Competitive ELISA Protocol and Animation

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying specific molecules, such as proteins, hormones, or antibodies, in a sample. ELISA is based on the principle of antigen-antibody binding and enzyme-mediated signal amplification.

There are different types of ELISA, depending on the format of antigen and antibody immobilization and detection. One of them is Competitive ELISA, which is a technique used for the estimation of antibodies present in a specimen, such as serum, plasma, saliva, or urine.

Competitive ELISA is especially useful for measuring low concentrations of antibodies or antigens that have only one epitope (the part of the antigen that is recognized by the antibody). Competitive ELISA can also overcome some of the limitations of other ELISA formats, such as interference from complex samples or cross-reactivity of antibodies.

In this blog article, we will explain the principle and protocol of Competitive ELISA, and show you an animation that illustrates the process step by step. We will also discuss the advantages and considerations of using Competitive ELISA for your research or diagnostic applications.

Competitive ELISA is based on the principle of competition between two specific antibodies for the same antigen. One antibody is present in the test sample (if it contains the target antibody), and the other antibody is conjugated with an enzyme and added to the reaction. The antigen is coated on the surface of a microtiter plate, and both antibodies compete to bind to it. The amount of enzyme-conjugated antibody that binds to the antigen is inversely proportional to the amount of antibody in the test sample. This means that if the test sample has a high concentration of antibody, less enzyme-conjugated antibody will bind to the antigen, and vice versa.

The enzyme-conjugated antibody that binds to the antigen can be detected by adding a substrate that reacts with the enzyme and produces a color or a fluorescence signal. The intensity of the signal is inversely proportional to the amount of antibody in the test sample. This means that if the test sample has a high concentration of antibody, the signal will be low, and vice versa. A positive test result is indicated by a low signal, while a negative test result is indicated by a high signal.

Competitive ELISA can be performed using either direct or indirect detection methods. In direct detection, the enzyme-conjugated antibody is specific to the target antibody in the test sample. In indirect detection, the enzyme-conjugated antibody is specific to a secondary antibody that binds to the target antibody in the test sample. The indirect detection method can increase the sensitivity and flexibility of competitive ELISA, as different secondary antibodies can be used for different target antibodies.

Competitive ELISA is a type of enzyme-linked immunosorbent assay that relies on the competition between two specific antibodies for the same antigen. The steps and process of Competitive ELISA are as follows:

• Step 1: The primary antibody (unlabeled) is incubated with the sample antigen. This allows the formation of antibody-antigen complexes in solution. The amount of complex formed depends on the concentration of antigen in the sample. If the sample has a high amount of antigen, more complex will be formed and less antibody will be available for binding to the antigen-coated wells. Conversely, if the sample has a low amount of antigen, less complex will be formed and more antibody will be available for binding to the antigen-coated wells.
• Step 2: The antibody-antigen complexes are then added to the microtiter plate wells that are pre-coated with the same antigen. The antibody-antigen complexes compete with the antigen-coated wells for binding to the primary antibody. The unbound antibody and complex are removed by washing the plate. The amount of antibody bound to the wells is inversely proportional to the amount of antigen in the sample. If the sample has a high amount of antigen, less antibody will bind to the wells and vice versa.
• Step 3: The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added to the wells. The secondary antibody binds to any primary antibody that is bound to the wells. The unbound secondary antibody is removed by washing the plate. The amount of secondary antibody bound to the wells is directly proportional to the amount of primary antibody bound to the wells, which is inversely proportional to the amount of antigen in the sample.
• Step 4: A substrate that can be converted by the enzyme into a chromogenic or fluorescent signal is added to the wells. The enzyme catalyzes the reaction and produces a color or fluorescence change that can be measured by a spectrophotometer or a fluorometer. The intensity of the signal is inversely proportional to the amount of antigen in the sample. If the sample has a high amount of antigen, less signal will be generated and vice versa.

The results of competitive ELISA are based on the principle that the amount of color produced is inversely proportional to the amount of antibodies in the sample. This means that the more antibodies present in the sample, the less color will be generated by the enzyme-substrate reaction. Conversely, the less antibodies present in the sample, the more color will be produced.

To interpret the results, a standard curve is usually plotted using known concentrations of antibodies and their corresponding optical densities (OD) measured by an ELISA reader. The OD values of the unknown samples are then compared with the standard curve to estimate their antibody concentrations.

A positive result is indicated by a low OD value, which means that there is a high amount of antibodies in the sample that competed with the enzyme-labeled antibodies for binding to the antigen-coated wells. A negative result is indicated by a high OD value, which means that there is a low amount of antibodies in the sample that allowed more enzyme-labeled antibodies to bind to the antigen-coated wells and produce color.

The cutoff value for determining a positive or negative result can be calculated by taking the mean OD value of the negative control samples (usually blank or non-specific samples) and subtracting two or three times the standard deviation of these values. Any sample with an OD value lower than the cutoff value is considered positive, and any sample with an OD value higher than the cutoff value is considered negative.

Here is an example of a standard curve and a cutoff value for competitive ELISA:

In this example, the cutoff value is 0.5, which is calculated by taking the mean OD value of the negative control samples (0.6) and subtracting two times the standard deviation of these values (0.05). Therefore, any sample with an OD value lower than 0.5 is positive, and any sample with an OD value higher than 0.5 is negative.

The results of competitive ELISA can also be expressed as percent inhibition (%I), which is calculated by using the following formula:

%I = x 100

The percent inhibition reflects how much the sample antigen inhibits the binding of the enzyme-labeled antibody to the antigen-coated wells. A high percent inhibition indicates a high amount of antibodies in the sample, and a low percent inhibition indicates a low amount of antibodies in the sample.

Here is an example of percent inhibition values for competitive ELISA:

In this example, the percent inhibition values range from 0% to 100%, with higher values indicating higher antibody concentrations. The cutoff value for determining a positive or negative result can be set at 50%, which means that any sample with a percent inhibition higher than 50% is positive, and any sample with a percent inhibition lower than 50% is negative.

The interpretation of competitive ELISA results depends on several factors, such as the quality and specificity of the antibodies and antigens used, the accuracy and precision of the assay conditions and measurements, and the validity and reliability of the standard curve and cutoff value. Therefore, it is important to perform appropriate controls and validations to ensure accurate and consistent results.

Competitive ELISA is a versatile and reliable technique for measuring the presence and concentration of antibodies in a specimen. Some of the advantages of this technique are:

• High specificity: Competitive ELISA uses two antibodies that are specific to the same antigen, one conjugated with an enzyme and the other present in the test serum. This ensures that only the target antigen is captured and detected, reducing the chances of false positives or cross-reactivity with other antigens.
• Suitability for complex samples: Competitive ELISA does not require the purification or separation of the antigen from the sample before measurement. This makes it suitable for analyzing complex samples such as serum, plasma, urine, saliva, or tissue extracts, where the antigen may be present in low concentrations or bound to other molecules.
• Flexibility and sensitivity: Competitive ELISA can use both direct and indirect detection methods, depending on the availability and quality of the antibodies and conjugates. Direct detection involves using an enzyme-labeled antigen that competes with the sample antigen for binding to the primary antibody. Indirect detection involves using an unlabeled antigen that competes with the sample antigen for binding to the primary antibody, followed by adding a secondary antibody that is specific to the primary antibody and conjugated with an enzyme. Both methods can achieve high sensitivity and dynamic range by adjusting the concentration and incubation time of the antibodies and conjugates.

Competitive ELISA is a useful tool for studying the immune response, diagnosing diseases, detecting contaminants, or quantifying biomarkers. By following a detailed protocol and considering some important factors, competitive ELISA can produce accurate and reproducible results.

• For most applications, a polyvinylchloride (PVC) microtiter plate is best. Add 50 μL of diluted primary antibody (capture) to each microtiter well. Allow to incubate for 4 hrs. at room temperature or 4°C overnight.
• Note: If a purified capture antibody is not available, plate should first be coated with a purified secondary antibody, directed against the host of the capture antibody according to the following procedure:
  • Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 μL of antibody solution to each well (20 μg/mL in Phosphate buffered saline (PBS)).
  • Incubate the plate overnight at 4°C to allow complete binding.
  • Add primary capture antibody (as above).
• Wash the wells twice with PBS. The antibody solution washes can be removed by flicking the plate over a suitable container.
• The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature.
• Wash wells twice with PBS.
• Add 50 μL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20).
• Note: Sodium azide is an inhibitor of horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.
• Add 50 μL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.
• Wash the plate four times with PBS.
• Add substrate. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.

• When performing Competitive ELISA, it is essential to use a purified secondary antibody that is specific to the primary antibody and conjugated with an enzyme. This ensures that the secondary antibody will only bind to the primary antibody and not to any other proteins in the sample or the plate. If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody that is directed against the host of the capture antibody, as described in the protocol section.
• Another important consideration when performing Competitive ELISA is to avoid sodium azide in any buffers or wash solutions if using an HRP-labeled conjugate for detection. Sodium azide is an inhibitor of horseradish peroxidase (HRP), which is a commonly used enzyme for generating a colorimetric or chemiluminescent signal. Sodium azide can interfere with the enzyme-substrate reaction and reduce the sensitivity and accuracy of the assay. Therefore, sodium azide should be replaced with another preservative, such as thimerosal or gentamicin, in any solutions that will come in contact with the HRP-labeled conjugate.
• A third important consideration when performing Competitive ELISA is to optimize the concentration and incubation time of the antigen-conjugate solution. The antigen-conjugate solution consists of the same antigen that is coated on the plate, but conjugated with an enzyme. The amount and duration of antigen-conjugate added to the wells will affect the degree of competition between the sample antigen and the add-in antigen for binding to the primary antibody. If too much or too little antigen-conjugate is added, or if the incubation time is too short or too long, the assay may not produce a clear signal or a reliable standard curve. Therefore, it is recommended to perform a titration experiment to determine the optimal conditions for each antigen-conjugate solution.

These are some of the important considerations when performing Competitive ELISA. By following these tips, you can improve the quality and reliability of your results. For more information and guidance on Competitive ELISA, you can watch this animation that illustrates the principle and procedure of this technique.

Competitive ELISA is a powerful and versatile technique for measuring the presence and concentration of antibodies in a sample. It is based on the principle of competition between two specific antibodies for the same antigen, and the detection of the bound antibody by an enzyme-conjugated secondary antibody. Competitive ELISA has several advantages over other types of ELISA, such as high specificity, suitability for complex samples, and flexibility and sensitivity. However, it also requires careful optimization of the assay conditions, such as the concentration and purity of the antibodies, the antigen-coating procedure, the blocking and washing steps, and the choice of substrate and detection method. To perform a successful competitive ELISA, it is important to follow a detailed protocol and to consider some important notes on the factors that may affect the assay performance.

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