eoe sub articles

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
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	<li>Single transfer method (Figure 1a)
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		<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...

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

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

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