McGiven John A, Sawyer Jason, Perrett Lorraine L, Brew Simon D, Commander Nicola J, Fisher Alan, McLarnon Stuart, Harper Kate, Stack Judy A
Brucella Research Group, Statutory and Exotic Bacteria, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey, UK.
J Immunol Methods. 2008 Aug 20;337(1):7-15. doi: 10.1016/j.jim.2008.05.004. Epub 2008 Jun 2.
The control and eradication of brucellosis is highly desirable but heavily resource intensive as high throughput serological testing is required. The aim of this study was to meet the needs of high throughput screening laboratories involved in this process through the development of a new assay. An existing cELISA used for the serodiagnosis of brucellosis in ruminants was converted to an AlphaLISA homogenous proximity based assay. This assay requires no separation steps and can be performed in low volume microtitre format. The Brucella AlphaLISA was validated on a panel of bovine, ovine and caprine sera from infected and uninfected animals. The diagnostic sensitivities (>96%) and specificities (>98%) obtained compared well to those from cELISA, iELISA and FPA performed on the same samples. The AlphaLISA met the testing criteria set for ELISAs as defined by the OIEELISA standards and had an analytical sensitivity similar to that of the parent cELISA. The method was also used on a small panel of serum samples from cattle that were experimentally infected with Yersinia enterocolitica O:9. Some false positive reactions were obtained as was also the case with results from FPA, iELISA, cELISA, CFT and SAT. Despite this, the methodological advantages of the AlphaLISA mean that this assay is well suited to high throughput serodiagnosis. This report is the first description of the use of AlphaLISA to detect pathogen specific antibodies. Furthermore, the relative ease with which the cELISA was converted to this platform indicates that this technology is ready to meet the high throughput testing requirements for the diagnosis of many other diseases.
控制和根除布鲁氏菌病非常必要，但由于需要进行高通量血清学检测，资源投入巨大。本研究的目的是通过开发一种新检测方法，满足参与此过程的高通量筛查实验室的需求。一种用于反刍动物布鲁氏菌病血清诊断的现有竞争酶联免疫吸附测定（cELISA）被转化为基于均相邻近分析的AlphaLISA测定。该测定无需分离步骤，可在微量滴定板中以小体积进行。布鲁氏菌AlphaLISA在一组来自感染和未感染动物的牛、羊和山羊血清上进行了验证。与对相同样本进行的cELISA、间接酶联免疫吸附测定（iELISA）和荧光偏振分析（FPA）相比，所获得的诊断敏感性（>96%）和特异性（>98%）相当。AlphaLISA符合世界动物卫生组织（OIE）ELISA标准所定义的ELISA检测标准，其分析敏感性与原始cELISA相似。该方法还用于一小组实验感染小肠结肠炎耶尔森菌O:9的牛血清样本。与FPA、iELISA、cELISA、补体结合试验（CFT）和虎红平板凝集试验（SAT）的结果一样，也获得了一些假阳性反应。尽管如此，AlphaLISA的方法学优势意味着该测定非常适合高通量血清诊断。本报告首次描述了使用AlphaLISA检测病原体特异性抗体。此外，将cELISA转化为该平台相对容易，这表明该技术已准备好满足许多其他疾病诊断的高通量检测要求。
