Indirect ELISA analysis of Digoxin was performed by preparing a 10 µg/mL solution of BSA or Digoxin-labeled BSA in 0.2M Carbonate-Bicarbonate, pH 9.4. 100uL of each solution was added to separate wells of a clear 96-well plate, and incubated overnight at 4C. Each well was blocked with SuperBlock in PBS. The plate was then washed with PBST and incubated with 100uL per well of Digoxin monoclonal antibody in triplicate at ~1000 - 2pg/mL for 1 hour at room temperature. The plate was washed with PBST and incubated with 100ul per well of goat anti-mouse IgG-HRP secondary antibody in all test wells at 1:25,000 for 30 minutes at room temperature. Detection was performed using 1-Step TMB Ultra substrate for 15 minutes at room temperature. The plate was then stopped with 0.2M sulfuric acid. Absorbances were read on a spectrophotometer at 450-550nm.