Name: snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays
Uploaded: 2017-11-13T12:30:00
Description: Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Vi takker Dr. Rob Sladek til brug i inkjet spotter. Vi anerkender den endelige støtte fra den canadiske institutter for sundhed Research (CIHR), naturvidenskabelige og Engineering Research Rådet i Canada (NSERC), canadiske kræft samfund Research Institute og Canada Foundation for Innovation (CFI). D.J. tak støtte fra en Canada forskning stol.

1. fabrikation og opbevaring af snap chips <ol><li>enkelt overførselsmetode ( figur 1a) <ol><li>Spot cAb opløsninger indeholdende 400 µg/mL antistoffer og 20% glycerol i fosfatbufferet saltopløsning (PBS) på en nitrocellulose (eller en functionalized glas) assay dias med en inkjet microarray spotter 13 ved en relativ luftfugtighed på 60% (1,2 nL for hver plet) med 800 µm center til center afstand. Sørg for, at diasset er fast på spotter dækket efter et h...

<ol>
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I dette arbejde har vi præsenteret en snap chip-teknologi, der gør de gratis Cross-Country reactivity multiplex immunassays bredt tilgængelige for forskere med eksperimentelle grundopsætning. Adskiller sig fra eksisterende antistof microarrays, ingen microarray spotter er nødvendig for slutbrugere. Både enkelt og dobbelt overførselsmetoder er påvist, og dobbelt overførsel giver overlegen justering nøjagtighed ned til ~ 40 μm for 98% steder, med den største fejljustering af 63 µm14. En...

Et panel af biomarkører bestående af flere proteiner kan give højere følsomhed og specificitet end en enkelt biomarkør i diagnosticering af komplekse sygdomme såsom kræft1,2. Enzym-forbundet immunosorbent assay (ELISA) har været guldstandarden teknologi anvendes i kliniske laboratorier at nå en grænse på opdagelse på lav pg/mL i plasma, men grænser for ét mål pr. assay3,4,5. Antistof microarrays er blevet udviklet for imødekommende tusindvis af miniaturized assays gennemført parallelt på en enkelt mikroskop dias6,7,8. Men den multiplexing kapacitet af denne metode er begrænset af reagens-drevet krydsreaktivitet, der følger af anvendelsen af en blanding af ising, og bliver det mere problematisk med et stigende antal mål9,10 , 11. Pla mfl. har erklæret, at den resulterende sårbarhed af en multiplex sandwich assay skalaer som 4N(N-1), hvor N er antallet af mål12.
For at afbøde krydsreaktivitet i antistof microarrays, antistof colocalization microarray er (ACM) blevet udviklet i vores laboratorium for multiplex sandwich assay12. Capture antistoffer (førerhuse) er spottet på et underlag med et microarray spotter. Efter blokering prøver på overfladen, og derefter individuelle klatter er plettet på de samme steder med cAb-antigen kompleks. Alle krydsreaktivitet scenarier mellem antistoffer og antigener kan afbødes med ACM, og påvisningsgrænser ved pg/mL er blevet nået. Men assay protokollen kræver forberedelse og spotting klatterne under eksperimenter ved hjælp af en on-site microarray spotter med høj præcision for justering formål, som er dyrt og tidskrævende, begrænse den brede anvendelse af denne teknologi i andre laboratorier. En håndholdt ACM, opkaldt snap chip er blevet udviklet Cross-Country-reactivity-fri og spotter-fri multiplex sandwich immunassays13,14,15. cAbs og klatter er pre spottet på en analyse og en overførsel dias henholdsvis i microarray format og gemt. Under analysen, dias er hentet og et microarray af klatter overføres kollektivt diaset assay som blot snapping de to chips sammen. En snap apparatet bruges til pålidelige reagens overførsel. Nitrocellulose belagt dias med en relativt stor antistof bindende kapacitet har været brugt som assay dias til at absorbere flydende dråber og dermed fremme reagens overførsel, imidlertid diasene er dyrere end almindelige glas lysbilleder og microarray scannere kompatibel med uigennemsigtige dias er nødvendige for signal erhvervelse.
I dette arbejde viser vi udfører en multiplex sandwich immunassay med en snap chip-protokollen. En roman snap apparater er blevet udviklet for mere praktisk og pålidelig reagens overførsel fra microarray til microarray. Vigtigere, har her vi etableret reagens overførselsmetode på regelmæssig glas dias med snap chip. 1024 steder blev overført og tilpasset til et glas dias, væsentlig udvidelse af brugen af denne teknologi i de fleste laboratorier.

<table>
	<tbody>
		<tr>
			<td>Phosphate buffered saline tablet</td>
			<td>Fisher Scientific</td>
			<td>5246501EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-conjugated Cy5</td>
			<td>Rockland</td>
			<td>s000-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>p1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer</td>
			<td>SurModics, Inc</td>
			<td>SG02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose coated slides</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>305116</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane coated slides</td>
			<td>Schott North America</td>
			<td>1064875</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Device</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-SD01</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkjet microarray spotter</td>
			<td>GeSiM</td>
			<td>Nanoplotter 2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide module gasket</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>204862</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Stabilization Beads</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-HU60</td>
			<td></td>
		</tr>
		<tr>
			<td>Array-Pro Analyzer software</td>
			<td>Media Cybernetics</td>
			<td>Version 4.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microarray scanner</td>
			<td>Agilent</td>
			<td>SureScan Microarray Scanner</td>
			<td></td>
		</tr>
		<tr>
			<td>Biostatistics software</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB10972</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin protein</td>
			<td>R&#38;D Systems</td>
			<td>1097-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF1097</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6a (see Table 1)</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6b (see Table 1)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays

Multiplex proteinanalyse har vist superior diagnostiske følsomhed og nøjagtighed i forhold til enkelt proteiner. Antistof microarrays mulighed for tusindvis af mikro-skala immunassays udføres samtidigt på en enkelt chip. Sandwich assay format forbedrer assay specificitet ved at opdage hver målet med to antistoffer, men lider af krydsreaktivitet mellem reagenser, hvilket begrænser deres multiplexing kapaciteter. Antistof colocalization microarray (ACM) er blevet udviklet til Cross-Country-reactivity-fri multipleksede protein påvisning, men kræver et dyrt spotter på stedet for microarray fabrikation under assays. I dette arbejde udviser vi en snap chip-teknologi, som overfører reagens fra microarray til microarray af blot snapping to chips sammen, således ingen spotter kræves under prøven inkubation og efterfølgende anvendelse af påvisning antistoffer (klatter) på opbevaring af pre plettede dias, skaber dias forberedelse fra assay udførelse. Både enkelt og dobbelt overførselsmetoder præsenteres for at opnå præcise tilpasning mellem de to microarrays og dias fabrikation for begge metoder er beskrevet. Resultaterne viser, at &#60; 40 μm justering er opnået med dobbelt overførsel, at nå en array tæthed af 625 steder/cm2. En 50-plexed immunoassay har gennemført for at påvise anvendeligheden af snap chip i multipleksede proteinanalyse. Grænserne for påvisning af 35 proteiner er i den vifte af pg/mL.

Analyseprocedure for både enkelt og dobbelt overførselsmetoder er vist i figur 1. I enkelt overførsel, førerhuse er spottet direkte på diasset assay og klatterne overføres diaset assay ved brug i et spejlet mønster af cAbs (figur 1a). Kun én overførsel procedure er påkrævet, men denne metode lider af misforholdet mellem de to microarrays, hovedsageligt forårsaget af den kantede misforholdet mellem diasset og inkjet ga...

Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Vi demonstrere en snap chip teknologi til at udføre Cross-Country-reactivity-fri multipleksede sandwich immunassays ved blot snapping to dias. En snap apparatet bruges til at pålideligt overføre reagenser fra microarray til microarray. Snap chip kan bruges til enhver biokemiske reaktioner, der kræver colocalization af forskellige reagenser uden kontaminering.

Fastgør Chip for Cross-Country-reactivity-fri og Spotter multipleksede Sandwich immunassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

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