SELECTION AND APPLICATION OF HUMAN SINGLE-CHAIN FV ANTIBODY FRAGMENTS FROM A SEMISYNTHETIC PHAGE ANTIBODY DISPLAY LIBRARY WITH DESIGNED CDR3 REGIONS
SELECTION AND APPLICATION OF HUMAN SINGLE-CHAIN FV ANTIBODY FRAGMENTS FROM A SEMISYNTHETIC PHAGE ANTIBODY DISPLAY LIBRARY WITH DESIGNED CDR3 REGIONS
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DOI：
10.1006/jmbi.1995.0204
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发表时间：
1995-04-21
影响因子：
5.6
通讯作者：
LOGTENBERG, T
中科院分区：
文献类型：
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作者：
DEKRUIF, J;BOEL, E;LOGTENBERG, T
我们通过将49个种系V-H基因与合成的重链CDR 3（HCDR 3）区和7个轻链结合，构建了大的（3.6 × 10（8）克隆）人单链抗体（scFv）片段噬菌体展示文库。HCDR 3区的长度在6和15个残基之间变化，并且被设计为包含完全随机化的氨基酸残基段，所述氨基酸残基段两侧是由天然抗体中经常出现的氨基酸残基组成的有限残基可变性区域。我们推断，这种方法将增加我们文库中功能分子的频率，此外，还允许我们有效地利用可用的克隆空间。通过固相结合抗原的直接选择，我们获得了与13种不同抗原具有结合活性的噬菌体抗体，包括Von Willebrand因子、转录因子TCF-1的DNA结合HMG盒和肿瘤抗原EGP-2。此外，我们应用竞争性选择程序将噬菌体抗体靶向重组融合蛋白的所需部分，并选择能够区分两种高度同源框蛋白PBX 1a和PBX 2的噬菌体抗体。单克隆噬菌体抗体的功能能力进行了评估，在免疫组织化学染色的组织标本，Western印迹分析和免疫荧光分析的细胞通过流式细胞术的结果表明，这个大的人噬菌体抗体库包含了广泛的分类的结合特异性，可以应用于各种生化分析。
We have constructed a large (3.6x10(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline V-H genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens, Western blotting assays and immunofluorescent analysis of cells by flow cytometry The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.