Ask AI: write a protocol for using human serum in a sandwich ELISA to detect phosphorylated threonine 181 in steps

1. Prepare two sets of plates with the same coating antigen (e.g. phosphorylated threonine 181) overnight at 4°C. 2. Remove the coating antigen and wash the plates 3 times with TBS + milk. 3. Block the plates with a blocking buffer (e.g. milk or BSA) for 1 hour at room temperature. 4. Add human serum samples to the wells of one set of plates at 1:2 dilution. 5. Incubate the serum samples for 2-3 hours at 37°C. 6. Wash the plates 3 times with TBS + milk. 7. Add a primary antibody (e.g. polyclonal antibody specific to phosphorylated threonine 181) to the wells of both sets of plates and incubate for 1 hour at 37°C. 8. Wash the plates 3 times with TBS + milk. 9. Add a secondary antibody (e.g. HRP-conjugated anti-species IgG) to the wells of both sets of plates and incubate for 1 hour at 37°C. 10. Wash the plates 3 times with TBS + milk then incubate with substrate solution for 5-10 minutes. 11. Measure the absorbance at 450 nm for both sets of plates to calculate the difference in absorbance between the plate with human serum samples and the control plate. 12. A positive result is indicated if the absorbance of the sample plate is significantly higher than the control plate.