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Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirAVp and PirBVp released from Vibrio spp.
Journal of Microbiological Methods ( IF 2.2 ) Pub Date : 2020-07-10 , DOI: 10.1016/j.mimet.2020.106002
Hung Nam Mai 1 , Roberto Cruz-Flores 1 , Arun K Dhar 1
Affiliation  

An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp down to 0.008 ng/μl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.



中文翻译:

开发了一种间接酶联免疫测定(iELISA)技术,该技术使用的是抗弧菌属昆虫相关毒素,从弧菌属物种中释放的PirAVp和PirBVp的单克隆抗体。

急性肝胰腺坏死病(AHPND)对全球虾业造成严重损失。AHPND的病原体是弧菌。携带一个编码二元毒素PirAB的大质粒。当前,通过基于PCR的方法诊断AHPND,该方法检测pir A和pir B基因的存在。然而,含有pir A和pir B基因的细菌菌株并不总是表达二元毒素,导致对含pir A和pir B基因的细菌菌株的毒力的错误估计。因此,基于免疫的测定(即ELISA)是检测PirA Vp和PirB Vp的有前途的方法。在本研究中,通过western印迹分析筛选了针对PirA Vp(20 mAbs)和PirB Vp(20 mAbs)的总共四十个单克隆抗体克隆,以选择四个在间接ELISA(iELISA)中显示最强免疫反应性的mAb克隆)。四个选定的mAb(即针对PirA Vp的1B9和5E9 ;针对PirB Vp的7B7和7B9 )专门检测到弧菌。造成AHPND。此外,四个选定的mAb能够检测低至0.008 ng /μl的PirA Vp或PirB Vp。使用30种AHPND感染和6种SPF虾南美白对虾的双盲检测通过iELISA分析以确定测定的检测灵敏度。结果表明,iELISA能够准确检测出30只AHPND感染虾中的29只。这些发现表明,iELISA是检测受感染虾中PirA Vp和PirB Vp毒素的可靠方法,将成为诊断AHPND和研究二元毒素在AHPND发病机理中的有用工具。

更新日期:2020-07-16
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