Competitive ELISA protocol

Introduction to competitive ELISA

ELISAs are powerful tools for detecting targets of interest in biological samples. Standard ELISAs typically utilize an immunogen bound directly to the assay plate (Direct or Indirect ELISA) or bound to the plate and detected with an antibody pair specific to different epitopes on the protein or peptide of interest (Sandwich ELISA). For targets that are too small to bind multiple antibodies (or an antibody and a plate), and thus incompatible with other ELISA formats, a technique known as Competitive ELISA is used instead.

In a competitive ELISA, the sample antigen competes with a fixed concentration of identical antigen for binding to a single antibody (referred to here as the ‘fixed antigen’ for clarity). The assay's signal is proportional to the amount of fixed antigen bound to the antibody, so the more antigen there is in the test sample, the less the fixed antigen binds to the antibody, resulting in a lower overall signal. The protocols below outline the steps for establishing a competitive ELISA for quantifying small molecules, post-translational modifications (PTMs), and small peptides across a wide range of sample types. This detailed guide includes reagent recommendations, incubation times, and troubleshooting tips to help researchers achieve consistent and reproducible results. Whether you’re new to competitive ELISA or optimizing your workflow, this protocol offers a reliable foundation for accurate small molecule quantification.

Background and principles

Competitive ELISAs can be designed with the antibody and fixed antigen in one of two orientations: Antibody-Down or Antigen-Down. In an antigen-down assay, the fixed antigen is coated onto the plate, and the sample and an antibody conjugate to a detection enzyme, such as HRP, are added to the well. Coating the plate with the fixed antigen while retaining its epitopes for antibody binding may again require conjugation to a carrier protein (eg, BSA). For an antibody-down assay, the assay plate is instead coated with the antibody. The sample being tested is then added to the assay well along with a detection enzyme coupled to a fixed antigen to facilitate signal generation. Conjugation of the fixed antigen to a carrier protein, such as BSA, may be necessary at this step to facilitate enzyme coupling.

The choice of orientation is most often determined by the availability of the necessary components. For example, it may be easier to conjugate HRP to the antibody than to the fixed antigen, favoring an antigen-down format. Signal intensity or assay sensitivity can also be optimized by using an alternate orientation. Finally, depending on the enzyme substrate that is used to develop the finished assay, the signal for either of these orientations can be colorimetric, fluorescent, or chemiluminescent.

Choosing the right antibodies

Selecting the right antibodies is fundamental to successful ELISA development, especially in a competitive assay where specificity to the target of interest is vital. Many classes of small molecules are differentiated by minute changes in their molecular structure, and antibodies that fail to recognize these differences can cause a non-specific signal when running your assay. Our small-molecule antibodies are designed at the immunogen stage to achieve the highest possible specificity for each target of interest. Subsequent characterization of these antibodies in a functional assay for cross-reactivity with other relevant small molecules is key to achieving accurate, reproducible results you can trust.

View all His-tag ELISA kits.

Stage 1 - Sample preparation

Competitive 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

As discussed above, for competitive ELISA, you will likely need to conjugate your fixed antigen to a carrier protein, such as BSA, to facilitate binding to the assay plate. This carrier protein-conjugated small molecule can additionally be used to facilitate coupling the detection enzyme to the fixed antigen for an antibody-down assay.

Carrier protein conjugations should avoid altering the antigen's structure excessively, especially moieties that may differentiate the molecule from a similar target.

If developing an antigen-down formatted assay, the antibody should instead be conjugated to the detection enzyme.

Next steps

As discussed previously, these assays can be run in one of two orientations. Protocols for each orientation are provided below.

Stage 3 – Assay preparation

Materials required

• Coating buffer containing carbonates (for example, ab210899)
• Fixed Antigen: This is your target of interest that competes with the sample antigen for antibody binding. Depending on the target’s size, it will likely need to be conjugated to a carrier protein such as BSA to avoid epitope masking when bound to the plate.
• 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

Diluting fixed antigen

• Dilute your fixed antigen in coating buffer. We suggest a concentration of 1-10 μg/mL

Adsorb the fixed antigen to the wells.

• Add ~50 µL of your diluted fixed antigen conjugate to each well at the optimized concentration. Cover the plate.
• Incubate with gentle agitation for 2 h at room temperature, or 4°C overnight.

Wash each well three times with the wash buffer.

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

Stage 4 – Blocking and sample addition

Now that the wells are coated with the fixed antigen, you’re ready to add your sample and antibody. 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)
• Detection enzyme-conjugated antibody
• Your sample
• Your controls and standards
• 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.

Prepare samples

• 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.

Prepare antibody

• Dilute the antibody to the proper concentration in dilution buffer. This concentration can be determined by testing serial dilutions of the antibody with the fixed antigen as described previously.

Add the samples and reagents to the wells

• Remove the blocking buffer with a multipipettor, being sure not to let the wells dry out. Alternatively, the blocking buffer can also be removed by inverting over a sink and flicking gently, then carefully tapping out the excess buffer onto a clean paper towel or kim wipe.
• Add 50 µL of your diluted samples and standards to the wells.
• Add 50 µL of your diluted antibody to the wells and cover the plate.

Wash the plate

Wash each well three times with the 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.

Go to stage 5.

Stage 3 – Assay preparation

Materials required

• Coating buffer containing carbonates (for example, ab210899)
• Antibody
• 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

Diluting the antibody

• Dilute your antibody in coating buffer. We suggest a concentration of 1-10 μg/mL.

Adsorb the antibody to the wells

• Add ~50 µL of your diluted antibody to each well at the optimized concentration. Cover the plate.

Wash plate

Wash each well three times with the wash buffer.

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

Stage 4 – Blocking and sample addition

Now that the wells are coated with your antibody, you’re ready to add your sample and the enzyme-labeled fixed antigen. 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)
• Detector enzyme-conjugated fixed antigen
• Your sample
• Your controls and standards
• 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.

Prepare samples

• Dilute your samples, controls, and standards in dilution buffer.
• Ensure the antigen concentration falls within the assay's expected dynamic range.

Prepare fixed antigen conjugate

• Dilute the fixed antigen-enzyme conjugate to the proper concentration in dilution buffer. This concentration can be determined by testing serial dilutions of the antibody with the fixed-antigen conjugate, as described previously.

Add the samples and reagents to the wells

• Remove the blocking buffer using a multipipettor, ensuring the wells do not dry out. Alternatively, the blocking buffer can also be removed by inverting over a sink and flicking gently, then carefully tapping out the excess buffer onto a clean paper towel or kim wipe.
• Add 50 µL of your diluted samples and standards to the wells.
• Add 50 µL of your diluted fixed-antigen conjugate to the wells, then cover the plate.

Wash the plate

Wash each well three times with the 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.

Go to stage 5.

Stage 5 – Detection

Competitive ELISA typically uses antibodies or fixed antigens conjugated with enzymes such as horseradish peroxidase (HRP). These react with a substrate in oxidizing conditions to produce either a colored, fluorescent, or luminescent product. The signal generated is proportional to the concentration of the protein of interest. This signal can be measured at several time points during substrate incubation (kinetic mode) or at a single time point 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 ab171523or 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.

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

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.

Fluorescent substrates must be kept in the dark to avoid photobleaching.

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 endpoint 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.

The reaction time for endpoint reading may need some optimization.

Stage 6 - Data analysis

-Here we have provided step-by-step best-practice guidelines for 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 the 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.

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

Perform a spike recovery test.

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

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.

• 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

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.

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