Name: a simple cell-based immunofluorescence assay to detect autoantibody against the n-methyl-d-aspartate (nmda) receptor in blood
Uploaded: 2018-01-09T13:00:00
Description: 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

Dette arbejde blev støttet af Chang Gung medicinsk Foundation (grant nummer CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632 og CMRPG3E0633).

Undersøgelsen blev godkendt af det institutionelle Review Board af Chang Gung Memorial Hospital på Linkuo, Taoyuan, Taiwan (102-2577A3).
1. forberedelse af NR1-normal god landbrugspraksis udtryk Plasmid
<ol><li>Mix 10 ng af NR1-normal god landbrugspraksis plasmid med 100 µL af Escherichia coli kompetente celler stamme DH5α i en sterile 1,5 mL centrifugeres tube, afpipetteres blandingen forsigtigt op og ned 4 - 6 gange, og Inkuber røret på isen i 20 min.</li>
	<li>Inkuber tube ...

<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>

Der er flere cellebaserede immunologiske assays til skærmen forekomst af autoantistoffer mod NMDA receptor rapporteret i litteraturen, herunder levende celle-baserede immunofluorescens assay11, fast cellebaserede immunofluorescens assay9 , og flow flowcytometri-baserede assay14. Den levende celle-baserede immunofluorescens assay bør udføres kort efter udarbejdelsen af celler, der ectopically udtrykker NR1 protein, mens strømmen flowcytometri-base...

Anti-NMDA receptor autoimmune encephalitis er en nyligt anerkendt sygdom enhed, der kan forekomme hos patienter i alle aldre, og påvirker overvejende kvindelige patienter1,2. Det er en af de hyppigst diagnosticerede hjernebetændelse blandt patienter med indledende ukendt ætiologi af hjernebetændelse3. Patienter, der lider med anti-NMDA receptor encephalitis normalt har prodromale symptomer på en hovedpine eller feber, efterfulgt af en hurtig udvikling af bevidsthed niveau forandring og en række akutte neuropsykiatriske symptomer, herunder agitation, irritabilitet , angst, søvnløshed, hallucinationer, vrangforestillinger, aggression, bizar adfærd, bevægelse abnormaliteter, autonome dysregulering og beslaglæggelse angreb4,5. Tidlig erkendelse af denne betingelse og rettidig behandling med immunterapi er vigtige for et bedre resultat og endda fuld helbredelse i berørte patienter6. Det foreslås derfor, at anti-NMDA receptor autoimmune encephalitis bør betragtes som en vigtig differentialdiagnose af patienter med akut eller ny-debut psykotiske træk7,8.
Udover kliniske funktioner er påvisning af autoantistof mod NMDA-receptor i blod eller CSF afgørende for den præcise diagnose af anti-NMDA receptor autoimmune encephalitis9. De fleste af de immunologiske tests for at påvise anti-NMDA receptor autoantistof er tilgængelige i et par forskning laboratorier10,11, og der er kun et kommercielt tilgængelige cellebaserede immunofluorescens assay for screening af anti-NMDA receptor autoantistoffer12. Målet med denne undersøgelse er at udvikle en enkel in-house cellebaserede immunofluorescens assay, der bekvemt kan bruges i laboratoriet til at screene tilstedeværelsen af anti-NMDA receptor autoantistoffer til at lette den kliniske forskning af anti-NMDA receptor autoimmun encephalitis. NMDA-receptor er en heterotetramer ion-kanal proteinkompleks især udtrykt i hjernen. Det er lavet af to obligatoriske NR1 underenheder, og kombinationen af en eller to underenheder af NR2A, NR2B, NR2C eller NR2D13. En tidligere undersøgelse rapporterede, at de vigtigste epitop målrettet af antistoffer var på domænet ekstracellulære N-terminalen af NR1 subunit5. Derfor, i denne protokol, vi udtrykke den menneskelige rekombinant NR1-subunit protein af NMDA-receptor markeret med grønt fluorescerende proteiner (NGL) i den menneskelige embryonale nyre epitelcelle linje (HEK293) og udvikle en celle-baserede immunofluorescens assay til påvisning IgG-klasse af anti-NMDA receptor autoantistoffer i blodet.

<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&#252;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

Tilstedeværelsen af anti-NMDA receptor autoantistof kan forårsage forskellige neuropsykiatriske symptomer hos de berørte patienter, kaldt anti-NMDA receptor autoimmune encephalitis. Påvisning af specifikke autoantistof mod NMDA-receptor i blod eller cerebrospinalvæske (CSF) er afgørende for den præcise diagnose af denne betingelse. NMDA-receptor er en ion-kanal protein kompleks der indeholder fire underenheder, herunder to obligatoriske NMDA receptor subunit 1 (NR1) og en eller to NMDA-receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA-receptor underenhed 2C (NR2C), eller NMDA receptor subunit 2D (NR2D). Epitop af anti-NMDA receptor autoantistof blev rapporteret til at være til stede i den ekstracellulære N-terminale domæne af NR1 subunit af NMDA-receptor. Målet med denne undersøgelse er at udvikle en enkel cellebaserede immunofluorescens assay, der kan bruges som en screeningstest til påvisning af forekomst af autoantistoffer mod NR1 underenhed af NMDA-receptor i blodet til at fremme den kliniske og grundlæggende forskning i anti-NMDA receptor autoimmune encephalitis.

I gennemsnit kunne vi få 200-300 µg endotoxin-fri udtryk plasmid NR1-normal god landbrugspraksis og normal god landbrugspraksis fra 250 mL bakteriekulturen efter procedurerne i afsnit 1 i protokollen. Et beløb på 100 ng/brønd af udtryk plasmid blev brugt til Transfektion af HEK293 celler dyrkes i 48-godt plade som beskrevet i punkt 2 i protokollen. 24-30 h efter Transfektion, cellerne udtrykt NR1-normal god landbrugspraksis, og normal god landbrugspraksis rekombinante proteiner kunne...

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

Vi udtrykte ectopically NR1 underenhed af NMDA-receptor markeret med grønt fluorescerende protein i humane embryonale celler (HEK293) som antigen til at opdage autoantistoffer mod NMDA receptor i blodet hos patienter mistænkt med autoimmun encephalitis. Denne enkle metode kan være egnet til screening i klinisk indstillinger.

En simpel cellebaserede immunofluorescens Assay at opdage autoantistof mod N-Methyl-D-aspartat (NMDA) Receptor i blod

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

The overall goal of this assay is to detect autoantibodies against the NMDA receptor in the blood of patients with suspected autoimmune encephalitis. The measure help answer the key questions in differential diagnosis of patients with acute neuro-psychiatric symptoms. The main advantage of this technique is that it is a simple and reliable screening measure.Begin this procedure by preparing gelatin-coated culture plates. Aliquot 200 microliters of a 2%gelatin solution to each well of a 48-well culture plate and incubate the plate at 37 degrees Celsius for at least 30 minutes. After 30 minutes, aspirate the gelatin solution from the wells.Seed in each well 5 times 10 to the 4th HEK293 cells in 200 microliters of cell culture medium, containing 10%FBS. Incubate the plate overnight at 37 degrees Celsius in a humidified incubator supplied with 5%carbon dioxide. On the following day, replace the spent culture medium in each well with 200 microliters of fresh culture medium, containing 10%FBS.Scaling up according to the total number of wells in the experiment, Prepare solution A by mixing the NR1-GFP plasmid with culture medium. Prepare solution B by mixing the transfection reagent with culture medium. Prepare solution C by mixing solution A and solution B and incubating the mixture at room temperature for 20 to 30 minutes.Next, aliquot 40 microliters of solution C to each well containing the HEK293 cells. Swirl the plate gently and incubate the plate at 37 degrees Celsius in a humidified incubator supplied with 5%carbon dioxide overnight. Check the expression of the NR1-GFP recombinant protein in the host cells under a fluorescent microscope with 40 times to 200 times magnification, before proceeding to the cell-based immunofluorescence assay.To begin this assay, first aspirate the spent medium from the wells of the culture plate and then wash each well with 200 microliters of PBS three times. Add 200 microliters of 4%paraformaldehyde solution to each well and incubate the plate at room temperature for 15 minutes. Aspirate the paraformaldehyde solution from the wells and wash each well with 200 microliters of PBS three times.Next, add 200 microliters of 10%skim milk in PBST solution to each well and incubate the plate at room temperature with gentle shaking for one hour. Aspirate the 10%skim milk in PBST solution from the wells and wash the wells with 200 microliters of PBST once. Incubate the wells with plasma diluted one to 100 in PBST as the primary antibody with gentle shaking at room temperature for one hour.After one hour, aspirate the diluted plasma from the wells and wash each well with 200 microliters of PBST three times. To each well, add 200 microliters of goat anti-human IgG, conjugated with Alexa Fluor 594 and incubate the culture plate at room temperature with gentle shaking for one hour. Next, aspirate the goat anti-human IgG conjugated with Alexa Fluor 594 solution and wash each well with 200 microliters of PBST three times.After the third PBST wash, add 100 microliters of glycerol solution, containing DAPI, to each well. Observe and image the cells under a fluorescent microscope using the correct filter for each dye and a 10 times eye piece with four times, 10 times and 20 times objectives. These representative images taken 24 to 30 hours after transfection, show HEK293 cells that express NR1-GFP.Approximately 30%of the cells had the green signal under the fluorescent microscope. In the corresponding Alexa Fluor 594 images, the red signal in panel B indicates the binding of the anti-NMDA receptor autoantibodies to the NR1 expressed in the HEK293 cells. The weak red signal in panel E indicates the background noise of the Alexa Fluor 594 images.The cells expressing NR1-GFP were used for screening the presence of anti-NMDA receptor autoantibodies in a human plasma sample. In these merged images, the yellow color indicates colocalization of the NR1-GFP and anti-NMDA receptor autoantibodies. The arrows indicate examples of cells with prominent colocalization.A plasma sample that shows a greater than 30%overlap of green and red signals will be interpreted as positive in this case. In the merged image of the negative control, there is little overlap of green and red signals, which confirms the specific binding of the anti-NMDA receptor autoantibodies to the NR1-GFP in the assay. Once mastered, the technique can be done in four hours if it is performed properly.While attempting this technique, it is important to remember it is just a screening measure. Following these procedures, other measures such as western blot analysis, and tissue-based analysis can be performed to answer additional questions like specificity of positive cases. After development, the technique pave the way for researchers in the field of psychiatry to explore differential diagnosis of patients with acute mental health emergency.After watching this video, you should have a good understanding of how to perform the assay to screen for autoantibodies against NMDA receptor in the blood.

Research

JoVE Journal

Neuroscience

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

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