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Multiplex Analysis of Eicosanoids and Inflammatory Cytokines by Flow Cytometry
Sponsored,vendor-submitted protocol   Sponsored by Assay Designs, Inc.    Published in BioTechniques Protocol Guide 2009 (p.8) DOI: 10.2144/000112995 Abstract

Replacement of traditional ELISA with multiplex immunoassay arrays makes the simultaneous measurement of multiple analytes possible while preserving precious sample, resulting in the more cost-effective assessment of biomarkers. The Inflammation (human) 8-Plex MultiBead™ Panel from Assay Designs and corresponding singleplex kits enable the measurement of eicosanoids (PGE2 and TXB2) and inflammatory cytokines (IL-4, IL-6, IL-8, IL-1β, IFN-γ, and TNF-α) in a variety of matrices including tissue culture media, serum, plasma, and urine. The MultiBead platform is the first to facilitate detection of both large and small analytes in simultaneous immunometric and competitive immunoassays, with the added benefit of compatibility with commercially available dual-laser flow cytometers.

Replacement of traditional ELISA with multiplex immunoassay arrays makes the simultaneous measurement of multiple analytes possible while preserving precious sample, resulting in the more cost-effective assessment of biomarkers. The Inflammation (human) 8-Plex MultiBead™ Panel from Assay Designs and corresponding singleplex kits enable the measurement of eicosanoids (PGE2 and TXB2) and inflammatory cytokines (IL-4, IL-6, IL-8, IL-1β, IFN-γ, and TNF-α) in a variety of matrices including tissue culture media, serum, plasma, and urine. The MultiBead platform is the first to facilitate detection of both large and small analytes in simultaneous immunometric and competitive immunoassays, with the added benefit of compatibility with commercially available dual-laser flow cytometers.

Principle

MultiBead assay technology utilizes the ability of standard dual-laser flow cytometers to separate latex beads covalently coupled to antibodies on the basis of size and fluorescent intensity. MultiBead assays can simultaneously quantify up to 22 analytes in a single well by utilizing two sizes of beads (4.0 and 5.4 µm) and 11 different bead intensities. Each bead size is sub-fractioned into different populations based on their inherent fluorescent intensity by a 670 nm filter, while the analytes of interest are simultaneously quantified by a 575 nm filter that measures fluorescence associated with phycoerythrin (PE) conjugated detection antibody or competitive analyte. Materials

The Inflammation (human) 8-Plex MultiBead Panel (Catalog #980-001) contains the following components, sufficient for 96-tests.

Inflammation Human 8-Plex Capture Bead Cocktail

Extracellular Assay Buffer 1

Wash Buffer Concentrate

Standard Cocktail 1

Standard Cocktail 2

Eicosanoid-PE Cocktail

Cytokine Antibody-PE Cocktail

Setup Beads

96 Well Filter Plate

Plate Sealers

MultiBead Analysis Software (free download)Protocol Outline (Figure 1):

  1. Antibody coated beads, sample or standard, and eicosanoid-PE conjugates are added to wells of a microtiter filter plate. The plate is then incubated at room temperature for 1 hour with shaking.

  2. The plate is then washed 3X, leaving only bead-bound cytokines, eicosanoids, or eicosanoid-PE conjugate in the well. A solution of cytokine specific antibody-PE conjugates is then added, binding the cytokine captured on each analyte specific bead. The plate is then incubated at room temperature for 1 hour with shaking.

  3. Following incubation, the plate is washed 3X to remove excess antibody-PE conjugate. The beads are then re-suspended in wash buffer and analyzed on a flow cytometer. The amount of signal associated with each analyte specific bead is directly proportional to the level of each cytokine and inversely proportional to the level of each eicosanoid (Figure 2).

  4. The corresponding FCS or LMD data files are imported into the complimentary MultiBead Analysis Software for standard curve generation and data analysis.


Figure 1. Protocol Outline (Click to enlarge)



Figure 2. Bead Analysis (Click to enlarge)


Results

The concentration of IL-8 was quantified in human serum, plasma, and EDTA plasma sample matrices using the Assay Designs MultiBead IL-8 Singleplex Kit (Cat. #985-004). The results are shown in Figure 3 compared to values derived from a solid-phase IL-8 ELISA kit from another vendor. The MultiBead assay produces comparable results while offering improved sensitivity and dynamic range. Similar results were seen using the Inflammation (human) 8-Plex Panel (Cat. #980-001).


Figure 3. MultiBead Assay Comparison in Various Matrices (Click to enlarge)


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