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Competitive ELISA Protocol

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The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample. Unlike a sandwich ELISA, whereby the analyte binds to a primary antibody and bound analyte correlates with levels of antigen a competitive ELISA works by competing with a reference (bound analyte on the plate) for binding to a limited amount of labeled antibody or antigen in a sample. The higher the sample antigen concentration, the weaker the output signal, indicating that the signal output inversely correlates with the amount of antigen in the sample.

Mouse DHEA / Dehydroepiandrosterone ELISA Kit – Competitive ELISA assay. The amount of signal decreases in correlation with the amount of analyte in the sample which completes for binding of the primary antibody.

  1. Coat the microtiter plate well with antigen/analyte solution in coating buffer.
  2. Cover the plate and incubate overnight at 4°C.
  3. Wash the plate three times with 300ul of wash buffer.
  4. Add the blocking buffer to the plate
  5. Incubate for 1 hr at 37°C.
  6. Prepare the analyte antibody mixture for samples and standards.
  7. Add the analyte and standard mixture to the selected wells.
  8. Incubate for 1 hour at 37°C.
  9. Wash three times with 300ul of wash buffer.
  10. Add the enzyme-conjugated secondary antibody to each well.
  11. Incubate for 1 hr at 37°C.
  12. Wash three times with 300ul of wash buffer.
  13. Add the substrate solution to the selected wells.
  14. Incubate at room temperature until the desired colour change is observed.
  15. Add stop solution.
  16. Read the absorbance values.