Protocol of Sandwich ELISA with Direct Detection

This protocol represents an example of a standard sandwich ELISA using an enzyme-labeled detection antibody with commonly used reagents and TMB (tetramethyl benzene) substrate. For other ELISA variations, see the section entitled Enzyme-linked Immunosorbent Assay (ELISA).

Reagents

Coating Buffer
Anhydrous Na₂CO₃, 1.5 g

Anhydrous NaHCO₃, 2.93 g

Distilled water, 1 liter, pH to 9.6
Blocking Buffer
Phosphate buffered saline (PBS) containing 1% w/v BSA
Wash Buffer
PBS containing 0.05% v/v Tween-20
Substrates and Stop solution

TMB (3,3’,5,5’-Tetramethylbenzidine), for use with horseradish-peroxidase (HRP)-conjugated antibodies. Stop with 2M H₂SO₄. Absorbance Settings: 450 nm.
PNPP (Para-Nitro Phenyl Phosphate), for use with alkaline phosphatase (AP)-conjugated antibodies. Stop with 1M NaOH. Absorbance Settings: 405 nm.

Recommended Starting Concentrations

	Monoclonal Capture Antibody Polyclonal Detection Antibody	Monoclonal Capture Antibody Monoclonal Detection Antibody	Polyclonal Capture Antibody Polyclonal Detection Antibody
Capture Antibody	1-8 µg/mL	0.5-4 µg/mL	0.2-0.8 µg/mL
Detection Antibody	50-400 ng/mL	0.25-2 µg/ml	50-400 ng/mL

Notes:

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

Method

Coat 96-well plate wells with 100 µl of the appropriate coating capture 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.
Add 150 µl of blocking solution to each well. Incubate for 1 h at 37°C. Wash 4 times in wash buffer.
Dilute samples and/or standards in wash buffer and add 100 µl of suitably diluted samples and/or standards to the wells, respectively. Samples or standards should preferably be run in triplicate. Incubate for 1.5 h at 37°C. Wash 3 times in wash buffer.
Add 100 µl of appropriately diluted enzyme-conjugated detection antibody to each well. Incubate for 1 h at 37°C. Wash 3 times in wash buffer.
Add 100 µl of 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.
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.
Tip: The presence of air bubbles in wells may impact the results and it is advisable to spin the plate in a centrifuge.
Data analysis.

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