Name: human serum anti-aquaporin-4 immunoglobulin g detection by cell-based assay
Uploaded: 2019-04-05T12:30:00.000+00:00
Duration: 5 min 45 s
Description: Watch this Scientific Journal Video about Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay at JoVE.com

The authors are grateful for the support from grants from the National Science Foundation of China (No. 31600820), The Health and Family Planning Commission of Jilin Province (No. 2016Q036), and The Science and Technology Planning Project of Jilin Province (No. 20180520110JH).

This procedure was approved by the Ethics Committee of the First Hospital of Jilin University and was performed on approximately 1,500 subjects.
1. Patient Enrollment and Blood Sample Collection
<ol>
	<li>Apply the laboratory detection of serum 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. 
	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. 
	Acute brainstem syndrome: Brainstem lesions could result in different symptoms and physical signs depending on the location of the lesions. 
	Symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSD-typical diencephalic MRI lesions. 
	Symptomatic cerebral syndrome with NMOSD-typical brain lesions. 
	 
	NOTE: According to the international consensus diagnostic criteria for NMOSD5, 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 
	&#8203;NOTE: 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: Prolonged clotting can increase serum levels due to leakage from platelets.</li>
		<li>Centrifuge at 2100 x g 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: 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: 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 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 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). 
		NOTE: Avoid air bubbles.</li>
		<li>Remove the protective cover on the biochip slides. 
		NOTE: 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. 
		NOTE: 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 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: Do not dry out the reaction fields. Do not touch the biochips with 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 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. 
		NOTE: The detailed protocol is shown in Table 1. Interpretation of the results is discussed in the representative results section.</li>
	</ol>
	</li>
</ol>
3. Diagnosis of Patients
<ol>
	<li>If the patient is serum anti-AQP4 IgG-positive and shows at least one core clinical characteristic of NMOSD (see discussion section), diagnose the patient with NMSOD5. However, if the patient is anti-AQP4 IgG-negative, a diagnosis of NMOSD may not be excluded.</li>
</ol>

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A., 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=Aquaporin-4+serostatus+does+not+predict+response+to+immunotherapy+in+neuromyelitis+optica+spectrum+disorders.">Aquaporin-4 serostatus does not predict response to immunotherapy in neuromyelitis optica spectrum disorders.</a> Multiple Sclerosis. , (2017).</li><li>Yang, Y., 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=The+role+of+aquaporin-4+antibodies+in+Chinese+patients+with+neuromyelitis+optica.">The role of aquaporin-4 antibodies in Chinese patients with neuromyelitis optica.</a> Journal of Clinical Neuroscience. 20 (1), 94-98 (2013).</li><li>Kitley, 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=Prognostic+factors+and+disease+course+in+aquaporin-4+antibody-positive+patients+with+neuromyelitis+optica+spectrum+disorder+from+the+United+Kingdom+and+Japan.">Prognostic factors and disease course in aquaporin-4 antibody-positive patients with neuromyelitis optica spectrum disorder from the United Kingdom and Japan.</a> Brain. 135 (6), 1834-1849 (2012).</li></ol>

The authors have nothing to disclose.

We have described a widely accessible method to detect anti-AQP4 IgG in human serum. Anti-AQP4 IgG is closely related to NMOSD, and establishing a reliable anti-AQP4 IgG detection method is crucial for the clinical diagnosis of NMOSD. First, anti-AQP4 IgG is specific for NMOSD. Multiple sclerosis is also an immune-mediated disease of the central nervous system and shares many similarities with NMOSD12. However, anti-AQP4 IgG is only positive in NMOSD13. Second, anti-AQP4 IgG is common among NMOSD patients. Approximately two-thirds of NMOSD patients are serum anti-AQP4 IgG-positive4. Third, the most recent diagnosis criteria for NMOSD is based on serum anti-AQP4 IgG testing5. Taken together, serum anti-AQP4 IgG detection could facilitate the clinical diagnosis of NMOSD.
Among various anti-AQP4 IgG detection methods, CBA is widely used for clinical diagnosis due to its high sensitivity and specificity6,7. Although CBA is a quick and straightforward method, some key points should be addressed. First, experimenters should be careful when working with biochips, as AQP4-M1-transfected and untransfected cells are fixed on the biochips. During the whole procedure, biochips should not be touched by hand or dried out. Additionally, slides with biochips should be handled gently, otherwise the fixed cells on the biochips may fall down or become contaminated, which may result in fluorescence defective zones or a high fluorescent background. Second, when adding samples to the reaction fields on the reagent tray, researchers must ensure that the drops of samples are not mixed with each other. Air bubbles between the samples and biochips should be avoided.
This method has limitations; for example, when using CBA to detect anti-AQP4 IgG, it is hard to quantify fluorescence intensity and describe the anti-AQP4 IgG titer in the serum. Currently, in our diagnostic center, the intensity of fluorescence is subjectively reported by two clinicians after comparing photographs of samples vs. controls. In most cases, it is not hard to determine whether the serum is anti-AQP4 IgG-positive or negative. However, in rare cases, the fluorescence from samples may seem only slightly stronger than the negative control, and the sample is considered to be &#34;probable positive&#34;. In this situation, clinicians should be more careful in making a diagnosis. Another possible solution is repeating the CBA. A more accurate method to quantify the intensity of fluorescence and an international definition of &#34;anti-AQP4 IgG-positive&#34; are warranted. Notably, the positive rate of anti-AQP4 IgG in cerebrospinal fluid is much lower than in serum, and testing for anti-AQP4 IgG in cerebrospinal fluid is not informative14,15.
CBA can be applied to both clinical diagnosis and scientific studies of NMOSD. Clinical diagnosis of NMOSD is made based on the combination of clinical manifestations, serum anti-AQP4 IgG, and neuroradiological findings. Serum anti-AQP4 IgG-positive patients with at least one core clinical characteristic of NMOSD can be diagnosed with NMOSD5; however, if the patient is anti-AQP4 IgG-negative, a diagnosis of NMOSD should not be excluded5, and clinicians should further evaluate the clinical manifestations and neuroradiological findings5,16,17. Serum myelin oligodendrocyte glycoprotein antibody may be detected to assist the diagnosis18,19,20. Notably, although the serum anti-AQP4 IgG titer may be changed with rituximab treatment21, it is not predictive of disease course and response to immunotherapy22,23. CBA is also widely used for scientific research. For example, Yang et al.24 described the clinical features of anti-AQP4 IgG-positive NMOSD patients. Additionally, Kitley et al.25 analyzed the prognostic factors for anti-AQP4 IgG-positive NMOSD patients25. Both studies used CBA to detect serum anti-AQP4 IgG.
In summary, CBA is a widely used method to detect anti-AQP4 IgG and can be applied to clinical diagnosis and scientific research of NMOSD.

Serum AQP4 IgG is a core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD)1. The cell-based assay (CBA) is a widely used anti-AQP4 IgG detection method with high sensitivity and specificity. Here, a detailed protocol for CBA is introduced.
AQP4, a water channel protein, has six membrane-spanning units and two helical domains surrounding one aqueous pore2. Anti-AQP4 IgG is involved in the pathogenesis of NMOSD through binding to its target AQP4, which is mainly located on the endfeet of astrocytes3. It has been shown that anti-AQP4 IgG is positive in approximately two-thirds of NMOSD patients4. In the most recent international diagnostic criteria for NMOSD, anti-AQP4 IgG is considered to be a core diagnostic biomarker5. In this regard, it is crucial to establish a reliable protocol to detect human serum anti-AQP4 IgG and facilitate clinical diagnosis of NMOSD.
Currently, various anti-AQP4 IgG detection methods are available, such as CBA, tissue-based assay, enzyme-linked immunosorbent assay, and flow cytometry6,7. CBA employs EU90 cells, which are transfected with human AQP4, to capture anti-AQP4 IgG. The captured anti-AQP4 IgG is detected by fluorescent secondary antibodies and subsequently visualized by microscopy. Accumulating evidence has shown that CBA is more sensitive and specific than other anti-AQP4 IgG detection methods6,7. According to a meta-analysis, the sensitivity and specificity of CBA were shown to be 76% and 99%, which were higher than tissue-based and enzyme-linked immunosorbent assays6. Furthermore, a multicenter comparison of diagnostic assays of anti-AQP4 IgG was conducted7. A total of 193 study subjects from 15 European diagnostic centers were enrolled7. Four different methods were utilized to detect serum anti-AQP4 IgG7. It was demonstrated that CBA was more sensitive and specific than the other methods7. As AQP4 is expressed as two major isoforms (AQP4-M1 and AQP4-M23), anti-AQP4 IgG capture cell is transfected with either AQP4-M1 or AQP4-M23. However, which type of capture cell is better remains controversial. One investigation has supported AQP4-M1 based CBA8, while others have indicated that AQP4-M23 based CBA is better7,9,10. However, AQP4-M23-based CBA may yield false positive results due to unspecific IgG binding8. Jarius et al.11 reported that there was no significant difference in anti-AQP4 IgG detection rates between AQP4-M1 and AQP4-M23-based CBAs.
In summary, serum anti-AQP4 IgG is a core biomarker for NMOSD. CBA has higher specificity and sensitivity than other anti-AQP4 IgG detection methods. It remains controversial whether AQP4-M1- or AQP4-M23-based CBA is better. In this article, a detailed protocol for AQP4-M1-based CBA is described, which can apply to clinical diagnosis and studies of NMOSD.

<table><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.&amp;nbsp;</td></tr><tr><td>CellSens Dimension</td><td>OLYMPUS</td><td>N/A</td><td>photograph software</td></tr><tr><td>Gel &amp;amp; clot activator tube</td><td>Improve medical</td><td>623040202</td><td>From a local Chinese company</td></tr></tbody></table>

human serum anti-aquaporin-4 immunoglobulin g detection by cell-based assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Using the procedure described here, specific anti-AQP4 IgG in serum is detectable. During the procedure, pre-diluted samples, a positive control, and a negative control were added to the reaction fields, which contain transfected and untransfected areas (Figure 2). Fluorescence of the negative control in a transfected area mainly indicated the unspecific binding of secondary antibody to the transfected cells on biochips (Figure 3). Homogeneously weak fluorescence was visible (Figure 3). As for the positive control, anti-AQP4 antibody was added to the reaction field. Strong fluorescence was observed in the transfected area, which indicated the specific binding of anti-AQP4 IgG to AQP4-M1 in the transfected cells (Figure 4). Fluorescence of the positive control in an untransfected area showed hints of unspecific binding of anti-AQP4 IgG to fixed cells on biochips (Figure 4). For 10x diluted samples, anti-AQP4 IgG-negative serum showed a fluorescent pattern similar to the negative control, while the positive serum showed a pattern similar to the positive control (Figure 5). The intensity of fluorescence was associated with the anti-AQP4 IgG titer. Examples of different intensity of fluorescence are presented in Figure 5. Figure 5A and&#160;5B were from anti-AQP4 IgG-negative samples, while Figure 5C-H were from anti-AQP4 IgG-positive samples. As long as heterogeneous and granular fluorescence appeared in transfected areas, the sample was considered anti-AQP4 IgG-positive, regardless of the strength of the fluorescence. The diagnostic criteria for NMOSD, which is based on the serum anti-AQP4 IgG, will be further described in the discussion section.
In a few samples, only a small number of cells was strongly stained in transfected areas, and it was hard to determine whether the serum was anti-AQP4 IgG-positive or negative (Figure 6). In this case, &#34;probable positive&#34; can be reported. In rarer cases, the fluorescence of a sample in transfected areas was homogeneously stronger than in untransfected areas (Figure 7). In this case, the sample should be regarded as anti-AQP4 IgG-negative. The high intensity of background fluorescence might be nonspecific. To create a reliable report, all photographs should be blindly evaluated by at least two clinicians. The clinicians should be trained before evaluation. In this protocol, they were shown representative photographs of different sample types and were informed as to how to evaluate results. Then, they evaluated photographs together with an experienced clinician. Finally, the trained clinicians worked independently. In an ideal situation, it should be always the same clinicians who evaluate results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59014/59014fig1.jpg" /> 
Figure 1: Flow chart of anti-AQP4 IgG detection protocol. 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59014/59014fig2.jpg" /> 
Figure 2: Sample incubation. (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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59014/59014fig3.jpg" /> 
Figure 3: Representative figures for negative control. (A) An overview of the transfected area of the negative control was obtained by 4x magnification microscopy. Homogeneously weak fluorescence was observed throughout the area. (B,C) More details were observed in 10x (B) and x (C) magnification photographs. Generally, the cell membrane and plasma were slightly stained, and the cell nucleus was unstained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59014/59014fig4.jpg" /> 
Figure 4: Representative figures for positive control. (A,B) Transfected (A) and untransfected (B) areas of positive control are shown. Homogeneously weak fluorescence was observed in untransfected areas, indicating unspecific binding of anti-AQP4 IgG to fixed cells. Conversely, heterogeneous strong fluorescence was observed in transfected areas, indicating specific binding of anti-AQP4 IgG to AQP4-M1 expressed on fixed cells. (C-F) More details of transfected (C,E) and untransfected (D,F) areas are observed in with 10x (C, D) or 20x (E,F) magnification. Weak and even fluorescence appeared in untransfected areas. However, a large percentage of cells in transfected areas showed intense fluorescence. In the plasma of strongly stained cells, anti-AQP4 IgG showed smooth and granular fluorescence. The cell nucleus was unstained or slightly stained. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59014/59014fig5.jpg" /> 
Figure 5: Representative figures for anti-AQP4 antibody negative or positive samples. Samples were 10x diluted, and all figures shown were taken with a 10x objective. (A,B) Negative fluorescence sample: the fluorescence was homogeneously weak in both (A) transfected and (B) untransfected areas. (C,D) Weak fluorescence sample: when compared with untransfected areas (D), a small amount of cells (approximately 25% to 50%) in transfected areas (C) showed more intense fluorescence. (E,F) Moderate fluorescence sample: in transfected areas (E), strong granular fluorescence was observed in a medium amount of cells (approximately 50% to 75%). (G,H) Strong fluorescence sample: cells in untransfected areas (H) were weakly stained. However, in transfected areas (G), granular fluorescence with high intensity appeared in a large number of cells (approximately over 75%). Generally, as the titer of anti-AQP4 IgG increased, both fluorescence intensity and percentage of strongly stained cells elevated correspondingly. The fluorescence of untransfected areas was always homogeneously weak. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59014/59014fig6.jpg" /> 
Figure 6:&#160;Representative figures for anti-AQP4 antibody &#34;probable positive&#34; samples. (A,B) No significant differences in fluorescence patterns were observed between transfected (A) and untransfected (B) areas at 4x magnification. (C-E) With 10x (C,D) or 20x (E,F) magnification, a few cells in transfected areas (C,E) showed strong granular fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59014/59014fig7.jpg" /> 
Figure 7: Example of untypical anti-AQP4 IgG-negative sample. Both transfected (A) and untransfected (B) areas were homogeneously stained. However, the fluorescence in transfected areas was stronger than in untransfected areas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59014/59014fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<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>
Table 1: Fluorescence microscopy. It is recommended to use 4x, 10x, and 20x objectives for imaging of transfected and untransfected areas.

Watch this Scientific Journal Video about Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay at JoVE.com

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

human-serum-anti-aquaporin-4-immunoglobulin-g-detection-cell-based

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.0K Views.  The First Hospital of Jilin University. Volgens de recente internationale diagnostische criteria voor neuromyelitis optica spectrum stoornissen, anti-AQP4 IgG wordt beschouwd als een kern diagnostische biomarker. Anti-AQP4 IgG detectie door celgebaseerde test kan de klinische diagnose vergemakkelijken. De op cellen gebaseerde test is dus gevoeliger en specifieker dan andere detectiemethoden.En het kan worden toegepast op zowel klinische diagnose als de wetenschappelijke studies. Breng eerst de serummonsters van de patiënt, ...

Video: Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Medicine

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.

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

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

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.

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

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Biology

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

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.

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

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Paper-Based Immunoassay for the Detection of Immunoglobulin G

Source: Peng, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54111">Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.</a>&#160;J. Vis. Exp. (2016)
The video demonstrates a paper-based immunoassay, involving amine-functionalized cellulose discs cross-linked with glutaraldehyde. Covalent immobilization of IgG-Fc-specific capture antibodies, followed by the introduction of immunoglobulin G and horseradish peroxidase-conjugated detection antibodies, results in a color change confirming the presence of immunoglobulin G.

1. Grafting Amine Functional Groups on Cellulose Paper Discs

	Prepare one piece of square paper with a dimension of 1 cm &#215; 1 cm, and 100 paper discs ...

Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum

Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 105 cells/mL in pre-warmed complete DMEM media.
Then, seed 100 microliters of cells, per well, into clear 96-well flat-bottomed plates to the concentration of 1 x 104 cells per well. Incubate the plate at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 22 hours.
On day two, generate serial dilutions of the serum samples of interest in 1.5-milliliter microcentrifuge tubes using pre-warmed complete DMEM.
To each tube with diluted serum samples, add 66 microliters of the 7.5 x 106 viral genome per microliter virus working solution. Mix the virus/serum dilutions by pipetting, and then, place the tubes containing the virus/serum mixtures in an incubator at 37 degrees Celsius with 5% carbon dioxide for 30 minutes, to allow potential neutralization to occur.
After 30 minutes, pipette 100 microliters of the virus/serum mixture to each well on the 96-well plate containing 1 x 104 cells per well, then, wrap the plate in a foil to place in an incubator at 37 degrees Celsius with 5% carbon dioxide overnight for 16 to 24 hours.
On day three, aspirate the media from the wells of the 96-well plate using a fume hood vacuum without disrupting the adhered cells, and add 50 microliters of 4% paraformaldehyde to each well. After wrapping the plate in foil, leave it for 10 minutes at room temperature.
Later, wash and aspirate the cells twice with 200 microliters of PBS at room temperature. After the second wash, pipette 200 microliters of pre-warmed PBS into each well, and wrap the plate in foil followed by incubation at 65 degrees Celsius for 90 minutes to denature endogenous alkaline phosphatase activity.
Following incubation, add 50 microliters of the freshly prepared, dissolved BCIP/NBT into each well, and incubate the wrapped plates at room temperature for 2 to 24 hours. Later, take photos of each well using a 4x objective lens in a light microscope camera, ensuring the consistent use of the same exposure, white balancing, and light settings for all assays.

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

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