Phytopathology. 1999 Nov;89(11):1015-21. doi: 10.1094/PHYTO.1999.89.11.1015.
ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.
为了评估每个单链可变片段抗体（scFv）作为实用诊断工具的适用性，研究了一组 11 种不同的与马铃薯卷叶病毒（PLRV）结合的 scFv。这些 scFv 先前从原始噬菌体展示文库中获得，在大肠杆菌中作为融合蛋白表达。融合蛋白由 scFv 与人类轻链 κ 恒定域（C(L)）、两性螺旋（Zip）、C(L)和 Zip 的组合或碱性磷酸酶（AP/S）连接而成。通过酶联免疫吸附试验（ELISA）板检测融合蛋白是否能够检测或捕获感染马铃薯叶片提取物中的 PLRV ，从而测试融合蛋白的能力。用不同的 scFv 融合蛋白进行的测试与使用兔多克隆抗体制剂包被微量滴定板和单克隆抗体 SCR3 检测 PLRV 的标准三抗体夹心（TAS）-ELISA 进行了比较。在用作 TAS-ELISA 中的检测抗体时，11 种 scFvC(L)融合蛋白中有 7 种与 SCR3 一样容易检测到 PLRV。不同 scFvC(L)融合蛋白对纯化 PLRV 的检测限范围为 250 至 5ng/ml；SCR3 的检测限为 5ng/ml。在 11 种 scFv 中，有 4 种与其他一些杆状病毒发生交叉反应。几种 scFvC(L)和 scFvC(L)Zip 融合蛋白从感染马铃薯叶片的提取物中有效地捕获 PLRV，与多克隆抗体制剂一样有效。四种 scFv 融合蛋白用于茎印测定法检测 PLRV，结果与使用 SCR3 获得的结果相似。scFvC(L)融合蛋白在 4°C 下至少可保持 6 个月的活性，并且在冻干后重新配制时，所有 scFv 融合蛋白都完全具有活性。设计了一种完全重组的 ELISA ，用于检测感染马铃薯叶片提取物中的 PLRV，结果与使用标准 TAS-ELISA 获得的结果相当。使用 scFv 融合蛋白常规检测植物病毒的优点包括能够在细菌发酵罐中廉价地大量生产试剂，并且可以将其纳入标准化测试中。
