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 ...

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.

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