The Simple Science behind Sandwich ELISA

Enzyme-linked immunosorbent Assay (ELISA) is a sensitive laboratory test used for detecting and quantifying specific proteins. Proteins such as antibodies or antigens, are detected in a biological sample for specific purposes in research, medicine, and diagnostics. It works like a lock and key: a special plate is coated with the substance you’re looking for. If the substance is in your sample, it sticks to the plate. Then, a second substance that attaches only to the first one is added, like a key fitting into a lock. This second substance has a color-changing marker. If the substance you’re looking for is there, the colour changes, and you can measure it to see how much is present. The most common variant of this technique is Sandwich ELISA. The process of Sandwich ELISA is the most complicated of all. Let’s dive into the details. 

A must read- Types of ELISA – My Biology Dictionary

What are the types of ELISA?

There are several types of ELISAs, each designed to perform specific purposes. However, the four major types are:

Direct ELISA:

  • Direct ELISA is like a detective checking if a specific thing (let’s call it a target) is present in a sample. Imagine a plate with tiny wells, and the detective puts the sample in these wells. If the target is there, it sticks to the plate. Now, the detective adds a special coloured marker that only attaches to the target. If the colour changes, it means the target is present. So, Direct ELISA is a quick way to see if the “clue” (target) is in the sample by directly looking for it with a coloured marker.

Indirect ELISA:

  • Indirect ELISA is like a detective finding a hidden clue indirectly. First, the detective puts the sample on a plate with tiny wells. If the target is there, it sticks. Next, the detective adds a general marker that attaches to anything stuck on the plate. Then, a second special marker is added, which only sticks to the first marker. If the colour changes, it means the target is there because the second marker only sticks to the general marker attached to the target. So, Indirect ELISA indirectly finds the hidden target by using two markers, like following a trail to discover a hidden clue.

Sandwich ELISA:

  • The process of Sandwich ELISA is simple, like making a sandwich with layers to find something special. Imagine a plate with tiny wells as the bread. First, you put the sample on the plate. If the thing you’re looking for (let’s call it the filling) is there, it sticks. Then, you add a special marker (like a sauce) that attaches to the filling. Finally, you add another marker (like another sauce) that only sticks to the filling. If the colour changes, it means the filling is there between the two markers, like a sandwich. So, Sandwich ELISA is a way to “capture” and detect a specific thing by sandwiching it between two markers.

Competitive ELISA:

  • Competitive ELISA is like a competition among ingredients. Imagine a plate with tiny wells, and you’re looking for a specific thing (let’s call it the treasure). First, you put the sample on the plate, and if the treasure is there, it sticks. Then, you add a known amount of the treasure (but labelled) to compete with the treasure in your sample. They fight for space on the plate. If your sample had a lot of treasure, less labelled treasure sticks. If your sample had less treasure, more labelled treasure sticks. So, by seeing how much-labelled treasure sticks, you can figure out how much treasure was in your sample. Competitive ELISA is like a friendly competition to find the hidden treasure in your sample.

All in all, these variations cater to different experimental requirements. They offer flexibility and precision in detecting and quantifying specific proteins in diverse biological samples.

Process of Sandwich ELISA

Midhun, N., K, S. B. J., Chakrapani, K., Haripriya, G., Pathuri, C. K. V. S., & Ramalakshmi, N. S. (2021). Enzyme linked immunosorbantassay- lab diagnosis: A review. Indian Journal of Microbiology Research, 8(1), 10–14.

Why choose Sandwich ELISA?

Researchers might choose Sandwich ELISA over other techniques for specific reasons:

    • First of all, the process of Sandwich ELISA offers high specificity because it uses two antibodies that bind to different epitopes on the target protein. This dual recognition enhances accuracy in detecting and quantifying the specific protein of interest.
    • The two-antibody system in Sandwich ELISA enhances sensitivity, evidently allowing for the detection of lower concentrations of the target protein. This is particularly useful when working with samples that have limited amounts of the protein.
    • Furthermore, Sandwich ELISA is versatile and can be used to detect a wide range of proteins, including complex samples like serum, plasma, and cell lysates.
    • The colour change in the enzymatic reaction is easy to measure and interpret, making it a straightforward method for both qualitative and quantitative analyses.
    • The antibodies and reagents for many proteins are commercially available, making it convenient for researchers to apply Sandwich ELISA in their experiments.

While Sandwich ELISA has its advantages, the choice of technique depends on the specific requirements of the experiment, the characteristics of the target protein, and the goals of the researcher. Other ELISA formats, such as indirect or competitive ELISA, may be chosen based on factors like simplicity, cost, or the nature of the samples being analyzed.

How does Sandwich ELISA work?

Process of Sandwich ELISA

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Sandwich ELISA works by “sandwiching” the target protein between both an antibody attached to the plate and an antibody labelled with an enzyme. This dual-antibody binding enhances specificity and sensitivity, making Sandwich ELISA a powerful tool for detecting and quantifying specific proteins in biological samples. Undoubtedly, it is the most widely used and common technique for ELISA.

As stated, two antibodies are used for “sandwiching” the target protein. One that is attached to the plate can be referred to as the ‘capture antibody’, whereas the one labelled with an enzyme is called the ‘detection antibody’.  (See figure)

The Process of Sandwich ELISA

Sandwich ELISA begins with coating the ‘capture antibody’ on the walls of the plate. This is done by adding the antibodies to the plate, and then the plates are covered and incubated overnight at 4 degrees Celsius. The plates are washed with buffer to remove the unbound antibodies. The next step is the addition of the sample containing the antigen or the target protein is added and left to react with the capture antibodies. The well is washed again. Next, the detection antibodies that are linked to an enzyme, are added. These are specific for a different epitope on the antigen. The well is rewashed to remove all unbound free antibodies. Lastly, the substrate is added to give a coloured reaction. This reaction is eventually used to measure the amount of target protein.


Sandwich ELISA undoubtedly finds application in various fields due to its ability to detect and quantify specific proteins with high sensitivity and specificity. Here are some easy-to-understand applications:

Disease Diagnosis:

It helps in detecting markers of diseases, like measuring specific proteins related to infections or autoimmune disorders, and hence aids doctors in diagnosing illnesses.

Pregnancy Tests:

It is used in identifying pregnancy by detecting hormones related to pregnancy (such as human chorionic gonadotropin, hCG) in a woman’s urine or blood.

Food Allergy Testing:

This technique helps determine the presence of allergens in food products, ensuring food safety by identifying substances that could cause allergic reactions.

Quality Control in Biotechnology:

It is used in verifying the production of specific proteins in biotechnological processes, ensuring the quality of biopharmaceuticals and other biotech products.

Cancer Biomarker Detection:

It is also used in identifying proteins associated with cancer, aiding in cancer diagnosis, and monitoring treatment effectiveness.

Virus Detection:

It is used to detect viral proteins in research or clinical settings, helping in the study and diagnosis of viral infections.

Autoimmune Disease Research:

It can also be used to study autoimmune diseases by detecting antibodies or antigens involved in immune system responses.

Environmental Monitoring:

The process also finds its use in monitoring environmental samples for the presence of specific proteins, such as detecting pollutants or harmful substances.

Monitoring Immune Responses:

It is used for assessing the immune system’s response to vaccines or infections by measuring the levels of specific antibodies.

Drug Development:

It helps in screening for the presence of target proteins during drug development and helps researchers understand how drugs interact with biological systems.

In essence, these applications demonstrate the versatility of Sandwich ELISA in various scientific and medical contexts, providing a valuable tool for researchers and healthcare professionals to study, diagnose, and monitor different conditions. Some things to keep in mind while performing the process of Sandwich ELISA are the specificity of antibody-antigen interactions, the use of enzymes for signal amplification, careful plate coating and washing steps, and the ability to quantify target molecules using calibration standards.

Keep reading for more!

Team MBD


1 Response

  1. Saguna Sutar says:

    Impressive information….love it❤️

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