孕母血清游离β绒毛膜促性腺激素时间分辨荧光免疫试剂盒的研制与评价

Development and evaluation of time-resolved fluoroimmunoassay kit for maternal serum free human chorionic gonadotropin β subunit

目的研制一种用于产前筛查孕母血清游离β绒毛膜促性腺激素（freehCGβ）的时间分辨荧光免疫（TRFIA）试剂盒。方法采用2株freehCG13单克隆抗体（McAb），一株用于固相包被（3μg／ml），另一株用于标记铕（Eu3＋）制备Eu3＋-freehCG13MeAb，以β-萘甲酰三氟丙酮为主要成分的增强液，应用双抗体夹心法建立freehCGBTRFIA法，并对其进行性能评价。采用线性回归分析及配对t检验进行统计学分析。结果该试剂盒的剂量一反应曲线线性相关系数（r）达0．9990；分析内、分析间变异系数（CV）均小于10．00％；标准品各点实测值与标示值的偏差（DIFF）均小于5．00％；灵敏度不高于0．20ng／ml；以国家标准品为对照，标准品效价比在0．9130～1．100间；与人促甲状腺激素（hTSH）、人黄体生成素（hLH）、人卵泡刺激素（hFSH）和人绒毛膜促性腺激素（hCG）交叉反应低；线性范围为2．00—200ng／ml；与同类国外试剂比对，线性回归方程为y=x＋0．0565，r=0．9771，检测结果间差异无统计学意义（t=0．1839，P〉0．05）；国内外同类仪器比对检测，线性回归方程为y=0．9894x＋0．0957，r=0．9995，检测结果间差异无统计学意义（t=-0．9669，P〉0．05）；室间质量评价实测值与靶值偏倚（-11．93％～7．01％）符合标准；干扰实验表明乙二胺四乙酸钾（EDTA．K2）≥0．54mmol／L、柠檬酸钠≥1．06mmol／L、草酸钾（K2C204H20）≥2．12mmol／L、氟化钠（NaF）≥29．76mmo]／L时，自制试剂盒检测结果明显偏低（干扰程度均〉10％），肝素≤150U／L、血红蛋白≤12g／L、三酰甘油≤21．54mmol／L和胆红素≤818txmol／L对该试剂盒检测结果无明显干扰（干扰程度均〈10％）；温度升高会使样本检测结果偏高。结论该试剂盒具有灵敏度高、特异性强、准确度好、线性范围宽等优点，与国外的同类产品性能相仿，能满足临床需要。

Objective To promote the development of the prenatal screening, and to develop a time-resolved fluoroimmunoassay （TRFIA） for maternal serum free human chorionic gonadotropin β subunit （free hCG β） as an aid in prenatal screening, and to prepare its test reagent. Methods Solid-phase sandwich TRFIA was used for quantification of free hCG β, which involved two anti-free hCG β monoclonal antibodies （McAb）. One anti-free hCG β McAb was coated on microplates, the other was labeled by Europium. The main component of enhancement solution was 2-naphthalene trifluoroaeetone. The assay methodology was evaluated. Linear regression analysis and paired samples t-test were used. Results The correlation coefficient （r） of self-made kit dose-response curve reached 0.9990. The intra-assay and inter-assay coeffi- cient of variation （CV） was lower than 10.00%. The relative bias measured values of kit standard were lower than 5.00% difference from their labeled values. The sensitivity was better than 0.20 ng/ml. The ratio of self-made kit standard measured value over national standard value was 0.900 - 1. 100. There was little cross-reaction with human thyroid stimulating hormone （hTSH）, human luteinizing hormone （hLH）, human follicle stimulating hormone （hFSH） , and hCG. The linear range was 2.00 to 200 ng/ml. Compared with imported kit, y=x ＋ 0. 0565, r = 0. 977t, and the results showed no statistical significance （paired samples t-test, t =0. 1839, P 〉0.05）. The values of self-made kit tested by self-made equipmentand imported equipment showed no difference （ paired samples t-test, t = - 0. 9669, P 〉 0. 05 ）, y = 0. 9894x ＋0. 0957, r =0.9995. The results of external quality assessment achieved 100%. EDTA-K2 （ 0.54 mmol/L）, sodium citrate （ ≥1.06 mmol/L）, K2C2O4H2O （ ≥2. 12 mmol/L） or NaF （≥29.76 mmol/L） in the tested samples had interference in the assay （ interference 〉 10% ）. Sodium heparin （ ≤ 150 U/L）, haemoglobin （≤12 g/L）, triglyceride （ ≤21.54 mmol/L） or bilirubin （ ≤818 μmol/L） in the tested samples had no significant interference in the assay （ interference 〈 10% ）. The results of samples would be ascended due to the instability of hCG if the temperature was elevated. Conclusion The quality of selfmade kit was in keeping with imported kit.