非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2022.03.014

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (3): 813-821.doi: 10.11843/j.issn.0366-6964.2022.03.014

非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立

徐玲玉¹, 曹琛福², 李中圣⁴, 陈俊宏¹, 柳腾飞¹, 王新凯¹, 贾伟新^1,3*

1. 华南农业大学兽医学院, 国家非洲猪瘟区域实验室(广州)/人兽共患病防控制剂国家地方联合工程实验室/农业农村部人畜共患病重点实验室/农业农村部兽用疫苗创制重点实验室/广东省动物源性人兽共患病防控重点实验室, 广州 510642;
2. 深圳海关动植物检验检疫技术中心, 深圳 518045;
3. 岭南现代农业科学与技术广东省实验室, 广州 510642;
4. 广东海大畜牧兽医研究院有限公司, 广州 511400

收稿日期:2021-06-10 出版日期:2022-03-23 发布日期:2022-03-31
通讯作者: 贾伟新,主要从事动物传染病研究,E-mail:jiaweixin@scau.edu.cn
作者简介:徐玲玉(1997-),女,四川成都人,硕士生,主要从事动物传染病研究
基金资助:
广东省重点领域研发计划资助(2019B020211003);广东省科技计划项目(2021B1212030015);国家质量监督检验检疫总局科技项目(2016IK233);国家"863"高新技术项目(2012AA101301)作者简介:徐玲玉(1997-),女,四川成都人,硕士生,主要从事动物传染病研究

Preparation of Monoclonal Antibody against African Swine Fever Virus p54 Protein and Development of Blocking ELISA

XU Lingyu¹, CAO Chenfu², LI Zhonsheng⁴, CHEN Junhong¹, LIU Tengfei¹, WANG Xinkai¹, JIA Weixin^1,3*

1. African Swine Fever Regional Laboratory of China (Guangzhou), National Local Joint Engineering Laboratory of Zoonosis Prevention and Control Agents, Key Laboratory of Zoonoses of Ministry of Agriculture and Rural Affairs, Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs, Guangdong Key Laboratory for Prevention and Control of Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center, Shenzhen 518045, China;
3. Lingnan Laboratory of Modern Agricultural Science and Technology of Guangdong Province, Guangzhou 510642, China;
4. Guangdong Haid Institute Co. Ltd. of Animal Husbandry & Veterinary, Guangzhou 511400, China Husbandry & Veterinary, Guangzhou 511400, China

Received:2021-06-10 Online:2022-03-23 Published:2022-03-31

摘要/Abstract

摘要： 旨在建立检测非洲猪瘟病毒(ASFV)血清抗体的阻断ELISA方法。采用原核表达的ASFV p54蛋白作为包被抗原,并制备了针对p54蛋白的单克隆抗体,采用方阵滴定法确定了阻断ELISA方法的最佳反应条件,并对建立的方法进行了敏感性、特异性、重复性和符合性评价。结果显示,抗原最佳包被浓度为2.0 μg·mL^-1,抗原包被温度及时间为4℃过夜,被检血清稀释度为1:40,酶标单抗稀释度为1:1 000,被检血清作用时间为1 h,酶标单抗作用时间为30 min,底物作用时间为10 min。经阴、阳性样品检测和特异性、敏感性试验,当临界值阻断率≥ 55.2%时,判定为阳性,当阻断率<43.4%时,判定为阴性,介于两者之间判定为可疑。该方法与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、口蹄疫病毒、猪流感病毒、猪伪狂犬病病毒阳性血清不发生交叉反应,特异性好。批内重复试验及批间重复试验的变异系数(CV)均在10%以内。将该方法进行初步应用,并与商品化ELISA试剂盒进行符合性试验,两者的检测符合率为98%。综上表明,建立的阻断ELISA方法具有良好的特异性、敏感性和重复性,可用于ASFV临床样品的抗体检测。

关键词: 非洲猪瘟病毒, ASFV, 阻断ELISA, p54蛋白, 单克隆抗体

Abstract: To develop a blocking ELISA for the detection of antibodies against African swine fever virus (ASFV), ASFV P54 protein expressed in E. coli was used as the coating antigen, and monoclonal antibody against P54 protein was prepared. The optimum reaction conditions of blocking ELISA were determined by square matrix titration, and the sensitivity, specificity, repeatability and conformity were evaluated. The results showed that the optimum conditions of the assay were as follows:the antigen coating concentration was 2.0 μg·mL^-1, antigen coating temperature and time were 4℃ overnight, the dilution of tested serum was 1:40, the dilution of enzyme-labelling mAb was 1:1 000, the action time of tested serum was 1 h, the action time of enzyme-labelling mAb was 30 min, and the action time of substrate was 10 min. Through the detection of negative and positive samples, as well as specificity and sensitivity tests, it was determined that the criteria for determining the negativity and positivity of the assay were:Results determined to be positive at cut-off blocking rate ≥ 55.2%, negative at blocking rate <43.4%, and suspicious between the two values. The assay had no cross reaction with positive sera of classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, foot-and-mouth disease virus, swine influenza virus and porcine pseudorabies virus. The coefficient of variation (CV) of intra- and inter-batch repeated tests were both within 10%. The coincidence rate between the assay and commercial ELISA kit was 98%. In conclusion, the blocking ELISA has ideal specificity, sensitivity and repeatability, and can be used for the antibody detection of ASFV clinical samples.

Key words: African swine fever virus, ASFV, blocking ELISA, p54 protein, mAb

中图分类号:

S852.659.1

徐玲玉, 曹琛福, 李中圣, 陈俊宏, 柳腾飞, 王新凯, 贾伟新. 非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立[J]. 畜牧兽医学报, 2022, 53(3): 813-821.

XU Lingyu, CAO Chenfu, LI Zhonsheng, CHEN Junhong, LIU Tengfei, WANG Xinkai, JIA Weixin. Preparation of Monoclonal Antibody against African Swine Fever Virus p54 Protein and Development of Blocking ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 813-821.

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非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立

Preparation of Monoclonal Antibody against African Swine Fever Virus p54 Protein and Development of Blocking ELISA

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