Quality enhancement of the non-immune phage scFv library to isolate effective antibodies
Quality enhancement of the non-immune phage scFv library to isolate effective antibodies
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DOI：
10.1248/bpb.29.1325
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发表时间：
2006-07-01
影响因子：
2
通讯作者：
Tsunoda, Shin-ichi
中科院分区：
文献类型：
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作者：
Imai, Sunao;Mukai, Yohei;Tsunoda, Shin-ichi
非免疫噬菌体抗体库系统是目前治疗、诊断和基础科学研究中最具吸引力的技术之一。该系统可以快速分离感兴趣的抗体，这些抗体随后可以直接应用于药物输送系统和抗体治疗。以前，我们报道了引物集包含抗体库，从而提高文库质量。然而，大量不同的引物集合导致抗体基因的扩增效率降低。在本研究中，我们重新生成了文库引物集，并从非免疫小鼠中构建了一个在种类和质量上都远远优于其他小鼠的改进文库。这个新的文库包含了24亿个独立克隆。此外，我们优化了从该文库中分离高亲和力抗体的选择步骤。通过结合自动化微流体仪器优化亲和筛选方案，成功分离出三种不同的人血管内皮生长因子受体2 （KDR）单克隆抗体。这些抗体被证明具有高特异性，并且能够通过点印迹分析检测仅0.6 fmol的KDR。以前报道的荧光素酶抗体也从这个文库中成功分离出来。我们的结果清楚地证明了改进的方案对抗体库制备和由此产生的抗体分离的临床和研究应用的重要性。
The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.