Direct ELISA using fluorescent substrate protocol
Procedure and tips for direct ELISA assay using a fluorescent conjugated primary antibody.
Last edited Tue 31 Jan 2023
Stage 1 - Day 1
Steps
Coat each well with 100 μl/well of coating antibody diluted in filtered PBS. Incubate the plate overnight at 4°C, covered with a plate sealer.
Stage 2 - Day 2
Steps
Block each well with 200 μl/well of 4 g Block ACE powder, diluted in 100 ml of deionized water (1:4 dilution) for 3 h at room temperature and the plate with a plate sealer.
Wash plates with PBS-T (0.05% Tween20) 250 μl/well; 3 times for 30 seconds each time.
Load 100 μl of standards or samples freshly diluted in 10% BlockACE in PBS-T overnight at 4°C and cover with a plate sealer.
On the day of application to the plate (day 2) standards are freshly diluted eg in 10% BSA in PBS-T from 10 ng/ml to 500 pg/ml
Stage 3 - Day 3
Steps
Incubate each well with 100 μl/well of biotinylated reporter antibody diluted in PBS for 2 hours at room temperature and cover with a plate sealer.
Incubate each well with 100 μl/well of streptavidin alkaline phosphatase (1:5,000 dilution) in PBS for 1 hour at room temperature and cover with a plate sealer.
Wash the plates three times with 250 μl/well TBS for 30 seconds each time.
Amplify signal by adding 100 μl/well AttoPhos Fluorescent substrate system, for 5-10 min at RT.
- 36 mg of AttoPhos substrate should be mixed with 60 ml of AttoPhos buffer 24 hours prior to use.
Measure the signal on a Fluorometer, with an excitation wavelength of 440 nm and emission wavelength of 550 nm