Sandwich ELISA protocol

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

• Your sample
• Extraction buffer (for example ab260490)
• Protease inhibitor cocktail (for example ab65251)
• Phosphatase inhibitor cocktail (optional – for example ab201112)
• Wash buffer (for example ab206977)
• A BCA or Bradford assay kit (for example ab102536 or ab102536)

Steps

Prepare the extraction buffer as recommended by the manufacturer.

• Be sure to add protease inhibitors if not included.
• Add phosphatase inhibitors for phosphorylated proteins.

Isolate the cells and suspend them in extraction buffer.

• You should prepare a suspension with ~ 1 mL of buffer per 10⁷ cells.
• Adherent cells can be isolated by scraping directly into extraction buffer with PBS.
• Suspension cells can be isolated by washing with PBS, spinning down in a centrifuge, and resuspending the pellet in extraction buffer.

Lyse the cells.

• Agitate the cell suspension in lysis buffer for 15 – 30 min at 4°C.

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

• Centrifuge at 18,000 x g for 20 mins at 4 °C.
• Keep the supernatant and discard the pellet.

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

Aliquot supernatant into several tubes.

• If not using immediately, store samples at -80 °C.

Materials required

• Your sample in cell culture
• Microcentrifuge

Steps

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

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4 °C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80 °C

Materials required

• Your sample
• Extraction buffer (for example ab260940)
• Protease inhibitor cocktail (for example ab65621)
• Phosphatase inhibitor cocktail (optional – for example ab201112)
• PBS (for example ab64026)

Steps

Prepare the extraction buffer as recommended by the manufacturer.

• Be sure to add protease inhibitors if not included.
• Add phosphatase inhibitors for phosphorylated proteins.

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

Add extraction buffer to the dissected tissue.

• For a ~ 5 mg piece of tissue, add 300 µL of extraction buffer.

Homogenize the suspension with a homogenizer.

Agitate the suspension for 2 h at 4 °C.

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

• Centrifuge at 18,000 x g for 20 min at 4 °C.
• Keep the supernatant and discard the pellet.

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

Aliquot extract into several tubes

• If not using immediately, store samples at -80 °C.

Materials required

• Your sample
• An assay-validated anti-coagulant (Sodium citrate, EDTA, heparin)
• Microcentrifuge

Steps

Collect blood samples in tubes with anti-coagulant.

• Anti-coagulant should be diluted to a final concentration of 0.1 M.

Spin down samples in a centrifuge and remove the pellet.

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4 °C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80 °C.

Materials required

• Your sample
• Microcentrifuge

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.

• Centrifuge at 1,000 – 10,000 x g for 10 min at 4 °C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80 °C.

Materials required

• Your samples
• Microcentrifuge

Steps

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

• Centrifuge at 1,000 – 10,0000 x g for 2 min at 4 °C.
• Keep the supernatant and discard the pellet.

Aliquot supernatant into several tubes.

• Aliquots should have a minimum volume of 50 µL.
• If not using immediately, store samples at -80 °C

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

• Coating buffer containing carbonates (for example ab210899)
• Capture antibody (for example, from our matched antibody pair kits)
• Microplate - uncoated (for example ab210903)
• A cover for the microplate (some plate come with seals, or adhesive plastic film can be used)
• Automatic wash system (optional)

Steps

Dilute your capture antibody in coating buffer.

• We suggest a concentration of 1 – 10 µg/mL.

Adsorb the capture antibody to the wells.

• Add ~ 50 µL of your diluted capture antibody solution to each well. Then cover the plate.
• Incubate with gentle agitation for 2 h at room temperature, or 4°C overnight.

Wash each well three times with wash buffer.

• If washing manually, remove the solution from wells after each wash by flicking over a sink.
• Alternatively, an automatic wash system can be used.

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

• Dilution buffer (for example PBS with 3 – 5 % w/v BSA)
• Wash buffer (for example ab206977)
• Your sample
• Your controls and standards
• Microplate – uncoated (for example ab210903) or pre-coated with a capture antibody
• A cover for the microplate (some plate come with seals, or adhesive plastic film can be used)
• Automatic wash system (optional)

Steps

Perform background blocking.

• Add 200 µL of blocking buffer to each well and cover the plate.
• Block with gentle agitation for 1 – 2 h at room temperature, or 4°C overnight.

Dilute your samples, controls and standards in dilution buffer.

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

Adsorb the samples to the wells.

• Add 100 µL of your diluted samples and standards to the wells and cover the plate.
• Adsorb with gentle agitation for 2 h at room temperature, or 4°C overnight.

Wash each well three times with wash buffer.

• If washing manually, remove the solution from wells after each wash by flicking over a sink.
• Alternatively, an automatic wash system can be used.

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

• Unconjugated detection antibody
• Conjugated secondary antibody
• Blocking buffer (for example ab126587)
• Wash buffer (for example ab172375)
• ELISA plate with your samples adsorbed

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.

• Optimum dilutions will often be suggested on the antibody datasheet.

Add the unconjugated detection antibody to the wells.

• Add 100 µL of the pre-diluted antibody to each well and cover the plate.
• Incubate for 2 h at room temperature or overnight at 4°C.

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.

• Add 100 µL of the secondary antibody to each well and cover the plate.
• Incubate for 1 – 2 h at room temperature.

Wash each well three times with wash buffer.

Materials required

• Conjugated detection antibody
• Blocking buffer (for example ab126587)
• Wash buffer (for example ab172375)
• ELISA plate with your samples adsorbed

Steps

Dilute the antibody in blocking buffer.

Optimum dilutions will often be suggested on the antibody datasheet.

Add the detection antibody to the wells.

• Add 100 µL of pre-diluted antibody to each well and cover the plate.
• Incubate for 2 h at room temperature or overnight at 4°C.

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

• Your samples and standards in a microplate
• Plate shaker
• Enzyme substrate (for example for HRP: TMB ab171523 or Stoplight Red)
• Stop solution (for example TMB stop solutions: ab171529 or ab171531)
• Plate reader

Steps

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

• If using kinetic mode, configure the plate reader to detect at specific time intervals.

Bring all reagents to room temperature.

• Allow around 10 minutes for all wells to equilibrate to room temperature.

Add the enzyme substrate solution to each well.

• Add 50 – 100 µL of enzyme substrate to each well and incubate with gentle agitation on a plate shaker, as directed by the manufacturer. See below for specific substrates that can be used.

Add the stop solution to each well.

• Add 100 µL of stop solution to each well.
• Shake the plate on a shaker for 1 min to mix.

Read the signal development in the plate reader.

• If reading in kinetic mode, measure the signal at the pre-defined time points during the reaction.
• If reading in end-point mode, allow the reaction to proceed at room temperature and measure at the end of the time-course. For colorimetric detection, you can add a stop solution to terminate the reaction and stabilize the signal.

Stage 6 - Data analysis

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

Materials required

• Plate reader with curve-fitting software

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.

• Plot concentration of standards used on the x-axis.
• Plot the signal minus any blanks on the y-axis.

Determine the curve fit and regression coefficient.

• Take note of the regression coefficient (R²) and the equation generated from the curve-fitting model.
• Acceptable R²  > 0.99.

Perform a spike recovery test.

• Compare the signals of standards in buffer against standards spiked in sample matrix. The difference between the signals, expressed as a percentage, gives sample recovery.
• Acceptable recovery range = 80 – 120 %

Calculate the coefficient of variation.

• Calculate the mean (µ) and standard deviation (σ) of replicates. Use the following formula to determine the coefficient of variation (CV) = σ / µ
• Acceptable intra-assay CV < 10
• Acceptable inter-assay CV < 15

Calculate the sample concentration.

• Use the equation from the curve-fit generated in step 2 to determine the concentration of samples.