Liu Xin'er, Zhao Yinzhen, Niu Nannan, Li Lingke, DU Xueli, Guo Jinxiang, Zhang Yingfu, Wang Jichuang, Zhang Yiqing, Wang Yunlong
The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China.
Henan Bioengineering Technology Research Center, Zhengzhou 450000, Henan, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Nov 25;40(11):4266-4276. doi: 10.13345/j.cjb.240104.
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.
本研究旨在建立一种时间分辨荧光免疫层析法，用于同时测定人乳头瘤病毒16型（HPV16）E6和L1蛋白浓度。对镧系元素微球标记抗体的用量、包被抗体的浓度及反应时间进行优化，进而制备出可同时测定蛋白浓度的检测试纸条。依据临床实际结果的一致性对该检测方法的性能进行评估。最佳条件分别为HPV16 L1和E6标记抗体8 μg和10 μg、包被抗体1.5 mg/mL以及反应10分钟。采用所建立的方法对L1和E6蛋白进行检测，其线性范围分别为5 - 320 ng/mL和2 - 64 ng/mL，最低检测限分别为1.78 ng/mL和1.09 ng/mL。与人免疫缺陷病毒（HIV）、梅毒螺旋体（TP）或HPV18 E6和L1蛋白均无交叉反应。所建立方法的平均回收率在97%至107%之间。本研究制备的检测试纸条在区分宫颈癌及癌前病变患者与健康受试者时，灵敏度、特异性及诊断准确率分别为97.46%、90.57%和95.32%，曲线下面积（AUC）为0.980 1，95%置信区间（CI）为0.956 5至1.000 0。本研究制备的检测试纸条结合时间分辨荧光免疫层析法在定量检测HPV16 E6和L1蛋白时，具有灵敏度高、准确性高、操作简便、反应快速的特点。因此，可作为宫颈癌及癌前病变诊断、早期筛查及病程评估的辅助方法。
