Recombinant human monoclonal Fab fragments against rotavirus from phage display combinatorial libraries
Recombinant human monoclonal Fab fragments against rotavirus from phage display combinatorial libraries
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DOI：
10.1093/oxfordjournals.jbchem.a022248
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发表时间：
1999-01-01
影响因子：
2.7
通讯作者：
Suzuki, T
中科院分区：
文献类型：
--
作者：
Itoh, K;Nakagomi, O;Suzuki, T
我们从两名健康人的外周血淋巴细胞(PBL)构建的免疫球蛋白G(1)x组合文库(简称N和O)中制备并鉴定了抗轮状病毒的人源单抗Fab片段。在对兔多克隆抗体捕获的人轮状病毒(HRV)Wa株进行5轮淘洗后，这些文库中的洗脱噬菌体获得了约30倍的浓缩。来自每个图书馆的48个克隆。用酶联免疫吸附试验(ELISA)检测对HRV Wa的反应性，并用BstNI指纹图谱鉴定阳性克隆的身份，结果分离到8个克隆(5个来自N文库，3个来自O文库)。在测试Fab对一组自身或非自身抗原的交叉反应中，所有Fab克隆都是针对HRV Wa的，来自两个文库的Fab克隆在与Wa的反应模式和与轮状病毒株的交叉反应方面表现出不同的特征，并显示出可变的重链(V-H)链基因使用，尽管它们识别免疫印迹确定的VP6蛋白，但来自两个文库的Fab对不同的表位识别表明在两个人感染轮状病毒期间对轮状病毒的体液免疫应答过程不同。
We prepared and characterized human monoclonal Fab fragments to rotavirus from IgG(1) x combinatorial libraries (designated as N and O) constructed from peripheral blood lymphocytes (PBLs) from two healthy individuals. Approximately 30-fold enrichment in eluted phage was obtained in these libraries after five rounds of panning against rabbit polyclonal antibody-captured human rotavirus (HRV) Wa strain. Forty-eight clones from each library. were tested for reactivity to HRV Wa in an enzyme-linked immunosorbent assay (ELISA), and the identities of positive clones were determined by BstNI fingerprinting, As a result, eight individual clones (five from N library and three from O library) were isolated. In testing the cross-reactivity of Fabs against a panel of self- or non-self antigens, all Fab clones were found to be specific for HRV Wa, Fab clones from the two libraries showed distinct characteristics with respect to their reaction patterns with Wa and crossreactivities with rotavirus strains, and displayed variable heavy (V-H) chain gene usage, although they recognized the VP6 protein as determined by immunoblotting, The distinct epitope recognition by Fabs from two libraries suggests different courses of humoral immune response to rotavirus during infection in the two individuals.