基于单克隆抗体捕获的猪血清和乳汁中PEDV IgA抗体间接ELISA方法的建立和评估

doi:10.16431/j.cnki.1671-7236.2020.07.031

摘要/Abstract

摘要： 为快速准确地评价猪血清和乳汁中猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）IgA抗体水平，本研究从4株PEDV单克隆抗体中筛选得到1株可高效捕获PEDV全病毒粒子（灭活的病毒细胞培养原液）的特异性IgG单克隆抗体8A3，将其用作检测孔内的包被抗体以捕获病毒粒子制备抗原板，从而建立了用于检测母猪血清和乳汁中PEDV特异性IgA抗体的间接ELISA方法。该方法中，单克隆抗体8A3的最适包被浓度为6.0 μg/mL，阴性判定临界值（D_{450 nm}）为0.34，且在检测中不与常见的猪病毒阳性血清反应。与传统的免疫过氧化物酶单层检测（immuno-peroxidase monolayer assay，IPMA）相比，所建立的ELISA方法检测阳性和阴性血清的符合率分别为98.7%（152/154）和98.0%（145/148）；检测初乳和乳汁样品的阳性和阴性符合率分别为100%（60/60）和95.8%（23/24）；检测初乳和泌乳期样品中的IgA水平与中和效价的趋势高度相关（kappa=0.835）。本研究建立的检测PEDV IgA的间接ELISA方法灵敏度、敏感性和特异性与传统方法高度一致，可简化检测步骤，适用于大规模评价临床样本乳汁和血清中PEDV IgA抗体的快速检测。

关键词: 猪流行性腹泻病毒(PEDV); 单克隆抗体(MAb); IgA抗体; 间接ELISA

Abstract: For the rapid and accurate evaluation of the IgA antibody level of porcine epidemic diarrhea virus (PEDV) in pig serum and milk,a specific PEDV IgG monoclonal antibody (MAb) 8A3 was screened from four strains of PEDV MAb,which could capture all virus particles (inactivated virus cell culture medium) of PEDV efficiently.In this method,the coating concentration of 6.0 μg/mL showed the optimal performance of MAb 8A3,the cut-off value (D_{450 nm}) was settled as 0.34,it had no cross-reactivity with the positive serums of common porcine viruses.Compared with immune-peroxidase monolayer assay (IPMA),the concordance rates of established ELISA for positive and negative serum detection were 98.7% (152/154) and 98.0% (145/148),respectively.For positive and negative samples of colostrum and milk,the concordance rates of the established ELISA compared with IPMA were 100% (60/60) and 95.8% (23/24),respectively.IgA levels in colostrum and milk samples during lactation detected by established ELISA were highly correlated with trends in neutralizing titers (kappa=0.835).Collectively,the indirect ELISA in this study had high sensitivity and specificity,it was a rapid and objective method suitable for large-scale detection of PEDV IgA in clinical samples.

Key words: porcine epidemic diarrhea virus (PEDV); monoclonal antibody (MAb); IgA antibody; indirect ELISA

中图分类号:

S852.65⁺9.6

王治平, 张云静, 孙玉洁, 徐鑫, 汪志艳, 黄柏成, 田克恭. 基于单克隆抗体捕获的猪血清和乳汁中PEDV IgA抗体间接ELISA方法的建立和评估[J]. 中国畜牧兽医, 2020, 47(7): 2248-2255.

WANG Zhiping, ZHANG Yunjing, SUN Yujie, XU Xin, WANG Zhiyan, HUANG Baicheng, TIAN Kegong. Development and Evaluation of an Indirect ELISA for Detection of Porcine Epidemic Diarrhea Virus IgA in Pig Serum and Milk Captured by Monoclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2248-2255.

参考文献

[1] JUNG K,SAIF L J.Porcine epidemic diarrhea virus infection:Etiology,epidemiology,pathogenesis and immunoprophylaxis[J].Veterinary Journal,2015,204(2):134-143.
[2] AYUDHYA S N,ASSAVACHEEP P,THANAWONGNUWECH R.One world——one health:The threat of emerging swine diseases.An Asian perspective[J].Transboundary and Emerging Diseases,2012,59(Suppl 1):9-17.
[3] PENSAERT M B,DE BOUCK P.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[4] KUSANAGI K,KUWAHARA H,KATOH T,et al.Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate[J].Journal of Veterinary Medical Science,1992,54(2):313-318.
[5] CHOI J C,LEE K K,PI J H,et al.Comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in Korea[J].Infection,Genetics and Evolution,2014,26:348-351.
[6] CHEN J,WANG C,SHI H,et al.Molecular epidemiology of porcine epidemic diarrhea virus in China[J].Archives of Virology,2010,155(9):1471-1476.
[7] LI W,LI H,LIU Y,et al.New variants of porcine epidemic diarrhea virus,China,2011[J].Emerging Infectious Diseases,2012,18(8):1350-1353.
[8] STEVENSON G W,HOANG H,SCHWARTZ K J,et al.Emergence of porcine epidemic diarrhea virus in the United States:Clinical signs,lesions,and viral genomic sequences[J].Journal of Veterinary Diagnostic Investigation,2013,25(5):649-654.
[9] VLASOVA A N,MARTHALER D,WANG Q,et al.Distinct characteristics and complex evolution of PEDV strains,North America,May 2013-February 2014[J].Emerging Infectious Diseases,2014,20(10):1620-1628.
[10] CHATTHA K S,ROTH J A,SAIF L J.Strategies for design and application of enteric viral vaccines[J].Annual Review Animal Biosciences,2015,3:375-395.
[11] OUYANG K,SHYU D L,DHAKAL S,et al.Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions[J].Veterinary Research,2015,46:140.
[12] ZIMMERMAN J J,KARRIKER L A,RAMIREZ A,et al.Diseases of Swine[M].11th Edition.New Jersey:Wiley-Blackwell,2019.
[13] LIN H,ZHOU H,GAO L,et al.Development and application of an indirect ELISA for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein[J].BMC Veterinary Research,2018,14(1):243.
[14] GERBER P F,GONG Q,HUANG Y W,et al.Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA[J].Veterinary Journal,2014,202(1):33-36.
[15] SOZZI E,MORENO A,LELLI D,et al.Development and validation of a monoclonal antibody-based competitive ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV)[J].Research in Veterinary Science,2018,121:106-110.
[16] HARWOOD M D,RUSSELL M R,NEUHOFF S,et al.Lost in centrifugation:Accounting for transporter protein losses in quantitative targeted absolute proteomics[J].Drug Metabolism and Disposition:The Biological Fate of Chemicals,2014,42(10):1766-1772.
[17] MUNROE W H,PHILLIPS M L,SCHUMAKER V N.Excessive centrifugal fields damage high density lipoprotein[J].Journal of Lipid Research,2015,56(6):1172-1181.
[18] WANG Y,GAO X,YAO Y,et al.The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets[J].Virus Research,2015,208:82-88.
[19] GERBER P F,GARROCHO F M,LANA A M,et al.Fetal infections and antibody profiles in pigs naturally infected with porcine circovirus type 2(PCV2)[J].Canadian Journal of Veterinary Research,2012,76(1):38-44.
[20] PARK J G,ALFAJARO M M,CHO E H,et al.Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea[J].Veterinary Research,2019,50(1):2.
[21] QING Y,LIU J,HUANG X,et al.Immunogenicity of transmissible gastroenteritis virus (TGEV) M gene delivered by attenuated Salmonella Typhimurium in mice[J].Virus Genes,2016,52(2):218-227.
[22] VANDERPOOTEN A,GODDEERIS B,DE ROOSE P,et al.Evaluation of parenteral vaccination methods with glycoproteins against Aujeszky's disease in pigs[J].Veterinary Microbiology,1997,55(1-4):81-89.
[23] ZHANG Y,YAO Y,GAO X,et al.Development of a neutralizing monoclonal antibody against porcine epidemic diarrhea virus S1 protein[J].Monoclon Antib Immunodiagn Immunother,2016,35(1):37-40.
[24] MAZANEC M B,KAETZEL C S,LAMM M E,et al.Intracellular neutralization of virus by immunoglobulin A antibodies[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(15):6901-6905.
[25] SUZUKI T,KAWAGUCHI A,AINAI A,et al.Relationship of the quaternary structure of human secretory IgA to neutralization of influenza virus[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(25):7809-7814.
[26] LANGEL S N,PAIM F C,LAGER K M,et al.Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV):Historical and current concepts[J].Virus Research,2016,226:93-107.
[27] AMARO F,SANCHEZ-SECO M P,VAZQUEZ A,et al.The application and interpretation of IgG avidity and IgA ELISA tests to characterize zika virus infections[J].Viruses,2019,11(2):E179.
[28] GONCALVES A K,MACHADO P R,DE SOUZA L C,et al.Detection of immunoglobulin IgA and IgG against human papilloma virus[J].Viral Immunology,2014,27(9):471-477.
[29] SENTHILKUMARAN C,BITTNER H,AMBAGALA A,et al.Use of oral fluids for detection of virus and antibodies in pigs infected with swine vesicular disease virus[J].Transboundary and Emerging Diseases,2017,64(6):1762-1770.
[30] KO Y J,JEOUNG H Y,LEE H S,et al.A recombinant protein-based ELISA for detecting antibodies to foot-and-mouth disease virus serotype Asia 1[J].Journal of Virological Methods,2009,159(1):112-118.
[31] OH J S,SONG D S,YANG J S,et al.Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection[J].Journal of Veterinary Science,2005,6(4):349-352.
[32] GERBER P F,LELLI D,ZHANG J,et al.Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains[J].Preventive Veterinary Medicine,2016,135:87-94.
[33] SONG Q,STONE S,DREBES D,et al.Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions[J].Virus Research,2016,226:85-92.