Department of Clinical Immunology and Allergy, Royal Prince Alfred Hospital, Sydney, NSW, Australia.
Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
Front Immunol. 2021 Jun 23;12:705292. doi: 10.3389/fimmu.2021.705292. eCollection 2021.
Autoimmune Autonomic Ganglionopathy (AAG) is an uncommon immune-mediated neurological disease that results in failure of autonomic function and is associated with autoantibodies directed against the ganglionic acetylcholine receptor (gnACHR). The antibodies are routinely detected by immunoprecipitation assays, such as radioimmunoassays (RIA), although these assays do not detect all patients with AAG and may yield false positive results. Autoantibodies against the gnACHR exert pathology by receptor modulation. Flow cytometric analysis is able to determine if this has occurred, in contrast to the assays in current use that rely on immunoprecipitation. Here, we describe the first high-throughput, non-radioactive flow cytometric assay to determine autoantibody mediated gnACHR immunomodulation. Previously identified gnACHR antibody seronegative and seropositive sera samples (RIA confirmed) were blinded and obtained from the Oxford Neuroimmunology group along with samples collected locally from patients with or without AAG. All samples were assessed for the ability to cause gnACHR immunomodulation utilizing the prototypical gnACHR expressing cell line, IMR-32. Decision limits were calculated from healthy controls, and Receiver Operating Characteristic (ROC) curves were constructed after unblinding all samples. One hundred and ninety serum samples were analyzed; all 182 expected negative samples (from healthy controls, autonomic disorders not thought to be AAG, other neurological disorders without autonomic dysfunction and patients with Systemic Lupus Erythematosus) were negative for immunomodulation (<18%), as were the RIA negative AAG and unconfirmed AAG samples. All RIA positive samples displayed significant immunomodulation. There were no false positive or negative samples. There was perfect qualitative concordance as compared to RIA, with an Area Under ROC of 1. Detection of Immunomodulation by flow cytometry for the identification of gnACHR autoantibodies offers excellent concordance with the gnACHR antibody RIA, and overcomes many of the shortcomings of immunoprecipitation assays by directly measuring the pathological effects of these autoantibodies at the cellular level. Further work is needed to determine the correlation between the degree of immunomodulation and disease severity.
自身免疫性自主神经节病（AAG）是一种罕见的免疫介导的神经疾病，导致自主功能衰竭，并与针对神经节乙酰胆碱受体（gnACHR）的自身抗体有关。这些抗体通常通过免疫沉淀测定法（如放射免疫测定法（RIA））来检测，尽管这些测定法并非能检测出所有的 AAG 患者，并且可能会产生假阳性结果。针对 gnACHR 的自身抗体通过受体调节发挥病理学作用。流式细胞分析能够确定是否发生了这种情况，而当前使用的依赖免疫沉淀的测定法则不能。在这里，我们描述了第一个高通量、非放射性流式细胞术测定法，用于确定自身抗体介导的 gnACHR 免疫调节。以前确定的 gnACHR 抗体阴性和阳性血清样本（RIA 确认）被盲法处理，并从牛津神经免疫学小组以及从当地有无 AAG 的患者中收集。所有样本均利用原型 gnACHR 表达细胞系 IMR-32 评估引起 gnACHR 免疫调节的能力。从健康对照者中计算出决策界限，并在对所有样本进行去盲后构建了接收器工作特征（ROC）曲线。分析了 190 个血清样本；所有 182 个预期的阴性样本（来自健康对照者、不被认为是 AAG 的自主障碍、无自主功能障碍的其他神经障碍以及红斑狼疮患者）的免疫调节均为阴性（<18％），RIA 阴性的 AAG 和未经证实的 AAG 样本也是如此。所有 RIA 阳性样本均显示出明显的免疫调节作用。没有假阳性或假阴性样本。与 RIA 相比，具有完美的定性一致性，ROC 的面积为 1。通过流式细胞术检测 gnACHR 自身抗体的免疫调节可与 gnACHR 抗体 RIA 具有极好的一致性，并通过直接在细胞水平上测量这些自身抗体的病理作用克服了免疫沉淀测定法的许多缺点。需要进一步的工作来确定免疫调节程度与疾病严重程度之间的相关性。
