猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

doi:10.16431/j.cnki.1671-7236.2017.06.034

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (6): 1818-1824.doi: 10.16431/j.cnki.1671-7236.2017.06.034

猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

王园园, 潘丽, 吕建亮, 张中旺

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

收稿日期:2016-11-30 出版日期:2017-06-20 发布日期:2017-06-28
通讯作者: 潘丽 E-mail:panli@caas.cn
作者简介:王园园(1989-),女,河南许昌人,硕士生,研究方向:动物疫苗与分子免疫学,E-mail:13919180464@163.com
基金资助:
"十三五"国家重点研发计划（2016YFD0501503）；甘肃省科技支撑计划（1604NKCA045-2）；国家生猪现代产业技术体系项目（CARS-36-06B）

Development and Primary Application of Indirect ELISA Method for Detecting the IgA Antibody against Foot-and-mouth Disease Virus Type A in Porcine

WANG Yuan-yuan, PAN Li, LV Jian-liang, ZHANG Zhong-wang

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

Received:2016-11-30 Online:2017-06-20 Published:2017-06-28

摘要/Abstract

摘要：

为建立一种检测口蹄疫病毒（foot-and-mouth disease virus，FMDV）特异性IgA抗体的检测方法，本研究以原核表达系统表达纯化的FMDV结构蛋白VP1作为包被抗原，以鼠抗猪IgA单克隆抗体为二抗，辣根过氧化酶标记的羊抗鼠IgG抗体为三抗，建立猪A型FMDV特异性IgA抗体间接ELISA检测方法。确定抗原包被浓度为3.50 μg/mL，二抗与三抗的最佳稀释度为1∶10 000，二抗和三抗作用时间均为30 min。所建立的方法与抗猪瘟病毒、猪繁殖与呼吸道综合征病毒等病原的特异性IgA抗体间无交叉反应，A型口蹄疫感染样品的阳性检出率在90%以上，批内和批间重复性试验的变异系数介于3.16%~9.76%。该方法为监测FMDV特异性IgA抗体水平变化规律及猪的黏膜免疫效果评价及口蹄疫的早期诊断提供了一种新方法。

关键词: 口蹄疫; 黏膜免疫; IgA; 间接ELISA

Abstract:

An indirect ELISA for detecting the IgA antibody against porcine foot-and-mouth disease virus (FMDV) type A was developed by using purified FMDV structural protein VP1 as coating antigen, mouse anti-pig IgA monoclonal antibody as second antibody and HRP-conjugated goat anti-mouse IgG as third antibody.The concentration of coating antigen was optimized as 3.50 μg/mL,the dilution and reaction time of second antibody and third antibody were optimized as 1:10 000 and 30 min, respectively.There was no cross-reactivity with anti-CSFV, PRRSV and other pathogen specific IgA antibodies.The positive detection rate of FMDV type A infectedsamples was above 90%.The coefficient variation of intra-and inter-assay was ranged from 3.16% to 9.76%.The ELISA method described in this study was proved to be specific and rapid for the detection of FMDV specific IgA antibody.It was potential to be applied for detection the level of FMDV specific IgA and evaluate the effect of mucosal immunity.Besides,it provided a new method for clinical diagnosis of foot-and-mouth disease.

Key words: foot-and-mouth disease (FMD); mucosal immunity; IgA; indirect ELISA

中图分类号:

S852.65

王园园, 潘丽, 吕建亮, 张中旺. 猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用[J]. 《中国畜牧兽医》, 2017, 44(6): 1818-1824.

WANG Yuan-yuan, PAN Li, LV Jian-liang, ZHANG Zhong-wang. Development and Primary Application of Indirect ELISA Method for Detecting the IgA Antibody against Foot-and-mouth Disease Virus Type A in Porcine[J]. , 2017, 44(6): 1818-1824.

参考文献

[1] Morello J A.Clinical microbiology reviews:Genesis of a journal[J].Clin Microbiol Rev,1999,12(2): 183-186.
[2] Baxi M K,Baxi S,Clavijo A,et al.Microarray-based detection and typing of foot-and-mouth disease virus[J].Vet J,2006,172(3):473-481.
[3] SaltJ S,Mulcahy G,Kitching R P,et al.Isotype-specific antibody responses to foot-and-mouth disease virus in sera and secretions of ‘arrier’ and ‘non-carrier’ cattle[J].Epidemiol Infect,1996,117(2): 349-360.
[4] Mahapatra M,Seki C,Upadhyaya S,et al.Characterisation and epitope mapping of neutralising monoclonal antibodies to A24 Cruzeiro strain of FMDV[J].Vet Microbiol,2011,149(1-2):242-247.
[5] Zaman M,Ozberk V,Langshaw E L,et al.Novel platform technology for modular mucosal vaccine that protects against streptococcus[J].Sci Rep,2016,20(6):39274.
[6] 赵雪,张辉,刘禹,等.分泌型IgA对肠道黏膜免疫的研究进展[J].中国畜牧兽医,2013,40(6):96-99.
[7] 王晓东,黄志华.SIgA在肠道免疫中的作用[J].国际消化病杂志,2006,26(5):339-341.
[8] Mathias A,Corthesy B.N-Glycans on secretory component: Mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis[J].Gut Microbes,2011,2(5):287-293.
[9] Wang M,Pan L,Zhou P,et al.Protection against foot-and-mouth disease virus in guinea pigs via oral administration of recombinant Lactobacillus plantarum expressing VP1[J].PLoS One,2015,10(12):0143750.
[10] 王淼,周鹏,潘丽,等.重组A型FMDV VP1基因乳酸杆菌穿梭表达质粒的构建及在大肠杆菌BL21中的表达[J].安徽农业科学,2015,43(21):25-27.
[11] 冯志新,张亚,华利忠,等.猪鼻拭子样品采集及制备方法的标准化[J].中国农学通报,2013,29(5):17-20.
[12] Feng Z X, Shao G Q, Liu M J,et al.Development and validation of a SIgA-ELISA for the detection of Mycoplasma hyopneumoniae infection[J].Veterinary Microbiology,2010,143(2-4):410-416.
[13] Hodinka R L,Nagashunmugam T,Malamud D.Detection of carriers of foot-and-mouth disease virus among vaccinated cattle[J].Vet Microbiol,2004,103(3-4):151-160.
[14] Cox S J,Voyce C,Parida S,et al.Protection against direct-contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx[J].Vaccine,2005,23(9):1106-1113.
[15] Pacheco J M,Arzt J,Rodriguez L L.Early events in the pathogenesis of foot-and-mouth disease in cattle after controlled aerosol exposure[J].Vet J,2010,183(1):46-53.
[16] Parida S,Anderson J,Cox S J,et al.Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance[J].Vaccine, 2006, 24(8):1107-1116.
[17] 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].Transbound Emerg Dis,2016,21(2):102-110.
[18] Pacheco J M,Butler J E,Jew J,et al.IgA antibody response of swine to foot-and-mouth disease virus infection and vaccination[J].Clin Vaccine Immunol,2010,17(4):550-558.
[19] Eble P L, Bouma A, Weerdmeester K, et al.Serological and mucosal immune responses after vaccination and infection with FMDV in pigs[J].Vaccine,2007,25(6):1043-1054.
[20] Wang J H, Liang C M, Peng J M, et al.Induction of immunity in swine by purified recombinant VP1 of foot-and-mouth disease virus[J].Vaccine,2003,21(25-26):3721-3729.

猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

Development and Primary Application of Indirect ELISA Method for Detecting the IgA Antibody against Foot-and-mouth Disease Virus Type A in Porcine

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