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

We thank Dr. Rob Sladek for the use of the inkjet spotter. We acknowledge the final support from the Canadian Institutes for Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC), the Canadian Cancers Society Research Institute and the Canada Foundation for Innovation (CFI). D.J. thanks support from a Canada Research Chair.

1. Fabrication and storage of snap chips
<ol>
	<li>Single transfer method (Figure 1a)
	<ol>
		<li>Spot cAb solutions containing 400 &#181;g/mL antibodies and 20% glycerol in phosphate-buffered saline (PBS) onto a nitrocellulose (or a functionalized glass) assay slide with an inkjet microarray spotter13 at a relative humidity of 60% (1.2 nL for each spot) with 800 &#181;m center-to-center spacing. Make sure the slide is fixed on the spotter deck according to one corn...

<ol>
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In this work, we have presented a snap chip technology that makes the cross-reactivity-free multiplex immunoassays widely available for the researchers with basic experimental setup. Different from existing antibody microarrays, no microarray spotter is needed for end-users. Both single and double transfer methods are demonstrated, and double transfer affords superior alignment accuracy down to ~ 40 &#956;m for 98% spots, with the largest misalignment of 63 &#181;m14. A novel snap apparatus was de...

A panel of biomarkers comprising multiple proteins may provide higher sensitivity and specificity than a single biomarker in the diagnosis of complex diseases such as cancers1,2. The enzyme-linked immunosorbent assay (ELISA) has been the gold standard technology used in clinical laboratories achieving a limit of detection at low pg/mL in plasma, but limits to one target per assay3,4,5. Antibody microarrays have been developed for accommodating thousands of miniaturized assays conducted in parallel on a single microscope slide6,7,8. However, the multiplexing capability of this method is limited by reagent-driven cross-reactivity, arising from the application of a mixture of dAbs, and it becomes more problematic with an increasing number of targets9,10,11. Pla et al. have stated that the resulting vulnerability of a multiplex sandwich assay scales as 4N(N-1) where N is the number of the targets12.
To mitigate cross-reactivity in antibody microarrays, antibody colocalization microarray (ACM) has been developed in our laboratory for multiplex sandwich assay12. Capture antibodies (cAbs) are spotted on a substrate with a microarray spotter. After blocking samples are applied on the surface, and then individual dAbs are spotted on the same spots with the cAb-antigen complex. All cross-reactivity scenarios between antibodies and antigens can be mitigated with ACM, and limits of detection at pg/mL have been achieved. However, the assay protocol requires preparing and spotting the dAbs during the experiments using an on-site microarray spotter with high precision for alignment purpose, which is expensive and time consuming, limiting the wide application of this technology in other laboratories. A handheld ACM, named snap chip has been developed for cross-reactivity-free and spotter-free multiplex sandwich immunoassays13,14,15. cAbs and dAbs are pre-spotted onto an assay slide and a transfer slide respectively in microarray format and stored. During the assay, the slides are retrieved and a microarray of dAbs are transferred collectively onto the assay slide by simply snapping the two chips together. A snap apparatus is used for reliable reagent transfer. Nitrocellulose coated slides with a relatively large antibody binding capacity have been used as the assay slides to absorb the liquid droplets and thus facilitating reagent transfer, however, the slides are more expensive than regular glass slides and microarray scanners compatible with non-transparent slides are needed for signal acquisition.
In this work, we demonstrate the protocol of performing a multiplex sandwich immunoassay with a snap chip. A novel snap apparatus has been developed for more convenient and reliable reagent transfer from microarray-to-microarray. Importantly, here we have established the reagent transfer method onto regular glass slides with the snap chip. 1024 spots were successfully transferred and aligned onto a glass slide, significantly expanding the use of this technology in most laboratories.

<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

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

The assay procedure for both single and double transfer methods is shown in Figure 1. In single transfer, the cAbs are spotted directly on the assay slide and the dAbs are transferred onto the assay slide upon use in a mirror pattern of the cAbs (Figure 1a). Only one transfer procedure is required, but this method suffers from misalignment between the two microarrays, mainly caused by the angular misalignment between the slide an...

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

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

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

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