血液中 n-甲基-d-天门冬氨酸 (NMDA) 受体自身抗体的简易细胞免疫荧光检测
Summary
我们 ectopically 表达了以绿色荧光蛋白标记的 nmda 受体在人胚细胞 (HEK293) 中的 NR1 亚基作为抗原, 以检测疑似自身免疫性脑炎患者血液中 nmda 受体的自身抗体。这种简单的方法可能适用于临床设置的筛选目的。
Abstract
抗 nmda 受体自身抗体的存在可导致各种神经精神症状的患者, 称为抗 nmda 受体自身免疫性脑炎。对血液或脑脊液中 NMDA 受体的特定自身抗体的检测是准确诊断这一病症的必要条件。nmda 受体是一个离子通道蛋白复合体, 包含四亚基, 包括两个强制性 nmda 受体亚基 1 (NR1) 和一个或两个 nmda 受体亚基 2A (NR2A), nmda 受体亚基 2B (NR2B), nmda 受体亚基 2C (NR2C), 或 nmda 受体亚基 2D (NR2D)。报道了 nmda 受体 NR1 亚单位的胞外 N 端区存在抗 nmda 受体自身抗体的表位。本研究的目的是开发一种简单的细胞免疫荧光检测方法, 可作为筛选试验, 检测血液中 NMDA 受体 NR1 亚基自身抗体的存在, 以促进临床和基础研究抗 NMDA 受体自身免疫性脑炎。
Introduction
抗 NMDA 受体自身免疫性脑炎是一种新的公认的疾病实体, 可以发生在所有年龄的患者, 并影响主要女性患者1,2。它是临床上最常见的脑炎之一, 最初不明病因的脑炎3。感染抗 NMDA 受体脑炎的患者通常有前驱症状的头痛或发烧, 其次是意识水平的快速发展变化和各种急性神经精神症状, 包括躁动, 烦躁, 焦虑, 失眠, 幻觉, 妄想, 侵略, 怪异行为, 运动异常, 自主失调, 和癫痫发作4,5。早期认识到这种情况和及时治疗的免疫疗法是重要的结果, 甚至完全恢复在受影响的患者6。因此, 建议抗 NMDA 受体自身免疫性脑炎应被认为是一个重要的鉴别诊断病人呈现急性或新发精神病功能7,8。
除临床特点外, 检测血液或脑脊液中 nmda 受体的自身抗体, 对于准确诊断抗 nmda 受体自身免疫性脑炎是必不可少的9。大多数检测抗 NMDA 受体自身抗体的免疫学试验在几个研究实验室中都是可用的10,11, 并且只有一个商业可用的细胞免疫荧光检测方法来筛选抗 NMDA 受体抗体12。本研究的目的是开发一种简单的 in-house 细胞免疫荧光分析法, 可以方便地用于实验室筛选抗 nmda 受体自身抗体的存在, 促进抗 nmda 受体的临床研究自身免疫性脑炎。NMDA 受体是一种 heterotetramer 的离子通道蛋白复合体, 在大脑中明确表达。它由两个强制 NR1 子单元组成, 以及 NR2A、NR2B、NR2C 或 NR2D13的一个或两个子单元的组合。先前的一项研究报告, 抗体靶向的主要表位位于 NR1 亚基5的胞外 N 端域。因此, 在本协议中, 我们表达了人类胚胎肾上皮细胞系 (HEK293) 中标记有绿色荧光蛋白 (GFP) 的 NMDA 受体的人重组 NR1-subunit 蛋白, 并建立细胞免疫荧光法检测血液中抗 NMDA 受体自身抗体的 IgG 类。
Protocol
Representative Results
Discussion
有几种细胞免疫分析筛选的存在自身抗体对 NMDA 受体的文献报道, 包括活细胞免疫荧光法检测11, 固定细胞免疫荧光检测9和流 cytometry-based 检测14。在 ectopically 表达 NR1 蛋白的细胞制备后不久, 应进行活体细胞免疫荧光检测, 而流 cytometry-based 检测则需要训练有素的人员和专用设备。固定细胞免疫荧光法是一种更方便的化验, 可以进行临床设置。因…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, CMRPG3E0633) 的支持。
Materials
| GRIN1 (GFP-tagged) – Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1 | Origene (Rockville, MD, USA) | RG219368 | NR1-cDNA clone tagged with C-terminal tGFP sequences |
| pCMV6-AC-GFP | Origene (Rockville, MD, USA) | PS100010 | mammalian vector with C-terminal tGFP tag |
| EndoFree Plasmid Maxi Kit | Qiagen (Hilden Germany) | 12362 | |
| Lipofectamine 2000 Transfection Reagent | Invitrogen (Carlsbad, CA, USA) | 11668-019 | |
| Opti-MEM I Reduced Serum Medium | Thermo Fisher | 31985-070 | |
| DMEM, High Glucose, Pyruvate | Thermo Fisher | 11995-065 | |
| Characterized Fetal Bovine Serum, US Origin | GE Healthcare Life Sciences | SH30071.01 | |
| Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate | Thermo Fisher | A-11014 | |
| Positive control: anti-glutamate receptor (type NMDA) | Euroimmun AG, Lübeck, Germany | CA 112d-0101 | |
| Euroimmun Assay Kit |
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