Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), the etiology of which is poorly understood. The most common laboratory abnormality associated with MS is increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands (OCBs) in the brain and cerebrospinal fluid (CSF). However, the major antigenic targets of these antibody responses are unknown. The risk of MS is increased after infectious mononucleosis (IM) due to EBV infection, and MS patients have higher serum titers of anti-EBV antibodies than control populations. Our goal was to identify disease-relevant epitopes of IgG antibodies in MS; to do so, we screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. Using a highly sensitive phage-mediated immuno-PCR assay, we determined specific bindings of the two EBV epitopes to IgG from CSF from 46 MS and 5 inflammatory control (IC) patients. MS CSF IgG have significantly higher bindings to EBNA1 epitope than to EBNA2 epitope, whereas EBNA1 and EBNA2 did not significantly differ in binding to IC CSF IgG. Further, the EBNA1 epitope was recognized by OCBs from multiple MS CSF as shown in blotting assays with samples separated by isoelectric focusing. The EBNA1 epitope is reactive to MS intrathecal antibodies corresponding to oligoclonal bands. This reinforces the potential role of EBV in the etiology of MS.

多发性硬化症 (MS) 是一种中枢神经系统 (CNS) 的慢性炎症性脱髓鞘疾病，其病因知之甚少。与 MS 相关的最常见的实验室异常是鞘内免疫球蛋白 G (IgG) 合成增加以及大脑和脑脊液 (CSF) 中存在寡克隆带 (OCB)。然而，这些抗体反应的主要抗原靶点是未知的。由于 EBV 感染，感染性单核细胞增多症 (IM) 后 MS 的风险增加，并且 MS 患者的抗 EBV 抗体血清滴度高于对照人群。我们的目标是识别 MS 中与疾病相关的 IgG 抗体表位。为此，我们使用从急性 MS 患者大脑中纯化的总 IgG 抗体筛选了噬菌体展示的随机肽库（12 聚体）。我们通过 ELISA、噬菌体介导的免疫 PCR 和等电聚焦鉴定并表征了与 MS 患者和对照的鞘内 IgG 结合特异性的噬菌体肽。我们鉴定了两种分别与 EBV 核抗原 1 和 2（EBNA1 和 EBNA2）共享序列同源性的噬菌体肽。通过 ELISA 和竞争性抑制测定证实了通过用 MS 脑 IgG 淘选发现的 EBV 表位的特异性。使用高度敏感的噬菌体介导的免疫 PCR 测定，我们确定了两个 EBV 表位与来自 46 名 MS 和 5 名炎症对照 (IC) 患者的 CSF 中的 IgG 的特异性结合。MS CSF IgG 与 EBNA1 表位的结合显着高于与 EBNA2 表位的结合，而 EBNA1 和 EBNA2 在与 IC CSF IgG 的结合方面没有显着差异。进一步，EBNA1 表位被来自多个 MS CSF 的 OCB 识别，如印迹分析所示，样品通过等电聚焦分离。EBNA1 表位对与寡克隆带相对应的 MS 鞘内抗体具有反应性。这加强了 EBV 在 MS 病因学中的潜在作用。