这项工作得到了 CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, CMRPG3E0633) 的支持。

这项研究获得了台湾桃园 Linkuo 昌起劲纪念医院机构审查委员会 (102-2577A3) 的批准。
1. NR1-GFP 表达质粒的制备
<ol><li>将 10 ng 的 NR1-GFP 质粒与100µL 的大肠埃希氏菌 DH5α在无菌1.5 毫升离心管中, 吸管混合均匀地上下4-6 次, 并在冰上孵育20分钟。</li>
	<li>在42° c 的水浴中孵育1分钟的试管, 从水浴中取出管子, 将1毫升的性肉汤 (LB) 培养基放入试管中, 并在37° c 的孵化箱中孵育, 并摇动 1 h。</li>
	<li>将50µL 的混合物放在含有氨苄青霉素 (0.1 毫克/毫升) 的 lb 琼脂板上, 在一夜间孵化箱中在37° c 处孵育 lb 琼脂板。</li>
	<li>使用无菌尖端从隔夜 lb 琼脂板中挑选一个菌落, 并在一个含有5毫升培养基和氨苄西林 (0.1 毫克/毫升) 的细菌培养管中旋转尖端。在恒温箱中孵育37° c 的试管, 隔夜摇动。</li>
	<li>分3毫升的夜间细菌培养成500毫升烧瓶, 其中包含250毫升无菌 LB 培养基和氨苄西林 (0.1 毫克/毫升)。在37° c 的晃动下孵育烧瓶。使用光谱仪定期检查600纳米波长的细菌培养的光学密度 (OD), 直到 OD600 达到 0.6-1.0。 注: 混合800µL 的隔夜细菌培养与200µL 甘油准备甘油库存, 并存储在-80 ° c 下的甘油库存, 以备将来使用。</li>
	<li>250毫升细菌培养制备 NR1-GFP 表达质粒<ol>
<li>通过离心在 6000 x g 收集细胞15分钟在4° c。</li>
		<li>重10毫升悬浮缓冲液中含有核糖核酸的细菌颗粒;漩涡彻底, 直到没有看到丛。</li>
		<li>在细菌悬浮液中加入10毫升裂解缓冲液, 轻轻倒置混合物4-6 次, 室温 (RT) 孵育裂解物5分钟。</li>
		<li>将10毫升 pre-chilled 沉淀缓冲液添加到裂解物中, 轻轻翻转混合物4-6 次。</li>
		<li>将裂解液倒入带盖的过滤筒的桶中;在 RT 中等待10分钟。</li>
		<li>卸下墨盒插座喷嘴的盖子, 将柱塞插入墨盒, 然后轻轻按下柱塞。将过滤的裂解液收集到50毫升的管子中。</li>
		<li>添加2.5 毫升专有缓冲从制造商到过滤的裂解物, 混合混合物通过倒置管大约10次, 并且在冰孵育混合物 30 min。</li>
		<li>将过滤过的裂解混合物应用到 pre-equilibrated 10 mL 平衡缓冲器的过滤器柱上。允许柱在重力作用下排出。</li>
		<li>用30毫升洗涤缓冲器两次洗涤柱, 然后用15毫升洗脱缓冲器从柱洗 DNA。</li>
		<li>将10.5 毫升 (0.7 卷) 异丙醇添加到洗 DNA 缓冲液中, 混合均匀, 并将混合物离心 2万 x g, 在4° c 处为30分钟。</li>
		<li>仔细醒酒上清液, 用5毫升内毒素-70% 乙醇一次性清洗 DNA 颗粒, 在 2万 x 克上离心10分钟。</li>
		<li>醒酒上清液, 在化学罩中干燥5-10 分钟的颗粒, 并在300-500 µL 内毒素释放缓冲剂中溶解颗粒。</li>
		<li>用分光光度计测定紫外260/280 纳米溶液的吸收量, 确定质粒浓度。 注: 使用同一协议制备表达质粒 GFP。</li>
	</ol>
</li>
</ol>2. NR1-GFP 表达质粒转染 HEK293 细胞
<ol><li>分200µL 2% 明胶溶液对48井培养板的每一个井, 并在37° c 下孵育至少30分钟的板材。</li>
	<li>从井中抽出明胶溶液, 并在每井的200µL 中含有10% 胎牛血清 (FBS) 的细胞培养基中, 5 x 104 HEK293 细胞。在37° c 的湿润恒温箱中孵育盘, 5% CO2过夜。 注意: 使用例计数单元格。</li>
	<li>第二天, 用200µL 的新鲜培养基取代含有 10% FBS 的培养基。</li>
	<li>为每一个井, 准备溶液 A 混合 100 ng NR1-tGFP 质粒与20µL 培养基, 和溶液 B 混合0.8 µL 转染试剂与20µL 培养基。将每个实验的总容积所需的井数乘以。</li>
	<li>通过混合溶液 a 和溶液 B 制备溶液 C, 并在 RT 处孵育20-30 分钟的混合物。</li>
	<li>分40µL 的溶液 C 的每一个井含有 HEK293 细胞, 轻轻地旋转板, 并孵化在37° c 在一个湿化孵化器提供 5% CO2过夜的板块。</li>
	<li>在荧光显微镜下检查 NR1-GFP 重组蛋白在 40 x 200 x 放大 (激发/发射: 482/502 nm) 下的表达, 并进行细胞免疫荧光检测。 注: 染表达质粒 GFP 进入 HEK293 细胞使用相同的协议。</li>
</ol>3. 细胞免疫荧光法
<ol><li>从水井中抽出所用的培养基, 然后用200µL 磷酸缓冲盐水 (PBS) 三次冲洗每一个井。</li>
	<li>每井加200µL 4% 甲醛溶液;在 RT 上孵育板15分钟。</li>
	<li>从井中吸取甲醛溶液, 用200µL PBS 三次冲洗每口井。</li>
	<li>在 PBST (PBS 的 0.1% Tween-20) 溶液中加入200µL 10% 脱脂牛奶, 并在 RT 中孵育 1 h, 轻轻摇晃。</li>
	<li>从井中抽出 PBST 溶液中的10% 脱脂牛奶, 然后用200µL PBST 冲洗一次。</li>
	<li>用稀释过的等离子体 (1:100 在 PBST) 孵育水井, 作为在 RT 1 小时内温和摇动的主要抗体。 注: 血浆是从疑似有自身免疫性脑炎的患者血液中获得的。</li>
	<li>从水井中吸取稀释后的等离子体, 用200µL PBST 三次洗涤每一个井。</li>
	<li>添加200µL 山羊人 IgG 共轭与 Alexa 594 (1:1, 000 稀释 PBST) 每井, 并孵化培养板在 RT 与温和晃动 1 h。</li>
	<li>吸取山羊人 IgG 共轭的 Alexa 594 溶液, 并洗涤每井与200µL 的 PBST 三倍。</li>
	<li>添加100µL 甘油溶液 (PBS 50% 甘油和 1:100,000 4 ", 6-diamidino-2-吲 (DAPI)) 每井。</li>
	<li>使用10X 目镜、4X、10X 和20X 的物镜观察和成像荧光显微镜下的细胞。 注意: 为每染料使用正确的过滤器: 为 NR1-GFP (励磁或放射 = 482/502 毫微米), 为 Alexa 流体 594 (励磁或放射 = 561/594 毫微米), 为 DAPI (励磁或放射 = 358/461 毫微米)。用同样的方法对 HEK 细胞进行负控制。</li>
</ol>

<ol>
	<li>Linnoila, J. J., Rosenfeld, M. R., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+surface+antibody-mediated+autoimmune+encephalitis.">Neuronal surface antibody-mediated autoimmune encephalitis.</a> Semin Neurol. 34 (4), 458-466 (2014).</li><li>Lancaster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+and+treatment+of+autoimmune+Encephalitis.">The diagnosis and treatment of autoimmune Encephalitis.</a> J Clin Neurol. 12 (1), 1-13 (2016).</li><li>Pruss, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+analysis+of+NMDA+receptor+antibodies+in+encephalitis+of+unknown+origin.">Retrospective analysis of NMDA receptor antibodies in encephalitis of unknown origin.</a> Neurology. 75 (19), 1735-1739 (2010).</li><li>Leypoldt, F., Armangue, T., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+encephalopathies.">Autoimmune encephalopathies.</a> Ann N Y Acad Sci. 1338, 94-114 (2015).</li><li>Dalmau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+case+series+and+analysis+of+the+effects+of+antibodies.">Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.</a> Lancet Neurol. 7 (12), 1091-1098 (2008).</li><li>Titulaer, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prognostic+factors+for+long-term+outcome+in+patients+with+anti-NMDA+receptor+encephalitis:+an+observational+cohort+study.">Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study.</a> Lancet Neurol. 12 (2), 157-165 (2013).</li><li>Chapman, M. R., Vause, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA+receptor+encephalitis:+diagnosis,+psychiatric+presentation,+and+treatment.">Anti-NMDA receptor encephalitis: diagnosis, psychiatric presentation, and treatment.</a> Am J Psychiatry. 168 (3), 245-251 (2011).</li><li>Gable, M., Glaser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-n-methyl-d-aspartate+receptor+encephalitis+appearing+as+a+new-onset+psychosis:+disease+course+in+children+and+adolescents+within+the+california+encephalitis+project.">Anti-n-methyl-d-aspartate receptor encephalitis appearing as a new-onset psychosis: disease course in children and adolescents within the california encephalitis project.</a> Pediatr Neurol. 72, 25-30 (2017).</li><li>Dalmau, J., Lancaster, E., Martinez-Hernandez, E., Rosenfeld, M. R., Balice-Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+experience+and+laboratory+investigations+in+patients+with+anti-NMDAR+encephalitis.">Clinical experience and laboratory investigations in patients with anti-NMDAR encephalitis.</a> Lancet Neurol. 10 (1), 63-74 (2011).</li><li>Dalmau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraneoplastic+anti-N-methyl-D-aspartate+receptor+encephalitis+associated+with+ovarian+teratoma.">Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma.</a> Ann Neurol. 61 (1), 25-36 (2007).</li><li>Irani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methyl-D-aspartate+antibody+encephalitis:+temporal+progression+of+clinical+and+paraclinical+observations+in+a+predominantly+non-paraneoplastic+disorder+of+both+sexes.">N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes.</a> Brain. 133 (Pt 6), 1655-1667 (2010).</li><li>Suh-Lailam, B. B., Haven, T. R., Copple, S. S., Knapp, D., Jaskowski, T. D., Tebo, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+antibody+encephalitis:+performance+evaluation+and+laboratory+experience+with+the+anti-NMDA-receptor+IgG+assay.">Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.</a> Clin Chim Acta. 421, 1-6 (2013).</li><li>Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+glutamate+receptor+ion+channel+complexes.">Structural biology of glutamate receptor ion channel complexes.</a> Curr Opin Struct Biol. 41, 119-127 (2016).</li><li>Ramberger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+diagnostic+accuracy+of+microscopy+and+flow+cytometry+in+evaluating+n-methyl-d-aspartate+receptor+antibodies+in+serum+using+a+live+cell-based+assay.">Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating n-methyl-d-aspartate receptor antibodies in serum using a live cell-based assay.</a> PLoS One. 10 (3), e0122037 (2015).</li><li>Wandinger, K. P., Saschenbrecker, S., Stoecker, W., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+a+severe,+multistage,+treatable+disorder+presenting+with+psychosis.">Anti-NMDA-receptor encephalitis: a severe, multistage, treatable disorder presenting with psychosis.</a> J Neuroimmunol. 231 (1-2), 86-91 (2011).</li><li>Dahm, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seroprevalence+of+autoantibodies+against+brain+antigens+in+health+and+disease.">Seroprevalence of autoantibodies against brain antigens in health and disease.</a> Ann Neurol. 76 (1), 82-94 (2014).</li><li>J&#233;z&#233;quel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-+and+single+molecule-based+methods+to+detect+anti-n-methyl-d-aspartate+receptor+autoantibodies+in+patients+with+first-episode+psychosis+from+the+OPTiMiSE+project.">Cell- and single molecule-based methods to detect anti-n-methyl-d-aspartate receptor autoantibodies in patients with first-episode psychosis from the OPTiMiSE project.</a> Biol Psychiatry. , (2017).</li></ol>

作者没有什么可透露的。

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。因此, 许多研究采用商业间接固定细胞免疫荧光法筛选血清和脑脊液标本中的抗 NMDA 受体自身抗体12,15。使用的试剂盒 (见材料表) 含有 ectopically 表达 NMDA 受体重组 NR1 亚基的固定 HEK293 细胞。对血清和脑脊液标本中的抗 NMDA 受体 IgG 抗体检测结果进行了评价, 结果12。在我们的协议中, 我们使用了与套件制造商推荐的相同的方法, 只是我们使用了 NR1-GFP 表达质粒。NR1-GFP 蛋白可用于监测 NR1 蛋白在每个实验中的转染效率和分布。因此, 它可以用来作为质量保证的化验。
NR1-GFP 表达质粒在 HEK293 细胞中的转染效率约为 30%, 这对于免疫荧光检测是足够的。然而, 我们发现在转染的细胞中, 绿色荧光的强度不均匀分布, 这就表明了个体细胞在表达能力上的差异。当绿色图像与 Alexa 氟594的红色图像合并时, 不同细胞中的重组 NR1-GFP 的表达水平会引起异质性的强度变化。以 Alexa 594 的山羊人 igg 为第二抗体, 检测人体自身抗体 igg 类的存在。我们发现, Alexa 594 的亮度相对较弱的绿色荧光蛋白。我们试图通过增加与亚历克斯氟594共轭的人 IgG 的浓度来优化信号强度, 但是, 背景也增加了。因此, 与亚历克斯氟594共轭的人 IgG 的最佳浓度是 1:1, 000 在这个实验室。此外, 一些等离子样品有一个显着高的背景信号在 Alexa 的594图像。对不同稀释的等离子体进行了测试, 发现在大多数样品中, 需要最少稀释1:100 才能获得干净的背景。因此, 我们建议1:100 稀释等离子体作为本协议的标准步骤。与其他能检测到1:10 和1:20 稀释血液样本的血清样本相比,11,16, 此当前协议没有足够的灵敏度来检测低滴度抗 NMDA 受体自身抗体,这是协议的一个限制。
目前的协议是一个固定细胞免疫荧光检测。该培养基在4° c 的冰箱内固定后可存放1月, 与活细胞法比较, 不能长期贮存。然而, 在固定过程中, NR1 蛋白会变性并失去其天然构象, 这可能会影响与抗 NMDA 受体自身抗体结合的表位的构象。因此, 一些研究采用了活细胞 immunofluorescene11,17。我们的协议可以交替修改, 以成为一个活细胞免疫荧光法, 以保持自然构象的 NR1 蛋白, 如果我们孵化的等离子体样品与活体细胞的第一, 并修复后的细胞。因此, 该协议有灵活性, 以满足实验的需要。
NMDA 受体是一种 heterotetramer 复合蛋白, 由 NR1、NR2A、NR2B、NR2C 和 NR2D 组成。本协议旨在检测 NMDA 受体 NR1 亚单位的自身抗体 IgG 类。该协议也可以修改, 以检测不同的 isotypes 抗体, 只要人 IgG 二级抗体取代其他特定类别的二级抗体。此外, 如果将 NR1-GFP 表达质粒分别替换为相应的表达质粒, 则可以对当前协议进行修改, 以检测对 NR2A、NR2B、NR2C 和 NR2D 的自身抗体的存在。
在这个协议中, 我们使用了血浆标本而不是血清, 因为我们经常收集血液样本与抗凝的各种研究目的, 但该协议可以适用于血清或脑脊液标本。血浆中自身抗体的滴度可以通过样品的连续稀释来定量。在实验室中, 我们还测试了非洲绿猴肾成纤维细胞系 (COS1) 作为宿主细胞使用相同的协议。结果与使用 HEK293 细胞的方法相同, 只是 COS1 细胞的转染效率比 HEK293 细胞略低。
为了确定抗 NMDA 受体自身抗体的存在, 建议进行额外的实验, 如使用啮齿动物脑组织免疫组化检测和使用原代培养的啮齿动物神经元免疫细胞化学.5 ,9。因此, 目前的协议只能被视为一个筛选试验。本研究采用阳性对照样品对其实验有效性进行了验证, 但在今后的研究中需要建立该方法的灵敏度和特异性。

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。

<table><tbody><tr><td>GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1</td><td>Origene (Rockville, MD, USA)</td><td>RG219368</td><td>NR1-cDNA clone tagged with C-terminal tGFP sequences</td></tr><tr><td>pCMV6-AC-GFP</td><td>Origene (Rockville, MD, USA)</td><td>PS100010</td><td>mammalian vector with C-terminal tGFP tag</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen (Hilden Germany)</td><td>12362</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>11668-019</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher</td><td>31985-070</td><td></td></tr><tr><td>DMEM, High Glucose, Pyruvate</td><td>Thermo Fisher</td><td>11995-065</td><td></td></tr><tr><td>Characterized Fetal Bovine Serum, US Origin</td><td>GE Healthcare Life Sciences</td><td>SH30071.01</td><td></td></tr><tr><td>Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td><td>Thermo Fisher</td><td>A-11014</td><td></td></tr><tr><td>Positive control: anti-glutamate receptor (type NMDA)</td><td>Euroimmun AG, L&amp;uuml;beck, Germany</td><td>CA 112d-0101</td><td></td></tr><tr><td>Euroimmun Assay Kit</td><td></td><td></td><td></td></tr></tbody></table>

a simple cell-based immunofluorescence assay to detect autoantibody against the n-methyl-d-aspartate (nmda) receptor in blood

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。

根据本议定书1节所述的程序, 平均可从250毫升细菌培养中获得200-300 µg 内毒素-游离表达质粒 NR1-GFP 和 GFP。本议定书2节所述的 48-井板中培养的 HEK293 细胞的转染量为 100 ng/很好的表达质粒。24-30 h 在转染后, 细胞表达 NR1-GFP, 荧光显微镜下可检测 GFP 重组蛋白。图 1A显示了表达 NR1-GFP 的宿主单元格的图像, 而图 1D显示了表达 GFP 的单元格。绿色信号可以作为检测的质量控制器: 大约30% 的细胞在荧光显微镜下有绿色信号。图 1A和1D显示了表达 NR1-GFP 的主机单元格的图像。绿色信号可以作为检测的质量控制器: 大约30% 的细胞在荧光显微镜下有绿色信号。用表达 NR1-GFP 的细胞在人血浆样品中筛选抗 NMDA 受体自身抗体的存在。
在《议定书》3节所述的细胞免疫荧光测定法中, 阳性对照样本 (从商业来源获得, 见材料表) 被添加到池中的等离子体中, 根据1:10 稀释制造商的说明。汇集血浆由5成年男性和5成年女性准备。图 1B是在与细胞表达式 NR1-GFP 孵育后, Fluor-594 阳性对照样品的 alexa 图像, 而图 1E是 Fluor-594 阳性对照样品在孵化后与细胞表达 GFP 的图像。图 1C是图 1A和图 1B的合并图像: 同一单元格中的绿色信号和红色信号有很大的重叠, 表明 NR1-GFP 的共存和对 NMDA NR1 亚基的抗体受体 (箭头)。在这种情况下, 一个显示大于30% 的绿色和红色信号重叠的等离子体样本将被解释为正值。图 1F是图 1D和图 1E中的合并图像, 用作图 1C的负控制。弱红颜色是 Alexa Fluor-594 图像的背景荧光。绿色和红色信号几乎没有重叠, 表明抗 NMDA 受体自身抗体对重组蛋白没有约束力。
在实验过程的建立过程中, 我们观察了 Fluor-594 图像在等离子体样品测试时的信噪比低。我们试图通过对等离子体形式1:10、1:50、1:100 和1:200 进行连续稀释来优化信噪比, 并测试不同浓度的亚历克斯 Fluor-594 标记的二级抗体从1:500 到 1:2, 000。我们发现, 1:100 稀释的血浆和 1:1, 000 或 1:2, 000 稀释的 Alexa Fluor-594 是最佳的条件解释的数据。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56676/56676fig1v3.jpg"> 图 1: 细胞免疫荧光法检测抗 NMDA 受体自身抗体的代表性图像.(A) 表示 NR1-GFP 的 HEK293 单元格的图像。(B) 在与阳性对照样本进行孵育后, 从A的同一字段中取出的 Alexa Fluor-594 图像。红色信号表明抗 NMDA 受体自身抗体与 HEK293 细胞中表达的 NR1 的结合。(C) A和B的合并图像。黄色表示共存的 NR1 (绿色信号) 和抗 NMDA 受体自身抗体 (红色信号)。箭头表明一些突出的细胞显示共存 NR1-GFP 和抗 NMDA 受体自身抗体的例子。(D) 表达 NR1-GFP 的 HEK293 细胞的图像。(E) 在与阳性对照样本进行孵育后, 从D的同一字段中取出的 Alexa Fluor-594 图像。微弱的红色信号表示 Alexa Fluor-594 图像的背景噪声。(F) D和E的合并图像。这个图像作为一个负控制, 因为有很少的黄色信号观察。阴性对照图像显示抗 NMDA 受体抗体对 NR1-GFP 的特异结合。所有的图像都是在10X 眼透镜和20X 透镜物镜下拍摄的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56676/56676fig1v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood at JoVE.com

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

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。

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

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

9.8K 次观看.  Chang Gung Memorial Hospital-Linkou. 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。

视频: A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

科研

免疫与感染

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

医学

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

人血清抗水药物蛋白-4 免疫球蛋白 g 的细胞检测

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

神经科学

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

生物学

研究自身免疫性脱髓鞘中的体液反应

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

作者聚焦:在多发性硬化症研究中研究 B 细胞反应的新型检测方法

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

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

摘要

探索更多视频

此视频中的章节

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

人血清抗水药物蛋白-4 免疫球蛋白 g 的细胞检测

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

抗核抗体筛选使用Hep-2细胞

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量

实验性自身免疫性脑脊髓炎后小鼠自身反应性抗体的定量