Itoh K, Nakagomi O, Suzuki K, Inoue K, Tada H, Suzuki T
Department of Pharmaceutical Science, Akita University Hospital, Akita University School of Medicine, Akita, 010-8543, Japan.
J Biochem. 1999 Jan;125(1):123-9. doi: 10.1093/oxfordjournals.jbchem.a022248.
We prepared and characterized human monoclonal Fab fragments to rotavirus from IgG1 kappa 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 (VH) 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.
我们从两名健康个体的外周血淋巴细胞（PBL）构建的IgG1 κ组合文库（命名为N和O）中制备并鉴定了针对轮状病毒的人源单克隆Fab片段。在针对兔多克隆抗体捕获的人轮状病毒（HRV）Wa株进行五轮淘选后，这些文库中洗脱噬菌体的富集倍数约为30倍。通过酶联免疫吸附测定（ELISA）检测每个文库中的48个克隆对HRV Wa的反应性，并通过BstNI指纹图谱确定阳性克隆的身份。结果，分离出了8个单克隆（5个来自N文库，3个来自O文库）。在测试Fab片段对一组自身或非自身抗原的交叉反应性时，发现所有Fab克隆均对HRV Wa具有特异性。来自两个文库的Fab克隆在与Wa的反应模式以及与轮状病毒株的交叉反应性方面表现出不同的特征，并且尽管通过免疫印迹确定它们识别VP6蛋白，但它们显示出可变重链（VH）基因的使用情况不同。来自两个文库的Fab片段对不同表位的识别表明，这两个个体在感染轮状病毒期间体液免疫反应的过程不同。
