Sandwich ELISA – Definition, Principle, Steps, Advantages

What is Sandwich ELISA?

• The sandwich ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used technique in immunology and biochemistry to quantify the concentration of specific antigens in a sample. It derives its name from the fact that the antigen of interest is sandwiched between two layers of antibodies, known as the capture antibody and the detection antibody.
• In a sandwich ELISA, the antigen being measured must possess at least two antigenic epitopes that can bind to different antibodies. This is because the assay requires the use of two antibodies that recognize different epitopes on the antigen. The capture antibody is immobilized on a solid support, such as a plastic microtiter plate, in a fixed quantity. This antibody is typically a polyclonal antibody, which can efficiently capture as much of the antigen as possible.
• To perform the sandwich ELISA, test solutions containing the antigen at an unknown concentration are added to the wells containing the capture antibody. The antigen molecules in the sample bind to the capture antibody. After allowing sufficient time for binding, unbound molecules are washed away, ensuring that only the antigen of interest remains attached to the capture antibody.
• Next, a detection antibody is added to the wells. This detection antibody is linked to an enzyme, which serves as the indicator system of the test. The detection antibody recognizes a different epitope on the antigen and binds to it, forming a sandwich complex with the capture antibody. The enzyme-linked detection antibody allows for the detection and quantification of the antigen.
• The more antigen present in the test solution, the more enzyme-linked detection antibodies will bind to the captured antigen. This is because the antigen acts as a bridge, facilitating the binding of the detection antibody. The intensity of the enzyme-linked signal is directly proportional to the concentration of the antigen in the sample.
• To determine the concentration of the antigen in the test sample, a series of standard solutions with known concentrations of the antigen are included in the assay. These standard solutions serve as controls and references. The results obtained from the standard solutions are used to construct a binding curve of the enzyme-linked detection antibody as a function of the antigen concentration. By measuring the absorbance of the enzyme-linked signal in the test sample and comparing it to the binding curve, the concentration of the antigen can be inferred.
• The sandwich ELISA is highly sensitive and specific, allowing for the detection and quantification of antigens even at low concentrations. It is a versatile technique that can be applied to various fields, including medical diagnostics, biotechnology research, and pharmaceutical development. The use of monoclonal antibodies as the capture and detection antibodies allows for precise detection and quantification of small differences in antigen concentrations.
• In summary, the sandwich ELISA is a powerful tool in the field of immunology, enabling researchers to measure the concentration of specific antigens. By employing two different antibodies that recognize different epitopes on the antigen, the sandwich ELISA provides a reliable and accurate method for antigen quantification.

Sandwich ELISA Principle

The principle of sandwich ELISA involves the detection of antigens using a combination of specific antibodies. The test begins by coating and immobilizing a known antibody onto the wells of a microtiter plate. This immobilized antibody serves as the capture antibody, which will bind to the antigen of interest.

Next, the test sample containing the suspected antigen is added to the wells and allowed to react with the immobilized antibodies. The antigen binds to the capture antibody, forming an antigen-antibody complex.

After incubation, the wells are washed to remove any unbound or nonspecifically bound substances. This step ensures that only the antigen of interest, bound to the capture antibody, remains in the wells.

Following the washing step, a second antibody is introduced. This antibody is conjugated with an enzyme and is specific for a different epitope on the antigen compared to the capture antibody. The enzyme-linked antibody is added to the wells and allowed to incubate. It binds to the antigen-antibody complex, forming a sandwich-like structure.

After another round of washing to remove any unbound enzyme-linked antibodies, a specific substrate is added to the wells. The substrate is a substance that the enzyme can act upon, leading to a chromogenic reaction. In the presence of the enzyme, the substrate undergoes a chemical reaction that produces a color.

The intensity of the chromogenic reaction, corresponding to the color produced, can be measured using a spectrophotometer or an ELISA reader. These instruments quantify the color intensity, providing a numerical value for the reaction.

To determine the exact amount of antigen present in the test sample, the measured color intensity is compared to a standard curve. The standard curve is generated using known concentrations of the antigen. By plotting the color intensity against the known concentrations, a calibration curve is obtained. This curve allows for the interpolation or extrapolation of the antigen concentration in the test sample based on its corresponding color intensity.

In a positive test, the enzyme acts on the substrate to produce a color, indicating the presence of the antigen. The intensity of the color is directly proportional to the concentration of the antigen in the test sample. This color change can be observed visually, even without specialized instruments, providing a qualitative assessment of the test result.

The sandwich ELISA principle offers high sensitivity and specificity, as it relies on the binding of two different antibodies to different epitopes on the antigen. This approach ensures a robust and reliable detection of the target antigen in various applications, including medical diagnostics and research.

Sandwich ELISA Principle Animated Video

Steps/ Method of Sandwich ELISA

The sandwich ELISA method involves several key steps for the detection and quantification of antigens. Here is a breakdown of the process:

1. Prepare the surface: A solid support, such as a microtiter plate, is used as the surface for the assay. The wells of the plate are coated with a known quantity of the capture antibody, which specifically binds to the antigen of interest.
2. Block nonspecific binding sites: To prevent any nonspecific binding, the plate is treated with a blocking agent, such as bovine serum albumin (BSA) or casein. This step helps to reduce background noise and ensures that the antibodies bind specifically to their respective targets.
3. Add the antigen-containing sample: The test sample, suspected to contain the antigen, is added to the wells. The antigen present in the sample will bind to the immobilized capture antibody, forming an antigen-antibody complex.
4. Wash the plate: After a suitable incubation period, the plate is washed to remove any unbound or nonspecifically bound substances. This step helps to eliminate any interfering substances and ensures that only the antigen-antibody complexes remain in the wells.
5. Add the detection antibody: A second antibody, known as the detection antibody, is added to the wells. This antibody recognizes a different epitope on the antigen compared to the capture antibody. It binds specifically to the antigen, creating a sandwich-like structure where the antigen is sandwiched between the capture and detection antibodies.
6. Add enzyme-linked secondary antibodies: To enable detection, enzyme-linked secondary antibodies are introduced. These antibodies bind specifically to the Fc region of the detection antibody. The enzyme linked to these secondary antibodies will generate a signal that can be measured later in the assay.
7. Wash the plate: The plate is washed again to remove any unbound detection antibodies and enzyme-linked secondary antibodies. This step ensures that only the specific antigen-antibody complexes, along with the associated enzyme, remain in the wells.
8. Add the substrate: A suitable substrate is added to the wells. The enzyme linked to the secondary antibodies converts the substrate into a detectable signal. The type of substrate used depends on the enzyme employed in the assay. For example, if the enzyme is horseradish peroxidase (HRP), a chromogenic substrate that produces a color change can be used.
9. Measure the signal: The intensity of the signal generated by the enzyme-substrate reaction is measured using appropriate instrumentation. This may involve spectrophotometry to measure absorbance, fluorometry to measure fluorescence, or electrochemical techniques to measure current changes. The signal is directly proportional to the quantity of antigen present in the sample.

By comparing the signal obtained from the test sample to that of known standards with known concentrations of the antigen, the exact amount of antigen in the sample can be determined. This allows for the quantitative measurement of the antigen concentration using the sandwich ELISA method.

Sandwich ELISA Advantages

The sandwich ELISA (Enzyme-Linked Immunosorbent Assay) offers several advantages that contribute to its widespread use in various fields. Here are some key advantages of the sandwich ELISA:

1. High specificity: The sandwich ELISA employs the use of two different antibodies that recognize different epitopes on the antigen. This dual antibody approach increases the specificity of the assay, as it requires both antibodies to bind to the antigen for detection. This enhances the accuracy of antigen quantification and reduces the risk of false positive or false negative results.
2. Suitable for complex samples: One of the significant advantages of the sandwich ELISA is its compatibility with complex samples, such as biological fluids or crude extracts. Since the antigen does not need to be purified prior to measurement, the assay can be performed directly on the sample, saving time and resources. This is particularly useful when dealing with samples containing a mixture of different components or contaminants.
3. Flexibility in detection methods: The sandwich ELISA allows for both direct and indirect detection methods to be employed. In the direct method, the detection antibody is conjugated directly to an enzyme, such as horseradish peroxidase (HRP), which generates a detectable signal. In the indirect method, an enzyme-conjugated secondary antibody is added after the primary detection antibody. This flexibility provides options for optimizing the assay based on the specific requirements and available reagents.
4. Sensitivity: The sandwich ELISA is known for its high sensitivity, enabling the detection and quantification of antigens even at low concentrations. The dual antibody configuration and the amplification provided by the enzymatic reaction contribute to the assay’s sensitivity. This is particularly advantageous when working with samples that contain low levels of the target antigen or when studying analytes with limited availability.
5. Quantitative measurement: The sandwich ELISA allows for the quantitative measurement of antigen concentration. By utilizing a standard curve generated using known concentrations of the antigen, the absorbance or fluorescence signal obtained from the test sample can be correlated to the corresponding antigen concentration. This quantitative aspect makes the sandwich ELISA suitable for applications requiring accurate measurement and comparison of antigen levels across different samples.
6. Versatility and applicability: The sandwich ELISA can be applied to various fields, including medical diagnostics, research, and pharmaceutical development. It can be used to detect and quantify a wide range of analytes, including proteins, hormones, viruses, and other biomarkers. The versatility of the assay makes it a valuable tool in many scientific disciplines.

In summary, the sandwich ELISA offers several advantages, including high specificity, suitability for complex samples, flexibility in detection methods, sensitivity, quantitative measurement, and versatility. These advantages contribute to the wide adoption of the sandwich ELISA as a reliable and robust technique for antigen detection and quantification in diverse applications.

FAQ