Direct ELISA using fluorescent substrate protocol

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Procedure and tips for direct ELISA assay using a fluorescent conjugated primary antibody.

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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 h at RT covered with plate sealer.

3) Wash plates: PBS-T (0.05% Tween20) 250 μl/well; 3x 30 s. 

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 eg in 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 s.

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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