Phytopathology. 1998 Mar;88(3):230-3. doi: 10.1094/PHYTO.1998.88.3.230.
ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.
摘要 无需动物免疫，我们从合成噬菌体展示抗体基因文库中获得了与黑醋栗返祖相关病毒（BRAV）结合的单链可变片段（scFv）抗体。经过三轮针对固定化病毒抗原的筛选，很快就获得了几种不同的 BRAV 特异性噬菌体 scFv。噬菌体 scFv 的酶联免疫吸附测定（ELISA）吸光度值大于对照健康植物提取物的 7 倍。相比之下，使用兔抗血清进行的比较测试失败了，因为健康植物提取物的背景值过高。将其中两个 scFv 亚克隆到 pDAP2 载体中，可快速高效地生产 scFv-碱性磷酸酶融合蛋白。在大肠杆菌中表达后获得了功能性融合蛋白，并用从周质提取物中制备的融合蛋白在 ELISA 中检测到了 BRAV。结果表明，从合成噬菌体展示文库中获得的抗体片段是有用的研究工具，当传统抗血清未能检测到 BRAV（一种弱免疫原）时，它们被证明是一种可行的实用替代方法。此外，抗体片段与碱性磷酸酶的遗传融合避免了进一步的化学偶联步骤，并且可以廉价地获得融合蛋白。
