Protocol of Indirect ELISA

This protocol provides an initial set of conditions when you perform indirect ELISA. It's important to note in advance that further optimization may be required on an individual basis. If there are any questions regarding indirect ELISA, please contact us.

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

1. Coat microtiter plate wells with 100 µl of the antigen/analyte solution at a concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate for 1-2 hours at room temperature or overnight at 4°C. Wash the plate 3 times in wash buffer and blot on paper towels after last wash.

2. Add 150 µl of blocking solution to each well. Incubate for 2 hours at 37°C. Wash 4 times in wash buffer and blot on paper towels after last wash.

3. Add 100 µl of the appropriately diluted unconjugated detection antibody (diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.

4. 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 and blot on paper towels after last wash.

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. Analysis of data.

Troubleshooting of Indirect ELISA is also available for you.