检测口蹄疫病毒非结构蛋白3ABC抗体的单克隆抗体阻断ELISA的建立与效果评价
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摘要
单克隆抗体被认为是最佳的分子探针,而非结构蛋白3ABC抗体被认为是在免疫动物群体中检测口蹄疫感染动物的最佳指示器。
建立了一种以区分疫苗免疫动物与康复带毒动物或持续感染动物为目的,检测口蹄疫非结构蛋白3ABC抗体的单克隆抗体阻断ELISA (FMDV NSP B-ELISA)。
本方法使用组氨酸单克隆抗体作为捕获抗体,口蹄疫病毒非结构蛋白3B单克隆抗体作为检测抗体,原核表达的组氨酸标签口蹄疫病毒非结构蛋白3ABC作为抗原。
使用本ELISA检测了大量牛、羊及猪的血清,并用公式(1-样品OD值/阴性对照OD值)来计算血清阻断率(percentage inhibition, PI),最终确定：PI≥0.46判为阳性,PI<0.46判为阴性。根据此标准检测实验感染牛、羊及猪血清敏感性为90.4%、96.1%及85.4%,检测免疫健康牛、羊及猪血清特异性≥99.2%。通过比较发现本ELISA与Ceditest NS ELISA试剂盒有很高的符合率,对感染动物血清与疫区田间牛血清的阳性检出率均高于同类试剂盒。
本ELISA可以作为田间疫情监测方法,在口蹄疫灭活疫苗免疫动物群体中使用。
The monoclonal antibody (mAb) is considered to be the best molecular probes, and non-structural protein (NSP)3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals.
In this report, a non-structural proteins (NSP)3B monoclonal antibody(mAb) trapping3ABC-blocking ELISA was developed to detect FMDV NSP3ABC antibody. The histidine mAb and NSP3B mAb were used as capture antibody and detection antibody respectively, and purified His-tagged3ABC fusion protein were used as antigen.
With statistics, the critical value0.46was defined as blocking rate after hundreds sera samples of cattle, sheep and pigs were detected. According to this criteria, the sensitivity of the positive serum>99.2%, the specificity of the negative serum>85.4%.
This method not only has a higher coincident rate compared with Ceditest NS ELISA kits, but also has a highter positive detection rate.
建立了一种以区分疫苗免疫动物与康复带毒动物或持续感染动物为目的,检测口蹄疫非结构蛋白3ABC抗体的单克隆抗体阻断ELISA (FMDV NSP B-ELISA)。
本方法使用组氨酸单克隆抗体作为捕获抗体,口蹄疫病毒非结构蛋白3B单克隆抗体作为检测抗体,原核表达的组氨酸标签口蹄疫病毒非结构蛋白3ABC作为抗原。
使用本ELISA检测了大量牛、羊及猪的血清,并用公式(1-样品OD值/阴性对照OD值)来计算血清阻断率(percentage inhibition, PI),最终确定：PI≥0.46判为阳性,PI<0.46判为阴性。根据此标准检测实验感染牛、羊及猪血清敏感性为90.4%、96.1%及85.4%,检测免疫健康牛、羊及猪血清特异性≥99.2%。通过比较发现本ELISA与Ceditest NS ELISA试剂盒有很高的符合率,对感染动物血清与疫区田间牛血清的阳性检出率均高于同类试剂盒。
本ELISA可以作为田间疫情监测方法,在口蹄疫灭活疫苗免疫动物群体中使用。
The monoclonal antibody (mAb) is considered to be the best molecular probes, and non-structural protein (NSP)3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals.
In this report, a non-structural proteins (NSP)3B monoclonal antibody(mAb) trapping3ABC-blocking ELISA was developed to detect FMDV NSP3ABC antibody. The histidine mAb and NSP3B mAb were used as capture antibody and detection antibody respectively, and purified His-tagged3ABC fusion protein were used as antigen.
With statistics, the critical value0.46was defined as blocking rate after hundreds sera samples of cattle, sheep and pigs were detected. According to this criteria, the sensitivity of the positive serum>99.2%, the specificity of the negative serum>85.4%.
This method not only has a higher coincident rate compared with Ceditest NS ELISA kits, but also has a highter positive detection rate.
引文
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2.曹轶梅,卢曾军,刘在新,等.大肠埃希氏菌表达FMDV3ABC非结构蛋白的纯化复性与活性检测.中国兽医科技,2003,33：22-25.
3.曹轶梅,卢曾军,刘在新,等.用大肠杆菌表达FMDV NSP3ABC鉴别感染与注苗动物ELISA方法的建立.畜牧兽医学报,2005,36：381-386.
4.陈波,尤永进,潘洁,等.区分动物疫苗免疫与病毒感染的口蹄疫非结构蛋白3AB鉴别诊断试剂盒的研制.中国预防兽医学报,2007,29：52-57.
5.董志强,张志,李晓成,等.口蹄疫检测技术研究进展.中国动物检疫,2007,24：48-50.
6.弗朗西斯科·索布林那,埃斯特班·多明戈.口蹄疫：现状与未来(朱彩珠,张强,卢永干).北京：中国农业科学技术出版社,2009,367-375.
7.郭粉粉,梁文波,张学梅,单克隆抗体技术及应用的研究进展.医学综述,2011,17：1129-1131.
8.何忠效,浅谈单克隆抗体.生物学通报,2004,39：1-4.
9.花群义,周晓黎.蓝舌病病毒单克隆抗体的制备及生物学特性鉴定.中国兽医科技,2000,30：8-10.
10.林密,马军武,祁淑芸,等.O型口蹄疫病毒固相竞争ELISA抗体检测方法的建立.中国预防兽医学报,2008,30：627-630.
11.李波.曲妥株单克隆抗体治疗卵巢癌的研究进展.中华乳腺病杂志：电子版,2010,4：63-69.
12.李莉,王丽花,孔宪涛.单克隆抗体治疗的过去、现在和未来.免疫学杂志,2005,21：13-16.
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