Laboratory of Experimental Immunology, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2013 Jul 23;8(7):e68902. doi: 10.1371/journal.pone.0068902. Print 2013.
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.
我们最近开发了一种高密度的光刻肽阵列技术，该技术的理论上限为每个 2cm² 的阵列可包含 200 万个不同的肽。在这里，我们以人血清白蛋白 (HSA) 为例，使用该技术对中等大小蛋白质靶标的线性 B 细胞表位进行了全面和详尽的分析。所有可能的重叠 15 肽均来自 HSA，并使用市售的多克隆兔抗 HSA 抗体制剂进行探测。为了能够识别即使是最微弱的表位，同时详细描述与抗体结合相关的关键残基，该阵列还包括在每个 15 肽的每个位置上进行完整的单取代扫描（即包括 20 种常见氨基酸中的每一种）。作为特异性对照，还包括来自牛血清白蛋白 (BSA) 和兔血清白蛋白 (RSA) 的所有可能的 15 肽。由此产生的布局包含超过 200000 个肽字段，并且可以在显微镜载玻片上的单个阵列中合成。以高分辨率鉴定和表征了 20 多个线性表位候选物，即确定了抗体相互作用所需或不需要的哪些氨基酸在哪些位置。正如预期的那样，鉴定到与 BSA 中的一些肽有中等交叉反应，而与 RSA 中的肽没有观察到交叉反应。我们得出结论，高密度肽微阵列是一种非常强大的方法，可用于鉴定和表征线性抗体表位，并且应该在单个抗体水平上详细描述个体特异性，以及在整个蛋白质组水平上进行血清学分析。
