Indirect ELISA (Protocol)

Indirect ELISA Process

Indirect ELISA is a two-step enzyme assay which involves binding of primary antibody and labeled secondary antibody. Primary antibody is incubated with antigen followed by incubation with secondary antibody.



The major steps involved in Indirect ELISA includes:

  1. Coating micro titer plate wells with antigen.
  2. Blocking unbound sites- It is done to prevent false positive results.
  3. Addition of sample containing antibody, Incubation (370C)
  4. Washing to remove unbound antibody.
  5. Addition of secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).
  6. Washing to remove unbound enzyme-linked antibodies.
  7. Addition of substrate which is converted by enzyme to produce a colored product.
  8. Reaction of a substrate with enzyme to produce a colored product and Reading of results.


Indirect ELISA Process



The common procedure for Indirect ELISA type is outlined as follows:


  1. Coat micro titer plate with antigen: Dispense 50 μl antigen solution (coating reagent) into wells of a micro titer plate.
  2. Wrap coated plates in plastic wrap to seal and incubate for 2 hr at 37 0C in an incubator.
  3. After incubation, uncover micro titer plate and discard the solution into a container.


  1. Washing Step: Remove coating solution and wash plate twice by filling wells with 200 µl PBS. The solutions or washes are removed by pipetting and remaining drops are removed by patting plate on a paper towel.
  2. Blocking step: Block remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer and incubate 30 min at room temperature.


  1. Discard the solution and perform washing step. Gently flick microplate onto paper towel.
  2. Add 50 μl of antibody solution using micropipette from the vial to wells.
  3. Wrap plate in plastic wrap, and incubate for 2 hr at room temperature. Discard liquid and pat bottom of plate with dry absorbent paper.


Indirect ELISA protocol


  1. Repeat washing step (Step 4).
  2. Repeat blocking step (Step 5).
  3. Repeat washing step (Step 4).


  1. Add 50 μl secondary antibody reagent to wells.
  2. Wrap micro titer plate in plastic wrap, and incubate 2 hr at room temperature.
  3. Repeat washing step (Step 4).


  1. Add 75 µl substrate solution from vial to the wells on micro titer plates.
  2. Wrap micro titer plates in plastic wrap and incubate for 1 hr at room temperature.
  3. Add 25 µl stop solution (0.5 M NaOH) from vial to the wells on micro titer plates.
  4. Using a micro titer plate reader to measure NPP hydrolysis, use a 405-nm filter.


Download Link:Indirect ELISA Protocol.pdf

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