A Multiplex Bead-Based Assay for Analyzing Cytokines in Human Tear Samples

Add human tear samples and an antibody-bead cocktail into a washed assay plate.

The antibodies are bound to color-coded magnetic beads, impregnated with varying proportions of fluorophores corresponding to distinct spectral signatures, facilitating multiple cytokine detection.

The tear cytokines bind to their corresponding bead-bound capture antibodies.

Using a magnetic plate washer, settle the antibody-bead complexes. Using buffer, remove the unbound biomolecules.

Add biotinylated detection antibodies that attach to the bound cytokines.

Pipette streptavidin-phycoerythrin conjugate, which binds to the biotinylated antibodies.

Using the plate washer, remove the unbound well contents. Resuspend the cytokine-bound complexes in sheath fluid.

During sample analysis, the fluidic system transports the bead complexes to the optics assembly.

The first laser activates the internal bead dyes, with the resulting signal identifying specific cytokines.

The second laser activates the reporter molecule bound to the bead complex, whose signal intensity represents the cytokine concentration, enabling simultaneous quantification of multiple cytokines.

To carry out the bead-based multiplex assay, bring all reagents to room temperature, and vortex them for five to 10 seconds. If eluted tear samples were stored at minus 80 degrees Celsius prior to the assay, thaw the frozen tear extracts on ice, and centrifuge the samples at 1,000 times g for five minutes. Prepare an assay worksheet in a vertical configuration for working human cytokine standards QC-1, QC-2, and samples.

Add 200 microliters of 1x wash buffer to each well of the plate, and seal it with a plate sealer. Then, incubate the plate on a plate shaker at room temperature for 10 minutes. Next, decant the 1x wash buffer by inverting the plate, and tap it onto absorbent towels several times to remove any residual amount of wash buffer in the wells.

Then, add 25 microliters of each working human cytokine standard QC-1, QC-2, and the blank with just assay buffer and samples into the appropriate wells. Add 25 microliters of assay buffer into each well. Then to the wells, add 25 microliters of 1x antibody bead cocktail solution.

As the antibody bead solution is light sensitive, seal the plate, and cover it with aluminum foil and plate cover to protect from light during the assay. Incubate the plate at four degrees Celsius on a shaker overnight. The following day, place the plate on the plate rack of an automatic magnetic plate washer, and let it sit for one minute to settle the magnetic beads at the bottom of the well.

Aspirate the well contents, and add 200 microliters of wash buffer per well using automatic magnetic plate washer. Let the plate sit for one minute, and then, aspirate the well contents as before, and repeat the wash one more time. Add 25 microliters of detection antibodies solution into each well. Seal the plate, cover it with aluminum foil and plate cover, and incubate at room temperature on a shaker for 60 minutes. Then, add 25 microliters of streptavidin-phycoerythrin solution into each well, and after sealing and covering the plate, incubate at room temperature on a shaker for 30 minutes.

Following the incubation, place the plate on a magnetic plate washer, and let it sit for one minute. Then, aspirate the well contents and add 200 microliters of wash buffer per well. Let it sit for one minute, then, aspirate the well contents before repeating the wash once again. Add 150 microliters of sheath fluid to each well. Then, place the plate on a shaker for five minutes at room temperature to resuspend the antibody beads before proceeding to read the plate and analyze the cytokine concentrations.