Protocol of Competition (Inhibition) ELISA

This protocol provides an initial set of conditions for competitive ELISA. However, further optimization may be required on individual demands. If there are any questions regarding competition ELISA, please contact us for help.

Reagents

1. Coating Buffer

   Na₂CO₃, 1.5 g

   NaHCO₃, 2.93 g

   Distilled water, 1 liter, pH to 9.6

2. Blocking Buffer

   Phosphate buffered saline (PBS) containing 1% w/v BSA

3. Wash Buffer

   PBS containing 0.05% v/v Tween-20

4. Substrates and Stop solution

• TMB, for use with horseradish-peroxidase (HRP)-conjugated antibodies. Stop with 2M H₂SO₄
• pNPP, for use with alkaline phosphatase (AP)-conjugated antibodies. Stop with 1M NaOH

Notes:

1. Do not use sodium azide in any buffers or solutions as sodium azide inactivates the HRP.
2. All solutions should be at ambient temperature prior to use.

Method

Direct Competitive ELISA

1. Coat microtiter plate wells with 100 µl of the antibody solution at a concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate overnight at 4°C or 2 hours at 37°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 mixture by adding 50 µl of samples to 50 µl of enzyme-conjugated antigen for each well. 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 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.
6. 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.
7. Data analysis.

Indirect Competitive ELISA

• Method 1

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 or 2 hours at 37°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 primary antibody for each well in the assay. 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.
8. Data analysis.

• Method 2

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 or 2 hours at 37°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 enzyme-conjugated antibody for each well in the assay. 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 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.
6. 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.
7. Data analysis.

Troubleshooting of Competition (Inhibition) ELISA is also available for you.