Mapping the C terminal epitope of the Alzheimer's disease specific antibody MN423
Mapping the C terminal epitope of the Alzheimer's disease specific antibody MN423
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DOI：
10.1016/s0022-1759(02)00006-6
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发表时间：
2002-04-01
影响因子：
2.2
通讯作者：
Novak, M
中科院分区：
文献类型：
--
作者：
Khuebachova, M;Verzillo, V;Novak, M
单克隆抗体表位的定位现在主要使用分子多样性技术，特别是噬菌体展示来进行。然而，直到最近。噬菌体展示方法不适合于分析需要游离羧基末端的表位。在这里，我们描述了使用两种不同的技术来分析已知的MN 423的C末端表位特异性，MN 423是一种特异性染色阿尔茨海默氏症脑中截短的tau蛋白的单克隆抗体。使用基于λ噬菌体的C-末端随机肽文库和基于截短的tau的细胞内表达文库，我们表明该抗体具有相对于C末端在位置-3处的甘氨酸的绝对要求。这两种方法给出了相似的结果，并确定了结合位点中的其他重要残基。然而，合成肽的亲和力分析显示，抗体对鉴定的三肽的亲和力远低于天然靶标中的五肽序列，并且这又大大低于对天然靶标本身的亲和力。这表明，分子多样性方法可以定义最小的，但不一定是完整的表位。本文所述的方法一般应用于分析怀疑在C末端发现的抗体表位。(C)2002 Elsevier Science B. V.保留所有权利。
The mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. However, until very recently. phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. Here we describe the use of two different techniques to analyze the known C terminal epitope specificity of MN423, a monoclonal antibody specifically staining truncated tau in Alzheimer's brain. Using a lambda phage based C-terminal random peptide library, and an intracellular expression library based on truncated tau, we show that this antibody has an absolute requirement for a glycine at position - 3 with respect to the C terminus. Both methods give similar results, and identify other important residues in the binding site. However, affinity analysis of synthetic peptides revealed that the affinity of the antibody for identified tripeptides was far lower than the pentapeptide sequence in the native target, and that this in turn was considerably below the affinity for the native target itself. This suggests that molecular diversity methods may define minimum, but not necessarily complete epitopes. The methods described here have a general application to the analysis of antibody epitopes suspected to be found at the C terminus. (C) 2002 Elsevier Science B.V. All rights reserved.