Multiplexed Cytokines

Assay Principle

The multiplex cytokine assay format differs from conventional ELISA in one significant way— the multiplex capture antibody is attached to a bead whereas the ELISA capture antibody is attached to the microplate well.

The use of the suspension bead-based technology enables the multiplexing capabilities of the Luminex® assays. The technology uses 5.6 micron polystyrene or magnetic microspheres, which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another. Beads covalently bound to different antibodies can be mixed in the same assay, utilizing a 96-well microplate format.

At the completion of the sandwich immunoassay, beads can be read, using the Bio-Plex Array Reader (Bio-Rad Laboratories Inc., Hercules, CA) detection system, in single-file by dual lasers for classification and quantification of each analyte.

How multi-plex cytokine assays work

(from Invitrogen's website)


DartLab Luminex assay request form

Click here to download request form.

 

DartLab Protocols

BioPlex protocol

MilliPlex protocol

Millipore human 42plex measures:

EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNF-α, TNF-β, VEGF, sCD40L

 

Millipore mouse 32plex measures:

Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC-like, LIF, LIX, M-CSF, MCP-1, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, VEGF

Examples

Polar plot showing the concentration of 42 cytokines (pg/ml; log scale) measured in human plasma using a Milliplex kit.

 

Methods (for publication)

Multiplex cytokine assays. Cytokines were measured using Millipore human cytokine multiplex kits (EMD Millipore.Corporation, Billerica, MA). Calibration curves from recombinant cytokine standards were prepared with threefold dilution steps in the same matrix as the samples.  High and low spikes (supernatants from stimulated human PBMCs and dendritic cells) were included to determine cytokine recovery.  Standards and spikes were measured in triplicate, samples were measured once, and blank values were subtracted from all readings. All assays were carried out directly in a 96-well filtration plate (Millipore, Billerica, MA) at room temperature and protected from light. Briefly, wells were pre-wet with 100 μl PBS containing 1% BSA, then beads together with a standard, sample, spikes, or blank were added in a final volume of 100 μl, and incubated together at room temperature for 30 min with continuous shaking. Beads were washed three times with 100 μl PBS containing 1% BSA and 0.05% Tween 20. A cocktail of biotinylated antibodies (50 μl/well) was added to beads for a further 30 min incubation with continuous shaking. Beads were washed three times, then streptavidin-PE was added for 10 min. Beads were again washed three times and resuspended in 125 μl of PBS containing 1% BSA and 0.05% Tween 20. The fluorescence intensity of the beads was measured in using the Bio-Plex array reader. Bio-Plex Manager software with five-parametric-curve fitting was used for data analysis.

How to acknowledge Shared Resources

 

Links

Challenges in comparing results to those of traditional methods

Multiplex cytokine assay handbook - includes sample preparation

Selection of standards

Principles of curve fitting

Parameter logistics

Bio-Plex magnetic bead assay validation

Luminex extracellular assay protocol (from Invitrogen)

Assay performance of Bio-Plex human multiplex cytokine assay kits