摘要
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.
出处
《中华核医学杂志》
CAS
CSCD
北大核心
2010年第2期75-79,共5页
Chinese Journal of Nuclear Medicine
基金
基金项目:广州经济技术开发区、广州高新技术产业开发区、广州出口加工区、广州保税区科技项目(2008Ss-P126)