[1]刘 烨,张家林,王潇博,等.猪流行性腹泻病毒IgA抗体间接ELISA方法的建立[J].中国预防兽医学报,2019,(09):918-923.[doi:10.3969/j.issn.1008-0589.201812021]
 LIU Ye,ZHANG Jia-lin,WANG Xiao-bo,et al.Development of an indirect ELISA for the detection of specific IgA antibodies against porcine epidemic diarrhea virus[J].Chinese journal of preventive veterinary medicine,2019,(09):918-923.[doi:10.3969/j.issn.1008-0589.201812021]
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猪流行性腹泻病毒IgA抗体间接ELISA方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年09
页码:
918-923
栏目:
诊断和检测技术
出版日期:
2019-10-25

文章信息/Info

Title:
Development of an indirect ELISA for the detection of specific IgA antibodies against porcine epidemic diarrhea virus
文章编号:
1008-0589(2019)09-0918-06
作者:
 

刘 烨张家林王潇博石 达时洪艳陈建飞张 鑫冯 力*

 (中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室,黑龙江 哈尔滨 150069)
Author(s):
 

LIU Ye ZHANG Jia-lin WANG Xiao-bo SHI Da SHI Hong-yan CHEN Jian-fei ZHANG Xin FENG Li*

 (State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
PEDV间接ELISAS1蛋白IgA抗体
Keywords:
 PEDV  indirect ELISA  S1 protein  IgA antibody
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.201812021
文献标志码:
A
摘要:
为建立检测猪流行性腹泻病毒(PEDV)血清特异性IgA抗体的间接ELISA方法,本研究利用融合PCR的方法,将PEDV G2型LNct2a株S蛋白的S1基因片段与人源IgG分子的Fc基因片段融合,克隆于真核表达质粒pAAV-IRES-hrGFP中,构建真核表达质粒pAAV-LNCT2-S1-Fc并转染293T细胞,表达并纯化了S1蛋白。以纯化的S1蛋白作为包被抗原,以羊抗猪IgA-HRP为二抗,通过优化反应条件,建立了检测PEDV G2型特异性IgA抗体的间接ELISA方法,并对该方法进行了特异性、敏感性及重复性试验。特异性试验结果显示该方法对猪传染性胃肠炎病毒、轮状病毒、猪德尔塔冠状病毒和圆环病毒的阳性血清均无交叉反应;敏感性试验结果显示该方法能够检测出400倍稀释的阳性血清;批内和批间重复性变异系数均小于10 %。利用该间接ELISA方法和间接免疫荧光方法(IFA)同时对临床样品检测,结果显示二者总符合率为98.6 %。本研究建立的ELISA方法特异性强、敏感性高、重复性好,能够用于检测及评价血清中PEDV特异性IgA抗体的水平,为PEDV流行情况及免疫效果评价提供了有效的手段。
  
Abstract:
In order to developan indirect enzyme-linked immunosorbent assay (iELISA) for detection of the specific IgA antibodies against porcine epidemic diarrhea virus (PEDV), the PEDV (G2 type) LNct2a isolate S1 protein was cloned by fusing the S1 gene and human IgG Fc segment using fusion PCR, and expressed by transfecting the recombinant pAAV-LNCT2-S1-Fc plasmid into 293T cells. The purified and identified S1 protein was used as the coating antigen and the HRP-conjugated goat anti-pig IgA was used as the secondary antibody. An iELISA was developed and optimized for the detection of anti-PEDV G2 genotype IgA antibody, followed by the specificity, sensitivity and repetitive tests. The specificity test showed no cross-reaction with those positive sera of porcine transmissible gastroenteritis virus (TGEV), rotavirus (PoRV), porcine delta coronavirus (PDCoV) and circovirus (PCV2). The sensitivity test showed that a 400-fold dilution of positive serum can be detected. The coefficients of variation (CV) of the intra- and inter-batch repetitive tests were less than 10%. The iELISA and indirect immunofluorescence assay (IFA) were used to simultaneously detect clinical samples and the agreements between the iELISA and IFA was 98.6%. The method developed in this study can be used to detect and evaluate the level of anti-PEDV IgA antibodies in serumand provide an effective method for evaluating the prevalence and vaccination efficiency of PEDV.

参考文献/References:

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(本文编辑:李 娜;英文编辑:王 刚)

 

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更新日期/Last Update: 2019-10-29