Selection and Characterization of Human Serum Albumin-specific Porcine scFv Antibodies Using a Phage Display Library
Selection and Characterization of Human Serum Albumin-specific Porcine scFv Antibodies Using a Phage Display Library
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DOI：
10.1089/mab.2013.0068
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发表时间：
2014-02-01
影响因子：
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通讯作者：
Ito, Yuji
中科院分区：
文献类型：
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作者：
Muraoka, Junko;Ozawa, Takuya;Ito, Yuji
构建了一个新的单链可变区抗体库，并对人血清白蛋白（HSA）特异性克隆进行了表征，以研究猪抗体的有效性。从用模型抗原HSA免疫的猪开发噬菌体文库。文库大小对于κ（V-L）为1.5x10（7），对于λ片段为1.4x10（7）。使用亲和选择分离来自κ文库的八个HSA特异性克隆和来自λ文库的一个克隆。使用噬菌体酶联免疫吸附测定（ELISA）确认这些克隆的结合特异性。在大肠杆菌中表达scFv，并从周质级分中纯化用于进一步研究。根据ELISA和Western blot分析结果，筛选出4个活性高、产量高的单链抗体克隆，并进行了纯化。来自9个克隆中的4个的纯化的scFv表现出约10（-8）M的K-D。这是第一份描述从抗体噬菌体库中分离HSA特异性猪单链抗体并表征其结合特性的报告。
A new single-chain variable fragment (scFv) antibody library was generated and human serum albumin (HSA)-specific clones were characterized to investigate the usefulness of porcine antibodies. Phage libraries were developed from pigs immunized with the model antigen HSA. The library size was 1.5x10(7) for kappa (V-L) and 1.4x10(7) for lambda fragments. Eight HSA-specific clones from the kappa library and one clone from the lambda library were isolated using affinity selection. The binding specificity of these clones was confirmed using a phage enzyme-linked immunosorbent assay (ELISA). The scFvs were expressed in Escherichia coli and purified from the periplasm fraction for further investigation. Based on the results of ELISA and Western blot analysis, four scFv clones with high activity and high yield were selected and purified. The purified scFvs from four of the nine clones exhibited an approximate K-D of 10(-8) M. This is the first report describing isolation of HSA-specific porcine scFv antibodies from an antibody phage library and characterization of their binding properties.