What Is Enzyme-Linked Immunosorbent Assay (ELISA)?

Updated on January 26, 2023

Enzyme-Linked Immunosorbent Assay (ELISA) is a type of immunoassay that uses the principles of immunology to detect the presence of a specific substance. It’s a widely used laboratory technique for detecting and quantifying particular proteins, glycoproteins, antigens, and antibodies in biological samples.   

The origins of ELISA can be traced back to the 1940s when Albert H. Coons and his team developed a technique called immunofluorescence, which involved labeling antibodies with a fluorescent dye and using them to identify antigens in tissue samples.  

In the 1960s, a technique called radioimmunoassay was introduced in scientific literature. Still, because it involved radioactivity, which posed health risks, scientists began searching for a safer alternative method.  

In 1971, Eva Engvall and Peter Perlman created a groundbreaking technique called the ELISA test, which utilizes antibodies to detect the presence of hormones or viruses in a sample. Since its introduction, this method has been modified and improved for various medical uses.  

It’s essential to note that ELISA troubleshooting is critical, as any lab error can affect the results’ accuracy. Therefore, it’s necessary to carefully follow the protocols and troubleshoot any issues that arise during the assay to ensure accurate results.   

Read on to learn more about ELISA, particularly how it works, its types, and its applications.   

How ELISA Works   

The assay utilizes the principles of immunology and biochemistry to detect and measure the presence and amount of a specific substance in a sample. The ELISA process can be divided into several critical steps, including plating, incubating, washing, and detecting.  

In particular, the first process begins with coating a plate with an antigen or the substance being detected. The sample is then added to the plate and allowed to incubate. During this incubation period, the antigen in the sample binds to the antibodies on the plate, forming an antigen-antibody complex.   

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After allowing the sample to incubate, any unbound material is removed by washing the plate. This secondary antibody specifically attaches to the antigen-antibody complex that was formed previously.  

The last step is detection. It’s carried out by introducing a substrate to the plate specific to the enzyme attached to the secondary antibody. When the substrate comes in contact with the enzyme, it leads to a change in color or other observable signals confirming the antigen’s existence and concentration.  

Types Of ELISA   

There are several different types of ELISA, each with its advantages and disadvantages. These types include:   

  • Direct ELISA   

In the direct ELISA method, the antigen in the sample is linked to an enzyme and added to multiwell plates. This approach is useful when a kit for measuring the specific protein isn’t available.  

The direct ELISA method is quick and eliminates the possibility of cross-reactivity with the secondary antibody. However, it can potentially impact the reactivity of the primary antibody, and labeling the primary antibody in each ELISA system is time-consuming and costly.  

  • Indirect ELISA   

In the indirect ELISA, the sample is first combined with a primary antibody that attaches to the antigen, making it possible to be captured. Then, a secondary antibody, labeled with an enzyme, is added and binds to the capture antibody.  

The enzyme-labeled antibody can then be detected by adding a substrate converted by the enzyme into a colored product. This method is less sensitive than the sandwich ELISA but is still very detailed and often used when the antigen is present in high concentrations.   

  • Sandwich ELISA   

The sandwich ELISA technique typically employs a pair of matched antibodies, each specific to a distinct, non-overlapping portion of the antigen molecule. The wells of the plate are coated with a capture antibody (primary antibody), and the sample is added.  

When the primary antibody binds to the antigen, a secondary antibody, also called a detection antibody, is introduced to determine the amount of antigen present in the sample.  

This method is very precise and works well for intricate or impure samples. It also gives a choice of direct and indirect detection methods, which provide both accuracy and flexibility.  

  • Competitive ELISA   

A competitive ELISA is a laboratory technique used to measure the presence and quantity of an antigen in a sample. The procedure begins by coating the wells of a microtiter plate with a known quantity of the antigen of interest.  

The sample antigen is mixed with a primary antibody, creating an antibody-antigen complex. This complex is added to the wells and competes with the antigen already bound to the plate for binding sites on the primary antibody.   

After incubation, the plate is washed to remove any unbound antibodies. The quantity of the primary antibody bound to the antigen on the plate is measured, and the concentration of the antigen is determined by comparing it to a standard curve.  

While this method can be useful for measuring antigen levels, it could be more sensitive and precise than the sandwich ELISA method.  

Applications Of ELISA   

Here are some of the applications of ELISA:   

  • Diagnosis Of Infectious Diseases   

ELISA is commonly used to diagnose infectious diseases like HIV, Hepatitis B and C, and Lyme disease. It works by detecting the presence of specific antibodies in a patient’s blood produced in response to a particular infectious agent.  

The test is susceptible and straightforward, and results can be obtained relatively quickly. It’s also relatively simple to perform, making it a widely-used diagnostic tool in clinical settings.   

  • Allergen Detection   

ELISA is a widely used diagnostic tool in cancer diagnosis and monitoring. The technique involves the detection of specific biomarkers or protein markers associated with different types of cancer. These biomarkers are typically present in elevated levels in the blood or other bodily fluids of individuals with cancer.  

One example of a cancer marker that can be detected using ELISA is the protein CA-125, which is often elevated in women with ovarian cancer.    

  • Cancer Diagnosis And Monitoring   

In cancer diagnosis and monitoring, ELISA is often used to detect the presence of specific biomarkers, or protein markers, associated with various cancer types. One example of a cancer marker that can be detected using ELISA is the protein CA-125, which is often elevated in women with ovarian cancer.   

Other examples include prostate-specific antigen (PSA) in prostate cancer and carcinoembryonic antigen (CEA) in the colon and different types of cancer.   

  • Food Testing   

ELISA is a sensitive and specific method for detecting gluten, making it a valuable tool in ensuring the safety of food products for individuals with dietary restrictions. It’s essential for individuals with gluten sensitivities or celiac disease, as exposure to gluten can cause severe health problems.   


All in all, the ELISA technique is a powerful and versatile tool that’s essential to modern biomedical research and clinical practice. Its ability to detect specific substances in a sample has made it a valuable tool in diagnosing various diseases and developing new therapeutics. With proper troubleshooting, it can be a reliable and accurate tool for researchers and clinicians.