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Direct ELISA Protocol

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Direct ELISA Protocol

The below protocol is a sample direct ELISA protocol using streptavidin-biotin detection.

In a direct ELISA a conjugated primary antibody binds directly to the analyte

  1. Coat the microtiter plate with antigen/analyte.
  2. Cover the plate and incubate overnight at 4°C.
  3. Wash the plate three times with 300ul of wash buffer.
  4. Add blocking buffer and incubate for 1 hr at 37°C.
  5. Wash four times with 300ul of wash buffer.
  6. Add samples and standards to the selected wells at the appropriate concentrations.
  7. Incubate for 90 minutes at 37°C or overnight at 4°C.
  8. Wash three times with 300ul of wash buffer.
  9. Add the biotin-conjugated streptavidin in wash buffer.
  10. Incubate for 1 hr at 37°C.
  11. Wash three times with wash buffer.
  12. Add the substrate solution to the selected wells.
  13. Incubate at room temperature until the desired colour change is observed.
  14. Add stop solution.
  15. Read the absorbance values.