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.

Antigen Coating Buffer, 5X maximizes the adsorption of antigens onto polystyrene plates for use in an indirect ELISA. By stabilizing the tertiary structure of the antigen, Antigen Coating Buffer helps the antigen retain its binding reactivity with the target antibody in the sample. This product is best for coating proteins ranging from 6 kDa to > 200 kDa onto polystyrene plates. Stabilizing the coated antigen allows for better analyte detection and enhances the specific signal of the assay. By generating a higher positive signal, a lower concentration of coating antigen may be used, saving valuable reagents. Under proper storage conditions, coated plates may last for years, enabling the manufacture of large batches for inclusion in diagnostic test kits. This also proves useful in non-manufacturing settings, where large batches of plates may be prepared at once to be stored for future experiments.