非洲猪瘟病毒抗体化学发光检测方法的建立与应用

doi:10.16431/j.cnki.1671-7236.2024.04.004

摘要/Abstract

摘要： 【目的】建立一种特异性强、敏感度高、操作简捷、高通量的非洲猪瘟病毒(African swine fever,ASFV)抗体化学发光检测方法,用于早期监测ASFV的流行情况。【方法】本研究以羧基磁珠作为固相载体偶联ASFV重组p72蛋白,用适量的牛血清白蛋白(BSA)进行封闭,加入检测样品进行反应,以兔抗猪IgG-AP抗体作为酶标抗体,加入相应底物进行显色,优化各项反应条件,建立ASFV抗体化学发光检测方法,并对该方法的特异性、敏感度、重复性和检出率进行验证。【结果】建立的ASFV抗体化学发光检测方法蛋白与羧基磁珠偶联最适pH为7.0,最佳蛋白偶联浓度为5 μg/mL,使用10% BSA溶液封闭效果最佳,反应磁珠终浓度为1.00 mg/mL,酶标二抗最佳工作稀释度为1∶40 000。同时该方法反应时间为30 min,血清稀释度为1∶512,敏感度高于商品化ASFV抗体ELISA检测试剂盒,与猪瘟病毒、A型口蹄疫病毒、O型口蹄疫病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪伪狂犬病病毒gE、猪伪狂犬病病毒gB抗体阳性标准血清均无交叉反应。重复性试验中,批内变异系数为4.41%~8.70%,批间变异系数为2.43%~8.07%,均＜10%。通过检测120份血清样品并与商业化ASFV抗体ELISA检测试剂盒进行对比分析,商业化ELISA试剂盒检出率为96.7%,本研究建立的化学发光检测方法检出率为100%。【结论】本研究建立的ASFV抗体化学发光检测方法可用于ASFV感染后抗体水平检测,为后续试剂盒的开发提供参考。

关键词: 非洲猪瘟病毒(ASFV); 重组p72蛋白; 化学发光; 抗体

Abstract: 【Objective】 The study was conducted to establish a chemiluminescent antibody detection method for African swine fever virus (ASFV) with high specificity,high sensitivity,simple operation and high throughput,which could be used for the early monitoring of the prevalence of ASFV.【Method】 In this study,we used carboxylated magnetic beads as a solid-phase carrier to couple ASFV recombinant p72 protein,which was closed with an appropriate amount of bovine serum albumin (BSA),and then added to the test sample for the reaction.Rabbit anti-porcine IgG-AP antibody was used as the enzyme-labeled antibody,and the corresponding substrate was added for the chromatography to optimize the conditions of each reaction, establish the chemiluminescent detection method of the ASFV antibody,and validate the specificity,sensitivity,reproducibility and detection rate of the method.【Result】 The chemiluminescence detection method for ASFV antibody was established,the optimal pH for protein coupling with carboxyl beads was 7.0,the optimal protein coupling concentration was 5 μg/mL,the best effect was achieved by using 10% BSA solution for closure,the final concentration of the reaction beads was 1.00 mg/mL,and the optimal dilution of enzyme-labeled secondary antibody was 1∶40 000.At the same time,the reaction time of this method was 30 min,and the serum dilution was 1∶512.The sensitivity of this method was higher than that of the commercial ASFV antibody ELISA detection kit,and there was no cross-reaction with the antibody-positive standard sera of Classical swine fever virus,Foot-and-mouth disease virus type A,Foot-and-mouth disease virus type O,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Porcine pseudorabies virus gE,Porcine pseudorabies virus gB.In the reproducibility test,the coefficients of variation ranged from 4.41% to 8.70% in intra-assay and from 2.43% to 8.07% in inter-assay,which were both ＜10%.By testing 120 serum samples and analyzing them in comparison with commercial ASFV antibody ELISA detection kit,the detection rate was 96.7% for commercial ELISA kit and 100% for the chemiluminescent detection method established in this study.【Conclusion】 The ASFV chemiluminescent antibody detection method established in this study could be used to detect the antibody level after ASFV infection and provide a reference for the development of subsequent kits.

Key words: African swine fever virus (ASFV); recombinant p72 protein; chemiluminescence; antibody

中图分类号:

S852.65

向国庆, 孙菁晗, 宋帅, 温肖会, 吕殿红, 贾春玲, 牛瑞辉, 顾有方, 罗胜军. 非洲猪瘟病毒抗体化学发光检测方法的建立与应用[J]. 中国畜牧兽医, 2024, 51(4): 1362-1371.

XIANG Guoqing, SUN Jinghan, SONG Shuai, WEN Xiaohui, LYU Dianhong, JIA Chunling, NIU Ruihui, GU Youfang, LUO Shengjun. Establishment and Application of a Chemiluminescent Detection Method for African Swine Fever Virus Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1362-1371.

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