Indirect ELISA- Introduction, Steps, Advantages and Protocol

What is Indirect ELISA?

• Indirect ELISA, short for Enzyme-Linked Immunosorbent Assay, is a widely used technique in immunology and diagnostic laboratories for the detection and quantification of antibodies in various biological samples, such as serum or other body fluids. This method involves a two-step process that utilizes a primary antibody and a labeled secondary antibody to detect and measure the presence of specific antibodies.
• The procedure of indirect ELISA begins with the immobilization of the antigen of interest onto a solid surface, typically a microtiter plate. The antigen-coated plate is then incubated with the sample containing the antibodies being tested. If the antibodies of interest are present in the sample, they will bind specifically to the immobilized antigen, forming an antigen-antibody complex.
• After an incubation period, the plate is thoroughly washed to remove any unbound molecules. This step helps to eliminate any nonspecific binding that could interfere with the accuracy of the assay. Next, a secondary antibody, which is labeled with an enzyme and recognizes the primary antibody’s constant region, is added to the plate. This secondary antibody binds specifically to the primary antibody, forming a secondary antibody-primary antibody complex.
• Again, after a washing step to remove any unbound secondary antibodies, an enzyme substrate chromogenic reagent is added to the plate. This chromogenic substrate reacts with the enzyme attached to the secondary antibody, resulting in the development of a colored product. The intensity of the color is directly proportional to the amount of bound primary antibody present in the sample.
• To quantify the results, the optical density (OD) values of the wells are measured using a spectrophotometer. A higher OD value indicates a greater amount of primary antibody bound to the antigen, reflecting a higher concentration of specific antibodies in the sample.
• The indirect ELISA format offers several advantages. First, it allows for the detection and measurement of total antibody levels in the sample, as the secondary antibody can bind to different isotypes of the primary antibody. This is especially useful when determining the overall immune response or the presence of multiple antibodies targeting the same antigen.
• Additionally, indirect ELISA offers greater sensitivity than direct ELISA, as the secondary antibody can amplify the signal through the enzymatic reaction. This amplification step enhances the detection limit of the assay, making it more suitable for samples with low antibody concentrations.
• However, it’s important to note that indirect ELISA may also introduce some limitations. The use of a secondary antibody increases the risk of cross-reactions or nonspecific binding, which can lead to false-positive results. Thus, careful selection and optimization of the secondary antibody are crucial to ensure the assay’s specificity and reliability.
• In conclusion, indirect ELISA is a valuable tool for detecting and quantifying antibodies in biological samples. By employing a two-step process involving a primary antibody and a labeled secondary antibody, this technique allows for the measurement of total antibody levels and provides a sensitive and versatile approach in immunological research, clinical diagnostics, and other applications requiring the detection of specific antibodies.

Indirect ELISA Principle

The principle of an indirect ELISA involves the use of two binding steps to detect and quantify specific antibodies in a sample. The first step involves the incubation of the primary antibody with the antigen of interest. The primary antibody specifically recognizes and binds to the target antigen, forming an antigen-antibody complex.

After the incubation, the plate is thoroughly washed to remove any unbound primary antibodies, reducing the chances of nonspecific binding. The second step involves the addition of a labeled secondary antibody that recognizes the constant region of the primary antibody. This secondary antibody is conjugated with an enzyme or a reporter molecule, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).

During this incubation step, the secondary antibody binds specifically to the primary antibody, forming a secondary antibody-primary antibody complex. Again, the plate is washed to remove any unbound secondary antibodies.

To visualize the presence of the secondary antibody bound to the primary antibody, a substrate specific to the enzyme or reporter molecule is added. This substrate undergoes a reaction with the enzyme, resulting in the generation of a detectable signal, such as a colored or fluorescent product.

The intensity of the signal is directly proportional to the amount of primary antibody present in the sample, which in turn reflects the concentration of the target antibody in the sample. By measuring the signal, typically using a spectrophotometer or a specialized ELISA reader, the quantity of the specific antibody can be determined.

One advantage of the indirect ELISA principle is the versatility it offers in terms of secondary antibody selection. Different secondary antibodies can be used depending on the requirements of the assay. For example, a secondary antibody that recognizes all antibody isotypes (e.g., IgG, IgM, IgA) can be employed to detect total antibody levels. Alternatively, specific secondary antibodies can be used to detect individual antibody isotypes, such as IgG-specific secondary antibodies for the detection of IgG antibodies.

However, it is important to be cautious of potential nonspecific signals that may arise due to cross-reactions of the secondary antibody. These nonspecific signals can lead to false-positive results. Careful optimization and validation of the assay, including the selection of appropriate secondary antibodies and control samples, are necessary to ensure the specificity and accuracy of the indirect ELISA.

In summary, the indirect ELISA principle involves a two-step process of primary antibody binding to the target antigen, followed by the binding of a labeled secondary antibody to the primary antibody. This method provides a versatile and sensitive approach for detecting and quantifying specific antibodies in a sample, making it a valuable tool in various research, diagnostic, and biomedical applications.

Indirect ELISA Protocol

1. Coating antigen to the microplate

Coating the antigen onto the microplate is a crucial step in the process of setting up an ELISA assay. It involves immobilizing the purified antigen onto the wells of a 96-well ELISA plate, creating a solid surface for subsequent antibody binding.

To coat the wells with the purified antigen, follow the steps outlined below:

1. Prepare the antigen solution: Dilute the purified antigen to the desired concentration in a suitable buffer. In this example, 2 mg/mL of purified A/PR/8 Influenza A virus is diluted in 0.05M Tris-HCl buffer (pH 9.5).
2. Pipette the antigen solution: Using a multichannel pipette or a repeating pipette, add 50 µL of the diluted antigen solution to each well of the ELISA plate. Ensure that each well is properly filled with the antigen solution. It is recommended to include appropriate control wells, such as blank wells without antigen or wells with a known concentration of antigen, for comparison purposes.
3. Cover and incubate the plate: Place an adhesive cover or seal over the top of the plate to prevent evaporation and contamination. Incubate the plate overnight at 4°C or at the optimal temperature specified for the particular antigen being used. The extended incubation period allows the antigen to bind to the surface of the microplate wells, ensuring its immobilization.
4. Remove the coating solution: After the overnight incubation, carefully remove the coating solution from the wells. One common method is to hold the plate upside down and gently flick it over a sink or suitable waste container to discard the solution. Be cautious not to dislodge the antigen coating from the wells during this step.

At this point, the microplate is ready for the subsequent steps of the ELISA assay, such as sample or antibody incubation. The coated wells now provide a solid surface for specific antibody binding, enabling the detection and quantification of the target antigen or antibodies of interest.

Proper coating of the antigen is essential for the success of the ELISA assay, as it ensures the presence of the immobilized antigen for subsequent interactions with specific antibodies in the sample.

2. Blocking

Blocking is an essential step in an ELISA assay that helps prevent nonspecific binding and reduce background noise. It involves saturating any remaining protein-binding sites on the microplate wells, effectively blocking them from interacting with unwanted proteins or antibodies. Here’s how the blocking process can be carried out:

1. Prepare the blocking buffer: In this example, 5% donkey serum in 1X PBS is used as the blocking buffer. However, alternative blocking reagents such as 5% non-fat dry milk or BSA in PBS, or normal serum from an animal in which the secondary antibody was generated, can also be used. The choice of blocking buffer depends on the specific assay requirements and the compatibility with the detection system.
2. Add the blocking buffer: Pipette 200 µL of the blocking buffer into each well of the coated microplate. Ensure that each well is completely filled with the blocking buffer. It is recommended to include control wells without any blocking reagent for comparison purposes.
3. Incubate the plate: Incubate the plate for a minimum of 2 hours at room temperature or, for even better blocking, incubate overnight at 4°C. This extended incubation period allows sufficient time for the blocking buffer to saturate the remaining protein-binding sites on the microplate surface, reducing the chances of nonspecific interactions.
4. Remove the blocking buffer: After the incubation, remove the blocking buffer from the wells by gently flicking the plate over a sink or suitable waste container. Take care not to dislodge the antigen coating during this step.
5. Wash the plate: Wash the plate with PBS containing 1% Tween-20 to remove any residual blocking buffer, unbound proteins, or antibodies. Washing helps eliminate nonspecific binding and reduces background noise. It is common to perform several washes, typically 3-5, by filling each well with the wash solution, gently agitating the plate, and then aspirating the liquid.

3. Incubation with the primary antibody

After the blocking step, the next crucial stage in an ELISA assay is the incubation with the primary antibody. The primary antibody binds specifically to the target antigen, allowing for the detection and quantification of the desired analyte. Here’s how the incubation with the primary antibody can be performed:

1. Prepare a dilution series: To determine the optimal dilution of the serum sample containing the primary antibody, prepare a dilution series. In this example, a serial dilution is performed using 1X PBS. Start by diluting the serum 1:12.5 (e.g., 10 µL serum + 115 µL 1X PBS) and then perform subsequent 4-fold dilutions to obtain a dilution range from 1:12.5 to 1:204,800. It is recommended to include appropriate positive and negative controls alongside the dilution series.
2. Add the diluted serum samples: Pipette 100 µL of each dilution of the serum sample into the corresponding wells of the microplate. Ensure that each well is properly filled with the diluted serum. It is common practice to perform duplicates or triplicates of each sample dilution to ensure reproducibility and accuracy.
3. Cover and incubate the plate: Place an adhesive cover or seal over the top of the plate to prevent evaporation and contamination. Incubate the plate at room temperature for 1-2 hours. The specific incubation time can vary depending on the nature of the primary antibody and the assay requirements. It is recommended to follow the manufacturer’s instructions or optimize the incubation time for your specific assay.
4. Flick and wash the plate: After the incubation period, carefully flick the plate over a sink or suitable waste container to remove the contents. This step helps to remove unbound primary antibodies and reduce background noise. Subsequently, wash the plate with PBS containing 1% Tween-20. Perform several washes, typically 3-5, to ensure thorough removal of any unbound antibodies.

4. Incubation with the secondary antibody

After the incubation with the primary antibody, the next step in an ELISA assay is the incubation with the secondary antibody. The secondary antibody recognizes and binds specifically to the primary antibody, allowing for the detection and amplification of the signal. Here’s how the incubation with the secondary antibody can be carried out:

1. Prepare the secondary antibody: Dilute the enzyme-conjugated secondary antibody, such as horseradish peroxidase (HRP)-conjugated donkey anti-mouse secondary antibody in this example, according to the manufacturer’s instructions or as optimized for your specific assay. Dilute the secondary antibody in a suitable diluent or buffer, usually provided with the antibody.
2. Add the secondary antibody: Pipette 100 µL of the diluted secondary antibody solution into each well of the microplate. Ensure that each well is properly filled with the secondary antibody solution. It is common practice to include appropriate positive and negative controls alongside the samples.
3. Incubate the plate: Cover the plate with an adhesive cover or seal to prevent evaporation and contamination. Incubate the plate at room temperature for 1 hour. The specific incubation time may vary depending on the secondary antibody and the assay requirements. It is advisable to follow the manufacturer’s instructions or optimize the incubation time for your particular assay.
4. Flick and wash the plate: After the incubation, gently flick the plate over a sink or suitable waste container to remove the contents. This step helps to remove any unbound secondary antibodies and minimize background noise. Subsequently, wash the plate with PBS containing 1% Tween-20. Perform several washes, typically 3-5, to ensure thorough removal of any unbound antibodies.

The incubation with the secondary antibody is a critical step in an ELISA assay as it enables the detection and amplification of the primary antibody signal. The secondary antibody recognizes the primary antibody, which is bound to the immobilized antigen, and brings along an enzyme, such as HRP, that catalyzes a reaction to generate a detectable signal.

Following the incubation with the secondary antibody, removing unbound antibodies through flicking and washing helps to minimize nonspecific interactions and reduce background signal. It ensures that only specific secondary antibodies, bound to the primary antibody-antigen complex, contribute to the signal generation.

Once this step is completed, the microplate is ready for further steps in the ELISA, such as the addition of substrate or chromogenic reagents, which initiate a colorimetric or fluorescent reaction, allowing for the quantification of the target analyte.

5. Detection

The detection step in an ELISA assay allows for the measurement of the signal generated by the enzymatic reaction, providing quantitative or qualitative information about the target analyte. Here’s how the detection process can be carried out:

1. Add the indicator substrate: Pipette 100 µL of the indicator substrate, such as 3,3′,5,5′-tetramethylbenzidine (TMB), at a concentration of 1 mg/mL, into each well of the microplate. Ensure that each well is properly filled with the substrate solution. The indicator substrate is typically colorless or pale in its unreacted form.
2. Incubate the plate with the substrate: Allow the plate to incubate with the substrate for 5-10 minutes at room temperature. During this incubation, the enzyme conjugated to the secondary antibody catalyzes a reaction with the indicator substrate, leading to the generation of a detectable signal. The reaction is typically accompanied by a color change, resulting in the development of a blue color.
3. Stop the enzymatic reaction: After the desired incubation time, add 100 µL of 2N sulfuric acid (H2SO4) to each well to stop the enzymatic reaction. The acid stops the reaction and stabilizes the color development.
4. Read the plate: Within 30 minutes of adding the stop solution, use a microplate reader to measure the absorbance of the wells. Set the reader to a wavelength of 405 nm, which is the typical wavelength used for detecting the signal generated by TMB. The microplate reader measures the intensity of the developed color, which is directly proportional to the amount of target analyte present in the sample.

By measuring the absorbance at 405 nm, the microplate reader provides quantitative data about the presence and concentration of the target analyte in the samples. A higher absorbance reading indicates a higher concentration of the analyte, while a lower reading indicates a lower concentration or absence of the analyte.

The detection step in an ELISA assay allows for the measurement of the signal generated by the enzymatic reaction, enabling the quantification or qualitative analysis of the target analyte in the samples.

Summerize Steps of Indirect ELISA

The indirect ELISA is a multi-step assay used to detect and quantify specific antibodies or antigens in a sample. Here are the steps involved in performing an indirect ELISA:

1. Coat the ELISA plate: Add the desired testing antigens to each well of the ELISA plate. Seal the plate and incubate it overnight at 4°C. This step allows the antigens to adhere to the plate surface.
2. Remove coating solution and wash: Discard the coating solution and wash the plate two times with a suitable buffer. The washing helps remove any unbound or non-specifically bound antigens.
3. Block the plate: Add a blocking solution, such as a protein-based blocking buffer, to the wells. Incubate the plate at 4°C for 1 hour. Blocking helps prevent non-specific binding of antibodies to the plate surface, reducing background noise.
4. Wash the plate: Wash the plate two times with the buffer to remove the blocking solution and any unbound blocking agents.
5. Incubate with primary antibody: Add the unconjugated primary antibody to the wells and incubate the plate at room temperature for 1 hour. The primary antibody binds specifically to the target antigen, forming an antigen-antibody complex.
6. Wash the plate: Wash the plate four times with the buffer to remove any unbound primary antibodies.
7. Incubate with HRP-labeled secondary antibody: Add the HRP-labeled secondary antibody, specific to the primary antibody species, to the wells. Incubate the plate at room temperature for 1 hour. The secondary antibody recognizes and binds to the primary antibody.
8. Wash the plate: Wash the plate four times with the buffer to remove any unbound secondary antibodies.
9. Incubate with substrate solution: Add a suitable substrate solution, such as ReadiUse™ TMB Substrate Solution, to each well. Incubate the plate at room temperature for 15-30 minutes. The substrate reacts with the HRP enzyme conjugated to the secondary antibody, leading to the development of a colored or fluorescent signal.
10. Stop the reaction: Add a stopping solution, such as Signal Guard™ HRP reaction stopping solution, to each well. The stopping solution halts the enzymatic reaction, preserving the color or fluorescence generated.
11. Measure the absorbance: Use an ELISA microplate reader to measure the absorbance of the wells at a specific wavelength, such as 650 nm. The absorbance is directly proportional to the amount of antigen-antibody complex present in the wells, allowing for the quantification of the target antibodies or antigens in the sample.

By following these steps, the indirect ELISA enables the detection and quantification of specific antibodies or antigens in a sample, making it a valuable tool in research, diagnostics, and various other applications.

Advantages of Indirect ELISA

The indirect ELISA method offers several advantages that make it a popular choice in antibody detection and quantification. Here are some of the key advantages of indirect ELISA:

1. Accessibility: Indirect ELISA benefits from a wide selection of commercially available pre-labeled secondary antibodies. These antibodies are specifically designed to recognize and bind to different primary antibodies, allowing for easy and efficient detection of target antigens.
2. Economy: Compared to direct ELISA, indirect ELISA requires fewer labeled antibodies. This reduces the cost of the assay, making it more economical, especially when large-scale or high-throughput testing is involved.
3. High sensitivity: Indirect ELISA exhibits high sensitivity due to signal amplification. The primary antibodies used in the assay contain multiple epitopes, which can be recognized and bound by several labeled secondary antibodies. This amplifies the signal, resulting in a stronger and more detectable response.
4. Flexibility: The indirect ELISA method offers great flexibility. A single labeled secondary antibody can be used to detect multiple primary antibodies, making it suitable for detecting a variety of analytes. This flexibility simplifies assay development and allows for customization based on specific experimental requirements.
5. Maximum immunoreactivity: Indirect ELISA preserves the maximum immunoreactivity of the primary antibody. The primary antibody is incubated with the antigen, allowing for optimal binding and formation of the antigen-antibody complex. This enhances the accuracy and reliability of the assay results.
6. Versatile visualization markers: Indirect ELISA enables the use of different visualization markers with the same primary antibody. For example, the primary antibody can be conjugated with biotin, and then detected using streptavidin linked to an enzyme or a fluorescent molecule. This versatility allows for a wide range of detection options and compatibility with various downstream applications.

Disadvantages of Indirect ELISA

While indirect ELISA offers several advantages, it is important to consider the potential disadvantages associated with this technique. Here are some of the key disadvantages of indirect ELISA:

1. Potential for cross-reactivity: Indirect ELISA involves the use of a secondary antibody, which may exhibit cross-reactivity with other proteins present in the sample. This cross-reactivity can lead to non-specific staining and result in false-positive signals. Careful selection and optimization of the secondary antibody are crucial to minimize this issue.
2. Lengthier protocol: Indirect ELISA generally requires more steps and a longer protocol compared to the direct ELISA format. This is because an additional incubation step is needed for the secondary antibody to bind to the primary antibody. The longer protocol can increase the overall assay time and may require more resources.
3. Possibility of background noise: Non-specific binding of the secondary antibody to components in the sample or the plate surface can contribute to background noise in the assay. This can affect the accuracy and specificity of the results. Appropriate blocking and washing steps are necessary to minimize background noise and optimize the signal-to-noise ratio.
4. Increased complexity: The use of an additional antibody in indirect ELISA introduces an additional layer of complexity to the assay. It requires careful selection and optimization of both primary and secondary antibodies, as well as their concentrations and incubation conditions. This complexity may require additional troubleshooting and optimization steps to achieve reliable and reproducible results.
5. Longer procedure compared to direct ELISA: Due to the requirement of an extra incubation step for the secondary antibody, indirect ELISA generally has a longer procedure compared to the direct ELISA technique. The longer procedure can be a limitation when rapid assay results are desired or when processing large sample volumes.

FAQ

References

1. Lin, A. V. (2015). Indirect ELISA. ELISA, 51–59. doi:10.1007/978-1-4939-2742-5_5
2. https://www.jove.com/v/10496/elisa-assays-indirect-sandwich-and-competitive
3. http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/indirect-elisa.html
4. https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html#Indirect
5. https://www.aatbio.com/resources/faq-frequently-asked-questions/What-is-an-Indirect-ELISA
6. https://www.abcam.com/kits/elisa-principle
7. https://www.sinobiological.com/category/indirect-elisa