Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, Honegger A, Klinger B, Mocikat R, Plückthun A
Universität Zürich, Switzerland.
Biotechniques. 1997 Jan;22(1):140-9. doi: 10.2144/97221rr01.
Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.
利用一种细胞结合免疫原，我们制备了一种单克隆抗体3D5，它能识别多种蛋白质上的羧基末端寡聚组氨酸标签（His标签）。基于这种单克隆抗体，我们制备了可变区单链片段（scFv）、二聚体scFv-碱性磷酸酶融合蛋白和多价scFv展示噬菌体。不同形式的抗体是用于荧光激活细胞分选分析、BIAcore方法、蛋白质免疫印迹和酶联免疫吸附测定（ELISA）的有效工具。蛋白质免疫印迹和ELISA可以直接使用产生scFv-碱性磷酸酶融合蛋白的大肠杆菌细胞粗提物来开展，从而提供了一种取之不尽且方便的检测试剂来源。或者，为此目的可直接从培养上清液中使用多价scFv展示噬菌体。测定了肽KGGHHHHH（KD = 4×10⁻⁷ M）和咪唑（KD = 4×10⁻⁴ M）的解离常数KD。Fv片段的分子模拟表明，肽的质子化组氨酸侧链与抗体中的酸性基团之间存在两个盐桥，这解释了为什么抗体或底物可能在弱碱性条件下被洗脱。
