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

Dit werk werd gesteund door de Chang Gung Medical Foundation (subsidie nummer CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632 en CMRPG3E0633).

De studie werd goedgekeurd door de institutionele Review Board van Chang Gung Memorial Hospital in Linkuo, Taoyuan, Taiwan (102-2577A3).
1. bereiding van de NR1-GFP expressie plasmide
<ol><li>Mix 10 ng van NR1-GFP plasmide met 100 µL van Escherichia coli bevoegde cellen stam DH5α in een steriele 1,5 mL Centrifugeer buis, Pipetteer het mengsel voorzichtig omhoog en omlaag 4 - 6 keer, en de buis op ijs gedurende 20 minuten uit te broeden.</li>
	<li>Incubeer de buis bij 42 ° C gedur...

<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>

Er zijn verschillende cel-gebaseerde immunologische tests op het scherm van de aanwezigheid van Autoantilichamen tegen NMDA receptor gemeld in de literatuur, met inbegrip van de levende cel-gebaseerde immunofluorescentie assay11, vaste cel-gebaseerde immunofluorescentie assay9 , en stroom cytometry gebaseerde assay14. De levende cel-gebaseerde immunofluorescentie bepaling moet kort na de voorbereiding van cellen die zijn ectopically uiting geven aan ...

Anti-NMDA receptor auto-immune encefalitis is een nieuw herkende ziekte-entiteit die kan optreden bij patiënten van alle leeftijden, en beïnvloedt overwegend vrouwelijke patiënten1,2. Het is een van de meest frequent gediagnosticeerde encefalopathie bij patiënten met eerste onbekende etiologie van encefalitis3. Patiënten getroffen met anti-NMDA receptor encefalitis meestal hebben prodromal symptomen van een hoofdpijn of koorts, gevolgd door de snelle ontwikkeling van de wijzigingen in bewustzijn en een verscheidenheid aan acute neuropsychiatrische symptomen, met inbegrip van agitatie, prikkelbaarheid , angst, slapeloosheid, hallucinaties, wanen, agressie, bizar gedrag, afwijkingen van de beweging, autonome disregulatie en inbeslagneming aanvallen4,5. Vroege erkenning van deze aandoening en tijdige behandeling met immunotherapie zijn belangrijk voor een beter resultaat en zelfs volledig herstel in getroffen patiënten6. Vandaar, wordt voorgesteld dat de anti-NMDA receptor auto-immune encefalitis moet worden beschouwd als een belangrijke differentiële diagnose van patiënten met acute of nieuwe-onset psychotische kenmerken7,8te presenteren.
Naast klinische eigenschappen is detectie van autoantibody tegen de NMDA-receptor in het bloed of LIQUOR essentieel voor de accurate diagnose van anti-NMDA receptor auto-immune encefalitis9. De meeste van de immunologische tests voor de opsporing van de anti-NMDA receptor autoantibody zijn beschikbaar in een paar onderzoek laboratoria-10,11, en er is slechts één verkrijgbare cel-gebaseerde immunofluorescentie assay voor screening de anti-NMDA receptor autoantilichamen12. Het doel van deze studie is het ontwikkelen van een eenvoudige in-house cel-gebaseerde immunofluorescentie-bepaling die op het scherm van de aanwezigheid van anti-NMDA receptor autoantilichamen ter vergemakkelijking van het klinische onderzoek van anti-NMDA receptor gunstig in het laboratorium kan worden gebruikt auto-immune encefalitis. De NMDA-receptor is een heterotetramer ion kanaal eiwit complex specifiek uitgedrukt in de hersenen. Het bestaat uit twee verplichte NR1 subeenheden, en de combinatie van een of twee subeenheden van NR2A, NR2B, NR2C of NR2D13. Een eerdere studie gemeld dat de belangrijkste epitoop door de antilichamen gericht op het extracellulaire N-terminale domein van de NR1 subeenheid5 was. Vandaar, in dit protocol, we uiten het menselijke recombinante eiwitten van de NR1-subunit van NMDA receptor gelabeld met groen fluorescente proteïne (GFP) in de menselijke embryonale nier epitheliale cellijn (HEK293), en ontwikkelen van een cel-gebaseerde immunofluorescentie assay te detecteren de IgG-klasse van anti-NMDA receptor autoantilichamen in het bloed.

<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

De aanwezigheid van anti-NMDA receptor autoantibody kan verschillende neuropsychiatrische symptomen veroorzaken in de betrokken patiënten, genoemd anti-NMDA receptor auto-immune encefalitis. Opsporing van de specifieke autoantibody tegen de NMDA-receptor in het bloed of de cerebrospinale vloeistof (CSF) is essentieel voor de accurate diagnose van deze aandoening. De NMDA-receptor is een ion kanaal eiwit complex dat vier subeenheden bevat, waaronder twee verplichte NMDA receptor subeenheid 1 (NR1) en één of twee NMDA receptor subeenheid 2A (NR2A), NMDA receptor subeenheid 2B (NR2B), NMDA receptor subeenheid 2C (NR2C), of NMDA receptor subeenheid 2D (NR2D). De epitoop van anti-NMDA receptor autoantibody werd gemeld om aanwezig te zijn bij het extracellulaire N-terminale domein van de NR1 subeenheid van de NMDA-receptor. Het doel van deze studie is het ontwikkelen van een eenvoudige cel-gebaseerde immunofluorescentie-test die kan worden gebruikt als een screeningtest voor het detecteren van de aanwezigheid van Autoantilichamen tegen NR1 subeenheid van de NMDA-receptor in het bloed te vergemakkelijken van de klinische en fundamentele onderzoek van anti-NMDA receptor auto-immune encefalitis.

We kunnen gemiddeld 200-300 µg endotoxine-vrije expressie plasmide NR1-GFP en GFP krijgen van 250 mL bacteriecultuur na de procedures in sectie 1 van het protocol. Een bedrag van 100 ng/putje van de expressie plasmide werd gebruikt voor de transfectie van de HEK293 cellen gekweekt in de 48-well plaat zoals beschreven in de sectie 2 van het protocol. 24-30 uur na de transfectie, de cellen uitgesproken de NR1-GFP en GFP recombinante eiwitten onder fluorescente microscoop kon worden opgespo...

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

Wij hebben ectopically NR1 subeenheid van NMDA receptor gelabeld met groen fluorescent proteïne in menselijke embryonale cellen (HEK293) als antigeen te detecteren autoantilichamen tegen NMDA receptor in het bloed van patiënten verdacht met auto-immune encefalitis. Deze eenvoudige methode kan geschikt voor screeningonderzoek in klinische instellingen zijn.

Een eenvoudige cel-gebaseerde immunofluorescentie Assay voor het detecteren van Autoantibody tegen de N-Methyl-D-aspartaat (NMDA)-Receptor in het bloed

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

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Neuroscience

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

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