Chiu Nan-Chang, Lin Yi-Jie, Tzang Ruu-Fen, Li Ying-Syuan, Lin Hui-Ju, Das Subir, Chen Caleb G, Chen Chiao-Chicy, Hsu Kate
Division of Pediatric Neurology, Department of Pediatrics, MacKay Children's Hospital, Taipei, Taiwan.
MacKay Junior College of Medicine, Nursing and Management, New Taipei City, Taiwan.
Front Neurol. 2018 Aug 22;9:661. doi: 10.3389/fneur.2018.00661. eCollection 2018.
Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is one of the most frequently encountered autoimmune encephalitis. The pathogenesis of both anti-NMDAR encephalitis and schizophrenia involve down-regulation of NMDA receptors. Whether autoantibody-mediated destruction of neuronal NMDA receptors is associated with schizophrenia or first-episode psychosis (FEP) remains unclear, as the current findings from different groups are inconsistent. The main culprits are likely due to heterogeneity of autoantibodies (autoAbs) in a patient's blood or cerebrospinal fluid (CSF), as well as due to limitation of the current detection methods for anti-NMDAR autoAbs. Here, we optimized the current diagnostic method based on the only commercially-available anti-NMDAR test kit. We first increased detection sensitivity by replacing reporter fluorophore fluorescein isothiocyanate (FITC) in the kit with Alexa Fluor 488, which is superior in resisting photobleaching. We also found that using an advanced imaging system could increase the detection limit, compared to using a simple fluorescence microscope. To improve test accuracy, we implemented secondary labeling with a well-characterized mouse anti-NR1 monoclonal antibody (mAb) after immunostaining with a patient's sample. The degree of colocalization between mouse and human antisera in NMDAR-expressing cells served to validate test results to be truly anti-NMDAR positive or false-positive. We also incorporated DNA-specific DAPI to simultaneously differentiate autoAbs targeting the plasma membrane from those targeting cell nuclei or perinuclear compartments. All the technical implementation could be integrated in a general hospital laboratory setting, without the need of specialized expertise or equipment. By sharing our experience, we hope this may help improve sensitivity and accuracy of the mainstream method for anti-NMDAR detection.
抗N-甲基-D-天冬氨酸受体（抗NMDAR）脑炎是最常见的自身免疫性脑炎之一。抗NMDAR脑炎和精神分裂症的发病机制都涉及NMDAR受体下调。自身抗体介导的神经元NMDAR受体破坏是否与精神分裂症或首发精神病（FEP）相关仍不清楚，因为不同研究小组的当前研究结果不一致。主要原因可能是患者血液或脑脊液（CSF）中自身抗体（autoAbs）的异质性，以及当前抗NMDAR自身抗体检测方法的局限性。在此，我们基于唯一可商购的抗NMDAR检测试剂盒优化了当前的诊断方法。我们首先通过将试剂盒中的报告荧光团异硫氰酸荧光素（FITC）替换为抗光漂白性能更优的Alexa Fluor 488来提高检测灵敏度。我们还发现，与使用简单荧光显微镜相比，使用先进的成像系统可提高检测限。为提高检测准确性，在用患者样本进行免疫染色后，我们用特征明确的小鼠抗NR1单克隆抗体（mAb）进行二次标记。在表达NMDAR的细胞中，小鼠和人抗血清之间的共定位程度用于验证检测结果是真正的抗NMDAR阳性还是假阳性。我们还加入了DNA特异性的4',6-二脒基-2-苯基吲哚（DAPI），以同时区分靶向质膜的自身抗体与靶向细胞核或核周区室的自身抗体。所有这些技术操作都可在综合医院实验室环境中实现，无需专业知识或设备。通过分享我们的经验，我们希望这可能有助于提高抗NMDAR检测主流方法的灵敏度和准确性。
