人乳头瘤病毒16型E6和L1蛋白荧光免疫层析检测方法的建立

A fluorescence immunochromatography method for detection of human papillomavirus type 16 E6 and L1 proteins

摘要:

本研究旨在建立同时测定人乳头瘤病毒(human papilloma virus, HPV)16型E6蛋白和L1蛋白浓度的时间分辨荧光免疫层析定量检测方法。通过优化载有镧系元素荧光微球标记抗体量、包被抗体浓度和反应时间，制备联合检测试纸条，对所建立的联合检测方法进行性能及临床一致性评价。HPV16 L1及E6抗体最佳标记量分别为8 μg、10 μg，包被划膜量均为1.5 mg/mL；最佳检测时间为加样后10 min；检测HPV16 L1及E6蛋白线性范围分别为5–320 ng/mL、2–64 ng/mL；最低检测限分别为1.78 ng/mL、1.09 ng/mL；与人类免疫缺陷病毒(human immunodeficiency virus, HIV)、梅毒螺旋体(Treponema Pallidum, TP)、HPV18 E6及L1蛋白无交叉反应；回收率在97%至107%之间；采用本研究制备的试纸条检测样本，区分宫颈癌及癌前病变患者与健康人的灵敏度为97.46%，特异性为90.57%，诊断准确率为95.32%，受试者工作特征曲线下面积(area under curve, AUC)为0.980 1，95%置信区间(confidence interval, CI) (0.956 5, 1.000 0)。本研究制备的时间分辨荧光免疫层析联合定量检测HPV16型E6蛋白与L1蛋白试纸条可为宫颈癌及癌前病变患者的早期筛查及病程评估提供辅助诊断方法。

Abstract:

This study aims to establish a time-resolved fluorescence immunochromatography method for simultaneous determination of human papillomavirus (HPV) type 16 E6 and L1 protein concentrations. The amount of lanthanide microsphere-labeled antibodies, the concentration of coated antibodies, and the reaction time were optimized, and then a test strip for the simultaneous determination of the protein concentrations was prepared. The performance of the detection method was evaluated based on the concordance of the results from clinical practice. The optimal conditions were 8 μg and 10 μg of HPV16 L1 and E6-labeled antibodies, respectively, 1.5 mg/mL coated antibodies, and reaction for 10 min. The detection with the established method for L1 and E6 proteins showed the linear ranges of 5–320 ng/mL and 2–64 ng/mL and the lowest limits of detection of 1.78 ng/mL and 1.09 ng/mL, respectively. There was no cross reaction with human immunodeficiency virus (HIV), treponema pallidum (TP), or HPV18 E6 and L1 proteins. The average recovery rate of the established method was between 97% and 107%. The test strip prepared in this study showed the sensitivity, specificity, and diagnostic accuracy of 97.46%, 90.57%, and 95.32%, respectively, in distinguishing patients with cervical cancer and precancerous lesions from healthy subjects, with the area under the curve (AUC) of 0.980 1 and 95% Confidence Interval (CI) of 0.956 5 to 1.000 0. The time-resolved fluorescence immunochromatography combined with the test strips prepared in this study showed high sensitivity, high accuracy, simple operation, and rapid reaction in the quantitation of HPV16 E6 and L1 proteins. It thus can be used as an auxiliary method for the diagnosis and early screening of cervical cancer and precancerous lesions and the assessment of disease course.