Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agriculture Technology , Suranaree University of Technology , Nakhon Ratchasima 3000 , Thailand.
Department of Biochemistry, Faculty of Science , Kasetsart University , 50 Ngam Wong Wan Road, Chatuchak , Bangkok 10900 , Thailand.
J Agric Food Chem. 2018 Jun 6;66(22):5713-5722. doi: 10.1021/acs.jafc.8b01141. Epub 2018 May 29.
A human antiaflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naı̈ve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable-heavy and variable-light (VH/VL)-shuffled library was constructed from the VH of 25 preselected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these libraries demonstrated that the VH domain played an important role in the binding of scFv to free AFB1. Therefore, in the next step, VH-shuffled scFv library was constructed from variable-heavy (VH) chain repertoires, amplified from the naı̈ve library, recombined with the variable-light (VL) chain of the clone yAFB1-c3. This library was then used to select a specific scFv antibody against soluble AFB1 by a standard biopanning method. Three clones that showed improved binding properties were isolated. Amino acid sequence analysis indicated that the improved clones have amino acid mutations in framework 1 (FR1) and the complementarity determining region (CDR1) of the VH chain. One clone, designated sAFH-3e3, showed 7.5-fold improvement in sensitivity over the original scFv clone and was selected for molecular binding studies with AFB1. Homology modeling and molecular docking were used to compare the binding of this and the original clones. The results confirmed that VH is more important than VL for AFB1 binding.
一种人抗黄曲霉毒素 B1（AFB1）单链抗体（yAFB1-c3），从一个原始的人噬菌体展示单链抗体文库中筛选出来，被用作模板，使用链改组技术改进和分析抗体-配体相互作用。将 25 个预先选择的克隆的 VH 与 yAFB1-c3 的 VL 进行重组，或者反过来，构建了 VH/VL 改组文库。从这些文库中进行的亲和选择表明，VH 结构域在 scFv 与游离 AFB1 的结合中起重要作用。因此，在下一步中，从原始文库扩增的 VH 链的可变重链（VH）库中构建了 VH 改组 scFv 文库，然后与克隆 yAFB1-c3 的可变轻链（VL）链进行重组。然后，使用该文库通过标准的生物淘选方法筛选针对可溶性 AFB1 的特异性 scFv 抗体。分离出三个显示出改善的结合特性的克隆。氨基酸序列分析表明，这些改良的克隆在 VH 链的框架 1（FR1）和互补决定区（CDR1）中具有氨基酸突变。一个名为 sAFH-3e3 的克隆，其灵敏度比原始 scFv 克隆提高了 7.5 倍，被选为与 AFB1 进行分子结合研究的候选物。同源建模和分子对接用于比较该克隆和原始克隆的结合情况。结果证实，VH 比 VL 更重要，对于 AFB1 的结合。
