Name: zika virus specific diagnostic epitope discovery
Uploaded: 2017-12-12T13:00:00
Description: Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Nuværende støtte fra en SBIR (Small Business Innovation Research) administrative supplement tilskud fra NIDCRR44 DE024456. NIDCR HIV grant, udviklet sig ud af NIDCR grant U01 DE017855 for udviklingen af en bekræftende punkt af pleje diagnostiske for HIV. Vi parlamentsarbejdet Silke Weisenburger(PEPperPRINT Heidelberg, Germany) for hendes teknisk bistand og venlige støtte. Vi takker også NYU Langone Medical Center for LI-COR Odyssey Imaging systemet.

Disse data er en del af en igangværende forskningsundersøgelse foretaget på New York University College of Dentistry og blev godkendt af det institutionelle Review Board på New York University School of Medicine, IRB # H10-01894. Kliniske prøver, der indgår i denne undersøgelse blev de identificerede prøver tidligere anvendt til diagnosticering og med tilladelse fra Wadsworth Center i New York State Department af sundhed, Albany, NY.
1. installation ZIKV High-density peptid Microarray gl...

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Vi har udviklet en protokol, der udnytter en high-density peptid microarray indeholdende hele Zika virus protein sekvens (franske polynesiske stamme). Microarray blev fremstillet af udskrivning 3,423 forskellige overlappende lineær peptider. Hver peptid blev 15 aminosyrer og varieret ved kun en rest fra nærmeste nabo i rækkefølge (dvs., 14 rester overlapning). Selv om kortere overlapninger kan printes, er epitop kortlægning mere præcise med længere overlapninger. Hver peptid blev trykt i to eksemplarer ti...

Zika virus (ZIKV) diagnose baseret på kliniske symptomer udfordrende, fordi den deler vektorer, geografiske fordeling og symptomer med denguefeber og Chikungunya-virus infektion1. I betragtning af risikoen for utilsigtede graviditet resultater i kvinder smittet med ZIKV under graviditet, er det vigtigt at skelne mellem de 3 vira. Selv om de nuværende Molekylær diagnostisk test er specifikke, er de kun nyttig i blod eller spyt i den relativt korte periode af akut infektion2,3. Serologiske analyser er afgørende for diagnosen uden for denne indledende periode af infektion4.
Udvikling af en specifik serologisk analyse ZIKV er udfordrende for to grunde: først, de Zika antigener, som det menneskelige immunsystem reagerer på i øjeblikket kendes ikke; og andet, bevares flavivira aminosyresekvenser inducerer antistof krydsreaktivitet. Vores mål var at opdage unikke ZIKV bestemte peptider anvendes i diagnostik. Forskellige metoder er blevet udviklet til skærmen peptid biblioteker dækker hele proteiner herunder phage, bakteriel, og gær overflade vise5,6,7,8,9, 10. Vores strategi var at bruge en high-density peptid microarray, der tillader hurtig og billig høj overførselshastighed serologisk screeninger11,12 og efterfølgende de identificerede peptider kan bruges til at forbedre nuværende serologiske assays til påvisning af ZIKV infektion.
Denne protokol giver mulighed for opdagelsen af Zika virus specifikke diagnostiske peptider ved hjælp af en high-density peptid microarray (figur 1). High-density peptid microarray blev produceret ved hjælp af peptid laser trykning teknologi. Hele ZIKV protein sekvens bestående af 3,423 aminosyrerester baseret på den franske polynesiske stamme (GenBank: KJ776791.2), blev trykt på et standard glas dias i blokke af 15 lineær restprodukter med et overlap på 14 aminosyrerester i to eksemplarer til en i alt 6,846 peptid steder. Ud over de hele Zika protein sekvens peptider, udnytter microarray Influenza hemagglutinin (HA) peptider for interne kontroller.
En Zika valideret positivt serum prøve fremstillet af Wadsworth Center (Albany, NY), blev brugt til at identificere specifikke Immunoglobulin M (IgM) og Immunoglobulin G (IgG) reaktive peptider. Efter inkubering med prøven natten over, var microarray farves med en sekundær fluorokrom konjugeret antistof (antihumant IgM eller -anti-human IgG), og analyseret på en microarray scanner. Kvantificering af spot Støtteintensiteter og peptid anmærkning blev udført med en specifik software leveret af samme selskab, der fremstilles af microarray.

<table>
	<tbody>
		<tr>
			<td>PEPperCHIP Custom Peptide Microarray</td>
			<td>PEPperPRINT</td>
			<td>PPC.001.001</td>
			<td>Custom peptide micarray: our microarray contains the entire Zika virus protein sequence</td>
		</tr>
		<tr>
			<td>PEPperCHIP incubation tray 3/1</td>
			<td>PEPperPRINT</td>
			<td>PPC.004.001</td>
			<td></td>
		</tr>
		<tr>
			<td>PEPperCHIP Staining Kit (680 nm) (anti-HA, DyLight labeling)</td>
			<td>PEPperPRINT</td>
			<td>PPC.037.002</td>
			<td></td>
		</tr>
		<tr>
			<td>PepSLide Analyzer</td>
			<td>PEPperPRINT</td>
			<td>PSA.004.001</td>
			<td>14-days free License for Windows</td>
		</tr>
		<tr>
			<td>Rockland Blocking buffer</td>
			<td>Rockland</td>
			<td>MB-070</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgM (mu chain) DyLight 800</td>
			<td>Rockland</td>
			<td>609-145-007</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgG Fc DyLight 680</td>
			<td>Thermo Scientific</td>
			<td>SA5-10138</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey Imaging System</td>
			<td>LI-COR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker device</td>
			<td>IKA</td>
			<td>MTS 2/4 digital microtiter shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deltagraph</td>
			<td>Redrocks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

zika virus specific diagnostic epitope discovery

High-density peptid microarrays tillade screening af mere end seks tusinde peptider på en enkelt standard mikroskopi dias. Denne metode kan anvendes for drug discovery, terapeutiske målet identifikation og udvikling af diagnostik. Vi præsenterer her, en protokol for at opdage specifikke Zika virus (ZIKV) diagnostiske peptider ved hjælp af en high-density peptid microarray. En human serum prøve valideret for ZIKV infektion var inkuberes med en high-density peptid microarray indeholdende den hele ZIKV protein oversat til 3,423 unikke 15 lineær aminosyre (aa) restprodukter med en 14-aa rester overlapning udskrevet i to eksemplarer. Farvning med forskellige sekundære antistoffer i samme array, opdaget vi peptider, der binder til Immunoglobulin M (IgM) og Immunoglobulin G (IgG) antistoffer i serum. Disse peptider blev udvalgt til yderligere validering eksperimenter. I denne protokol, vi beskriver den strategi, der følges for at designe, behandle og analysere en high-density peptid microarray.

Resultaterne ved hjælp af protokollen beskrevet er vist i figur 2 og figur 3. Ingen baggrund interaktioner blev konstateret når pre farvning microarray med sekundære antihumant IgM (data ikke vist). Farvning med en anti-menneskelige IgM resulterede konjugat i flere områder over baggrunden grønne fluorescens intensiteter, der angiver bindingen af disse peptider med IgM i serumprøven vært (<strong class="xf...

Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Zika Virus Specific Diagnostic Epitope Discovery

I denne protokol beskriver vi en teknik til at opdage Zika virus specifikke diagnostiske peptider ved hjælp af en high-density peptid microarray. Denne protokol kan readly tilpasses til andre smitsomme sygdomme.

Zika Virus specifikke diagnostiske epitop opdagelse

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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