Al-Mrabeh Ahmad, Ziegler Angelika, Cowan Graham, Torrance Lesley
Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.
J Virol Methods. 2009 Aug;159(2):200-5. doi: 10.1016/j.jviromet.2009.03.025. Epub 2009 Apr 5.
A recombinant antibody fusion protein, V3HCL, which was shown previously to have specific reactivity for potato leafroll virus (PLRV), was labeled with biotin using standard chemical coupling procedures and by an in vivo method. The in vivo method proved superior giving reproducible V3HCL-biotin preparations. A fully recombinant ELISA was devised incorporating V3HCL, V3HCL-biotin and streptavidin alkaline phosphatase conjugate. This assay gave comparable results for PLRV detection in potato to an assay based on immunoglobulins. The V3HCL-biotin preparations were stable and retained specific activity for more than 1 year when stored at 4 degrees C or -20 degrees C. The results demonstrate that scFv reagents derived from synthetic phage display platforms can provide effective alternatives to assays incorporating immune reagents.
一种重组抗体融合蛋白V3HCL，先前已证明其对马铃薯卷叶病毒（PLRV）具有特异性反应，采用标准化学偶联程序和体内方法用生物素进行标记。体内方法被证明更具优势，可得到可重复的V3HCL-生物素制剂。设计了一种完全重组的ELISA，其中包含V3HCL、V3HCL-生物素和链霉亲和素碱性磷酸酶缀合物。该检测方法在马铃薯中检测PLRV的结果与基于免疫球蛋白的检测方法相当。V3HCL-生物素制剂稳定，在4℃或-20℃储存时，其比活性可保持1年以上。结果表明，源自合成噬菌体展示平台的单链抗体片段试剂可为包含免疫试剂的检测方法提供有效的替代方案。
