Direct ELISA analysis of hCG proteins was performed by coating wells of a plate with a purified hCG protein extracted from human urine at a concentration of 10 µg/mL overnight at 4C. The plate was washed 3 times with ELISA Wash Buffer and blocked with 300 ul of StartingBlock (PBS) Blocking Buffer for at least 1 hour at room temperature. After washing 100ul of hCG monoclonal antibody was added to wells in duplicate at 10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, 9.5, 0.078, 0.039, 0.019 and 0 µg/mL concentrations, and the samples were incubated for 2 hours at room temperature. The plate was washed, and then incubated with 100ul per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 for 1 hour at room temperature, and washed again with ELISA Wash Buffer. The plate was developed by incubating 100ul per well of 1-Step Ultra TMB substrate per well for 10 minutes at room temperature in the dark. The reaction was stopped with 100ul per well of 0.16M sulfuric acid. Absorbances were read on a spectrophotometer at 450-550nm.