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目的:建立一种简便、快速和高敏感度检测甲胎蛋白(alpha-fetoprotein,AFP)变异体AFP-L3的新方法。方法:采用薄膜蒸发法制备扁豆凝集素(lens culinaris agglutinin,LCA)修饰的纳米功能磁球(LCA-纳米功能磁球),并通过磁性作用分离结合AFP-L3的LCA-纳米功能磁球,从而富集AFP-L3。结合人AFP定量检测试纸(时间分辨荧光法)检测AFP-L3的浓度,建立基于纳米功能磁球的AFP-L3检测方法。采用AFP-L3标准品比较LCA-纳米功能磁球法和已商品化的亲和离心层析柱法对AFP-L3的回收率,并应用上述2种方法分别检测了64例AFP阳性肝癌患者血清中AFP-L3的浓度并比较检测结果。选择830例血清样品,包括100例正常健康人群,300例慢性肝炎患者、228例肝硬化患者和202例肝癌患者,采用LCA-纳米功能磁球检测AFP-L3浓度,并比较各组的差异。结果:成功制备获得LCA偶联的纳米功能磁球,并建立了纳米磁球提取AFP-L3的方法。与亲和层析离心柱提取AFP-L3的方法进行比较,在AFP-L3浓度较高(4 000 ng/m L)时,LCA-纳米功能磁球法对AFP-L3的回收率显著优于亲和层析离心柱法(P<0.05),而在低浓度时两者的回收率相当。64例AFP阳性肝癌患者血清中AFP-L3的浓度检测结果显示,2种检测方法检测AFP-L3的阳性率均为37.5%,差异无统计学意义(P>0.05)。830例临床样本的检测结果显示,正常健康人群、慢性肝炎患者、肝硬化患者以及肝癌患者血清中AFP和AFP-L3的双阳性率分别为0.0%、3.3%、19.7%和60.9%,肝癌患者的阳性率明显高于良性肝病患者或正常人健康人群,和已报道结果一致。结论:采用LCA修饰的纳米功能磁球通过自动化提取AFP-L3的方法是一种准确、高效而简单的检测AFP-L3的方法。
OBJECTIVE: To establish a simple, rapid and sensitive method for the detection of alpha-fetoprotein (AFP) variant AFP-L3. Methods: Nanofunctional magnetic spheres (LCA-nanospheres) modified by LCA were prepared by thin-film evaporation. LCA-nanofunctional magnetic spheres bound to AFP-L3 were separated by magnetic interaction. Enriched AFP-L3. Combined with AFP quantitative test strip (time-resolved fluorescence method) to detect AFP-L3 concentration, the establishment of nano-functional magnetic ball AFP-L3 detection method. The AFP-L3 standard was used to compare the recoveries of AFP-L3 by LCA-nano-functional magnetic ball method and the commercial affinity centrifugation column method, and 64 cases of AFP positive hepatocellular carcinoma In the concentration of AFP-L3 and compare the test results. A total of 830 serum samples were collected, including 100 normal healthy subjects, 300 chronic hepatitis patients, 228 patients with liver cirrhosis and 202 hepatocellular carcinoma patients. The concentrations of AFP-L3 in LCA-Nano-functional magnetic sphere were measured and compared among groups. Results: LCA-coupled nanofunctional magnetic spheres were successfully prepared and the method of nanosphere extraction of AFP-L3 was established. Compared with the method of extracting AFP-L3 from affinity chromatography column, the recovery of AFP-L3 by LCA-NIFS was significantly better than that of AFP-L3 when the concentration of AFP-L3 was higher (4 000 ng / mL) Affinity chromatography with spin column method (P <0.05), while at low concentrations, the recovery rate of both was comparable. The results of AFP-L3 concentration in serum of 64 patients with AFP positive hepatocellular carcinoma showed that the positive rate of AFP-L3 detected by both methods was 37.5%, the difference was not statistically significant (P> 0.05). The results of 830 clinical samples showed that the positive rates of AFP and AFP-L3 in normal healthy people, chronic hepatitis patients, cirrhosis patients and liver cancer patients were 0.0%, 3.3%, 19.7% and 60.9%, respectively. The positive rate was significantly higher than that in patients with benign liver disease or healthy people, and reported the same result. Conclusion: The method of automated extraction of AFP-L3 by nanofunctional magnetic sphere modified by LCA is an accurate, efficient and simple method for the detection of AFP-L3.