Indirect ELISA- Introduction, Steps, Advantages and Protocol

ELISA stands for enzyme-linked immunosorbent assay, which is a technique to detect the presence of antigens in biological samples using antibodies. There are different types of ELISA, such as direct, indirect, sandwich and competitive ELISA, depending on the format of antigen and antibody binding.

Indirect ELISA is a two-step ELISA that involves two binding processes of primary antibody and labeled secondary antibody. The primary antibody is an antibody that recognizes and binds to the specific antigen of interest. The secondary antibody is an antibody that recognizes and binds to the primary antibody, and is conjugated with an enzyme or a fluorophore that can produce a detectable signal.

The basic principle of indirect ELISA is as follows:

• The antigen is immobilized on a solid surface, such as a microtiter plate well, either directly or via a capture antibody that is coated on the surface.
• The sample containing the primary antibody is added to the well and incubated. If the primary antibody is present in the sample, it will bind to the antigen on the surface. The unbound primary antibody is then washed away.
• The secondary antibody conjugated with a detection molecule is added to the well and incubated. If the primary antibody has bound to the antigen, the secondary antibody will bind to the primary antibody. The unbound secondary antibody is then washed away.
• A substrate for the detection molecule is added to the well and incubated. If the secondary antibody has bound to the primary antibody, the detection molecule will catalyze a reaction with the substrate, producing a color change or a fluorescence signal that can be measured by a spectrophotometer or a fluorometer.

The indirect ELISA can be used for various purposes, such as:

• Quantifying the amount of antibodies in a sample by comparing the signal intensity with a standard curve.
• Detecting the presence or absence of antibodies in a sample by comparing the signal intensity with a cut-off value.
• Comparing the relative levels of antibodies in different samples by normalizing the signal intensity with a reference value.

The indirect ELISA has some advantages and disadvantages over other types of ELISA, such as:

• High sensitivity: More than one labeled secondary antibody can bind to each primary antibody, amplifying the signal.
• Flexibility: Different primary antibodies can be used with the same labeled secondary antibody, reducing the cost and increasing the versatility.
• Cross-reactivity: The secondary antibody may bind to other antibodies or proteins in the sample that are not specific to the antigen, causing nonspecific signals and background noise.
• Additional step: The indirect ELISA requires an extra incubation and washing step compared to direct ELISA, increasing the time and complexity of the assay.

Indirect ELISA is a two-step ELISA that involves the binding of a primary antibody to the antigen, followed by the binding of a labeled secondary antibody to the primary antibody. The secondary antibody is conjugated to an enzyme that can produce a colorimetric or fluorescent signal when a substrate is added. The intensity of the signal is proportional to the amount of antigen-antibody complex formed in the wells.

The general steps of Indirect ELISA are as follows:

1. Coating antigen to microplate: The antigen solution is diluted in a buffer (usually PBS or carbonate buffer) and dispensed into the wells of a microtiter plate. The plate is incubated for 2 hours at room temperature or overnight at 4°C to allow the antigen to bind to the plastic surface. The plate is then washed with PBS to remove any unbound antigen.
2. Blocking: The remaining protein-binding sites on the plate are blocked with a blocking buffer, such as 5% non-fat dry milk or serum in PBS, to prevent non-specific binding of antibodies. The plate is incubated for at least 2 hours at room temperature or overnight at 4°C with the blocking buffer. The plate is then washed again with PBS.
3. Incubation with primary antibody: The primary antibody, which is specific for the antigen, is diluted in the blocking buffer and added to each well. The plate is incubated for 2 hours at room temperature or overnight at 4°C to allow the primary antibody to bind to the antigen. The plate is then washed four times with PBS to remove any unbound primary antibody.
4. Incubation with secondary antibody: The secondary antibody, which is specific for the primary antibody and conjugated to an enzyme (such as HRP or ALP), is diluted in the blocking buffer and added to each well. The plate is incubated for 1-2 hours at room temperature to allow the secondary antibody to bind to the primary antibody. The plate is then washed four times with PBS to remove any unbound secondary antibody.
5. Detection: A substrate solution for the enzyme is added to each well. The enzyme catalyzes a reaction that produces a colorimetric or fluorescent signal that can be measured by a microplate reader. The signal intensity reflects the amount of antigen-antibody complex formed in the wells.

Indirect ELISA is a two-step ELISA that uses a primary antibody to bind the antigen of interest and a secondary antibody conjugated to an enzyme or a fluorophore to detect the primary antibody. This method has several advantages over other types of ELISA, such as:

• High sensitivity: More than one labeled secondary antibody can bind to each primary antibody, resulting in signal amplification. This allows the detection of antigens at very low concentrations, such as picograms per milliliter.
• Flexibility: Different primary antibodies can be used with the same secondary antibody, as long as they are from the same species. This reduces the need for multiple labeled antibodies and allows the use of different detection systems (e.g., colorimetric, fluorescent, chemiluminescent) with the same secondary antibody.
• Cost-saving: Fewer labeled antibodies are required for indirect ELISA compared to direct ELISA, which uses a labeled primary antibody. This lowers the cost of the assay and increases its availability.

However, indirect ELISA also has some disadvantages, such as:

• Longer protocol: Indirect ELISA requires an additional incubation and washing step for the secondary antibody, which increases the time and labor involved in the assay.
• Potential high background: The secondary antibody may cross-react with other proteins or antibodies in the sample or on the plate, leading to nonspecific signals and reduced specificity. This can be minimized by using high-quality antibodies, blocking agents, and washing buffers.

Indirect ELISA is a widely used technique for the quantitative estimation of antibodies in various biological samples. It offers high sensitivity, flexibility, and cost-saving benefits over other types of ELISA. However, it also requires more steps and may produce higher background noise than direct ELISA. Therefore, the choice of ELISA format depends on the specific needs and goals of each experiment.

The protocol of indirect ELISA consists of the following steps:

1. Coating the plate with antigen: The antigen of interest is diluted in a coating buffer (such as carbonate-bicarbonate buffer) and added to the wells of a microtiter plate. The plate is then incubated at a suitable temperature (usually 37°C) for a certain time (usually 2 hours) to allow the antigen to bind to the surface of the wells. The excess antigen is then discarded and the plate is washed with a washing buffer (such as phosphate-buffered saline or PBS) to remove any unbound antigen.
2. Blocking the plate: To prevent nonspecific binding of antibodies to the plate, the remaining protein-binding sites in the coated wells are blocked with a blocking buffer (such as bovine serum albumin or BSA) that contains a high concentration of an irrelevant protein. The plate is incubated with the blocking buffer at room temperature for about 30 minutes and then washed again with the washing buffer.
3. Adding the primary antibody: The primary antibody that is specific for the antigen is diluted in a dilution buffer (such as PBS with BSA) and added to the wells. The plate is incubated at room temperature for another 2 hours to allow the primary antibody to bind to the antigen. The excess primary antibody is then discarded and the plate is washed again with the washing buffer.
4. Adding the secondary antibody: The secondary antibody that is conjugated with an enzyme (such as horseradish peroxidase or HRP) and that recognizes the primary antibody is diluted in a dilution buffer and added to the wells. The plate is incubated at room temperature for another 2 hours to allow the secondary antibody to bind to the primary antibody. The excess secondary antibody is then discarded and the plate is washed again with the washing buffer.
5. Adding the substrate: The substrate that can be converted by the enzyme into a colored or fluorescent product is added to the wells. The plate is incubated at room temperature for about an hour to allow the enzyme-substrate reaction to occur. The intensity of the color or fluorescence is proportional to the amount of enzyme-bound secondary antibody, which reflects the amount of primary antibody bound to the antigen, which in turn reflects the amount of antigen in the sample.
6. Adding the stop solution: The stop solution that can terminate the enzyme-substrate reaction and stabilize the color or fluorescence is added to the wells. The plate is then read by a microtiter plate reader that can measure the optical density (OD) or fluorescence intensity (FI) of each well at a specific wavelength.

The protocol of indirect ELISA can be summarized in this table:

The washing step in indirect ELISA is a crucial step that removes any unbound or excess reagents from the wells of the microtiter plate. This step helps to reduce the background noise and increase the specificity and accuracy of the assay. The washing step is performed after each incubation step, such as antigen coating, primary antibody addition, secondary antibody addition, and substrate addition.

The washing step involves filling the wells with a washing buffer, such as phosphate-buffered saline (PBS) or Tris-buffered saline (TBS), and then discarding the buffer into a container. The washing buffer should be compatible with the antigen, antibody, and enzyme used in the assay. The washing buffer may also contain a detergent, such as Tween 20, to reduce nonspecific binding and increase solubility of the reagents. The washing buffer should be at room temperature or slightly warmer to avoid precipitation of the reagents.

The number of washes and the volume of washing buffer used depend on the size and shape of the wells, the concentration and affinity of the reagents, and the sensitivity and specificity required for the assay. Typically, two to five washes are performed per step, with 200 to 400 µl of washing buffer per well. The washing buffer should completely fill the wells and cover the bottom surface. The washing buffer should be discarded by pipetting or by inverting and tapping the plate on a paper towel. The remaining drops should be removed by patting the plate on a paper towel or by using a plate washer device.

The washing step should be performed carefully and consistently to avoid cross-contamination, incomplete removal of reagents, or damage to the antigen-antibody complex. Some tips for performing the washing step are:

• Use fresh washing buffer for each wash cycle and avoid reusing or recycling the buffer.
• Use a multichannel pipette or a plate washer device to dispense and remove the washing buffer quickly and uniformly.
• Avoid bubbles or splashes in the wells that may cause uneven distribution of reagents or cross-contamination.
• Avoid drying out the wells between washes or incubations that may cause loss of activity or binding of reagents.
• Avoid overwashing or underwashing that may affect the signal-to-noise ratio or the dynamic range of the assay.

The washing step is an essential part of indirect ELISA that ensures optimal performance and reliability of the assay. By following proper protocols and techniques, the washing step can improve the quality and accuracy of indirect ELISA results.

The blocking step is an important step in indirect ELISA to prevent non-specific binding of antibodies and other proteins to the microplate wells. Non-specific binding can result in high background signal and low signal-to-noise ratio, which can affect the accuracy and sensitivity of the assay.

The blocking step involves adding a solution of non-reactive protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells. The blocking agent should not interfere with the antigen-antibody interaction or the enzyme-substrate reaction. Common blocking agents include bovine serum albumin (BSA), non-fat dry milk, serum, casein, gelatin, and blockACE.

The blocking step is usually performed after the coating step and before the incubation with primary antibody. The blocking solution is added to each well and incubated for at least 30 minutes at room temperature or overnight at 4°C. The blocking solution is then discarded and the plate is washed twice with PBS or other washing buffer. The plate is now ready for the incubation with primary antibody.

The blocking step can improve the specificity and sensitivity of indirect ELISA by reducing non-specific binding and background noise. However, the optimal blocking agent and concentration may vary depending on the antigen, antibody, and detection system used. Therefore, it is advisable to test different blocking agents and concentrations to find the best conditions for each assay.

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