检测游离β-HCG时间分辨免疫荧光分析法的建立及临床应用

Development and application of a time-resolved fluoroimmunoassay for the determination of free β-HCG

目的建立一种快速灵敏的时间分辨免疫荧光分析法（TRFIA）,用于定量检测孕妇血清中游离β人绒毛膜促性腺激素（β-HCG）的浓度。方法包被游离β-HCG单克隆抗体,铕（Eu3＋）标记抗β-HCG单克隆抗体,建立双抗体夹心法游离β-HCG TRFIA,对该试剂的各项指标进行评价。结果最低检测量〈0.06 ng/mL;线性检测范围为0.052-400 ng/mL;分析内和分析间变异系数分别为1.0%-6.4%、3.3%-5.1%;人黄体生成素（HLH）、人促甲状腺素（HTSH）、人促卵泡激素（HFSH）和人绒毛膜促性腺激素（HCG）与本试剂的交叉反应率分别为0.04%、0.03%、0.05%和0.11%;1 043份孕妇血样用该试剂与进口同类试剂同时检测,2种试剂检测值具有显著相关性（r=0.990,P=0.000）;血清样本2-8℃储存超过3 d,室温储存1 d或冻融2次,与新鲜样本比较,游离β-HCG浓度显著性增加（P〈0.05）。结论该试剂敏感性高、特异性强、操作简便快捷,具有良好的精密度,可以替代进口试剂用于临床样本的检测。为保证结果的可靠性,应注意样本的保存条件。

Objective To develop a rapid and sensitive method for quantitative determination of free beta subunit of human chorionic gonadotropin （β-HCG） in pregnant women by time-resolved fluoroimmunoassay （TRFIA）. Methods Monoclonal antibodies against the free β-HCG and β-HCG marked by enropium （ Eu^3＋） were used to develop the sand- wich TRFIA, and the performance and their characteristics were analyzed and evaluated by detecting 1 043 serum samples. Results The assay sensitivity was less than 0.06 ng/mL. The linear detection range was 0. 052-400 ng/mL. The intra- and inter-assay coefficients of variation were 1.0%-6.4% and 3.3%-5.1%. The cross reactivity rates were 0. 04%, 0. 03%, 0. 05% and 0. 11% with human luteinizing hormone （ HLH）, human thyroid stimulating hormone （ HTSH）, human follicle stimulating hormone （HFSH） and human chorionic gonadotropin（HCG） , respectively. The results of developed TRFIA reagent and imported reagent showed excellent correlation （ r = 0. 990, P = 0. 000 ） by detecting 1 043 serum samples. Compared with fresh samples, a significant increase （P 〈 0.05 ） in free β-HCG concentration was observed when stored at 2-8 ℃ for 3 d, room-temperature for 1 d, or freeze-thaw twice. Conclusions The developed free β-HCG TRFIA, with high sensitivity, specificity and precision, is a valuable method for clinical application instead of imported reagents. In addition, proper saving conditions for serum samples are essential for reliable results.