Protocol of Sandwich ELISA with Streptavidin-biotin Detection

This method provides a general procedure for use with the majority of sandwich ELISA with streptavidin-biotin detection. 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 provided with 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. If there are any questions regarding sandwich ELISA with streptavidin-biotin detection, 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

1. Coat microtiter plate wells with 100 μl of the appropriate coating antibody, at a concentration 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. Add 100 μl of suitably diluted samples to the relevant wells. Ensure that appropriately diluted standards are included (dilute samples and standards in wash buffer). Samples or standards should preferably be run in triplicate. Incubate for 90 minutes at 37°C or overnight at 4°C. Wash 3 times in wash buffer.
4. Add 100 μl of biotin-conjugated detection antibody (appropriately diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
5. Add 100 μl of enzyme-conjugated streptavidin (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.

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