荧光微球时间分辨免疫层析技术定量检测甲胎蛋白的研究

Quantitation of alpha fetoprotein(AFP) in human serum using fluorescence microspheres time-resolved immunochromatographic technology

目的建立荧光微球时间分辨免疫层析技术检测血清中甲胎蛋白（AFP）的反应体系并对该法进行评价。方法以包裹有Eu螯合物的荧光微球作为标记物,共价偶联抗AFP的单克隆抗体AC18#,将抗AFP单克隆抗体AC17#和羊抗鼠抗体喷涂于硝酸纤维素膜上分别作为检测线和质控线,制作免疫层析试纸条。结果标准曲线经Log-Log B函数处理,检测AFP的线性范围为0.03~1 000 U/ml,批内和批间变异系数（CV）分别为5.35%和8.94%,平均回收率为98.45%。该法检测100例健康人员的AFP水平为（2.04±1.92）U/ml,正常参考值为5.80 U/ml,与时间分辨免疫分析法（TRFIA）检测AFP有较好的相关性,r=0.978（P〈0.05）。结论本研究建立的荧光微球时间分辨免疫层析具有快速、灵敏、简便、经济、可单人份操作等优点,为检测AFP提供了一种高效的新途径,符合我国国情,便于临床推广。

This study was designed to develop a fluorescent microspheres time-resolved immunochromatographicassay for detection of alpha fetoprotein（AFP） in human serum, and to evaluate the method performance. The Eu-time-resolved fluorescent polystyrene particle conjugated with the monoclonal antibody AC18#was used as a fluorescent label. The monoclonal antibody AC17#and goat anti-mouse Ig G were immobilized on the nitrocellulose membrane as the test line and control line. The linear response range of the method to detect the serum AFP was 0.03-1 000 U/ml. And the intra- and inter- assay coefficient were 5.35% and 8.94%,respectively, while average recovery rate was 98.45%. The means of 100 healthy volunteers was（2.04 ±1.92） U/ml,thus the normal ranges of serum AFP levels were less than 5.80 U/ml. Measurements obtained by the fluorescent microspheres time-resolved immunochromatographic strip showed a correlation coefficient（r） of 0.978（P 0.05） with measurements obtained by TRFIA. In conclusion, the fluorescent microspheres time-resolved immunochromatographic strip for assaying AFP provided high sensitivity, rapid, simple operation, low cost, single-servings and can be extensively used for clinical operations.