线性范围广、稳定性强的优点,各项指标均达到临床检测要求,可以推广使用.
Abstract
Objective To develop a high-sensitivity chemiluminescence immunoassay method for the determination of thyroid stimulating hormone (TSH)in human serum based on the principle of double-site sandwich chemiluminescence immunoassay.Methods The study samples were co-incubated with superparamagnetic beads pre-coated with anti-thyrotropin antibody (TSH)and anti-TSH-alkaline phosphate conjugate in the reaction tube.TSH in the sample was combined with magnetic beads and anti-TSH-alkaline phosphate conjugate by sandwich method to form a two-site complex.The complex bound to the magnetic bead was adsorbed to the magnetic field and the unbound material was washed away with a washing buffer.The luminescent substrate, 3-(2-spiroadamantane )-4-methoxy-4-(3-phosphoryloxy )-phenyl-1 ,2-dioxetane (AMPPD)was added,and the relative photon intensity (RLU)was measured.The concentration of TSH in the sample was calculated using a standard curve.Results TSH detection method established in this study showed that the minimum detection limit was 0.003μIU/mL,in-batch CV was less than 1％,and total CV was less than 3％.The relative deviation between the measured value and the target value of the sample was within the range of ±12.5％,while the linear range was from 0.005 to 100μIU/mL, and the correlation coefficient (r)was higher than 0.9900.The relative deviation of the stability was less than 10％.Conclusion The proposed method in this study demonstrates a high sensitivity,high accuracy,wide linear range and good stability for quantifying serum TSH,and all indicators meet the requirements of clinical testing and can be popularized for future applications.
基金项目
国家重点研发计划子课题(2017YFF0205401)
维普
万方数据