Department of Functional Chemistry and Engineering, Kyoto Institute of Technology, 1 Matsugasaki-Hashikami-Cho, Sakyo-ku, Kyoto 606-8585, Japan.
Department of Functional Chemistry and Engineering, Kyoto Institute of Technology, 1 Matsugasaki-Hashikami-Cho, Sakyo-ku, Kyoto 606-8585, Japan.
J Biosci Bioeng. 2021 Mar;131(3):299-304. doi: 10.1016/j.jbiosc.2020.10.007. Epub 2020 Nov 19.
We demonstrated an efficient screening method for rabbit scFv antibodies using antigen-coupled multi-lamellar vesicles (Ag-MLVs) as solid supports. Model phages displaying mouse anti-human IgG scFv at a probability of 10-10% were successfully isolated by Ag-MLVs after 3 or less rounds of biopanning, whereas they could not be isolated using conventional antigen-coated immunotubes. This screening method was applied to isolate rabbit antigen-specific scFvs from 4 different phage libraries. Biopanning procedures employing Ag-MLVs yielded positive phages in the 3rd round or earlier, and specific antigen-binding of scFvs was observed after the 1st round in two biopanning selections. The dissociation rate constants (k) of isolated scFv clones tended to decrease with progressing biopanning rounds. The average dissociation constants (K) of the isolated scFvs ranged between 1.7 and 87 nM, whereas the lowest K of 12 pM was recorded for anti-CRP scFv. Comprehensive characterization of 355 different clones of the isolated rabbit scFvs presented a relatively low isoelectric point, and most of these were more thermo-stable than the conventional mouse scFvs, based on their instability and aliphatic indices. These results clearly indicate the advantages and potential of a combination of rabbit scFv-displaying phage library and biopanning using Ag-MLVs for antibody discovery. In addition, the results obtained in this study support the suitability of rabbit scFvs for several applications, including the development of diagnostic agents and affinity ligands for molecular diagnosis and bioseparation.
我们展示了一种使用抗原偶联多层层囊（Ag-MLVs）作为固体支持物筛选兔 scFv 抗体的有效方法。在 3 轮或更少轮的生物淘选后，成功地从 Ag-MLVs 中分离出了展示概率为 10-10%的鼠抗人 IgG scFv 的模型噬菌体，而使用传统的抗原包被免疫管则无法分离到这些噬菌体。该筛选方法已应用于从 4 个不同噬菌体文库中分离兔抗原特异性 scFv。采用 Ag-MLVs 的生物淘选程序在第 3 轮或更早的轮次中获得了阳性噬菌体，并且在两轮生物淘选中有 2 轮在第 1 轮就观察到了 scFv 的特异性抗原结合。分离出的 scFv 克隆的解离速率常数（k）随着生物淘选轮次的增加而趋于减小。分离出的 scFv 的平均解离常数（K）在 1.7 和 87 nM 之间，而抗 CRP scFv 的最低 K 值为 12 pM。对分离出的 355 个不同克隆的 scFv 进行的综合表征表明，其等电点相对较低，并且大多数比传统的鼠 scFv 更耐热，这基于它们的不稳定性和脂肪指数。这些结果清楚地表明了兔 scFv 展示噬菌体文库和使用 Ag-MLVs 进行生物淘选相结合在抗体发现方面的优势和潜力。此外，本研究的结果支持兔 scFv 适用于包括开发诊断试剂和亲和配体在内的几种应用，用于分子诊断和生物分离。
