DIRECT ELISA USING FLUORESCENT SUBSTRATE PROTOCOL

Day 1:1. Coat with 100 μl/well of coating antibody diluted in filtered PBS. Incubate plate overnight at 4°C, coveredwith 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 1:4dilution 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.4. Load 100 μl of standards or samples ** freshly diluted in 10% BlockACE in PBS-T overnight at 4°C,covered with plate sealer.Day 3:5. Incubate 100 μl/well of biotinylated reporter antibody diluted in PBS for 2 hours at RT covered with platesealer6. 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 ofAttoPhos substrate should be mixed with 60 ml of AttoPhos buffer 24 hours prior to use. Make sure well isclean-no contamination.9. Signal measured on Fluorometer, (Victor2, Perkin Elmer); excitation: 440 nm; emission: 550 nm**• Prepare standards ahead of time.• On day of application to the plate, (day 2 in above procedures), standards are freshly diluted in 10% BSAin PBS-T from 10 ng/ml to 500 pg/ml, for example.• After washing or aspirating flip plate over onto kim wipes on bench to remove excess liquid.

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