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 37^◦C.
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.

Note:

• 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.