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

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.