Kretzschmar T, Zimmermann C, Geiser M
Ciba-Geigy Limited, Basel, Switzerland.
Anal Biochem. 1995 Jan 1;224(1):413-9. doi: 10.1006/abio.1995.1059.
Libraries of peptides and proteins can be displayed on the surface of filamentous bacteriophage. The efficient capturing of phage recognizing a defined target molecule remains a serious obstacle, in particular when the phage are present at a low frequency or have a reduced affinity like nonmatured phage antibodies and when the availability of target molecules is limited. We present theoretical considerations and experimental data which allowed us to substantially improve microselection under these conditions. We used a model phage displaying an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody fragment. Following standard protocols and aiming at a low nonspecific binding, only 3.6 x 10(-3)% of the phage input could be recovered from a single round of selection performed in the wells of a microtiter plate. Our results explain why this often employed panning in wells is not efficient, especially with high-molecular-weight target molecules. We devised a procedure which increased the probability of microselection by a factor of 34. An alternative capturing method using immunotubes with a new protocol decreased the amount of required work by a factor of 30. In the case of a nonlimited supply of target molecules, column-affinity chromatography is recommended.
肽和蛋白质文库可展示在丝状噬菌体表面。有效捕获识别特定靶分子的噬菌体仍然是一个严重障碍，特别是当噬菌体以低频率存在或具有降低的亲和力时，如未成熟的噬菌体抗体，以及当靶分子的可用性有限时。我们提出了理论思考和实验数据，使我们能够在这些条件下大幅改进微选。我们使用了一种展示抗（2-苯基-5-恶唑酮）单链Fv抗体片段的模型噬菌体。按照标准方案并旨在实现低非特异性结合，在微量滴定板孔中进行的一轮选择中，仅能回收3.6×10⁻³%的噬菌体输入量。我们的结果解释了为什么这种常用于孔中的淘选效率不高，尤其是对于高分子量靶分子。我们设计了一种程序，将微选概率提高了34倍。一种使用免疫管和新方案的替代捕获方法将所需工作量减少了30倍。在靶分子供应不受限的情况下，建议使用柱亲和色谱法。
