Name: measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells
Uploaded: 2017-11-22T15:00:00
Description: Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

Vi takker Dr. Xiuhua Lu, Dr. Feng Liu og Ms. Ashley Burroughs fra Influenza Division af CDC for deres kritiske review og assistance i forberedelsen af dette manuskript. Vi takker Dr. Adrian Reber fra Influenza Division af CDC for sin bistand til forberedelse grafik af figur 3. Endelig vil takke vi Dr. M. Matrosovich, Marburg, Tyskland for at give MDCK-SIAT1 celler.

Alle influenzavirus skal håndteres efter passende biosikkerhed niveau krav (BSL-2 eller højere) som defineret i biosikkerhed på mikrobiologiske og biomedicinske laboratorier (BMBL)12.

1. forberedelse af reagenser og råvare
<ol><li>
Forberede MDCK-SIAT1 celler og sterile cellekulturmedium
	<ol>
<li>Forberede MDCK-SIAT1 cellekulturmedium med 500 mL af Dulbeccos modificerede Eagle Medium (DMEM) med høje glukose, 10% v/v varm...

<ol>
	<li>Reber, A., Katz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+assessment+of+influenza+vaccines+and+immune+correlates+of+protection.">Immunological assessment of influenza vaccines and immune correlates of protection.</a> Expert Rev of Vaccines. 12, 519-536 (2013).</li><li>Li, C. K., Rappuoli, R., Xu, X. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlates+of+protection+against+influenza+infection+in+humans--on+the+path+to+a+universal+vaccine?.">Correlates of protection against influenza infection in humans--on the path to a universal vaccine?.</a> Curr Opin in Immunol. 25, 470-476 (2013).</li><li>WHO Global Influenza Surveillance Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for the labratory diagnosis and virological surveillence of influenza. , (2011).</li><li>Govorkova, E. A., Kodihalli, S., Alymova, I. V., Fanget, B., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+immunogenicity+of+influenza+viruses+cultivated+in+Vero+or+MDCK+cells+and+in+embryonated+chicken+eggs.">Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.</a> Dev Biological Stand. 98, 39-51 (1999).</li><li>Levine, M. Z., 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=Neutralizing+Antibody+Responses+to+Antigenically+Drifted+Influenza+A(H3N2)+Viruses+among+Children+and+Adolescents+following+2014-2015+Inactivated+and+Live+Attenuated+Influenza+Vaccination.">Neutralizing Antibody Responses to Antigenically Drifted Influenza A(H3N2) Viruses among Children and Adolescents following 2014-2015 Inactivated and Live Attenuated Influenza Vaccination.</a> Clin Vaccine Immunol : CVI. 23, 831-839 (2016).</li><li>Lin, 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+characteristics+and+antigenic+properties+of+recently+emerged+subclade+3C.3a+and+3C.2a+human+influenza+A(H3N2)+viruses+passaged+in+MDCK+cells.">The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells.</a> Influenza Other Respir Viruses. , (2017).</li><li>Lin, Y. 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=Neuraminidase+receptor+binding+variants+of+human+influenza+A(H3N2)+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site:+a+role+in+virus+attachment?.">Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?.</a> J of Virol. 84, 6769-6781 (2010).</li><li>Skowronski, 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=Mutations+acquired+during+cell+culture+isolation+may+affect+antigenic+characterisation+of+influenza+A(H3N2)+clade+3C.2a+viruses.">Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses.</a> Euro Surveillance. 21, 30112 (2016).</li><li>Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N. A., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+alpha-2,6-sialyltransferase+in+MDCK+cells+increases+influenza+virus+sensitivity+to+neuraminidase+inhibitors.">Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.</a> J of Virol. 77, 8418-8425 (2003).</li><li>Oh, D. Y., Barr, I. G., Mosse, J. A., Laurie, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDCK-SIAT1+cells+show+improved+isolation+rates+for+recent+human+influenza+viruses+compared+to+conventional+MDCK+cells.">MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells.</a> J Clin Microbiol. 46, 2189-2194 (2008).</li><li>US Department of Health and Human Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2009).</li><li>Reed, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> The American Journal of Hygiene. 27, (1938).</li><li>Laurie, K. L., 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+Laboratory+Comparison+of+Influenza+Microneutralization+Assays+for+A(H1N1)pdm09,+A(H3N2),+and+A(H5N1)+Influenza+Viruses+by+CONSISE.">International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE.</a> Clinical Vaccine Immunol : CVI. 22, 957-964 (2015).</li><li>Lin, Y., Gu, Y., McCauley, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+Quantitative+Micro-neutralization+Assay.">Optimization of a Quantitative Micro-neutralization Assay.</a> J Vis Exp : JoVE. , (2016).</li><li>van Baalen, C. 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=ViroSpot+microneutralization+assay+for+antigenic+characterization+of+human+influenza+viruses.">ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.</a> Vaccine. 35, 46-52 (2017).</li></ol>

MN-analysen er en af de vigtigste assays anvendes til influenza serologi til påvisning af antistof svar efter influenza-infektion eller vaccination. Titers genereret fra MN assays er ofte brugt som det primære resultat af mange influenza seroepidemiology undersøgelser. MN assays er også almindeligt brugt til sero-diagnose, og evalueringen af vaccine immunogenicitet. Internationale Inter lab undersøgelser er blevet gennemført for at sammenligne MN assays udføres i flere laboratorier14.
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Influenzavirus fortsætte med at forårsage sygelighed og dødelighed i den menneskelige befolkning hvert år. HA er den store overflade glycoprotein af influenzavirus. Neutraliserende antistoffer rettet mod HA er den vigtigste immune mekanisme, der korrelerer med beskyttelse af influenza-infektion1,2. Hemagglutination hæmning (HI) assays og MN assays er to metoder, der er almindeligt anvendt til at måle antistof reaktioner i menneskelige sera efter influenza-infektion eller vaccination3. HI assay måler antistof hæmning af virus hemagglutination af røde blodlegemer og betragtes som en surrogat assay. I modsætning til HI, kan MN assay direkte måle niveauet af antistoffer i menneskelige sera, der neutraliserer influenza-infektion i cellekulturer. MDCK celler bruges ofte i influenza virusisolation og MN-undersøgelser,4.
Influenzavirus undergår konstant antigene drift og skift, erhverve mutationer på HA proteiner, der kan ændre receptor binding specificiteten af virus. Siden 2014, nye klynger af A(H3N2) virus dukkede op og fortsatte med at cirkulere indtil den aktuelle sæson. Fleste af disse virus tilhører genetiske gruppe 3C.2a og 3C.3a baseret på Fylogenetisk analyse af HA proteiner. Mange af de cirkulerende 3C.2a vira havde reduceret evne til at hemagglutinate røde blodlegemer og derfor ikke kan være kendetegnet ved HI assays5. Neutralisering assays skal bruges til at måle antistof reaktioner på disse vira, der ikke hemagglutinate6. Desuden, undersøgelser har vist, at disse moderne A(H3N2) virus har ændret receptor bindende egenskaber sammenlignet med tidligere A(H3N2) virus og har tendens til at akkumulere kultur tilpasset mutationer og polymorfi når passaged i in vitro- celle kulturer7,8,9. Sammenlignet med konventionelle MDCK celler, er MDCK-SIAT1 en cellelinje, udviklet af Matrosovich et al. gennem stabile Transfektion af MDCK celler med cDNA af menneskelige en2, 6-sialtransferase (SIAT1). Denne cellelinie udtrykker øgede mængder af en2, 6-sialic galactose fraspaltning og nedsat beløb for en2, 3-sialic syre fraspaltning end overordnet MDCK celler10. MDCK-SIAT1 celler har vist sig for at forbedre isolering priser for A(H3N2) virus i forhold til MDCK celler11. For nylig, Lin et al. rapporterede, at for nyopstået 3C.2a og 3C.3a menneskelige A(H3N2) influenzavirus, mere trofaste virus replikeringer og bedre virus smitteevne blev opnået, når virus var kulturperler i MDCK-SIAT1 cellelinjer sammenlignet med MDCK celler7. MDCK-SIAT1-celler passer således bedre i MN assays til at karakterisere antistof svar til de seneste klynger af A(H3N2) virus.
Her beskriver vi en MN analyse ved hjælp af MDCK-SIAT1 celler til at måle antistof svar til moderne 3C.2a og 3C.3a A(H3N2) vira i menneskelige sera. Virus dyrkes i enten æg eller celler kan bruges i denne analyse. Varme inaktiveret sera indeholdende neutraliserende antistoffer er først seriefremstillede fortyndet, derefter inkuberes med 100 TCID50/godt af A(H3N2) influenzavirus tillade antistoffer i sera at binde sig til virussen. MDCK-SIAT1 celler er derefter føjes til virus-antistof blandingen og inkuberes i 18-20 timer ved 37 ° C, 5% CO2 til at tillade A(H3N2) virus at inficere MDCK-SIAT1 celler og kopiere. Efter natten inkubation, pladerne er faste og mængden af virus i hver brønd er kvantificeret ved en ELISA ved hjælp af anti-influenza A NP monoklonale antistoffer. Påvisning af NP indikerer tilstedeværelsen af virusinfektion og fraværet af neutraliserende antistoffer. Neutraliserende antistof titer er defineret som reciprokke af den højeste serumfortynding, der indeholder ≥ 50% hæmning af virus smitteevne.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM) with high Glucose</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">11965</td>
			<td style="width:308px;">A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal bovine serum (FBS)</td>
			<td style="width:181px;">Hyclone</td>
			<td style="width:185px;">SH30070.03</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Bovine Serum Albumin (BSA) Fraction V, Protase Free</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">3117332001</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">L-Glutamine</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">25030-081</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium pyruvate</td>
			<td style="width:181px;">Life Science</td>
			<td>11360-070</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Geneticin G-418 disulfate salt&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">A1720-5G&#160;&#160;</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">HEPES</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15630-080</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/Streptomycin</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15140-122</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:295px;">Acetone</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">67-64-1</td>
			<td style="width:308px;">Used at an 80% concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Phosphate-Citrate Buffer with Sodium Perborate&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBF2806V</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">O-Phenylenediamine Dihydrochloride tablet</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBQ1086V</td>
			<td style="width:308px;">1 tablet per 100ml of cell culture grade water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:295px;">Sulfuric Acid</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:185px;">A510-P500</td>
			<td style="width:308px;">Used 0.5M final concentration</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">Ethanol, Denatured, 4L</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">EM-AX0422-3</td>
			<td style="width:308px;">Used at an 70% concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Trypsin-EDTA</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">1748048</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RDE II &#34;Seiken&#34;</td>
			<td style="width:181px;">Denka Seiken</td>
			<td style="width:185px;">370013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tween 20</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">P1379-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-NP mouse monoclonal Ab</td>
			<td style="width:181px;">Millipore pool</td>
			<td style="width:185px;">MAB 8257 MAB 8258</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-mouse IgG HRP&#160;</td>
			<td style="width:181px;">KPL</td>
			<td style="width:185px;">074-1802</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">96-well flat-bottom plates</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:185px;">3455</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Plate reader</td>
			<td style="width:181px;">Molecular Device</td>
			<td style="width:185px;">Spectromax 384 plus</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell Culture Flask 162 cm2/Vent Cap</td>
			<td style="width:181px;">Corning/VWR</td>
			<td style="width:185px;">3151</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells

Neutraliserende antistoffer mod hemagglutinin (HA) influenza-vira betragtes som den vigtigste immune mekanisme, der korrelerer med beskyttelse af influenza-infektioner. Microneutralization (MN) assays er ofte bruges til at måle neutraliserende antistof reaktioner i menneskelige sera efter vaccination mod influenza eller infektion. Madine Darby Canine nyre (MDCK) celler er almindeligt anvendte celle substrat for MN assays. Men i øjeblikket cirkulerer 3C.2a og 3C.3a A(H3N2) influenza virus har fået ændret receptor binding specificitet. MDCK-SIAT1-cellelinie med øget α-2,6 sialic galactose fraspaltning på overfladen har vist sig for at give forbedret smitteevne og mere trofast replikationer end konventionelle MDCK celler efter disse moderne A(H3N2) virus. Her, beskriver vi en MN analyse ved hjælp af MDCK-SIAT1 celler, der er blevet optimeret for at kvantificere neutraliserende antistof titers til disse moderne A(H3N2) virus. I denne protokol fortyndes varme inaktiveret sera indeholdende neutraliserende antistoffer først seriefremstillede, derefter inkuberes med 100 TCID50/godt af A(H3N2) influenzavirus til at tillade antistoffer i sera at binde til virus. MDCK-SIAT1 celler er derefter føjes til virus-antistof blandingen og inkuberes i 18-20 timer ved 37 ° C, 5% CO2 til at tillade A(H3N2) virus at inficere MDCK-SIAT1 celler. Efter natten inkubation, plader er fast og mængden af virus i hver godt er kvantificeret ved en enzym-forbundet immunosorbent assay (ELISA) ved hjælp af anti-influenza en nucleoprotein (NP) monoklonale antistoffer. Neutraliserende antistof titer er defineret som reciprokke af den højeste serumfortynding, der giver ≥50% hæmning af virus smitteevne.

Bestemmelse af smitteevne af virus bestande er det første skridt i MN assay. Figur 2 illustrerer plade layout for at bestemme TCID50 af virus bestande. For viruslagrene med ukendt smitteevne, kan virus være titreres fra flere præ fortyndinger, for eksempel både 10-2 og 10-3, for at fange de bedste titreringskurven for at beregne smitteevne af virus. Den virus mængde anvendes i MN assay bør være standardiseret til 100 TC...

Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutraliserende antistoffer korrelerer med beskyttelse af influenza-infektioner. Microneutralization assays måling af neutraliserende antistoffer i menneskelige sera og bruges ofte til influenza human serum. Vi beskriver en microneutralization analyse ved hjælp af MDCK-SIAT1 celler til at måle neutraliserende antistof titers til moderne 3C.2a og 3C.3a A(H3N2) virus efter vaccination mod influenza eller infektion.

Måling af Influenza neutraliserende antistof svar til A(H3N2) virus i menneskelige Sera af Microneutralization undersøgelser, som ved hjælp af MDCK-SIAT1 celler

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

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Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

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Brug zebrafisk modeller af human influenza A-virus infektioner at Screen antivirale lægemidler og karakterisere værtsimmunsystem Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Fluorescensbaseret neuraminidase-inhiberingsassay i vurdering af den følsomhed over influenzavira til neuraminidaseinhibitoren Class of Antivirale

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generation af undslippe varianter af neutraliserende Influenza Virus monoklonale antistoffer

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

Identifikation af mus og humant antistof repertoirer af næste generations sekventering

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...