Name: an assay to detect serum anti-aquaporin-4 immunoglobulin g
Uploaded: 2024-03-29T12:50:35.000+00:00
Duration: 1 min 31 s
Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

an assay to detect serum anti-aquaporin-4 immunoglobulin g

An Assay to Detect Serum Anti-Aquaporin-4 Immunoglobulin G

First, bring the patient serum samples, the biochip slide, the PBS powder, and the Tween-20 from the refrigerated cell-based assay kit to the room temperature. Then, prepare more than 200 milliliters of PBS wash buffer containing 0.2% Tween-20 for each biochip slide.
Use the wash buffer to make 10-fold serial dilutions of each serum sample. Prepare positive and negative controls, according to the manufacturer&#39;s instructions. Add 30 microliters of 10-fold serial dilutions of the sample, the positive control, and the negative control to five individual reaction fields on the reagent tray.
Hold the biochip slide by its sides and remove its protective cover without touching its reaction fields. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer. Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the wash buffer, and leave it at the room temperature for 10 minutes.
Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields. Let the slide air-dry for 1 to 2 minutes.
In the dark room, add 25 microliters of fluorescent secondary antibody to each reaction field of the reagent tray. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer.
Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the fresh wash buffer, and leave it at room temperature for 10 minutes. Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields.
Let the slide air-dry for 1 to 2 minutes. Carefully add 1 drop of the embedding medium to each reaction field on the biochip slide. Finally, seal the biochip slide with a coverslip glass, while avoiding air bubbles, and proceed with imaging.

an-assay-to-detect-serum-anti-aquaporin-4-immunoglobulin-g

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Assay to Detect Serum Anti-aquaporin-4 Immunoglobulin G

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Patient Enrollment and Blood Sample Collection
<ol>
	<li>Apply the laboratory detection of serum anti-aquaporin-4 immunoglobulin G (anti-AQP4 IgG) to clinic patients with the chief complaints and symptoms listed below. Perform physical examinations as well. 
	Optic neuritis: Patients suffer with visual deficits, such as loss of visual fields and reduction of visual acuity.&#160;&#160;&#160;&#160;&#160;&#160; 
	Acute myelitis: Patients may present with paralysis, sensory deficits and autonomic dysfunction. 
	Area postrema syndrome: Patients may have symptoms of unexplained hiccups, nausea or vomiting.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	Acute brainstem syndrome: Brainstem lesions could result in different symptoms &#160;&#160;&#160; and physical signs depending on the location of the lesions.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	Symptomatic narcolepsy or acute diencephalic clinical syndrome with neuromyelitis optica spectrum disorders (NMOSD)-typical diencephalic MRI lesions.&#160; 
	Symptomatic cerebral syndrome with NMOSD-typical brain lesions. 
	NOTE:&#160;According to the international consensus diagnostic criteria for NMOSD, a diagnosis of NMOSD can be made if the patient is serum anti-AQP4 IgG-positive and has at least one of the core clinical characteristics mentioned above. However, we do not recommend testing of anti-AQP4 IgG in patients with optic neuritis only.</li>
	<li>Blood sample collection&#160; 
	NOTE:&#160;Patients do not need to be fasting.
	<ol>
		<li>Draw 2-3 mL of venous blood in a 4 mL vacuum blood collection tube.</li>
		<li>Allow the serum to clot for 30 min at room temperature (RT). 
		NOTE:&#160;Prolonged clotting can increase serum levels due to leakage from platelets.</li>
		<li>Centrifuge at 2100 x&#160;g&#160;for 5 min. Carefully transfer the serum to a new tube.</li>
		<li>Analyze the serum immediately or aliquot and store at -20 or -80 &#176;C. 
		NOTE:&#160;Storage at -20 &#176;C is for storage of months, while -80 &#176;C is for storage of years.</li>
	</ol>
	</li>
</ol>
2. Anti-AQP4 Antibody Detection
CAUTION:&#160;Patient samples and used kit reagents should be considered infectious materials. Sodium azide-containing reagents in the kit are toxic. A flow chart of the protocol is provided in&#160;Figure 1.
<ol>
	<li>Preparation
	<ol>
		<li>Bring the kit to RT before use. 
		NOTE: Store the kit at 2-8 &#176;C.</li>
		<li>Prepare at least 200 mL of phosphate-buffered saline (PBS) wash buffer containing 0.2% Tween 20. Pour 100 mL of wash buffer into a 500 mL beaker.</li>
		<li>Dilute the serum samples 10x with wash buffer. Prepare the positive and negative controls according to the instructions of the distributor.</li>
	</ol>
	</li>
	<li>Sample incubation
	<ol>
		<li>Add 30 &#181;L of the pre-diluted samples, positive control, or negative control to the reaction fields of the reagent tray (Figure 2).&#160;&#160;&#160; 
		NOTE:&#160;Avoid air bubbles.</li>
		<li>Remove the protective cover on the biochip slides. 
		NOTE:&#160;Do not touch the biochips to avoid the detachment and contamination of fixed cells. Hold the slide side-by-side before removing the cover.</li>
		<li>Apply the biochip slide to the reagent tray (Figure 2). Start a 30 min incubation at RT.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;Every sample should be an individual droplet connected to the corresponding biochip without mixing with other droplets.</li>
		<li>Gently rinse the biochip slide once with the wash buffer. Immerse the biochip slide into the 500 mL beaker filled with 100 mL of wash buffer for at least 5 min. Place the beaker on a shaker if possible.</li>
		<li>Take out the biochip slide from the wash buffer. Carefully wipe away the residual wash buffer outside of the reaction fields with a paper towel. Expose the biochip slide in open air for 1-2 min to evaporate the residual wash buffer on the reaction field. 
		NOTE:&#160;Do not dry out the reaction fields. Do not touch the biochips with a paper towel.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation
	<ol>
		<li>Prepare the secondary antibody solution according to the instructions of the distributor.</li>
		<li>Add 25 &#181;L of fluorescent secondary antibody to the reaction fields of a clean reagent tray.</li>
		<li>Apply the biochip slide to the reagent tray loaded with secondary antibody. Incubate for 30 min at RT.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Pour out the used wash buffer and add 100 mL of fresh wash buffer into the beaker. Repeat the washing as described in steps 2.2.4 and 2.2.5.</li>
		<li>Carefully add embedding medium to the reaction fields on a biochip slide (one drop per reaction field). Seal the biochip slide with one piece of cover glass. Avoid air bubbles.</li>
	</ol>
	</li>
	<li>Fluorescence detection
	<ol>
		<li>Turn on the fluorescent lamp of the microscope and pre-heat for 15 min. Choose a 488 nm filter.</li>
		<li>Open photograph software. Take pictures from both transfected and untransfected areas of all reaction fields with different magnifications. First, take one picture with 4x magnification for an overview, then take more pictures with 10x and 20x magnifications.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;The detailed protocol is shown in&#160;Table 1. Interpretation of the results is discussed in the representative results section.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-aquaporin-4 IIFT</td>
			<td>Euroimmun</td>
			<td>FA 1128-2005-50</td>
			<td>Contains biochip slides coated with AQP4-M1 transfected and untransfected EU 90 cells, fluorescein-labelled anti-human IgG, anti-AQP4 antibody as positive control, antibody negative sample, salt for PBS pH 7.2, Tween 20 and embedding medium.&#160;</td>
		</tr>
		<tr>
			<td>CellSens Dimension</td>
			<td>OLYMPUS</td>
			<td>N/A</td>
			<td>photograph software</td>
		</tr>
		<tr>
			<td>Gel &#38; clot activator tube</td>
			<td>Improve medical</td>
			<td>623040202</td>
			<td>From a local Chinese company</td>
		</tr>
	</tbody>
</table>

Source: Liu, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59014">Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay.</a>&#160;J. Vis. Exp.&#160;(2019)
The video demonstrates a cell-based assay for detecting anti-aquaporin-4 immunoglobulin G using a biochip containing untransfected cells and transfected cells expressing aquaporin-4. This method has applications in diagnosing neuromyelitis optica spectrum disorders, confirmed by strong green fluorescence on the biochip.

<img alt="Figure 1" src="/files/ftp_upload/22084/22084_Figure1.jpg" /> 
Figure 1: Flow chart of anti-AQP4 IgG detection protocol.&#160;AQP4-M1-transfected or untransfected EU 90 cells are fixed on the biochips. When adding serum to biochips, anti-AQP4 IgG in serum is captured by the fixed transfected cells. Then, fluorescein-labelled secondary antibody is applied to detect anti-AQP4 IgG. The fluorescence can be visualized by microscopy with various magnifications.
<img alt="Figure 2" src="/files/ftp_upload/22084/22084_Figure2.jpg" /> 
Figure 2: Sample incubation.&#160;(A) Top view of reagent tray. Every reagent tray contains five individual reaction fields. The positive control, samples, and negative control should be added to the separate reaction fields. (B) Top view of biochip slide. Every biochip slide contains five reaction fields, which have two subsections. The transfected subsection contains fixed AQP4-M1-transfected cells, while the untransfected subsection contains untransfected cells. (C) Front view of reagent tray and biochip slide. Reaction fields on the reagent tray and biochip slide are paired with each other. The sample added to the reaction field on reagent tray should be connected to the corresponding reaction field on the biochip slide.
Table 1: Fluorescence microscopy.&#160;It is recommended to use 4x, 10x, and 20x objectives for imaging of transfected and untransfected areas.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Magnification</td>
			<td>Transfected area</td>
			<td>Untransfected area</td>
		</tr>
		<tr>
			<td>4x</td>
			<td>1 picture</td>
			<td>1 picture</td>
		</tr>
		<tr>
			<td>10x</td>
			<td>4 pictures</td>
			<td>1 picture</td>
		</tr>
		<tr>
			<td>20x</td>
			<td>5 pictures</td>
			<td>1 picture</td>
		</tr>
	</tbody>
</table>

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

To detect anti-aquaporin-4 immunoglobulin-G or anti-AQP4 IgG &#8212; a biomarker of an autoimmune disorder, begin with a multi-well reagent tray.
Fill the wells individually with a positive control containing anti-AQP4 IgGs and pre-diluted serum samples.
Position a biochip with reaction fields over the reagent tray, ensuring direct contact.
Each biochip field contains two subsections, one coated with transfected cells expressing aquaporin-4 &#8212; a water-channel protein, and the other with untransfected cells lacking aquaporin-4.
The anti-AQP4 IgGs from the samples bind to the aquaporin-4 channels on transfected cells.&#160;
Remove the biochip and rinse with a detergent-supplemented wash buffer to eliminate non-specific interactions.
Overlay with fluorophore-labeled-secondary antibodies, interacting with pre-bound anti-AQP4 IgGs.
Wash, add a mounting medium, and observe under a fluorescence microscope.
The serum sample shows stronger fluorescence in the transfected subsections compared to the untransfected ones.
This fluorescence pattern aligns with that of the positive control, confirming the presence of anti-AQP4 IgGs in the serum.

26 ビュー. 血清抗アクアポリン-4免疫グロブリンGを検出するためのアッセイ

ビデオ: 血清抗アクアポリン-4免疫グロブリンGを検出するためのアッセイ - 実験

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

麻痺、視神経脊髄炎自己抗原アクアポリン 4 の T 細胞特定のマウスの視覚系障害の誘導

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

JoVE Journal

免疫・感染学

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

細胞に基づく試金の酵素補充療法の細胞内取り込みを中和する抗体の検出

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

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.

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.

血の N-メチル-D-アスパラギン酸 (NMDA) 受容体に対する自己抗体を検出する単純な細胞ベースの蛍光抗体法

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

An Assay to Detect Serum Anti-aquaporin-4 Immunoglobulin G

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An Assay to Detect Serum Anti-Aquaporin-4 Immunoglobulin G