Samples and reagents were thawed to room temperature and used according to manufacture protocol. Samples were diluted to 1:2 with assay buffer included in kit. Briefly, a provided lyophilized standard was reconstituted, diluted in serial fashion to create standard curves for each of the kit analytes. The standards provided quality control samples (reconstituted) and experiment samples were then added to a flat bottom 96 well plate included in the kit. Biotinylated detection antibodies were added, followed by the addition of the mixed magnetic beads. The plate was then sealed for overnight incubation at room temperature on orbital shaker (700rpm) protected from light. The next day, the plate was washed using a handheld magnet (EMD Millipore Catalog #40–285) to retain the magnetic beads during decanting. Next, Streptavidin-PE conjugate, a reporter molecule, was added to each well and incubated on a shaker at room temperature for 30min to allow each bead to be individually identified and quantified based on fluorescent signals. After an additional round of plate washing, a final volume of wash buffer was added to the plate in place of
