Direct ELISA using fluorescent substrate protocol
General Procedure
Day 1:
1.
Coat with 100 µl/well of coating antibody diluted in filtered PBS. Incubate plate overnight at 4°C, covered with plate
sealer.
Day 2:
2.
Block plates with 200 µl/well of 4 g Block ACE powder, diluted in 100 ml of deionized water (USE a 1:4 dilution of this)
for 3 hrs at RT covered with plate sealer.
3.
Wash plates: PBS-T (0.05% Tween20) 250 µl/well; 3x 30 seconds.
After washing or aspirating flip plate over onto kim wipes on bench to remove excess liquid.
4.
Load 100 µl of standards or samples ** freshly diluted in 10% BlockACE in PBS-T overnight at 4°C, covered with plate
sealer.
Prepare standards ahead of time.
On day of application to the plate, (day 2) , standards are freshly diluted in eg 10% BSA
in PBS-T from 10 ng/ml to 500 pg/ml
Day 3:
5.
Incubate 100 µl/well of biotinylated reporter antibody diluted in PBS for 2 hours at RT covered with plate sealer.
6.
Incubate 100 µl/well of streptavidin alkaline phosphatase, 1:5000 dilution in PBS for 1 hour at RT, covered with plate
sealer.
7.
Wash plates: TBS 250 µl/well; 3x 30 seconds.
8.
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. Make sure well is clean-no
contamination.
9.
Signal measured on Fluorometer, (Victor2, Perkin Elmer); excitation: 440 nm; emission: 550 nm.
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