Competitive ELISA Protocol

Protocol: Competitive ELISA

This is a general procedure for use with the majority of Bio-Rad reagents recommended for competitive ELISA. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information available for each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.


1. Coating Buffer
Anhydrous Na2CO3, 1.5 g
Anhydrous NaHCO3, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer use ELISA coating buffer (BUF030)
2. Blocking buffer
Phosphate Buffered Saline (PBS) containing 1% w/v BSA
For an alternative blocking buffer, use either ELISA BSA Block (BUF032), ELISA Ultrablock (BUF033), or ELISA Synblock (BUF034)
3. Wash buffer
Phosphate Buffered Saline containing 0.05% v/v Tween®-20
For an alternative wash buffer use ELISA wash buffer (BUF031)
Recommended Substrates and Stop Solutions
TMB Core+ (BUF062), for use with HRP-conjugated antibodies. Stop with 0.2M H2SO4.
pNPP (BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M NaOH.


  1. Coat microtiter plate wells with 100 µl of the antigen solution, at a concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate overnight at 4°C. Wash the plate 3 times in wash buffer.
  2. Add 150 µl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4 times in wash buffer.
  3. Prepare the antigen antibody mixture by adding 50 µl of antigen to 50 µl of antibody for each well in the assay (use a range of antigen concentrations appropriately diluted in wash buffer). Incubate for 1 hour at 37°C.
  4. Add 100 µl of the mixture to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
  5. Add 100 µl enzyme-conjugated secondary antibody (appropriately diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
  6. Add 100 µl of the appropriate substrate solution to each well. Incubate at room temperature (and in the dark if required) for 30 minutes, or until desired color change is attained.
  7. Read absorbance values immediately at the appropriate wavelength or add 50 µl of “stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance within 30 minutes.