ELISA validation

Briefly, standards were prepared and samples were diluted as determined by an optimization step performed previously, then 100 µL of these were added to the appropriate wells of the ELISA plate and incubated at 37°C for 2hs. Following aspiration of the samples and standards, 100 µL of biotin conjugated antibody was added and incubated for a further 1 h at 37°C. Wells were washed with the provided Wash Buffer, then HRP-avidin detection reagent was added and incubated at 37°C for 30 minutes (USCN kits) or 1h (Cusabio kits and VWA5B2 My Biosource kit). Detection reagent was then removed and the wells were washed again with Wash Buffer before adding substrate solution and incubation for another 15-30 minutes at 37°C. A stop solution was then added and the optical density of each well was read at 450 nm.

The TSR1 My Biosource kit procedure was different in that 50 µL standard and sample were used and were added to the plate followed by the addition of 100 µL of HRP-conjugate followed by a 60 minute incubation at 37°C. After four washes with the wash buffer, 50 µL each of Chromogen solution A and 50 µL of Chromogen solution B were added to the wells and this mixture was incubated at 37°C for 15 minutes. Finally, 50 µL of stop solution was added prior to the plate being read at 450 nm ²⁰.