Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naive antibody phage library
Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naive antibody phage library
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DOI：
10.1016/j.antiviral.2005.06.012
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发表时间：
2005-12-01
影响因子：
7.6
通讯作者：
Choi, IH
中科院分区：
文献类型：
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作者：
Park, SG;Jeong, YJ;Choi, IH
我们报道了一个单链可变区片段（scFv）格式的大型非免疫人噬菌体抗体文库的构建，该文库允许选择体外中和乙型肝炎病毒（HBV）的抗体。我们利用从50名未免疫的人类供者的外周血单个核细胞中纯化的重链和轻链功能变量(V)基因片段的cDNA，生成了1.1 x 1010个独立的scFv克隆。使用BlAcore，我们选择了两个识别pre-S1并中和与Chang肝细胞结合的pre-S1和HBV的克隆。克隆G10的亲和度最高（K-D = 1.69 × 10(-7) M），高于先前从重链洗牌免疫文库中获得的克隆1E4。BlAcore测定的克隆脱靶率在10(-3)s（-1）以内，与来自正常二次免疫应答的抗体的脱靶率相当。流式细胞术和聚合酶链反应检测G10对肝细胞前s1和病毒结合的抑制作用，结果表明G10比1E4具有更好的中和活性。新的噬菌体文库可能是针对不同靶点具有合理亲和力的抗体的宝贵来源，抗pre- s1 G10可能是抗HBV感染的免疫预防轴的良好候选者。(c) 2005年Elsevier B.V.出版
We report the construction of a large nonimmunized human phage antibody library in single-chain variable region fragment (scFv) format, which allowed the selection of antibodies that neutralize hepatitis B virus (HBV) in vitro. We generated 1.1 x 1010 independent scFv clones using the cDNA of functional variable (V) gene segments of heavy and light chains purified from the peripheral blood mononuclear cells of 50 nonimmunized human donors. Using BlAcore, we selected two clones that recognized pre-S1 and neutralized pre-S1 and HBV binding to Chang liver cells. Clone G10 had the highest affinity (K-D = 1.69 x 10(-7) M), which was higher than that of clone 1E4 that was generated previously from a heavy chain-shuffled immune library. The off-rates of clones were within 10(-3) s(-1) as determined by BlAcore and were comparable to those of antibodies derived from a normal secondary immune response. In the inhibition assays of pre-S1 and virus binding to Chang liver cells using flow cytometry and the polymerase chain reaction, G10 had better neutralizing activity than 1E4. The new phage library may be a valuable source of antibodies with reasonable affinities to different targets, and the anti-pre-S1 G10 may be a good candidate for immunoprophyl axis against HBV infection. (c) 2005 Published by Elsevier B.V.