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>

<ol>
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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. 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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. 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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 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:553px;" width="552">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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

Rilevamento di immunoglobulina Anti-acquaporina-4 G di siero umano dall'analisi Cell-based

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 ...

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Immunology and Infection

22.9K Views.  The First Hospital of Jilin University. Secondo i recenti criteri diagnostici internazionali per i disturbi dello spettro della neuromielite ottica, l'IgG anti-AQP4 è considerato un biomarcatore diagnostico di base. Il rilevamento IgG anti-AQP4 tramite saggio a base cellulare può facilitare la diagnosi clinica. Quindi, il saggio basato sulle cellule è più sensibile e specifico rispetto ad altri metodi di rilevamento.E può essere applicato sia alla diagnosi clinica che agli studi scientifici. In primo luogo, portare i ca...

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

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Determinazione della reattività e Titolo di siero usando un test dell&#39;emoagglutinazione

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

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Immunologia e infezioni

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Uso del mouse BLT umanizzato come cellule staminali basata su un Modello Tumore Terapia Genica

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Base di un semplice flusso Cytometry Assay per determinare In Vitro dell'anticorpo dipendente valorizzazione del Virus di febbre rompiossa utilizzando siero convalescente Zika Virus

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Un protocollo di aspirazione Blister per studiare T-cellula umana richiamo Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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.

Rilevazione degli anticorpi che neutralizzano l'assorbimento cellulare di terapie di sostituzione enzimatica con un'analisi Cell-based

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

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caratterizzazione delle risposte di isotipo dell'immunoglobulina timo-dipendenti e indipendenti del timo in topi usando l'analisi enzima-collegata dell'immunosorbente

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...