Anti-FITC抗体[9] (ab116639)

Chemiluminescent detection was performed using SuperSignal West Dura.

Direct ELISA analysis of FITC was performed by coating wells of a plate with FITC-BSA or BSA at a concentration of 1 µg/mL overnight at 4°C.

The plate was washed 3 times with ELISA Wash Buffer  and blocked with 300 µL of StartingBlock (PBS) Blocking Buffer for at least 1 hour at room temperature. After washing, 100 µL of ab116639 was added to wells in duplicate at 1000, 500, 250, 125, 62, 31, 15, 7.8, 3.9, 1.9 and 0.9 ng/mL concentrations, and the samples were incubated for 2 hours at room temperature. The plate was washed, and then incubated with 100 µL per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at 1/10000 dilution for 1 hour at room temperature, and washed again with ELISA Wash Buffer. The plate was developed by incubating 100 µL per well of 1-Step Ultra TMB substrate per well for 5 minutes at room temperature in the dark. The reaction was stopped with 100 µL per well of 0.16M sulfuric acid.

Absorbances were read on a spectrophotometer at 450-550 nm.