Sandwich ELISA protocol

Sandwich ELISA (also known as sandwich immunoassay) requires two antibodies specific to different epitopes of the antigen. One of the antibodies is coated on the surface of the assay plate and used as a capture antibody to facilitate the immobilization of the antigen. The assay plate's surface properties are important for optimal binding and assay performance. The sandwich ELISA is designed to detect the antigen of interest, which is the specific target protein or molecule present in the sample. The other antibody, often an enzyme-linked antibody, can be conjugated to facilitate the detection of the antigen by generating a measurable signal. Alternatively, this detection antibody can be bound to a further conjugated secondary antibody.

The sandwich ELISA is an immunoassay-based technique used to detect and quantify the target analyte in complex samples. This protocol, outlines each step from sample preparation to detection, ensuring high sensitivity and specificity. Two antibodies that bind to different epitopes on the target antigen should be used, forming a “sandwich” structure. This method is ideal for low-abundance targets and is widely used in diagnostics and research. This detailed guide includes reagent recommendations, incubation times, and troubleshooting tips to help researchers achieve consistent and reproducible results. Whether you’re new to ELISA or optimizing your workflow, this protocol offers a reliable foundation for accurate protein quantification.

Introduction to ELISA

Sandwich ELISA is a widely adopted ELISA method for detecting specific antigens in biological samples. Unlike direct or indirect ELISA formats, this approach uses two antibodies: a capture antibody immobilized on the plate and a detection antibody that binds to a different epitope of the same antigen. To ensure selective binding to the target molecule, specific antibodies are required for both capture and detection steps. This dual-recognition system enhances specificity and sensitivity, making it suitable for detecting the target molecule present in complex matrices like serum, plasma, or cell lysates.

Background and principles

The sandwich ELISA is a type of enzyme immunoassay that operates on the principle of antigen-antibody specificity. A capture antibody is first coated onto a microplate to immobilize the target antigen from the sample. A second, detection antibody then binds to a different epitope on the antigen, forming a sandwich complex. This detection antibody is either directly conjugated to an enzyme or recognized by an enzyme-conjugated secondary antibody, which facilitates signal generation. Upon substrate addition, a measurable signal is produced through an enzyme-substrate reaction, proportional to the antigen concentration. This format minimizes background noise and is ideal for detecting low-abundance proteins. Abcam’s protocol emphasizes the importance of using matched antibody pairs to ensure optimal binding and signal clarity.

Choosing the right antibodies

Selecting the right antibodies is fundamental to successful ELISA development. In ELISA assays, primary antibodies are responsible for binding directly to the target antigen, while secondary antibodies recognize and bind to the primary antibodies, often carrying an enzyme label for detection.

• Monoclonal antibodies provide high specificity by recognizing a single epitope, ensuring consistent results and minimal cross-reactivity.
• Polyclonal antibodies can bind multiple epitopes on the target antigen, increasing detection sensitivity but sometimes at the expense of specificity.
• Recombinant antibodies offer enhanced specificity and reproducibility, reducing batch-to-batch variation and improving assay reliability.

For sandwich ELISAs, it is crucial to use capture and detection antibodies that recognize different, non-overlapping epitopes on the target antigen. Our matched antibody pairs are specifically validated for this purpose, ensuring optimal performance and minimal background. When developing or optimizing an ELISA, always consider the affinity, specificity, and potential cross-reactivity of your antibody pairs. Using well-characterized primary and secondary antibodies, or validated matched antibody pairs, is key to achieving accurate and reproducible results.

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 ab102535)

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 plates 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 plates 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

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.

Other ELISA formats

ELISA is a versatile immunosorbent assay technique, available in several formats to suit different experimental needs. The main ELISA formats include direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA.

• Direct ELISA uses a labeled primary antibody that binds directly to the target antigen immobilized on the plate. This format is straightforward and quick, but may offer lower sensitivity due to limited signal amplification.
• Indirect ELISA involves an unlabeled primary antibody that binds to the target antigen, followed by a labeled secondary antibody for detection. This approach increases sensitivity and flexibility, as the same labeled secondary antibody can be used with different primary antibodies.
• Sandwich ELISA is the most widely used format, employing a capture antibody to immobilize the target antigen, followed by a detection antibody that binds to a different epitope. This dual-antibody approach enhances specificity and sensitivity, making it ideal for complex samples.
• Competitive ELISA measures antigen concentration by detecting the reduction in signal caused by competition between sample antigen and a labeled antigen for binding to a specific antibody. This format is particularly useful for small molecules or when only one antibody is available.

Selecting the appropriate ELISA format depends on the nature of the target antigen, the availability of antibodies, and the required sensitivity and specificity for your application.

ELISA kit selection and use

ELISA kits provide a convenient and reliable solution for detecting a wide range of targets, including cytokines, hormones, and disease biomarkers. These kits typically include pre-coated ELISA plates, matched antibody pairs, standards, and detection reagents, streamlining the workflow and reducing assay development time.

Quality control

Implementing robust quality control and lab safety practices is essential for reliable ELISA results. Quality control measures include the use of positive and negative controls, standards, and blanks to monitor assay performance and identify potential issues. Regular calibration and maintenance of equipment, such as pipettes and plate readers, help ensure accuracy and consistency.

Applications

Sandwich ELISA is extensively used in both research and clinical diagnostics. It is ideal for quantifying cytokines, growth factors, hormones, and other biomarkers in serum, plasma, cell culture supernatants, and tissue lysates. Its high specificity makes it suitable for detecting low-abundance proteins in complex biological matrices. Researchers use it to monitor disease progression, evaluate therapeutic responses, and validate biomarker candidates.

Limitations

Despite its advantages, sandwich ELISA has some limitations. It requires high-quality matched antibody pairs that recognize distinct epitopes on the target antigen, which may not be available for all proteins. The assay can be time-consuming due to multiple incubation and washing steps. Additionally, matrix effects from complex samples like serum or plasma can interfere with antigen-antibody binding, affecting accuracy. Cross-reactivity and non-specific binding may also occur if antibodies are not properly validated. Abcam’s protocol addresses these challenges by providing detailed guidance on reagent selection, sample preparation, and assay optimization to minimize variability and improve reliability.

Troubleshooting

Common issues in sandwich ELISA include weak signals, high background, and inconsistent results. Weak signals may result from insufficient antigen concentration, degraded antibodies, or improper incubation times. High background often stems from inadequate washing, non-specific binding, or contaminated reagents. To resolve these, ensure proper plate blocking, use fresh reagents, and optimize antibody concentrations. Inconsistent results may indicate pipetting errors or temperature fluctuations. Abcam’s protocol includes a troubleshooting guide that helps identify and correct these issues. It also recommends using controls and replicates to validate assay performance. Following these tips ensures reliable and reproducible ELISA results.

Conclusion

The enzyme-linked immunosorbent assay remains a cornerstone technique in biomedical research and diagnostics, offering sensitive and specific detection of proteins and other analytes. Mastery of ELISA formats, careful selection of antibodies, appropriate use of ELISA kits, and adherence to quality control and lab safety protocols are all critical for successful immunosorbent assay experiments. As ELISA technology continues to evolve, with advances in antibody engineering and detection methods, its role in identifying disease biomarkers and advancing our understanding of biological systems will only grow. By following best practices and leveraging high-quality ELISA kits, researchers and clinicians can achieve reliable, reproducible results that drive scientific discovery and improve patient care.

References

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