引用本文:宋 伟,刘玲珑,王鹏飞,姚清媚,周素芳.ELISA法检测血清中抗CNN2抗体的实验条件优化性探讨[J].中国临床新医学,2020,13(5):462-466.
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ELISA法检测血清中抗CNN2抗体的实验条件优化性探讨
宋 伟,刘玲珑,王鹏飞,姚清媚,周素芳
530021 南宁,广西医科大学基础医学院生物化学与分子生物学教研室
摘要:
[摘要] 目的 探讨血清抗钙调节蛋白Calponin 2(CNN2)抗体的最适酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)实验条件。方法 将BL21(DE3)pLysS-pColdⅢ-C基因工程菌重组表达而分离纯化的CNN2包被至酶标板。采集肝细胞癌患者血清和正常人血清,探讨蛋白包被浓度、血清稀释液的选择、封闭方式及条件的选择、HRP标记亲和素稀释度的最适条件。结果 CNN2蛋白浓度为10 μg/ml时为最适蛋白包被浓度。0.5%胎牛血清蛋白(BSA)为最适封闭方式。使用PBST来稀释血清,成本低,效果好。亲和素的最适稀释比为1∶9 000。最适实验条件的重复性良好。ROC曲线分析结果显示,最适实验条件下,该检测方法ROC曲线下面积(AUC)为0.978(95%CI:0.947~1.000,P=0.000);截断值为0.484,其对应的灵敏度为96.0%,特异度为93.0%。结论 构建了检测血清抗CNN2抗体的最适ELISA实验条件。
关键词:  肝细胞癌  Calponin 2  酶联免疫吸附测定
DOI:10.3969/j.issn.1674-3806.2020.05.08
分类号:R-331
基金项目:国家自然科学基金项目(编号:81460432)
Optimization of experimental conditions for detection of serum anti-CNN2 antibody by ELISA method
SONG Wei, LIU Ling-long, WANG Peng-fei, et al.
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Guangxi Medical University, Nanning 530021, China
Abstract:
[Abstract] Objective To explore the optimal enzyme-linked immunosorbent assay(ELISA) experimental conditions for detection of serum anti-calmodulin Calponin 2(CNN2) antibody. Methods BL21(DE3) pLysS-pColdⅢ-C genetically engineered bacteria were recombinantly expressed and CNN2 was isolated and purified, and coated onto a microtiter plate. Serum samples from the patients with hepatocellular carcinoma(HCC) and normal controls were collected to investigate the concentration of protein coating, the selection of serum diluents, the selection of sealing methods and conditions, and the optimal conditions of horseradish peroxidase(HRP)-labeled avidin dilution. Results When the concentration of CNN2 protein was 10 μg/ml, the concentration of protein coating was the optimal concentration. Using 0.5% bovine serum albumin(BSA) was the best sealing method. Using PBST to dilute serum had low costs and good results. The optimal dilution ratio of avidin was 1∶9 000. The repeatability of the optimal experimental conditions was good. The results of receiver operating characteristic(ROC) curve analysis showed that under the optimal experimental conditions, the area under the ROC curve(AUC) of the detection method was 0.978(95%CI: 0.947~1.000, P=0.000), and the cut-off value was 0.484, and the corresponding sensitivity was 96.0%, and the specificity was 93.0%. Conclusion The optimal ELISA experimental conditions for the detection of serum anti-CNN2 antibody is constructed.
Key words:  Hepatocellular carcinoma(HCC)  Calponin 2(CNN2)  Enzyme-linked immunosorbent assay(ELISA)