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 10⁻⁴–10⁻⁵% 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_off) of isolated scFv clones tended to decrease with progressing biopanning rounds. The average dissociation constants (K_D) of the isolated scFvs ranged between 1.7 and 87 nM, whereas the lowest K_D 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.

我们证明了使用抗原偶联的多层囊泡（Ag-MLVs）作为固体支持物的兔scFv抗体的有效筛选方法。经过3轮或更少的生物淘洗后，通过Ag-MLV成功分离出显示小鼠抗人IgG scFv的模型噬菌体，其概率为10 ^-4 –10 ^-5％，而无法使用常规的抗原包被的免疫管分离。该筛选方法适用于从4个不同噬菌体库中分离兔抗原特异性scFv。使用Ag-MLV的生物淘选程序在第3轮或更早的时间内产生了阳性噬菌体，并且在第1轮之后在两个生物淘选中观察到了scFv的特异性抗原结合。离解速率常数（k _off）分离的scFv克隆往往会随着生物淘斗的进行而减少。分离的scFv的平均解离常数（K _D）在1.7和87 nM之间，而最低的K _D记录了12 pM的抗CRP scFv。对分离的兔scFv的355个不同克隆的综合表征显示出较低的等电点，并且基于它们的不稳定性和脂肪族指数，其中大多数比常规的小鼠scFv更热稳定。这些结果清楚地表明了展示兔scFv的噬菌体文库和使用Ag-MLV进行生物淘选进行抗体发现相结合的优势和潜力。此外，本研究中获得的结果支持了兔scFvs在多种应用中的适用性，包括开发用于分子诊断和生物分离的诊断剂和亲和配体。