By-passing immunization

免疫球蛋白轻链 平移(音频) 半抗原 抗原 噬菌体展示 分子生物学 抗体 基因 生物 噬菌体 化学 大肠杆菌 遗传学 噬菌体 古生物学 缩放 镜头(地质)
作者
James D. Marks,Hennie R. Hoogenboom,Timothy P. Bonnert,John McCafferty,Andrew D. Griffiths,Greg Winter
出处
期刊:Journal of Molecular Biology [Elsevier]
卷期号:222 (3): 581-597 被引量:1536
标识
DOI:10.1016/0022-2836(91)90498-u
摘要

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.
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