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

Questo lavoro è stato supportato dalla Fondazione medica Chang Gung (concessione numero CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632 e CMRPG3E0633).

Lo studio è stato approvato dall'istituzionale Review Board di Chang Gung Memorial Hospital presso Linkuo, Taoyuan, Taiwan (102-2577A3).
1. preparazione del plasmide di espressione NR1-GFP
<ol><li>Miscelare 10 ng di plasmide NR1-GFP con 100 µ l del ceppo di cellule competenti di Escherichia coli DH5α in una sterile 1,5 mL Centrifuga tubo, dispensare la miscela delicatamente fino e giù 4 - 6 volte e incubare la provetta in ghiaccio per 20 min.</li>
	<li>Incubare la provetta a 42 ...

<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>

Ci sono parecchi saggi immunologici basati su celle a schermo la presenza di autoanticorpi diretti contro il recettore NMDA segnalati nella letteratura, compreso immunofluorescenza diretta basati su cellule test11, fissata il test di immunofluorescenza basati su cellule9 , e14di analisi basati su citometria a flusso. Il test di immunofluorescenza diretta basati su cellule deve essere eseguito subito dopo la preparazione delle celle ectopically esprim...

L'encefalite autoimmune di anti-NMDA del ricevitore è un'entità recentemente riconosciuta di malattia che può accadere in pazienti di tutte le età e colpisce i pazienti prevalentemente femminile1,2. È uno dell'encefalite più frequentemente diagnosticato fra i pazienti con eziologia sconosciuta iniziale di encefalite3. Pazienti affetti da encefalite di anti-NMDA receptor solitamente hanno sintomi prodromici di un mal di testa o febbre, seguita dallo sviluppo veloce del cambiamento di livello di coscienza e una varietà di sintomi neuropsichiatrici acuti, tra cui agitazione, irritabilità , ansia, insonnia, allucinazioni, deliri, aggressività, comportamenti bizzarri, anomalie del movimento, disregolazione autonomica e sequestro attacchi4,5. Il riconoscimento iniziale di questa condizione e il tempestivo trattamento con immunoterapia sono importanti per un risultato migliore e anche pieno recupero in pazienti colpiti6. Quindi, esso è suggerito che l'encefalite autoimmune di anti-NMDA del ricevitore dovrebbe essere considerata come un'importante diagnosi differenziale dei pazienti che presentano con caratteristiche psicotiche acute o nuova insorgenza7,8.
Oltre alle caratteristiche cliniche, rilevamento degli autoanticorpi contro il recettore NMDA nel sangue o CSF è essenziale per la diagnosi esatta di anti-NMDA receptor encefalite autoimmune9. La maggior parte dei test immunologici per rilevare il autoantibody del recettore anti-NMDA sono disponibile in qualche ricerca laboratori10,11, e c'è solo un test di immunofluorescenza di basati su cellule commercialmente disponibili per lo screening del anti-NMDA receptor autoantibodies12. L'obiettivo di questo studio è quello di sviluppare un test di immunofluorescenza di basati su cellule in-House semplice che può essere convenientemente utilizzato in laboratorio allo schermo anti-NMDA receptor autoanticorpi per facilitare la ricerca clinica del recettore NMDA anti encefalite autoimmune. Il recettore NMDA è una heterotetramer ione canale proteina complessa specificamente espressa nel cervello. È fatta di due unità secondarie NR1 obbligatorie e la combinazione di uno o due subunità NR2A, NR2B, NR2C o NR2D13. Un precedente studio riferito che l'epitopo principale mirato da parte degli anticorpi è presso il dominio N-terminale extracellulare della subunità NR15. Quindi, in questo protocollo, esprimiamo la proteina ricombinante umana di NR1-subunità del recettore NMDA etichettato come proteina fluorescente verde (GFP) nella linea cellulare epiteliale embrionali umane del rene (HEK293) e sviluppare un test di immunofluorescenza basati su cellule per rilevare la classe di IgG del anti-NMDA receptor autoanticorpi nel sangue.

<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

La presenza degli autoanticorpi del recettore anti-NMDA può causare vari sintomi neuropsichiatrici in pazienti commoventi, definiti l'encefalite autoimmune di anti-NMDA receptor. L'individuazione degli specifici autoanticorpi contro il recettore NMDA nel sangue o liquido cerebrospinale (CSF) è essenziale per la diagnosi esatta di questa condizione. Il recettore NMDA è un complesso di proteine canale dello ione che contiene quattro subunità, tra cui due subunità di ricevitore NMDA 1 (NR1) obbligatoria e recettore NMDA uno o due subunità 2A (NR2A), 2B di subunità del recettore NMDA (NR2B), unità secondaria del ricevitore NMDA 2C (NR2C), o del recettore NMDA subunità 2D (NR2D). L'epitopo di autoanticorpi anti-NMDA del ricevitore è stato segnalato per essere presente nel dominio N-terminale extracellulare della subunità NR1 del recettore NMDA. L'obiettivo di questo studio è quello di sviluppare un test di immunofluorescenza basati su cellule semplice che può essere utilizzato come test di screening per rilevare la presenza di autoanticorpi diretti contro NR1 subunità del recettore NMDA nel sangue per facilitare la ricerca di base e clinica di encefalite autoimmune di anti-NMDA receptor.

In media, potremmo ottenere 200-300 µ g plasmide endotossina-libera espressione NR1-GFP e GFP da coltura batterica 250ml seguendo le procedure descritte nella sezione 1 del protocollo. Un importo di 100 ng/pozzetto del plasmide di espressione è stato utilizzato per la transfezione delle cellule HEK293 coltivate nella piastra 48 pozzetti come descritto nella sezione 2 del protocollo. 24-30 h dopo trasfezione, le cellule hanno espresso la NR1-GFP, e proteine ricombinanti GFP potrebbero es...

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

Abbiamo ectopically espresso NR1 subunità del recettore NMDA etichettato come proteina fluorescente verde in cellule embrionali umane (HEK293) come antigene per rilevare autoanticorpi diretti contro il recettore NMDA nel sangue di pazienti con sospettata con l'encefalite autoimmune. Questo semplice metodo può essere adatto per scopi nelle regolazioni cliniche di screening.

Un semplice Cell-based immunofluorescenza per rilevare autoanticorpi contro il recettore N-metil-D-aspartato (NMDA) nel sangue

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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L&#39;applicazione di una conduttanza recettore NMDA nei neuroni dopaminergici mesencefalo Rat Utilizzando la tecnica dinamica morsetto

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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