Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854 Japan.
Structural Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
J Biotechnol. 2017 Aug 10;255:1-8. doi: 10.1016/j.jbiotec.2017.06.010. Epub 2017 Jun 20.
We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries.
我们描述了一种方法，即单步集落分析法，用于简单快速地筛选单链 Fv 片段（scFv）文库。在亲水滤膜上形成表达 scFv 文库的大肠杆菌集落，该滤膜与包被抗原的膜紧密接触。用诱导试剂处理集落后，scFv 表达被触发，随后 scFvs 从细胞中分泌出来并扩散到抗原包被的膜上。与抗原具有结合亲和力的 scFvs 被膜固定化的抗原捕获。最后，检测膜上抗原与 scFv 的结合情况，可鉴定出表达抗原特异性 scFv 的滤膜上的克隆。我们使用抗兔 IgG scFv（scFv(A10B)）和大鼠免疫 scFv 文库对该方法进行了测试。使用 scFv(A10B)进行的实验表明，该方法可提高集落分析过程中 scFv 的表达。通过使用我们的方法筛选 3×10 个 scFv 克隆的免疫文库，我们建立了几个对抗原具有亲和力的克隆。此外，我们还测试了 7 种其他抗原，包括肽，并成功鉴定了阳性克隆。我们相信，这种简单的单步集落分析程序和 scFv 的受控表达，可以使抗体筛选既快速又可靠，并从抗体文库中成功分离出阳性克隆。
