抗口蹄疫病毒A 型单克隆抗体的制备及其在竞争性ELISA 血清学诊断中的应用

Generation of mAbs to foot-and-mouth disease virus serotype A and application in a competitive ELISA for serodiagnosis.

作者信息

Yang Ming, Xu Wanhong, Bittner Hilary, Horsington Jacquelyn, Vosloo Wilna, Goolia Melissa, Lusansky Diana, Nfon Charles

机构信息

National Centre for Foreign Animal Disease, 1015 Arlington Street, Winnipeg, MB, R3E 3M4, Canada.

Australian Animal Health Laboratory, CSIRO, 5 Portarlington Road, Geelong, VIC, 3220, Australia.

出版信息

Virol J. 2016 Nov 28;13(1):195. doi: 10.1186/s12985-016-0650-z.

BACKGROUND

Foot-and-mouth disease (FMD) is an economically devastating disease that severely limits international trade of animals. Of the seven FMD virus (FMDV) serotypes, serotype A is one of the most widespread cross the world. Currently antibodies to FMDV are detected in animals using the virus neutralization test (VNT) and the enzyme-linked immunosorbent assay (ELISA). The VNT is laborious, time-consuming and reliant on live virus and cell cultures, while ELISA has the advantage of using inactivated antigens and often provides more reproducible results. The aim of this study was to develop a reliable and rapid competitive ELISA (cELISA) for the detection of antibodies to FMDV serotype A (FMDV/A).

RESULTS

A panel of FMDV/A specific monoclonal antibodies (mAbs) was generated and their ability to compete with a polyclonal serum from FMDV/A-infected cattle was examined. Two mAbs inhibited the binding of a polyclonal serum to FMDV/A viruses. The binding epitopes of each were determined as conformational and located on the VP2 viral capsid protein. The FMDV/A cELISA was developed using these two mAbs and FMDV/A inactivated virus as antigen. The diagnostic specificity and sensitivity were 99.7 and 99.3% (98.5-100%) respectively, based on a predetermined cut-off of 50% inhibition. When analysing sera from animals experimentally infected with FMDV/A, the cELISA detected antibodies from 5-days post infection (dpi) and remained positive for at least 21-28 days post infection. Comparison based on the Kappa coefficient showed strong agreement (90-94%) between cELISA and VNT.

CONCLUSION

The cELISA results are comparable to the VNT for antibody detection making it a simple and reliable test to detect antibodies against FMDV/A.

背景

口蹄疫（FMD）是一种具有经济破坏性的疾病，严重限制了动物的国际贸易。在七种口蹄疫病毒（FMDV）血清型中，A型是全球分布最广的血清型之一。目前，使用病毒中和试验（VNT）和酶联免疫吸附测定（ELISA）在动物中检测FMDV抗体。VNT费力、耗时且依赖活病毒和细胞培养，而ELISA具有使用灭活抗原的优势，且通常能提供更可重复的结果。本研究的目的是开发一种可靠、快速的竞争ELISA（cELISA），用于检测A型口蹄疫病毒（FMDV/A）抗体。

结果

制备了一组FMDV/A特异性单克隆抗体（mAb），并检测了它们与FMDV/A感染牛的多克隆血清竞争的能力。两种mAb抑制了多克隆血清与FMDV/A病毒的结合。每种mAb的结合表位被确定为构象表位，位于VP2病毒衣壳蛋白上。使用这两种mAb和FMDV/A灭活病毒作为抗原开发了FMDV/A cELISA。基于预定的50%抑制率临界值，诊断特异性和敏感性分别为99.7%和99.3%（98.5 - 100%）。在分析实验感染FMDV/A的动物血清时，cELISA在感染后5天（dpi）检测到抗体，并在感染后至少21 - 28天保持阳性。基于kappa系数的比较显示，cELISA和VNT之间具有高度一致性（90 - 94%）。