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EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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 spotter&#160;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 corner (here, the bottom left corner was used).</li>
		<li>Incubate the spotted assay slide at room temperature for 1 h with a relative humidity of 60%.</li>
		<li>Clamp a slide module gasket with 16 compartments on the assay slide to divide it into 16 wells. Rinse the slide three times by adding 80 &#181;L of PBS containing 0.1% Tween-20 (PBST) into each well, and shaking at 450 rpm on a shaker, 5 min each time.</li>
		<li>Add 80 &#181;L of blocking solutions to each well and shake for 1 h at 450 rpm.</li>
		<li>Remove the gasket and dry the assay slide with a stream of nitrogen.</li>
		<li>Fix a transfer slide on the spotter deck and push the bottom left corner of the slide against the bottom left corner of the spotter deck. Inkjet spots an array of alignment marks (a solution of polystyrene micro-beads) with the same layout as that for the capture antibodies (cAbs).</li>
		<li>Let the alignment marks dry. Flip the transfer slide and put it back onto the spotter deck, fixing it against the bottom left corner.</li>
		<li>Prepare the detection antibody (dAb) spotting solutions containing 20 &#181;g/mL antibodies, 20% glycerol, and 1% bovine serum albumin (BSA).</li>
		<li>Using the spotter&#39;s camera, take a picture of an alignment mark. Implement this picture into the spotting program as a fiducial and get the image recognition system of the inkjet spotter to identify the top left mark. Use its coordinate as the first spot of the dAb array. Spot 8 nL per droplet with a center-to-center spacing of 800 &#181;m.</li>
	</ol>
	</li>
	<li>Double transfer method (Figure 1b)
	<ol>
		<li>Place a transfer slide 1 on the inkjet spotter deck against the bottom left corner. Spot cAbs solutions containing 400 &#181;g/mL antibodies, 20% glycerol, and 1% BSA in PBS onto the slide with an inkjet microarray spotter at a relative humidity of 60% (0.4 nL for each spot and ~ 200 &#181;m in diameter). The center-to-center spacing is 400 &#181;m.</li>
		<li>Snap the transfer slide with a nitrocellulose coated (or a functionalized glass) assay slide using a snap apparatus (Figure 2) to transfer the cAb droplets onto the assay slide.
		<ol>
			<li>Turn all six buttons on the side of the snap apparatus by 90 degrees to pull and lock all the plungers in the open position. Insert the transfer slide into the slide holder in its receptacle with the clipped corner facing the fixed pogo pin. Turn the first set of three buttons to release the plungers with the golden pogo pin and make sure the slides are pushed against the alignment pins.</li>
			<li>Insert a nitrocellulose-coated slide in the snap apparatus upside-down sitting on the 4 pogo pins. Turn the second set of three buttons to release plungers with the silver pin and make sure the slides are pushed against the alignment pins.</li>
			<li>Close the snap apparatus by placing the top shell on the slide holder using the alignment pillars and holes for a precise positioning.</li>
			<li>Insert the closed snap apparatus into its cage and push the closure tab completely down to bring the microarrays face-to-face while applying the appropriate pressure. Keep closed for 1 min.</li>
		</ol>
		</li>
		<li>Separate the slides. Incubate the assay slide at room temperature for 1 h with a relative humidity of 60%. Wash, block, and dry the assay slide as described in steps 1.1.3 - 1.1.5.</li>
		<li>Place another transfer slide on the inkjet spotter deck and push against the bottom left corner. Spot dAb solutions containing 50 or 100 &#181;g/mL of antibodies (see Table 1), 20% glycerol, and 1% BSA in PBS. Ensure that each spot is 0.8 nL and the center-to-center spacing between spots is 400 &#181;m.</li>
		<li>Store the assay and the transfer slide. Seal both assay and transfer slides in an airtight bag containing desiccant and put in a -20 &#176;C freezer.</li>
	</ol>
	</li>
</ol>
2. Multiplexed immunoassays with snap chips
<ol>
	<li>Retrieve the assay slide from the freezer. Leave the bag sealed for 30 min until the slide comes to room temperature.</li>
	<li>Prepare 7-point serial diluted sample solutions by spiking proteins in PBS containing 0.05% Tween-20.&#160;&#160;&#160;&#160; 
	NOTE: Here, a 5-fold dilution factor is used. The starting concentrations for each protein are listed in Table 1.</li>
	<li>Prepare sample solutions as appropriate. For example, dilute human serum 4 times in PBST buffer.</li>
	<li>Clamp a 16-compartment gasket on the assay slide.</li>
	<li>Fill one column of 8 wells with the 7 protein dilution solutions and a protein-free PBST buffer solution by pipetting. Pipette the sample solutions in the other 8 wells on the same slide.&#160;&#160;&#160;&#160; 
	NOTE: 80 &#181;L solutions can be fit in each well. More slides can be used to measure additional samples when necessary.</li>
	<li>Incubate the samples on a shaker at 450 rpm either for 1 h at room temperature or for overnight at 4 &#176;C. Wash the slide three times with PBST on the shaker at 450 rpm, 5 min each time. Remove the gasket and dry the slide under a stream of nitrogen gas.</li>
	<li>Retrieve the transfer slide with dAbs from the freezer. Keep the bag sealed for 30 min at room temperature. Then, incubate the slide in a closed chamber (e.g.&#160;empty tips box) containing 60% humidity stabilization beads for 20 min for rehydration.</li>
	<li>Snap the transfer slide with the assay slide using a snap apparatus (Figure 2) to transfer the dAb droplets. See section 1.2.2 for the operation of the snap apparatus.</li>
	<li>Separate the slides. Incubate the assay slide in a closed chamber containing 60% humidity stabilization beads for 1 h. Clamp the assay slide with a 16-compartment gasket and rinse the slide 4 times using PBST on a shaker at 450 rpm, 5 min each time.</li>
	<li>Pipette 80 &#181;L of solutions containing 2.5 &#181;g/mL streptavidin fluorophore in PBS into each well. Incubate for 20 min on a shaker at 450 rpm.</li>
	<li>Rinse the slide 3 times with PBST and once with distilled water on the shaker at 450 rpm. Remove the gasket and dry the slide using nitrogen gas.</li>
</ol>
3. Slide scanning and data analysis
<ol>
	<li>Scan the assay slide with a fluorescence microarray scanner using the 635-nm-laser.</li>
	<li>Extract the net intensity of each spot using an analysis software (e.g. array-pro analyzer).</li>
	<li>Calculate the limit of detection (LOD) of each protein using a statistics software and determine the protein concentrations in the samples.&#160;&#160;&#160;&#160; 
	NOTE: The LOD is defined as the Y-intercept of the standard curve incremented by three times the standard deviation of three independent assays.</li>
</ol>
Table 1.&#160;Protein concentrations and LODs in buffer from the 50-plex assay. The LOD for CA 15-3 is in U/ml (*).&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein name</td>
			<td>Starting concentration (ng/ml)</td>
			<td>LOD (pg/ml)</td>
			<td>Protein name</td>
			<td>Starting concentration (ng/ml)</td>
			<td>LOD (pg/ml)</td>
		</tr>
		<tr>
			<td>ANG2</td>
			<td>500</td>
			<td>1.3 &#215; 104</td>
			<td>IL-6b</td>
			<td>1</td>
			<td>2.1 &#215; 102</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>500</td>
			<td>1.0 &#215; 102</td>
			<td>IL-5</td>
			<td>50</td>
			<td>77</td>
		</tr>
		<tr>
			<td>CA 15-3*</td>
			<td>1</td>
			<td>4.9 &#215; 103</td>
			<td>IL-4</td>
			<td>1000</td>
			<td>1.5 &#215; 104</td>
		</tr>
		<tr>
			<td>CEA</td>
			<td>1000</td>
			<td>5.4 &#215; 103</td>
			<td>IL-2</td>
			<td>50</td>
			<td>76</td>
		</tr>
		<tr>
			<td>CXCL10/IP-10</td>
			<td>50</td>
			<td>1.7 &#215; 102</td>
			<td>LEP</td>
			<td>200</td>
			<td>4.0 &#215; 102</td>
		</tr>
		<tr>
			<td>CRP</td>
			<td>200</td>
			<td>44</td>
			<td>MIG</td>
			<td>500</td>
			<td>11 &#215; 102</td>
		</tr>
		<tr>
			<td>ENG</td>
			<td>1000</td>
			<td>6.2 &#215; 102</td>
			<td>CCL3/MIP-1&#945;</td>
			<td>50</td>
			<td>3.3</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>50</td>
			<td>1.8 &#215; 102</td>
			<td>CCL4/MIP-1&#946;</td>
			<td>50</td>
			<td>12</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>200</td>
			<td>1.9 &#215; 102</td>
			<td>MMP-3</td>
			<td>500</td>
			<td>1.0 &#215; 102</td>
		</tr>
		<tr>
			<td>FAS-L</td>
			<td>500</td>
			<td>4.5 &#215; 102</td>
			<td>M-CSF</td>
			<td>500</td>
			<td>8.2 &#215; 103</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>500</td>
			<td>1.0 &#215; 103</td>
			<td>MMP-9</td>
			<td>200</td>
			<td>3.1 &#215; 102</td>
		</tr>
		<tr>
			<td>G-CSF</td>
			<td>500</td>
			<td>39</td>
			<td>CCL2/MCP-1</td>
			<td>50</td>
			<td>55</td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>50</td>
			<td>3.8</td>
			<td>NCAM-1</td>
			<td>500</td>
			<td>1.7 &#215; 103</td>
		</tr>
		<tr>
			<td>GRO-&#945;</td>
			<td>50</td>
			<td>3.0 &#215; 102</td>
			<td>&#946;-NGF</td>
			<td>200</td>
			<td>1.4 &#215; 103</td>
		</tr>
		<tr>
			<td>HER2</td>
			<td>500</td>
			<td>3.5 &#215; 103</td>
			<td>NT-3</td>
			<td>200</td>
			<td>5.8 &#215; 102</td>
		</tr>
		<tr>
			<td>PDGF-BB</td>
			<td>200</td>
			<td>73</td>
			<td>OPN</td>
			<td>500</td>
			<td>1.9 &#215; 103</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>500</td>
			<td>7.9 &#215; 102</td>
			<td>RBP4</td>
			<td>200</td>
			<td>1.2 &#215;102</td>
		</tr>
		<tr>
			<td>IL-1ra</td>
			<td>200</td>
			<td>1.1 &#215; 103</td>
			<td>SPARC</td>
			<td>1000</td>
			<td>4.6 &#215;104</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>50</td>
			<td>8.2 &#215; 102</td>
			<td>TNF-&#945;</td>
			<td>50</td>
			<td>4.4</td>
		</tr>
		<tr>
			<td>IL-12</td>
			<td>1</td>
			<td>30</td>
			<td>TNF-RI</td>
			<td>50</td>
			<td>1.3 &#215; 102</td>
		</tr>
		<tr>
			<td>IL-11</td>
			<td>500</td>
			<td>1.2 &#215; 103</td>
			<td>TNF-RII</td>
			<td>50</td>
			<td>12</td>
		</tr>
		<tr>
			<td>IL-10</td>
			<td>500</td>
			<td>6.1 &#215;102</td>
			<td>FAS/TNFRSF6</td>
			<td>200</td>
			<td>2.8 &#215;102</td>
		</tr>
		<tr>
			<td>IL-8</td>
			<td>50</td>
			<td>6.6</td>
			<td>uPA</td>
			<td>200</td>
			<td>24</td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>2.5</td>
			<td>26</td>
			<td>uPAR(CD87)</td>
			<td>200</td>
			<td>76</td>
		</tr>
		<tr>
			<td>IL-6a</td>
			<td>1000</td>
			<td>84</td>
			<td>VEGF</td>
			<td>200</td>
			<td>6.7 &#215; 102</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>

a snap chip technology for a cross-reactivity-free multiplexed sandwich immunoassay

Source: Li, H. et al., <a href="https://app.jove.com/v/56230">Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays.</a>&#160;J. Vis. Exp. (2017)
The video showcases the implementation of snap chip technology for cross-reactivity-free multiplexed sandwich immunoassays. The simple action of snapping two microarray slides, each containing different reagents, ensures reagent transfer without cross-contamination and helps to detect multiple proteins using sandwich immunoassay.

<img alt="Figure 1" src="/files/ftp_upload/22137/22137_Figure1.jpg" /> 
Figure 1. Schematic of assay procedure comparing (a) single and (b) double transfer methods.
<img alt="Figure 2" src="/files/ftp_upload/22137/22137_Figure2.jpg" /> 
Figure 2. Schematic showing differences between single- and double-transfer.&#160;(a) mirroring of the transfer reagents amplifies the angular misalign...

A Snap Chip Technology for a Cross-Reactivity-Free Multiplexed Sandwich Immunoassay

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Fabrication and storage of snap ...

Begin by assembling a pre-treated microarray assay slide with distinct spots containing protein-specific capture antibodies for multiple protein detection.
Load the dilutions of the protein standard and the pre-diluted serum samples into separate wells. These dilutions create a range of protein concentrations for accurate quantification.
Incubate to facilitate protein-antibody interactions. Wash to remove unbound proteins, disassemble, and dry the slide.
Invert the assay slide in a snap apparatus containing a rehydrated transfer slide spotted with protein-specific biotinylated detection antibodies in the same positions as the assay slide.
Close the apparatus and press to snap the slides over each other. This facilitates the transfer of the detection antibodies to corresponding spots on the assay slide, minimizing the cross-reactivity.
Separate the slides to allow the interaction of detection antibodies with bound proteins, forming immunocomplexes.
Reassemble the assay slide, rinse it, and introduce fluorophore-conjugated streptavidin, interacting with biotinylated antibodies.
Using a scanner, detect fluorescence spots at various locations, indicating multiple proteins in the serum sample. Compare the spot intensities with the standard for protein quantification.

a-snap-chip-technology-for-cross-reactivity-free-multiplexed-sandwich

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

38 Views. Źródło: Li, H. et al., Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoastests.J. Vis. Exp. (2017) Film prezentuje implementację technologii chipów snap do wielowarstwowych testów immunologicznych bez reaktywności krzyżowej. Prosta czynność polegająca na zatrzaśnięciu dwóch szkiełek mikromacierzy, z których każdy zawiera inne odczynniki, zapewnia transfer odczynnika bez zanieczyszczenia krzyżowego i pomaga w wykrywaniu wielu białek za pomocą t...

Video: Technologia Snap Chip do wielowarstwowego testu immunologicznego bez reaktywności krzyżowej - Concept

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

JoVE Journal

Immunology and Infection

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

A Snap Chip Technology for a Cross-Reactivity-Free Multiplexed Sandwich Immunoassay

Transkrypcja

A Snap Chip Technology for a Cross-Reactivity-Free Multiplexed Sandwich Immunoassay