Harper K, Toth R L, Mayo M A, Torrance L
Scottish Crop Research Institute, Dundee, UK.
J Virol Methods. 1999 Aug;81(1-2):159-68. doi: 10.1016/s0166-0934(99)00071-3.
Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.
从两个天然噬菌体展示文库中获得了12种与马铃薯卷叶病毒（PLRV）颗粒结合的单链可变片段（scFv）抗体。针对固定在试管上的PLRV颗粒或解离的PLRV颗粒筛选噬菌体。通过ELISA鉴定单个与PLRV结合的scFv，其表达形式为融合到噬菌体颗粒表面，或作为可溶性scFv（scFv-c-myc），或作为通过亚克隆到pSKAP/S中获得的scFv-碱性磷酸酶融合蛋白（scFv-AP）。这些程序导致分离出具有不同特性的scFv。例如，一些scFv与病毒颗粒强烈反应，但与解离的衣壳蛋白不反应，这表明它们与不连续表位反应。其他scFv与解离的衣壳蛋白和SDS变性蛋白反应，这表明它们与连续表位反应。scFv也被亚克隆到pC(L)中，以表达与人κ恒定区的融合蛋白（scFv-C(L)）。这些构建体在大肠杆菌中的表达每升细菌培养物产生0.2-1毫克蛋白质。在ELISA中评估不同的scFv融合蛋白以检测弗罗里达酸浆叶提取物中的PLRV。用融合蛋白获得的吸光度值大于用scFv-c-myc获得的吸光度值，并且与使用单克隆抗体或多克隆抗体进行的测定中获得的吸光度值相似。
