猪流行性腹泻病毒N蛋白阻断ELISA抗体检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2022.04.017

摘要/Abstract

摘要： 旨在建立一种猪流行性腹泻病毒(PEDV) N蛋白阻断ELISA抗体检测方法。本研究将纯化的N蛋白作为包被抗原，通过棋盘滴定法优化ELISA反应条件，建立了检测PEDV抗体的阻断ELISA方法，并对其进行特异性、敏感性和重复性试验。对140份临床血清样品进行检测，并将检测结果与市售IDvet PEDV间接ELISA抗体检测试剂盒试剂盒进行比较。结果显示，建立的阻断ELISA方法的抗原最佳包被浓度为625 ng·mL^-1，血清最佳稀释比例为1∶1；酶标抗体的最佳工作浓度为1∶5 000；检测健康猪血清以及猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)和传染性胃肠炎病毒(TGEV)等常见猪病病原的阳性血清，均无交叉反应；PEDV阳性血清的灵敏度为1∶16，与市售IDvet PEDV间接ELISA抗体检测试剂盒(效价1∶32)的敏感性相当；批内、批间重复性试验的变异系数均小于10%，说明具有良好的重复性和稳定性；对140份临床血清样品的检测并与商品化试剂盒检测结果进行比较，两者的kappa值为0.87，为高度一致。综上表明，本研究建立的阻断ELISA抗体检测方法可以应用于PEDV的防控、流行病学调查以及疫苗免疫后抗体水平的监测。

关键词: 猪流行性腹泻病毒, N蛋白, 抗体检测, 阻断ELISA

Abstract: The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus (PEDV). The purified N protein was used as the coating antigen, and the ELISA reaction conditions were optimized by the chess rboard titration. A blocking ELISA method for detecting PEDV antibodies was established, and its specificity, sensitivity and repeatability tests were carried out. One hundred and forty clinical serum samples were tested, and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit. The results showed that the best antigen coating concentration was 625 ng·mL^-1, and the best dilution ratio of serum was 1:1; The best dilution of the HRP-conjugated antibody working solution was 1:5 000; There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and transmissible gastroenteritis virus (TGEV). The sensitivity of PEDV positive serum was 1:16, which was equivalent to that of IDvet ELISA kit (titer 1∶32). The coefficient of variation of within-run and between-run repeatability test is less than 10%, so it showed that the blocking ELISA established in this study had good repeatability and stability; the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA. The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV, epidemiological investigation and the monitoring of antibody levels after vaccine immunization.

Key words: porcine epidemic diarrhea variant virus, N protein, antibodies detection, blocking ELISA

中图分类号:

S852.659.6

万颖, 周改静, 麻园, 石正旺, 肖书奇, 罗俊聪, 宋锐, 曹丽艳, 杨波, 王丽娟, 田宏, 郑海学. 猪流行性腹泻病毒N蛋白阻断ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2022, 53(4): 1173-1181.

WAN Ying, ZHOU Gaijing, MA Yuan, SHI Zhengwang, XIAO Shuqi, LUO Juncong, SONG Rui, CAO Liyan, YANG bo, WANG Lijuan, TIAN Hong, ZHENG Haixue. Development and Application of N-protein Blocking ELISA for Detecting Porcine Epidemic Diarrhea Virus Antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1173-1181.

参考文献 31

[1]	ADAMS M J, LEFKOWITZ E J, KING A M O, et al. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016)[J]. Arch Virol, 2016, 161(10):2921-2949.
[2]	LI Z L, CHEN F, YUAN Y, et al. Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China[J]. Arch Virol, 2013, 158(6):1267-1273.
[3]	DUARTE M, TOBLER K, BRIDGEN A, et al. Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF[J]. Virology, 1994, 198(2):466-476.
[4]	LEE C, PARK C K, LYOO Y S, et al. Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis[J]. Vet J, 2008, 178(1):138-140.
[5]	LI B X, GE J W, LI Y J. Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus[J]. Virology, 2007, 365(1):166-172.
[6]	朱卫霞, 袁万哲, 李丽敏, 等. 截短猪流行性腹泻病毒N蛋白的可溶性表达及其抗原活性[J]. 畜牧兽医学报, 2014, 45(9):1561-1566.ZHU W X, YUAN W Z, LI L M, et al. Prokaryotic soluble expression of trunked n protein of porcine epidemic diarrhea virus and its antigenic activity[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(9):1561-1566. (in Chinese)
[7]	PIJPERS A, VAN NIEUWSTADT A P, TERPSTRA C, et al. Porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs[J]. Vet Rec, 1993, 132(6):129-131.
[8]	MA Z Q, WANG T Y, LI Z W, et al. A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus[J]. J Nanobiotechnol, 2019, 17(1):96.
[9]	DIEL D G, LAWSON S, OKDA F, et al. Porcine epidemic diarrhea virus:an overview of current virological and serological diagnostic methods[J]. Virus Res, 2016, 226:60-70.
[10]	OKDA F, LIU X D, SINGREY A, et al. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus[J]. BMC Vet Res, 2015, 11:180.
[11]	KANG K J, KIM D H, HONG E J, et al. The carboxy terminal region on spike protein of porcine epidemic diarrhea virus (PEDV) is important for evaluating neutralizing activity[J]. Pathogens, 2021, 10(6):683.
[12]	LIN H X, 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 Vet Res, 2018, 14(1):243.
[13]	MARTELLI P, LAVAZZA A, NIGRELLI A D, et al. Pensaert MB. Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy[J]. Vet Rec, 2008, 162(10):307-310.
[14]	SONG D, PARK B. Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology, diagnosis, and vaccines[J]. Virus Genes, 2012, 44(2):167-175.
[15]	LI Z L, ZHU L, MA J Y, et al. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China[J]. Virus Genes, 2012, 45(1):181-185.
[16]	ISHIKAWA K, SEKIGUCHI H, OGINO T, et al. Direct and rapid detection of porcine epidemic diarrhea virus by RT-PCR[J]. J Virol Methods, 1997, 69(1-2):191-195.
[17]	SALEM A N B, SERGEI A C, OLGA P B, et al. Multiplex nested RT-PCR for the detection of porcine enteric viruses[J]. J Virol Methods, 2010, 165(2):283-293.
[18]	ZHOU X R, ZHANG T S, SONG D P, et al. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus[J]. Mol Cell Probes, 2017, 33:36-41.
[19]	LIU G P, JIANG Y H, OPRIESSNIG T, et al. Detection and differentiation of five diarrhea related pig viruses utilizing a multiplex PCR assay[J]. J Virol Methods, 2019, 263:32-37.
[20]	石明明. 猪流行性腹泻病毒(PEDV)及其抗体检测方法的研究与应用[D]. 南京:南京农业大学, 2012.SHI M M. Investigation and application of virus and serological detection methods of porcine epidemic diarrhea[D]. Nanjing:Nanjing Agricultural University, 2012. (in Chinese)
[21]	DE GENST E, SILENCE K, DECANNIERE K, et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies[J]. Proc Natl Acad Sci U S A, 2006, 103(12):4586-4591.
[22]	MUYLDERMANS S, BARAL T N, RETAMOZZO V C, et al. Camelid immunoglobulins and nanobody technology[J]. Vet Immunol Immunopathol, 2009, 128(1-3):178-183.
[23]	WANG P Y, LI G H, YAN J R, et al. Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin[J]. Toxicon, 2014, 92:186-192.
[24]	MAASS D R, SEPULVEDA J, PERNTHANER A, et al. Alpaca (Lama pacos ) as a convenient source of recombinant camelid heavy chain antibodies (VHHs)[J]. J Immunol Methods, 2007, 324(1-2):13-25.
[25]	LIU M J, DU G M, ZHANG Y, et al. Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65[J]. J Vet Med Sci, 2016, 78(8):1319-1322.
[26]	BAGHERI A, MADANI R, NAVIDPOUR S, et al. Serodiagnosis of Przhevalskiana spp. infestation in goats using a competitive ELISA[J]. Arch Razi Inst, 2020, 75(2):233-239.
[27]	张琪, 徐丽美, 周宏超, 等. 基于N蛋白的PEDV间接ELISA检测方法的建立与初步应用[J]. 中国兽医科学, 2018, 48(2):148-154.ZHANG Q, XU L M, ZHOU H C, et al. Establishment and preliminary application of an indirect ELISA based on recombinant N protein for the detection of antibodies against PEDV[J]. Chinese Veterinary Science, 2018, 48(2):148-154. (in Chinese)
[28]	ZHU M, GONG X, HU Y L, et al. Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5 N1[J]. J Transl Med, 2014, 12:352.
[29]	ZHANG Y A, LI Y N, ZHANG J L, et al. Nanocage-based capture-detection system for the clinical diagnosis of autoimmune disease[J]. Small, 2021, 17(25):2101655.
[30]	熊玲红, 黄亚兰, 孟君, 等. 化学发光和酶联免疫吸附法检测SARS-CoV-2 IgM/IgG抗体比较[J]. 热带医学杂志, 2020, 20(11):1399-1401, 1424.XIONG L H, HUANG Y L, MENG J, et al. Comparision of chemiluminescence and ELISA kits for SARS-CoV-2 IgM/IgG detection[J]. Journal of Tropical Medicine, 2020, 20(11):1399-1401, 1424. (in Chinese)
[31]	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)