Direct ELISA using fluorescent substrate protocol

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:

  1. 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.
  2. Wash plates: PBS-T (0.05% Tween20) 250 μl/well; 3x 30 s.

    Top tip Molly

    After washing or aspirating flip plate over onto kim wipes on bench to remove excess liquid.

  3. Load 100 μl of standards or samples freshly diluted in 10% BlockACE in PBS-T overnight at 4°C, covered with plate sealer.

    Top tip Molly

    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:

  1. Incubate 100 μl/well of biotinylated reporter antibody diluted in PBS for 2 hours at RT covered with plate sealer.
  2. Incubate 100 μl/well of streptavidin alkaline phosphatase, 1:5000 dilution in PBS for 1 hour at RT, covered with plate sealer.
  3. Wash plates: TBS 250 μl/well; 3x 30 s.
  4. 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.
  5. Signal measured on Fluorometer, (Victor2, Perkin Elmer); excitation: 440 nm; emission: 550 nm