Direct ELISA Protocol

  1. Coat antigen on the ELISA plates diluted in coating buffer for 2 hours at RT. Most protocols recommend using 100-300ng of purified protein per well. A maximum of 600-800ng can be added to each well in case the sample is more complex.
  2. Wash off any unbound antigen from the wells.
  3. Block the wells using 3-5% BSA prepared in 1x PBS, pH 7.5 and store the plate at 4°C overnight. Blocking can also be carried out at room temperature for 1 hour or 30 min at 37C.
  4. Wash off excess blocking. This step is optional as some antibodies do not need it.
  5. Incubate with primary antibodies diluted in blocking buffer for a minimum of 2 hours at RT or overnight in cold conditions (at a dilution recommended by the manufacturer).
  6. Wash off unbound primary antibodies.
  7. 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.
  8. Stop the enzymatic reaction using a stop solution (0.1N H2SO4) of 50-100ul per well.
  9. Read the plate in a plate reader.


  • All washes are to be done using 1x PBST (0.01-0.1% Tween 20 added) three times for 5-10 minutes each at RT.
  • The plate can also read without stopping the reaction, but it should be done immediately after incubation. The stopping step allows for extra time for delayed plate reading.
  • The detection is through a single antibody species; hence, an enzyme-tagged monoclonal antibody should be used.
  • The output will be a reading of total antibodies against the target antigen. Classes of antibodies cannot be distinguished.
  • Incubations can be carried out at 37◦C and in stationery conditions.