Luminex HLA antibody assay principle. The principle of the Luminex-based HLA antibody assay is depicted. (1) Bead coated with HLA molecules. Up to 100 different sets of color-coded beads, each bearing 1 or several HLA types can be tested simultaneously. Each bead set is internally dyed with differing ratios of 2 fluorochromes resulting in a unique signal. (2) After incubation with the test serum, the HLA antibody, if present, binds to the appropriate HLA molecule. Non-HLA antibodies are discarded after washing. (3) The bound HLA antibody is detected by a phycoerythrin-conjugated secondary antibody specific for human IgG. (4) The beads are analyzed on a dual-laser flow-based detection instrument. The red laser classifies the bead and determines the HLA molecule that is being detected. The green laser determines the magnitude of the phycoerythrin-derived signal, which is proportional to the amount of HLA antibody bound.