非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2022.12.019

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (12): 4337-4345.doi: 10.11843/j.issn.0366-6964.2022.12.019

非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

周改静, 罗俊聪, 石正旺, 万颖, 杨波, 曹丽艳, 宋锐, 田宏^*, 郑海学^*

中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室 OIE/国家口蹄疫参考实验室, 兰州 730046

收稿日期:2021-11-10 出版日期:2022-12-23 发布日期:2022-12-25
通讯作者: 田宏，主要从事动物病毒分子生物学及疫病诊断技术研究，E-mail：xibeitian0931@163.com；郑海学，主要从事动物传染病与流行病学研究，E-mail：zhenghaixue@caas.cn
作者简介:周改静(1994-)，女，河南新蔡人，硕士，主要从事动物传染病病原学与流行病学研究，E-mail:1870611792@qq.com
基金资助:
广东省重点领域研发计划资助(2019B020211003)；科技重大专项计划(21ZD3NA001)；所级基本科研业务费优秀青年科技人才项目(1610312021009)

Preparation of Monoclonal Antibody against African Swine Fever Virus p30 Protein and Establishment of a Blocking ELISA for Detection of Antibody against ASFV p30 Protein

ZHOU Gaijing, LUO Juncong, SHI Zhengwang, WAN Ying, YANG Bo, CAO Liyan, SONG Rui, TIAN Hong^*, ZHENG Haixue^*

State Key Laboratory of Veterinary Etiological Biology and OIE/National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2021-11-10 Online:2022-12-23 Published:2022-12-25

摘要/Abstract

摘要： 为建立检测非洲猪瘟病毒(ASFV)抗体的阻断ELISA方法，本研究利用原核表达的ASFV p30重组蛋白免疫BALB/c小鼠制备单克隆抗体。以重组p30蛋白作为包被抗原，以辣根过氧化物酶(HRP)标记的p30单克隆抗体作为检测抗体，经条件优化，建立了一种检测ASFV抗体的阻断ELISA方法。ROC曲线分析显示，该方法最佳阻断率临界值为16.63%。该方法与CSFV、FMDV-O/A、PRRSV、PEDV、SVA的阳性血清均无交叉反应；最低能检出1∶128稀释的阳性血清；批内和批间变异系数(CV)均＜10%。用本方法与商品化试剂盒平行检测208份血清样品，Kappa值为0.96，表明具有高度一致性。上述结果表明，本研究建立的阻断ELISA方法具有较高的特异性和敏感性，可用于血清ASFV抗体的检测，为ASFV流行病学调查及猪群疫情监控提供技术支持。

关键词: 非洲猪瘟病毒, p30蛋白, 单克隆抗体, 阻断ELISA

Abstract: In order to establish a blocking ELISA method for detection of African swine fever virus (ASFV) antibody, the prokaryotic expressed ASFV p30 protein was used to immunize BALB/c mice for preparation of monoclonal antibody (MAb). A blocking ELISA method was developed after optimization of the reaction conditions with the recombinant p30 protein as a coating antigen and the horseradish peroxidase (HRP) conjugated MAb as detecting antibody. The receiver operating characteristic (ROC) curve calculated an optimal cut-off value of 16.63%. This method had no cross-reaction with positive serum samples of CSFV, FMDV-O/A, PRRSV, PEDV or SVA. The sensitivity test showed that a 1∶128 dilution of positive serum can be detected by this method. The coefficient of variation (CV) of the intra- and inter-assay was less than 10%. A total of 208 serum samples were detected in parallel by the method and the commercial ELISA kit, the Kappa value was 0.96, which demonstrated its high consistency. In conclusion, the established blocking ELISA method with high sensitivity and specificity can be applied to the detection of ASFV antibodies in serum, which provides technical support for the epidemiological investigation and epidemic monitoring of ASFV.

Key words: African swine fever virus, p30 protein, monoclonal antibody, blocking ELISA

中图分类号:

S852.659.1

周改静, 罗俊聪, 石正旺, 万颖, 杨波, 曹丽艳, 宋锐, 田宏, 郑海学. 非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2022, 53(12): 4337-4345.

ZHOU Gaijing, LUO Juncong, SHI Zhengwang, WAN Ying, YANG Bo, CAO Liyan, SONG Rui, TIAN Hong, ZHENG Haixue. Preparation of Monoclonal Antibody against African Swine Fever Virus p30 Protein and Establishment of a Blocking ELISA for Detection of Antibody against ASFV p30 Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4337-4345.

参考文献 26

[1]	GALINDO I,ALONSO C.African swine fever virus:A review[J].Viruses,2017,9(5):103.
[2]	GAUDREAULT N N,MADDEN D W,WILSON W C,et al.African swine fever virus:an emerging DNA arbovirus[J].Front Vet Sci,2020,7:215.
[3]	COSTARD S,MUR L,LUBROTH J,et al.Epidemiology of African swine fever virus[J].Virus Res,2013,173(1):191-197.
[4]	JIA N,OU Y W,PEJSAK Z,et al.Roles of African swine fever virus structural proteins in viral infection[J].J Vet Res,2017,61(2):135-143.
[5]	TEKLUE T,SUN Y,ABID M,et al.Current status and evolving approaches to African swine fever vaccine development[J].Transbound Emerg Dis,2020,67(2):529-542.
[6]	杨文兵,邹亚文,蒋一凡,等.非洲猪瘟血清学诊断靶点的研究进展[J].畜牧兽医学报,2021,52(5):1208-1217.YANG W B,ZOU Y W,JIANG Y F,et al.Advances research on African swine fever serological diagnostic targets[J].Acta Veterinaria et Zootechnica Sinica,2021,52(5):1208-1217.(in Chinese)
[7]	MURGIA M V,MOGLER M,CERTOMA A,et al.Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus[J].Arch Virol,2019,164(2):359-370.
[8]	GÓMEZ-PUERTAS P,RODRÍGUEZ F,OVIEDO J M,et al.The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response[J].Virology,1998,243(2):461-471.
[9]	GIMÉNEZ-LIROLA L G,MUR L,RIVERA B,et al.Detection of african swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30 (p30) dual matrix indirect ELISA[J].PLoS One,2016,11(9):e0161230.
[10]	AFONSO C L,ALCARAZ C,BRUN A,et al.Characterization of p30,a highly antigenic membrane and secreted protein of African swine fever virus[J].Virology,1992,189(1):368-373.
[11]	OVIEDO J M,RODRÍGUEZ F,GÓMEZ-PUERTAS P,et al.High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents[J].J Virol Methods,1997,64(1):27-35.
[12]	郑海学,李攀,冯涛,等.非洲猪瘟基因缺失弱毒株的构建及其作为疫苗的应用:中国,111748563A[P].2020-10-09.ZHENG H X,LI P,FENG T,et al.Construction of a attenuated African swine fever virus with gene deletion and its application as a vaccine:CN,111748563A[P].2020-10-09.(in Chinese)
[13]	PETROVAN V,YUAN F F,LI Y H,et al.Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus[J].Virus Res,2019,269:197632.
[14]	ZANETTI M R D,PETRICELLI C D,ALEXANDRE S M,et al.Determination of a cutoff value for pelvic floor distensibility using the Epi-no balloon to predict perineal integrity in vaginal delivery:ROC curve analysis.Prospective observational single cohort study[J].Sao Paulo Med J,2016,134(2):97-102.
[15]	PARK S H,GOO J M,JO C H.Receiver operating characteristic (ROC) curve:Practical review for radiologists[J].Korean J Radiol,2004,5(1):11-18.
[16]	TANG W,HU J,ZHANG H,et al.Kappa coefficient:A popular measure of rater agreement[J].Shanghai Arch Psychiatry,2015,27(1):62-67.
[17]	麻园,石正旺,罗俊聪,等.猪瘟病毒化学发光抗体检测方法的建立与应用[J].畜牧兽医学报,2021,52(6):1744-1752.MA Y,SHI Z W,LUO J C,et al.Establishment and application of chemiluminescence detection method for antibody against Classical swine fever virus[J].Acta Veterinaria et Zootechnica Sinica,2021,52(6):1744-1752.(in Chinese)
[18]	WU K K,LIU J M,WANG L X,et al.Current state of global African swine fever vaccine development under the prevalence and transmission of ASF in China[J].Vaccines (Basel),2020,8(3):531.
[19]	张蕾,董春娜,李静,等.非洲猪瘟病毒间接ELISA抗体检测方法的建立[J].病毒学报,2020,36(4):670-674.ZHANG L,DONG C N,LI J,et al.Development of an ELISA based on synthetic peptides for detection of antibodies against the African swine fever virus[J].Chinese Journal of Virology,2020,36(4):670-674.(in Chinese)
[20]	王彩霞,冯春燕,肖颖,等.基于非洲猪瘟病毒p72蛋白的阻断ELISA检测方法的建立及初步应用[J].中国兽医科学,2021,51(11):1341-1347.WANG C X,FENG C Y,XIAO Y,et al.Establishment and preliminary application of a blocking ELISA based on p72 protein of African swine fever virus[J].Chinese Veterinary Science,2021,51(11):1341-1347.(in Chinese)
[21]	BARDERAS M G,WIGDOROVITZ A,MERELO F,et al.Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae[J].J Virol Methods,2000,89(1-2):129-136.
[22]	PÉREZ-FILGUEIRA D M,GONZÁLEZ-CAMACHO F,GALLARDO C,et al.Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae[J].J Clin Microbiol,2006,44(9):3114-3121.
[23]	于浩洋,王彩霞,吴绍强,等.非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA检测方法的建立[J].中国兽医科学,2022,52(1):48-55.YU H Y,WANG C X,WU S Q,et al.Preparation of monoclonal antibody against the African swine fever virus p30 protein and establishment of a blocking ELISA for detection of African swine fever virus[J].Chinese Veterinary Science,2022,52(1):48-55.(in Chinese)
[24]	HOO Z H,CANDLISH J,TEARE D.What is an ROC curve?[J].Emerg Med J,2017,34(6):357-359.
[25]	王庆敏,冯晨晨,蒋昵真,等.国内常用7家HBsAg酶免检测试剂盒灰区临界值的确定[J].中国输血杂志,2017,30(3):279-281.WANG Q M,FENG C C,JIANG N Z,et al.Analysis and verification of the cut-off value in domestic HBsAg ELISA kits during blood donation screening using Youden index[J].Chinese Journal of Blood Transfusion,2017,30(3):279-281.(in Chinese)
[26]	吴崇胜,胡立胜,陈家旭.中医证候诊断临界值的确定方法新探[J].中国中医基础医学杂志,2010,16(5):358-359,375.WU C S,HU L S,CHEN J X.A new exploration on method of determining the critical value of Chinese medical syndrome diagnosis[J].Chinese Journal of Basic Medicine in Traditional Chinese Medicine,2010,16(5):358-359,375.(in Chinese)

非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

Preparation of Monoclonal Antibody against African Swine Fever Virus p30 Protein and Establishment of a Blocking ELISA for Detection of Antibody against ASFV p30 Protein

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