Sandwich ELISA Protocol

  1. Coat the plate with the primary antibody in the coating buffer. This primary antibody is termed as ‘capture antibody’.
  2. Wash off unbound capture
  3. Block plate with blocking buffer.
  4. Wash off the blocking buffer.
  5. Add the sample containing antigen, diluted in blocking buffer.
  6. Wash off unbound antigen.
  7. Add detection ‘primary antibody’ diluted in blocking buffer.
  8. Wash off unbound primary antibody.
  9. Add secondary antibodies diluted in blocking buffer at a dilution recommended by the manufacturer. Incubate at RT for 1 hour.
  10. Wash off excess secondary antibody.
  11. Develop the plate using the chromogenic substrate (TMB substrate when using HRP-labelled antibodies, pNPP for ALP labelled). Most substrates take around 10-30 minutes of reaction time.
  12. Stop the enzymatic reaction using a stop solution (0.1N H2SO4).50-100ul of stop solution is recommended per well.
  13. Read the plate in a plate reader.

Depending upon the use of an enzyme labelled primary or enzyme labelled secondary antibody, sandwich ELISAs can be termed as direct sandwich ELISA and an indirect sandwich ELISA. The above protocol discusses an indirect sandwich ELISA though a direct one also utilizes the same protocol with a change in the use of a labelled primary antibody.


  • Caution should be taken to choose antibodies for different epitopes. A simple way can be choosing a polyclonal antibody as a capture antibody and a monoclonal one for detection.
  • In a certain cases, when the detection antibody is unavailable, the capture antibody can be used as an alternative. However, this can compromise the accuracy of the method.

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