AlphaLISA assay with conjugated beads

Samples were diluted in HBSS for optimal linearity for each antibody pair. For human samples, 300 cells/well were used for SOD-1 and 3000 cells/well for β-tubulin. The PDX-samples were diluted 10 x for tubulin and 500 times for SOD1. 3 µl of each sample was loaded in AlphaPlate (Perkin Elmer 6008350), in duplicate when possible. 2 µl biotinylated antibody was added to each well and the plate were then briefly spun down before addition of Acceptor beads. For both β-tubulin and SOD-1 the biotinylated antibody (biotin-AF3418 and biotin-ab6046 respectively) were used at a final concentration of 3 nM.

Acceptor breads (2 µl) were added and the plate was spun briefly and incubated on a shaker for 1 h at 60 rpm. For SOD-1, Acceptor beads were diluted to 45 µg/ml for a final concentration of 10 µg/ml and for β-tubulin T5201-conjugated Acceptor beads were used at a concentration of 180 µg to get at final concentration of 40 µg/ml. Then 2 µl Donor beads (Perkin Elmer 6760002S) was added at a concentration of 180 µg/ml for get at final concentration of 40 µg/ml. Handling of beads was only done in a room with green light. Plates were sealed with sticky film, covered in foil, briefly spun down, and incubated in the dark over night before being read in an EnSpire 2300 (Perkin Elmer).