Immunoprecipitation of hCG proteins was performed by spiking 5 ugs of purified hCG protein extracted from human urine into 1ml of 1:2 diluted human urine sample. Antigen-antibody complexes were formed by incubating with 5ug of hCG antibody overnight on end over end tube rotator at 4C. The immune complexes were captured by using 50ul Protein A/G Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% milk for at least 1 hour at room temperature. hCG was detected at ~32 and 22 kD using hCG monoclonal antibody at a concentration of 4 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.