Clackson T, Hoogenboom H R, Griffiths A D, Winter G
MRC Laboratory of Molecular Biology, Cambridge, UK.
Nature. 1991 Aug 15;352(6336):624-8. doi: 10.1038/352624a0.
To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.
为了绕过杂交瘤技术和动物免疫，我们试图通过模拟免疫选择的特征在细菌中构建抗体。最近，我们使用fd噬菌体展示与一种次要外壳蛋白融合的抗体片段，从而实现用抗原富集噬菌体。利用来自对半抗原2-苯基恶唑-5-酮（phOx）免疫的小鼠的重链（VH）和κ轻链（Vκ）重排的随机组合文库，我们现在已在fd噬菌体表面展示了多种抗体片段文库。经过一次通过半抗原亲和柱后，检测到具有一系列phOx结合活性的fd噬菌体，其中至少有一种具有高亲和力（解离常数，Kd = 10^(-8) M）。第二次通过以牺牲弱结合者为代价富集了强结合者。这些结合者由与抗phOx杂交瘤中发现的V基因相似但组合混杂的V基因编码（即在几种不同伙伴中发现相同的V基因）。通过将一个混杂的VH或Vκ基因与不同的伙伴文库组合以创建分层文库，我们引发了更多具有强结合活性的配对。噬菌体展示为从V基因文库制备抗体、改变V结构域配对以及选择具有良好亲和力的抗体提供了新方法。
