Sandwich ELISA protocol

Learn how to set up a sandwich ELISA, covering all steps from plate coating and blocking to incubations with primary and secondary antibodies.

Sandwich ELISA (also known as sandwich immunoassay) requires two antibodies specific to different epitopes of the antigen. These two antibodies are normally referred to as matched antibody pairs. One of the antibodies is coated on the surface of the multi-well plate and used as a capture antibody to facilitate the immobilization of the antigen. The other antibody can be conjugated to facilitate the detection of the antigen. Alternatively, this detection antibody can be bound to a further conjugated secondary antibody.

Stage 1 - Sample preparation

ELISAs can be run on a number of sample types. Here we provide ways to prepare different sample formats.

Materials required

Steps

Prepare the extraction buffer as recommended by the manufacturer.

Keep all buffers, reagents and equipment on ice at 4 °C.

Isolate the cells and suspend them in extraction buffer.

Spin-speeds and washes may require optimization.

Some adherent cells may require enzymatic or mechanical detachment.

Lyse the cells.

Spin down the suspension in a centrifuge to pellet insoluble contents.

Determine the concentration of protein in your extract using a Bradford or BCA assay.

Aliquot supernatant into several tubes.

Minimize freeze/thaw cycles.

Materials required

Steps

Collect the cell culture media, spin down in a centrifuge and remove the pellet.

Aliquot supernatant into several tubes

Minimize freeze / thaw cycles

Materials required

Steps

Prepare the extraction buffer as recommended by the manufacturer.

Keep all buffers, reagents and equipment on ice at 4 °C.

Dissect the tissue with clean tools on ice, as quickly as possible to prevent degradation by proteases.

If not homogenizing the tissue samples immediately, snap freeze in liquid nitrogen and store at -80°C.

Add extraction buffer to the dissected tissue.

Homogenize the suspension with a homogenizer.

Rinse the blade with extraction buffer when done.

Agitate the suspension for 2 h at 4 °C.

Spin down the suspension in a centrifuge to pellet insoluble contents.

Determine the concentration of protein in your extract using a Bradford or BCA assay.

Aliquot extract into several tubes

Materials required

Steps

Collect blood samples in tubes with anti-coagulant.

The anti-coagulant prevents the blood from clotting.

Spin down samples in a centrifuge and remove the pellet.

Aliquot supernatant into several tubes.

Minimize freeze/thaw cycles.

Materials required

Steps

Collect samples in untreated tubes and leave undisturbed at room temperature for 20 min.

Spin down samples in a centrifuge and remove the pellet.

Aliquot supernatant into several tubes.

Minimize freeze/thaw cycles.

Materials required

Steps

Collect the samples, spin down in a centrifuge and remove the pellet.

Keep all buffers, reagents and equipment on ice at 4 °C

Aliquot supernatant into several tubes.

Minimize freeze/thaw cycles.

Stage 2 - Adding capture antibody

At this stage, the capture antibody is added to the plate. This will later bind to the antigen when it is added.
Note: our SimpleStep® ELISA kits use a streamlined type of sandwich ELISA in which the microplate is pre-coated with an anti-tag antibody.

Materials required

Steps

Dilute your capture antibody in coating buffer.

The capture antibodies must recognize a different epitope to the detection antibodies. Our matched antibody pair kits can be used for this purpose.
You may need to perform serial dilutions to find the antibody concentration that works best.

Adsorb the capture antibody to the wells.

It’s important to keep the plate covered to prevent wells from drying out.
Ensure you’re using the correct microplate format for your detection method:
Colorimetric - clear plate
Fluorometric – black/clear-bottom plate
Luminometric – white plate

Wash each well three times with wash buffer.

Stage 3 - Antigen coating and blocking

Now the wells are coated with capture antibodies, you’re ready to add your sample. This is preceded by a blocking step to prevent non-specific binding.

Materials required

Steps

Perform background blocking.

Dilute your samples, controls and standards in dilution buffer.

Ensure the antigen concentration is within the expected dynamic range of the assay.

If you don’t know what dilutions to use, consult the literature and optimize by performing serial dilutions down the plate.

Adsorb the samples to the wells.

Wash each well three times with wash buffer.

Stage 4 - Antibody incubation

Now that the plate has been coated with capture antibodies and your sample, you’re ready to add detection antibodies.

The detection antibody will bind the antigen at an alternative site to the capture antibody, forming a sandwich. The detection antibody can be conjugated to an enzyme that facilitates detection of the target protein by itself (single sandwich). Alternatively, a conjugated secondary antibody can be added to bind the detection antibody (double sandwich).

Materials required

Note: The capture and detector antibodies must be different isoforms or from different species. The conjugated secondary must only recognize the detector antibody.

Steps

Dilute the antibodies in blocking buffer.

The detection antibodies must recognize a different epitope to the capture antibodies. Our matched antibody pairs can be used for this purpose.
If this information is not included, you may need to perform serial dilutions to find the antibody concentration

Add the unconjugated detection antibody to the wells.

Wash each well three times with wash buffer.

Add 100 µL of the conjuagted secondary antibody diluted in blocking buffer to each well. Then cover the plate.

It’s important to keep the plate covered to prevent wells from drying out.
Incubation time may need optimization.

Wash each well three times with wash buffer.

Materials required

Steps

Dilute the antibody in blocking buffer.

Optimum dilutions will often be suggested on the antibody datasheet.

The detection antibodies must recognize a different epitope to the capture antibodies. Our matched antibody pairs can be used for this purpose.
If this information is not included, you may need to perform serial dilutions to find the antibody concentration.

Add the detection antibody to the wells.

Incubation time may need optimization.

Wash each well three times with wash buffer.

Stage 5 - Detection

ELISA typically uses antibodies conjugated with enzymes such as horseradish peroxidase (HRP). These react with a substrate in oxidizing conditions to produce either a colored or fluorescent product. The signal generated is proportional to the concentration of the protein of interest. This signal can be measured at several time points throughout the substrate incubation (kinetic mode), or at a defined point in time after the reaction is complete (end-point mode).

Materials required

Steps

Set up your plate reader to observe the color change or fluorescence at the expected wavelength.

Bring all reagents to room temperature.

All wells need to reach an equal temperature to minimize edge effects.

Add the enzyme substrate solution to each well.

HRP substrates
The substrate for HRP is hydrogen peroxide. The cleavage of hydrogen peroxide is coupled to the oxidation of a hydrogen donor, which changes color during the reaction
TMB (3,3’,5,5’-tetramethylbenzidine)
Add TMB solution to each well, incubate for 15–30 min, add an equivalent volume of stopping solution (2 M H2SO4), and measure the optical density at 450 nm
OPD (o-phenylenediamine dihydrochloride)
Keep and store the substrate in the dark, as it is light-sensitive. Add the OPD solution and incubate for 10-30 min. The reaction can be stopped using sulfuric acid 2.5-3.0 M H2SO4. For stopped reactions, measure the reaction using a plate reader at 450 nm; for reactions that are not stopped, measure the reaction at 490 nm.
ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt)
ABTS is an HRP substrate. Add ABTS solution to each well and incubate for 20 min on a rocker. If color generation occurs rapidly, then reduce the concentration of the conjugated antibody in the assay. Do not dilute this solution. This highly sensitive substrate produces a green product. The end product is measured at 405 nm. Always handle carefully and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens)
AP substrate
P-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after a 15–30 min incubation at room temperature and stop the reaction by adding an equivalent volume of 0.75 M NaOH.
Fluorescent substrates must be kept in the dark to avoid photobleaching.

Add the stop solution to each well.

Read the signal development in the plate reader.

The reaction time for end-point reading may need some optimization.

Stage 6 - Data analysis

Here we have provided step-by-step best-practice guidelines to analyzing data for quantitative ELISAs.

Materials required

Tip: If the plate reader does not come with software, you can use a general statistical analysis tool such as GraphPad Prism

An in-depth guide on the analysis of data from ELISA can be found here.

Steps

Plot the standard curve from your standard controls using curve-plotting software.

A 4 or 5 parameter log plot (4/5-PL) tends to provide the best fit for most ELISA standard curves. However, you can try different models to see which one gives the best fit to your data.

Determine the curve fit and regression coefficient.

If  R2  < 0.99, your data cannot be reliably used for quantitative analysis. See our troubleshooting for possible causes.

Perform a spike recovery test.

If using an ELISA kit, expected recovery ranges will be listed on the datasheet.
At concentrations where the recovery falls outside the acceptable range, the assay cannot be reliably used for quantitative analysis of the given sample type.

Calculate the coefficient of variation.

If using an ELISA kit, expected CV values will be listed on the datasheet.
If the CV is > 15, there is too much variation in your data for quantitative analysis.  See our troubleshooting for possible causes of high CV.

Calculate the sample concentration.