Choi I H, Park S G, Chung J H, Kim I J, Hong H J
Department of Microbiology, College of Medicine, Inje University, Pusan, Korea.
Hybridoma. 1998 Dec;17(6):535-40. doi: 10.1089/hyb.1998.17.535.
Human monoclonal antibodies (MAbs) have considerable potential in the prevention and treatment of many viral diseases. A combinatorial antibody library of heavy (Fd)- and light-chain genes derived from peripheral blood lymphocytes of a volunteer with high antibody titer to preS1 of HBV was constructed and expressed on the surface of filamentous phages. The library contained 7 x 10(9) independent clones. A phage antibody population from the third panning against preS1 was converted to one expressing soluble Fabs by removal of the g3 sequences from the pComb3 phagemid vector and subsequent transformation into E. coli TG1 cells. Screening of the library led to the identification of two clones, 3DW and 8GW, showing high reactivity toward preS1. The authenticity of the Fabs was confirmed by immunoblot analysis which yielded approximately 60 and approximately 30 kDa bands under nonreducing and reducing conditions, respectively. The soluble Fabs of 3DW and 8GW exhibited relative affinities of 6 x 10(5) and 8 x 10(6) M(-1), respectively. The sequencing results demonstrate that all Fd sequences belong to subgroup II and all light chain sequences belong to subgroup I. There are differences in CDR length and composition, especially in the FW3 and CDR3 regions of the heavy- and light-chain genes. These human Fab MAbs specific to preS1, generated from a combinatorial library, represent prototypes of passive immunotherapy candidates for viral hepatitis B.
人单克隆抗体在许多病毒性疾病的预防和治疗中具有巨大潜力。构建了一个由一名对乙肝病毒前S1具有高抗体滴度的志愿者外周血淋巴细胞来源的重链（Fd）和轻链基因组成的组合抗体文库，并在丝状噬菌体表面表达。该文库包含7×10⁹个独立克隆。通过从pComb3噬菌粒载体中去除g3序列并随后转化到大肠杆菌TG1细胞中，将第三次针对前S1淘选得到的噬菌体抗体群体转化为一个表达可溶性Fab片段的群体。对该文库的筛选导致鉴定出两个克隆，3DW和8GW，它们对前S1表现出高反应性。通过免疫印迹分析证实了Fab片段的真实性，在非还原和还原条件下分别产生了约60 kDa和约30 kDa的条带。3DW和8GW的可溶性Fab片段的相对亲和力分别为6×10⁵ M⁻¹和8×10⁶ M⁻¹。测序结果表明，所有Fd序列属于II亚组，所有轻链序列属于I亚组。在互补决定区（CDR）长度和组成上存在差异，尤其是在重链和轻链基因的框架区3（FW3）和互补决定区3（CDR3）区域。这些从组合文库中产生的针对前S1的人Fab单克隆抗体代表了乙型病毒性肝炎被动免疫治疗候选物的原型。
