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

This work was supported by the Chang Gung Medical Foundation (grant number CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, and CMRPG3E0633).

The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital at Linkuo, Taoyuan, Taiwan (102-2577A3).
1. Preparation of the NR1-GFP Expression Plasmid
<ol>
	<li>Mix 10 ng of NR1-GFP plasmid with 100 &#181;L of Escherichia coli competent cells strain DH5&#945; in a sterile 1.5 mL centrifuge tube, pipet the mixture gently up and down 4-6 times, and incubate the tube on ice for 20 min.</li>
	<li>Incubate the tube at 42 &#176;C for 1 min in a water bath, remo...

<ol>
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There are several cell-based immunological assays to screen the presence of autoantibodies against NMDA receptor reported in the literature, including live cell-based immunofluorescence assay11, fixed cell-based immunofluorescence assay9, and flow cytometry-based assay14. The live cell-based immunofluorescence assay should be performed shortly after the preparation of cells ectopically expressing the NR1 protein, while the flow cytometry-based assay ...

Anti-NMDA receptor autoimmune encephalitis is a newly recognized disease entity that can occur in patients of all ages, and affects predominantly female patients1,2. It is one of the most frequently diagnosed encephalitis among patients with initial unknown etiology of encephalitis3. Patients affected with anti-NMDA receptor encephalitis usually have prodromal symptoms of a headache or fever, followed by the quick development of consciousness level change and a variety of acute neuropsychiatric symptoms, including agitation, irritability, anxiety, insomnia, hallucinations, delusions, aggression, bizarre behaviors, movement abnormalities, autonomic dysregulation, and seizure attacks4,5. Early recognition of this condition and timely treatment with immunotherapy are important for a better outcome and even full recovery in affected patients6. Hence, it is suggested that anti-NMDA receptor autoimmune encephalitis should be considered as an important differential diagnosis of patients presenting with acute or new-onset psychotic features7,8.
Besides clinical features, detection of autoantibody against the NMDA receptor in the blood or CSF is essential for the accurate diagnosis of anti-NMDA receptor autoimmune encephalitis9. Most of the immunological tests to detect the anti-NMDA receptor autoantibody are available in a few research laboratories10,11, and there is only one commercially available cell-based immunofluorescence assay for screening the anti-NMDA receptor autoantibodies12. The goal of this study is to develop a simple in-house cell-based immunofluorescence assay that can be conveniently used in the laboratory to screen the presence of anti-NMDA receptor autoantibodies to facilitate the clinical research of anti-NMDA receptor autoimmune encephalitis. The NMDA receptor is a heterotetramer ion channel protein complex specifically expressed in the brain. It is made of two compulsory NR1 subunits, and the combination of one or two subunits of NR2A, NR2B, NR2C, or NR2D13. A previous study reported that the main epitope targeted by the antibodies was at the extracellular N-terminal domain of the NR1 subunit5. Hence, in this protocol, we express the human recombinant NR1-subunit protein of NMDA receptor tagged with green fluorescent protein (GFP) in the human embryonic kidney epithelial cell line (HEK293), and develop a cell-based immunofluorescence assay to detect the IgG class of anti-NMDA receptor autoantibodies in the blood.

<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

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

On average, we could obtain 200-300 &#181;g endotoxin-free expression plasmid NR1-GFP and GFP from 250 mL bacterial culture following the procedures described in section 1 of the protocol. An amount of 100 ng/well of the expression plasmid was used for the transfection of the HEK293 cells cultured in the 48-well plate as described in the section 2 of the protocol. 24-30 h after transfection, the cells expressed the NR1-GFP, and GFP recombinant proteins could be detected under fluorescent ...

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

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

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

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

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