[1]李宛生?,李敏华?,杨建伟,等.北美型猪繁殖与呼吸综合征竞争ELISA抗体检测方法的建立及初步应用[J].中国预防兽医学报,2019,(12):1221-1226.[doi:0.3969/j.issn.1008-0589.201906026]
 LI Wan-sheng?,LI Min-hua?,YANG Jian-wei,et al. Development and preliminary application of a competitive ELISA for antibody detection of North American-type porcine reproductive and respiratory syndrome [J].Chinese journal of preventive veterinary medicine,2019,(12):1221-1226.[doi:0.3969/j.issn.1008-0589.201906026]
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北美型猪繁殖与呼吸综合征竞争
ELISA抗体检测方法的建立及初步应用
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年12
页码:
1221-1226
栏目:
诊断和检测技术
出版日期:
2020-01-25

文章信息/Info

Title:
 

Development and preliminary application of a competitive ELISA for antibody detection of North American-type porcine reproductive and respiratory syndrome

文章编号:
1008-0589(2019)12-1221-06
作者:
 

李宛生1?李敏华3?杨建伟1田志军2王 倩2*冷超粮1*

 (1. 南阳师范学院,河南 南阳 473061;2. 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猪病实验室,
黑龙江 哈尔滨 150069;3. 北京爱德士元亨生物科技有限公司,北京101318)
Author(s):
 LI Wan-sheng1? LI Min-hua3? YANG Jian-wei1 TIAN Zhi-jun2 WANG Qian2* LENG Chao-liang1*
 

(1. Nanyang Normal University, Nanyang 473061, China; 2. National Key Swine Disease Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
3. Beijing IDEXX Yuanheng Laboratories, Co., Ltd, Beijing 101318, China)

关键词:
猪繁殖与呼吸综合征病毒北美型M蛋白竞争ELISA
Keywords:
porcine reproductive and respiratory syndrome virus  North American-type  M protein  competitive ELISA
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201906026
文献标志码:
A
摘要:
为建立快速检测猪繁殖与呼吸综合征病毒(PRRSV)抗体的ELISA方法,本研究以纯化的PRRSV全病毒为包被抗原,利用抗北美型PRRSV M蛋白的特异性单克隆抗体为竞争抗体,经条件优化,建立了特异性检测北美型PRRSV的竞争ELISA(C-ELISA)方法。结果显示,最佳抗原包被量为660 ng/孔,待检血清样本稀释度为1∶2,竞争抗体最佳稀释度为1∶8。ELISA结果判定标准:血清样本抑制率PI≥25.65 %时判定为阳性,PI<25.65 %判定为阴性。特异性试验结果显示,该方法仅能检测北美型PRRSV,与PRV、PCV2、CSFV、PEDV、TGEV等猪病毒阳性血清无交叉反应;敏感性试验结果显示,该方法能检测出128倍稀释的阳性血清;批内和批间重复性试验的变异系数均小于10 %。利用该方法对黑龙江地区收集的546份临床血清样品进行检测,PRRSV抗体阳性率达95.1 %,与商品化IDEXX PRRSV试剂盒的符合率为95.8 %。本研究建立的C-ELISA方法为PRRS的流行病学调查、临床上疫苗评估及免疫前后抗体水平监测提供了有效的检测手段。
Abstract:
In order to establish an enzyme-linked immunosorbent assay(ELISA) method for detection of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV), a specifically competitive-ELISA (C-ELISA) method using purified PRRS whole virus as coating antigen and a monoclonal antibody against the M protein of the North American-type PRRS strain was developed with optimizing the reaction conditions. The results showed that the optimal antigen coating concentration was 660 ng/well, the optimal serum dilution was 1∶2, and the optimal dilution of the competitive antibody was 1∶8. The ELISA result criteria: if the serum sample percentage inhibition (PI) was not less than 25.65%, it was considered to be positive or it was negative. The specificity test showed that this method can only detect North American-type PRRS strain and no cross-reaction with those antibody positive serums against PRV, PCV2, CSFV, PEDV, TGEV. The sensitivity test showed that a 128-fold dilution of positive serum can be detected. And the coefficients of variation (CV) of the intra- and inter-assay repeated tests were less than 10%. Using this method, 546 clinical serum samples collected in Heilongjiang region were tested. The positive rate of PRRSV antibody was 95.1%, and the coincidence rate with the commercial IDEXX PRRSV kit was 95.8%. Therefore, the ELISA method established in this study provides an effective detection method for epidemiological investigation of PRRSV, clinical vaccine evaluation and antibody level monitoring before and after immunization.

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(本文编辑:彭永刚;英文编辑:孟凡丹)

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更新日期/Last Update: 2020-01-19