Fig1: Indirect ELISA analysis of MMAE was performed by coating wells of a 96-well plate with 50 μl per well of MMAE-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 MMAE monoclonal antibody serial diluted starting from a concentration of 20ug/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.

Fig2:Indirect ELISA analysis of MMAE was performed by coating wells of a 96-well plate with 50 μl per well of MMAE-ADC 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 MMAE monoclonal antibody serial diluted starting from a concentration of 20ug/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.

Fig3:Competitive ELISA analysis of MMAE was performed by coating wells of a 96-well plate with 50 μl per well of MMAE-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 MMAE monoclonal antibody at concentration of 1 μg/mL with serial diluted MMAE 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.

Fig4:Competitive ELISA analysis of MMAE was performed by coating wells of a 96-well plate with 50 μl per well of MMAE-ADC 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 MMAE monoclonal antibody at concentration of 1 μg/mL with serial diluted MMAE 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.