Indirect ELISA Protocol

  1. Coat antigen on the ELISA plates, diluted in coating buffer, for 2 hours at RT.
  2. Wash off any unbound antigen from the wells.
  3. Block the wells using 3-5% BSA prepared in 1x PBS.
  4. Wash off excess blocking.
  5. Incubate with primary antibodies diluted in blocking buffer for 2 hours at RT or overnight in cold conditions at a dilution recommended by the manufacturer
  6. Wash off unbound primary antibodies.
  7. Add secondary antibodies diluted in blocking buffer at a dilution recommended by the manufacturer. Incubate at RT for 1 hour.
  8. Wash off excess secondary antibody.
  9. 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.
  10. Stop the enzymatic reaction using a stop solution (0.1N H2SO4). 50-100ul of stop solution is recommended per well.
  11. Read the plate in a plate reader at specific wavelengths.


  • Caution must be exercised when selecting a secondary antibody. The tagged enzyme, the host it is grown in, and the organism it is raised against. If the antibody denotes ‘goat anti-rabbit’, the secondary antibody is raised in a goat but is against the rabbit. Similarly, if the label says goat anti-rabbit IgG, then the secondary antibody will only bind to Fc region of IgG antibodies in rabbit. In fact, the subclass IgG1 and IgG2 can be distinguished as conjugates of these subclasses are also available. Also the enzyme tagged with the secondary antibody should be noted as it determines the substrate to be used and directs the protocol. HRP-labelled secondary antibodies most commonly use TMB/H2O2 substrate.
  • Dilutions of both primary and secondary antibodies should always be made in the blocking reagent. This is to prevent non-specific binding of the antibody to the surface of the plate.
  • Incubation times can be minimized by incubating at 37°C for 30 min. Also, stationery conditions can be applied.

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