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Indirect ELISA Protocol

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Indirect ELISA protocol

Sample indirect ELISA protocol:

In an indirect ELISA the secondary antibody binds to the primary antibody similarly to a Western Blot

  1. Coat the microtiter plate with antigen/analyte
  2. Cover the plate and incubate overnight at 4°C
  3. Wash three times with 300ul of wash buffer
  4. Add blocking buffer
  5. Incubate for 1 hr at 37°C
  6. Wash four times with 300ul of wash buffer
  7. Add the samples and standards to the selected well in the plates. Samples and standards should be added in duplicate or triplicate
  8. Incubate for 90 minutes at 37°C or overnight at 4°C
  9. Wash three times in 300ul of wash buffer
  10. Add the detection antibody in wash buffer to the selected wells.
  11. Incubate for 1 hr at 37°C
  12. Wash three times in 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