Sandwich ELISA analysis of human IL-1 alpha was performed using a Human IL-1 alpha Colorimetric ELISA Kit (Product # EH2IL1A) by loading 50 µL per well of Human IL-1 alpha Recombinant Protein (Product # SIL1A) in dodecuplicate at 400, 160, 64, 25.6, 10.25 and 0 pg/mL across a 3 µg/mL mouse anti-human IL-1 alpha (Product # M421A) pre-coated plate and incubating for 1 hour at room temperature. The plate was washed, then incubated with 50 µL per well of a biotinylated-mouse anti-human IL-1 alpha (Product # M420AB) in dodecuplicate at 0.15 µg/mL for 1 hour at room temperature. The plate was washed and incubated with 100 µL per well of Ultra Streptavidin-HRP (Product # N504) in all test wells at 1:10 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was then stopped with 0.16M sulfuric acid. Absorbances were read on a spectrophotometer at 450-550 nm.