Figure 3.

Representative schematic of the AlphaLISA assay system

A total of 8 μL of acceptor bead solution, containing biotinylated antibodies as well as acceptor beads, were added to each well containing 2 μL of supernatant. After a 1 h incubation, a 10 μL solution of Streptavidin donor beads were added to this solution and incubated for 30 min. Both beads are manufactured to be specific to a binding site for the cytokine of interest. When read on the EnVision Plate Reader, bound donor and acceptor beads, due to their proximity, will emit a 615 nm luminescence when excited by a 680 nm wavelength. The intensity of this luminescence reflects the concentration of bound beads or the concentration of the cytokine.