A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Take a multiwell plate with filters at the base.

Add a test sample containing different cytokines.

Add different sets of microspheres differentiated by size and internal fluorescence intensity.

Each set is conjugated to an antibody targeting a specific cytokine.

Incubate the plate in the dark under agitation.

The antibodies bind to their target cytokines, immobilizing them on the microspheres.

Apply a vacuum to remove the unbound cytokines. Microsphere-bound cytokines are retained due to the smaller pore size of the membrane.

Add a cocktail of biotin-conjugated detection antibodies that bind to the microsphere-bound cytokines.

Add a fluorescent reporter-conjugated streptavidin and incubate in the dark under agitation. Streptavidin binds to biotin, immobilizing the reporter on the microsphere complex.

Remove unbound molecules and resuspend the microspheres.

Using flow cytometry, identify the bound cytokines by determining the size and internal fluorescence of the microspheres.

To determine the amount of a specific cytokine, measure the reporter's fluorescence intensity.

To perform the assay, allow all reagents to warm to room temperature before use.

Polypropylene filter plates are used in this demonstration. It is absolutely essential that the plate be kept upright during the entire assay procedure, including the washing steps, to avoid losing the beads.

Pre-wet the filter paper by adding 100 microliters of 1x wash buffer to each well, and let the plate sit for 1 minute at room temperature. Remove the buffer volume by using the vacuum manifold. Blot excess wash buffer from the bottom of the plate by pressing the plate on a stack of clean paper towels. Then place the plate on top of the inverted plate cover.

For cell culture supernatant samples, add 25 microliters of assay buffer to all wells. Add 25 microliters of each standard to the standard wells. Finally, add 25 microliters of each sample to the sample wells. Vortex the mixed beads for 30 seconds. Then, add 25 microliters of the mixed beads to each well, shaking the bead bottle intermittently to avoid beads' settling.

Seal the plate with a plate sealer. Once sealed, wrap the entire plate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker, secure it, and shake at approximately 500 RPM for two hours at room temperature. Without inverting, place the plate on the vacuum manifold and apply the vacuum as before. Then, add 200 microliters of 1x wash buffer to each well.

Remove the contents of the assay plate wells by vacuum filtration. Blot excess wash buffer from the bottom of the plate with an absorbent pad or paper towels before repeating this step one more time. Next, add 25 microliters of detection antibodies to each well. After sealing the plate with a fresh plate sealer, wrap the entire plate, including the inverted plate cover, with aluminum foil.

Place the plate on a plate shaker and shake at approximately 500 RPM for 1 hour at room temperature. Without vacuuming, add 25 microliters of streptavidin phycoerythrin reagent directly to each well. Seal and wrap the plate as before. Then. place the plate on a plate shaker and shake at approximately 500 RPM for 30 minutes at room temperature.

After repeating the vacuum filtration step twice, add 200 microliters of 1x wash buffer to each well. Resuspend the beads on a plate shaker for 1 minute. Using a multichannel pipette, transfer the samples from the filter plate to FACS tubes in order to read the samples on a flow cytometer.