Generation and application of a fluorescein-specific single chain antibody
Generation and application of a fluorescein-specific single chain antibody
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DOI：
10.1016/j.biochi.2007.06.008
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发表时间：
2007-11-01
期刊：
影响因子：
3.9
通讯作者：
Micheel, Burkhard
中科院分区：
文献类型：
--
作者：
Schenk, Joerg A.;Sellrie, Frank;Micheel, Burkhard
使用原始杂交瘤细胞作为可变抗体重链和轻链（VH和VL）基因的来源，生成鼠单克隆抗荧光素抗体B13-DE 1的重组单链抗体片段（命名为scDE 1）。在将可变基因克隆到噬菌体载体中之后，通过噬菌体展示淘选选择功能性抗体片段。重组抗体在大肠杆菌中可表达为噬菌体抗体和可溶性单链抗体。在细菌培养上清液中也可以检测到高产量的scDE 1。scDE 1显示出与亲本单克隆抗体相同的结合特异性，即它结合荧光素、荧光素衍生物和荧光素肽模拟表位。表面等离子体共振显示，与单克隆抗体的0.7 nM相比，scDE 1的K-D为19 nM。分离的可溶性scDE 1可以很容易地与辣根过氧化物酶结合，这使得该结合物可以用作在不同免疫测定中检测荧光素标记蛋白质的通用指示剂。使用scDE 1作为模型系统进行尿中hCG的检测。除E.在大肠杆菌中，scFv基因也能在真核细胞中转移表达。最后，我们产生了在细胞表面表达scDE 1的HEK 293细胞。(C)2007年，Elsevier Masson SAS。All rights reserved.
A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K-D of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface. (C) 2007 Elsevier Masson SAS. All rights reserved.