Identification and mapping of linear antibody epitopes in human serum albumin using high-density peptide arrays

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 cm2. 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.

本团队近期开发了一项高密度光刻肽阵列技术，在2平方厘米的阵列面积下，理论上限可达200万种不同肽段。本研究以此技术为平台，以人血清白蛋白（human serum albumin, HSA）为模式靶标，对中等分子量蛋白靶标的线性B细胞表位开展全面且无遗漏的穷尽式分析。我们合成了覆盖人血清白蛋白全序列的所有重叠15肽段，并使用商业化多克隆兔抗人血清白蛋白抗体制剂进行探针孵育检测。为精准识别最弱结合的表位，同时详细表征抗体结合所涉及的关键氨基酸残基，该阵列还针对每一条15肽段的每个位点，引入了完整的单氨基酸替换扫描设计——即对每个位点分别替换为20种常见氨基酸中的每一种。作为特异性对照，我们同时纳入了牛血清白蛋白（bovine serum albumin, BSA）与兔血清白蛋白（rabbit serum albumin, RSA）的所有可能15肽段。最终该阵列布局包含超过20万个肽段位点，可在单块显微镜载玻片上完成合成。本研究最终鉴定并高分辨率表征了二十余个线性表位候选物，明确了抗体结合所需或非必需的氨基酸位点与种类。正如预期，我们检测到该抗血清与牛血清白蛋白部分肽段存在轻度交叉反应，而未观察到与兔血清白蛋白肽段的交叉反应。综上，高密度肽微阵列是一种极具应用潜力的线性抗体表位鉴定与表征技术，其可推动单抗体水平下个体特异性的精准解析，以及蛋白质组层面的血清学分析研究。