Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays
Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays
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DOI：
10.1371/journal.pone.0068902
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发表时间：
2013-07-23
期刊：
影响因子：
3.7
通讯作者：
Schafer-Nielsen, Claus
中科院分区：
文献类型：
--
作者：
Hansen, Lajla Bruntse;Buus, Soren;Schafer-Nielsen, Claus
我们最近开发了一种高密度光刻肽阵列技术，理论上每个2厘米(2)的阵列上限为200万个不同的肽。在这里，我们使用这种方法以人血清白蛋白（HSA）为例，对中等大小蛋白靶点的线性B细胞表位进行了完整而详尽的分析。合成所有可能重叠的HSA 15-mers，并用市售兔抗HSA抗体进行探针检测。为了识别最弱的表位，同时对参与抗体结合的关键残基进行详细的表征，该阵列还包括在每个15-mer肽的每个位置进行完整的单取代扫描（即包括20个常见氨基酸中的每一个）。作为特异性对照，还包括牛血清白蛋白（BSA）和兔血清白蛋白（RSA）中所有可能的15-mer肽。所得到的布局包含超过200.000个肽场，可以在显微镜载玻片上以单一阵列合成。超过20个候选线性表位被鉴定并以高分辨率表征，即确定哪些氨基酸在哪些位置需要或不需要抗体相互作用。与预期的一样，与BSA中的一些肽有中度交叉反应，而与RSA中的肽没有交叉反应。我们得出结论，高密度肽微阵列是一种非常强大的方法来识别和表征线性抗体表位，并且应该在单个抗体水平上推进个体特异性的详细描述以及在蛋白质组水平上的血清学分析。
We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.