Competitive ELISA analysis of SN38 was performed by coating wells of a 96-well plate with 50 µl per well of SN38-BSA diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 1%BSA blocking buffer, and incubated with 100 µl per well of SN38 monoclonal antibody (HA721259) at concentration of 1 µg/mL with serial diluted SN38 starting from a concentration of 10ug/ml for 1 hours at room temperature. The plate was washed and incubated with 50 µl per well of an HRP-conjugated goat anti-Rabbit IgG secondary antibody at a dilution of 1:15,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

Western blot analysis of SN38 on different lysates with Rabbit anti-SN38 antibody (HA721259) at 1/1,000 dilution. Lane 1: SN38-BSA (50 ng/Lane) Lane 2: BSA (negative) (50 ng/Lane) Lane 3: 293T cell lysate (negative) (20 µg/Lane) Exposure time: 2 seconds; ECL: K1801; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721259) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.