流行性出血病多克隆抗体C-ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2018.07.013

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (7): 1440-1450.doi: 10.11843/j.issn.0366-6964.2018.07.013

流行性出血病多克隆抗体C-ELISA检测方法的建立

朱建波, 杨振兴, 肖雷, 高林, 苗海生, 何于雯, 孟锦昕, 杨恒, 李华春*

云南省畜牧兽医科学院, 云南省热带亚热带动物病毒病重点实验室, 昆明 650224

收稿日期:2017-12-06 出版日期:2018-07-23 发布日期:2018-07-19
通讯作者: 李华春,E-mail:Li_huachun@hotmail.com
作者简介:朱建波(1970-),男,云南红河人,研究员,硕士,主要从事动物病毒学研究,E-mail:zhujb70@126.com;杨振兴(1986-),男,云南昆明人,助理研究员,硕士,主要从事牛、羊虫媒病毒研究,E-mail:s300yn@163.com。
基金资助:
国家重点研发计划（2016YFD0500908）；国家公益性行业（农业）科研专项（201303035）

Establishment of a Rapid Competitive ELISA for Detecting Antibodies against Epizootic Haemorrhagic Disease Virus

ZHU Jian-bo, YANG Zhen-xing, XIAO Lei, GAO Lin, MIAO Hai-sheng, HE Yu-wen, MENG Jin-xin, YANG Heng, LI Hua-chun*

Yunnan Tropical and Subtropical Animal Virus Disease Laboratory, Yunnan Veterinary and Animal Science Institute, Kunming 650224, China

Received:2017-12-06 Online:2018-07-23 Published:2018-07-19

摘要/Abstract

摘要：

为能简便、快速地进行流行性出血病病毒（epizootic haemorrhagic disease virus，EHDV）抗体监测，采用纯化的6型EHDV抗原包被ELISA板，以豚鼠抗EHDV-2型多克隆抗体作为竞争抗体，建立了检测EHDV群特异性抗体的竞争ELISA（C-ELISA）方法。结果显示：抗原最佳包被浓度为5.12 μg·mL^-1（1：10 000），竞争抗体最佳稀释倍数为1：10 000；通过对各270份阴性和阳性的牛、羊血清检测，确定该检测方法临界值为50%；特异性试验表明该ELISA方法仅能检测出不同血清型EHDV抗体，具有较好的群特异性；对已知抗体效价的阳性血清检测表明，建立的C-ELISA方法敏感性好于血清中和试验（SNT）和琼脂扩散试验（AGID）；分别用C-ELISA、SNT、RT-PCR和病毒分离试验对监控动物采集的血清和抗凝血样品进行检测，ELISA结果与其他3种方法检测结果对应一致。结果表明本研究建立的C-ELISA为EHDV抗体的检测提供了一种快速、敏感、稳定的方法。

关键词: 流行性出血病病毒, 多克隆抗体, 竞争 ELISA

Abstract:

The objective of this research was to establish a simple and fast operation method for detecting epizootic haemorrhagic disease virus(EHDV) antibodies. In this research, a competitive ELISA(C-ELISA) method was developed, by using purified EHDV-6 coated ELISA plates and guinea pig anti-EHDV-2 antibody as a competitive antibody. The assay had a high specificity and reproducibility, with optimal dilution of the multi-clone antibody at 1:10 000 and virus antigen at 5.12 μg·mL^-1(1:10 000). The critical value of the detection method was 50%, through the serum testing of cows and sheep, including negative and positive 270 copies each. Specific tests showed that the ELISA method can only detect different serotypes of EHDV antibodies, with better group specificity. Positive serum tests on known antibody titers showed that the established C-ELISA method was more sensitive than the serum neutralization test(SNT) and Agar gel immunodiffusion test(AGID). While the serum and anticoagulant samples collected from monitoring animals were detected by C-ELISA, SNT, RT-PCR and virus isolation respectively, the results of ELISA were consistent with the other two methods. These results indicate that the C-ELISA is a rapid, sensitive and stable method for the detection of EHDV antibodies.

Key words: epizootic haemorrhagic disease virus (EHDV), multi-clone antibody, competitive ELISA (C-ELISA)

中图分类号:

S854.43

朱建波, 杨振兴, 肖雷, 高林, 苗海生, 何于雯, 孟锦昕, 杨恒, 李华春. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7): 1440-1450.

ZHU Jian-bo, YANG Zhen-xing, XIAO Lei, GAO Lin, MIAO Hai-sheng, HE Yu-wen, MENG Jin-xin, YANG Heng, LI Hua-chun. Establishment of a Rapid Competitive ELISA for Detecting Antibodies against Epizootic Haemorrhagic Disease Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1440-1450.

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流行性出血病多克隆抗体C-ELISA检测方法的建立

Establishment of a Rapid Competitive ELISA for Detecting Antibodies against Epizootic Haemorrhagic Disease Virus

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