Antigen Coating Buffer, 5X

1. Dilute Antigen Coating Buffer, 5X 1:5 using deionized water and mix for 15 minutes. As Antigen Coating Buffer is a 5X concentrate, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil. 2. Dilute your antigen into the coating buffer. Optimal coating concentration varies significantly from 0.2 μg/mL to 10 μg/mL. 3. Let the solution stir 10 – 15 minutes and pipette onto the plate. Optimal coating volume generally ranges from 50 – 200 μL per well. ICT recommends coating antibodies onto Immulon II HB plates. 4. Once added to the plate, incubate the coating solution from 8 – 24 hours at room temperature protected from light. Minimize evaporation by individually covering each plate with a plate sealing cover, wrapping a stack in plastic wrap, or placing plates in a humidified storage box and covering. 5. After incubation, dump or aspirate the coating solution out of the wells. 6. Wash the plate twice with ICT’s ELISA Wash Buffer. 7. Aspirate and pipette one of ICT’s Blocking Buffers onto the plate at a higher volume than the coating solution (300 – 400 μL per well) . 8. Once added to the plate, incubate the block buffer from 8 – 24 hours at room temperature protected from light. Minimize evaporation by individually covering each plate with a plate sealing cover, wrapping a stack in plastic wrap, or placing plates in a humidified storage box and covering. 9. Aspirate the block buffer. 10. The assay can be run at this point, or the plate can be dried and packaged for later use. 11. Dry the plate by letting it sit in the fume hood covered with foil overnight, or dry in a drying chamber under vacuum from 3 – 6 hours at room temperature. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at 2-8°C protected from light.