Padan E, Venturi M, Michel H, Hunte C
Division of Microbial and Molecular Ecology, The Hebrew University of Jerusalem, Israel.
FEBS Lett. 1998 Dec 11;441(1):53-8. doi: 10.1016/s0014-5793(98)01524-5.
Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins. In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA). Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter. The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay. With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form. The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography. Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein. We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation. Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay). Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes. Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter.
识别天然表位的单克隆抗体（mAb）是蛋白质功能和结构研究的重要工具，但它们很少用于转运蛋白研究。为了制备针对大肠杆菌NhaA钠/氢反向转运蛋白的单克隆抗体，我们在基于标准酶联免疫吸附测定（ELISA）的筛选过程中遇到了困难。在此，我们报告一种快速有效的筛选抗NhaA单克隆抗体的方法，该抗体可识别反向转运蛋白的天然表位。该方法基于使用His标签蛋白、镍离子-次氮基三乙酸包被的平板以及测定中的非变性条件。通过该程序获得了四种单克隆抗体，其中三种识别天然构象的NhaA，一种优先识别变性形式。后一种单克隆抗体在蛋白质印迹中呈阳性，其他单克隆抗体在蛋白质印迹中呈阴性，并通过凝胶过滤色谱法测定与去污剂溶解的NhaA结合。竞争实验表明，天然表位对于His标签蛋白和野生型蛋白是共同的。我们认为，在标准ELISA中，NhaA蛋白不是以天然构象呈现给抗体的，而基于His标签的方案有利于这种呈现。实际上，我们可以通过用抗NhaA单克隆抗体包被平板来显著提高标准ELISA的低反应性，并将其用于呈现NhaA（“夹心”ELISA或双抗体测定）。值得注意的是，两种结合天然NhaA的单克隆抗体（5H4、2C5）极大地抑制了由蛋白脂质体中重组的His标签NhaA介导的ΔpH驱动的22Na摄取。因此，这些单克隆抗体为研究反向转运蛋白的结构/功能关系提供了一种新工具。
