Name: human serum anti-aquaporin-4 immunoglobulin g detection by cell-based assay
Uploaded: 2019-04-05T12:30:00
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 acu...

<ol>
	<li>Zekeridou, A., Lennon, V. A. <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+autoimmunity.">Aquaporin-4 autoimmunity.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 2 (4), 110 (2015).</li><li>Ho, J. D., 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=Crystal+structure+of+human+aquaporin+4+at+1.8+A+and+its+mechanism+of+conductance.">Crystal structure of human aquaporin 4 at 1.8 A and its mechanism of conductance.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (18), 7437-7442 (2009).</li><li>Takeshita, 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=Effects+of+neuromyelitis+optica-IgG+at+the+blood-brain+barrier+in+vitro.">Effects of neuromyelitis optica-IgG at the blood-brain barrier in vitro.</a> in vitro.Neurology Neuroimmunology &amp; Neuroinflammation. 4 (1), 311 (2016).</li><li>Sato, D. K., 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=Distinction+between+MOG+antibody-positive+and+AQP4+antibody-positive+NMO+spectrum+disorders.">Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders.</a> Neurology. 82 (6), 474-481 (2014).</li><li>Wingerchuk, D. 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=International+consensus+diagnostic+criteria+for+neuromyelitis+optica+spectrum+disorders.">International consensus diagnostic criteria for neuromyelitis optica spectrum disorders.</a> Neurology. 85 (2), 177-189 (2015).</li><li>Ruiz-Gaviria, 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=Specificity+and+sensitivity+of+aquaporin+4+antibody+detection+tests+in+patients+with+neuromyelitis+optica:+A+meta-analysis.">Specificity and sensitivity of aquaporin 4 antibody detection tests in patients with neuromyelitis optica: A meta-analysis.</a> Multiple Sclerosis and Related Disorders. 4 (4), 345-349 (2015).</li><li>Waters, P., 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=Multicentre+comparison+of+a+diagnostic+assay:+aquaporin-4+antibodies+in+neuromyelitis+optica.">Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica.</a> Journal of Neurology, Neurosurgery, and Psychiatry. 87 (9), 1005-1015 (2016).</li><li>Fryer, J. P., 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=AQP4+autoantibody+assay+performance+in+clinical+laboratory+service.">AQP4 autoantibody assay performance in clinical laboratory service.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 1 (1), 11 (2014).</li><li>Long, 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=Aquaporin-4+antibody+in+neuromyelitis+optica:+re-testing+study+in+a+large+population+from+China.">Aquaporin-4 antibody in neuromyelitis optica: re-testing study in a large population from China.</a> The International Journal of Neuroscience. 127 (9), 790-799 (2017).</li><li>Pisani, F., 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+autoantibodies+in+Neuromyelitis+Optica:+AQP4+isoform-dependent+sensitivity+and+specificity.">Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.</a> PloS One. 8 (11), 79185 (2013).</li><li>Jarius, S., 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+antibody+testing:+direct+comparison+of+M1-AQP4-DNA-transfected+cells+with+leaky+scanning+versus+M23-AQP4-DNA-transfected+cells+as+antigenic+substrate.">Aquaporin-4 antibody testing: direct comparison of M1-AQP4-DNA-transfected cells with leaky scanning versus M23-AQP4-DNA-transfected cells as antigenic substrate.</a> Journal of Neuroinflammation. 11, 129 (2014).</li><li>Jury&#324;czyk, M., Craner, M., Palace, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+CNS+inflammatory+diseases:+differentiating+features+of+NMO+and+MS.">Overlapping CNS inflammatory diseases: differentiating features of NMO and MS.</a> Journal of Neurology, Neurosurgery, and Psychiatry. 86 (1), 20-25 (2015).</li><li>Chen, 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=Clinical+Features+of+Patients+with+Multiple+Sclerosis+and+Neuromyelitis+Optica+Spectrum+Disorders.">Clinical Features of Patients with Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorders.</a> Chinese Medical Journal. 129 (17), 2079-2084 (2016).</li><li>Majed, M., Fryer, J. P., McKeon, A., Lennon, V. A., Pittock, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utility+of+testing+AQP4-IgG+in+CSF:+Guidance+for+physicians.">Clinical utility of testing AQP4-IgG in CSF: Guidance for physicians.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 3 (3), 231 (2016).</li><li>Jarius, S., 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=Cerebrospinal+fluid+antibodies+to+aquaporin-4+in+neuromyelitis+optica+and+related+disorders:+frequency,+origin,+and+diagnostic+relevance.">Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance.</a> Journal of Neuroinflammation. 7, 52 (2010).</li><li>Asgari, N., 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=Disruption+of+the+leptomeningeal+blood+barrier+in+neuromyelitis+optica+spectrum+disorder.">Disruption of the leptomeningeal blood barrier in neuromyelitis optica spectrum disorder.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 4 (4), 343 (2017).</li><li>Kim, H. 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=MRI+characteristics+of+neuromyelitis+optica+spectrum+disorder:+an+international+update.">MRI characteristics of neuromyelitis optica spectrum disorder: an international update.</a> Neurology. 84 (11), 1165-1173 (2015).</li><li>Jarius, S., 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=MOG+encephalomyelitis:+international+recommendations+on+diagnosis+and+antibody+testing.">MOG encephalomyelitis: international recommendations on diagnosis and antibody testing.</a> Journal of Neuroinflammation. 15 (1), 134 (2018).</li><li>Narayan, 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=MOG+antibody+disease:+A+review+of+MOG+antibody+seropositive+neuromyelitis+optica+spectrum+disorder.">MOG antibody disease: A review of MOG antibody seropositive neuromyelitis optica spectrum disorder.</a> Multiple Sclerosis and Related Disorders. 25, 66-72 (2018).</li><li>Ogawa, 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=MOG+antibody-positive,+benign,+unilateral,+cerebral+cortical+encephalitis+with+epilepsy.">MOG antibody-positive, benign, unilateral, cerebral cortical encephalitis with epilepsy.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 4 (2), 322 (2017).</li><li>Valentino, P., Marnetto, F., Granieri, L., Capobianco, M., Bertolotto, A. <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+antibody+titration+in+NMO+patients+treated+with+rituximab:+A+retrospective+study.">Aquaporin-4 antibody titration in NMO patients treated with rituximab: A retrospective study.</a> Neurology Neuroimmunology &amp; Neuroinflammation. 4 (2), 317 (2016).</li><li>Kessler, R. 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=Anti-aquaporin-4+titer+is+not+predictive+of+disease+course+in+neuromyelitis+optica+spectrum+disorder:+A+multicenter+cohort+study.">Anti-aquaporin-4 titer is not predictive of disease course in neuromyelitis optica spectrum disorder: A multicenter cohort study.</a> Multiple Sclerosis and Related Disorders. 17, 198-201 (2017).</li><li>Mealy, M. 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>

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 Ig...

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.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:185px;">Anti-aquaporin-4 IIFT</td>
			<td style="width:96px;">Euroimmun</td>
			<td style="width:105px;">FA 1128-2005-50</td>
			<td style="width:167px;">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 height="40">
			<td height="40" style="height:40px;">CellSens Dimension</td>
			<td>OLYMPUS</td>
			<td>N/A</td>
			<td>photograph software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gel &#38; 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...

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 ...

According to the recent international diagnostic criteria for neuromyelitis optica spectrum disorders, anti-AQP4 IgG is considered to be a core diagnostic biomarker. Anti-AQP4 IgG detection by cell-based assay can facilitate clinical diagnosis. So, cell-based assay is more sensitive and specific than other detection methods.And it can be applied to both clinical diagnosis and the scientific studies. First, bring the patient's 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 percent 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'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 slides 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 one to two 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 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 one to two minutes. Carefully add one drop of the embedding medium to each reaction field on the biochip slide.Finally, seal the biochip slide with a cover glass while avoiding air bubbles and proceed with imaging. The weak fluorescence intensity of the negative control observed in the infected area indicates the unspecific binding of the secondary antibody. The comparison between the detected fluorescence intensities of the positive control in the infected and uninfected areas indicates the specific binding of the anti-AQP4 IgG to AQP4M1 in the infected cells.The anti-AQP4 IgG negative serum shows binding patterns similar to the negative control in both infected and uninfected areas. The positive serum shows binding patterns similar to the positive control in both infected and uninfected areas. Samples are considered anti-AQP4 IgG positive when fluorescence heterogeneously increases in the infected area.The intensity of fluorescence is correlated to anti-AQP4 IgG titer in the serum. Samples are considered probably positive when only a small number of cells in the transfected area show detectable fluorescence intensities. A sample should be regarded as anti-AQP4 IgG negative when its intensity in the infected area is only homogeneously stronger than in the uninfected area.Avoiding air bubbles is vital and difficult. To eliminate air bubbles, first use a pipette to avoid air bubbles in droplets. Second, while applying the biochip slide to the reagent tray, first let one side of the slide touch the droplets, and then level the slide slowly.In addition, do not touch the reaction fields throughout the experiment.

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