All About the ELISA

ELISA (enzyme-linked immunosorbent assay) is a common technique used to quantify or detect compounds like antigens and antibodies. ELISA can be used to diagnose a number of diseases, and usually is used for viral diagnostics. A few examples of diseases that use ELISA as a diagnosis tool includes: HIV, allergies, and lyme disease. There are four major types of ELISA: Direct, Indirect, Competitive, and Sandwich ELISA. 

How Does ELISA Work?

ELISA uses the relationship between antibodies and antigens, where antibodies have the capability to bind specifically to antigens and form a complex. A capture antibody, depending on the type of ELISA, is usually used to coat the plate in the first step, and to bind to an antigen of interest. Detection antibodies are conjugated (linked to) enzymes that will produce a visible color change in wells when it is exposed to a substrate, so that the compound of interest can be detected/quantified. The substrate catalyzes an enzymatic reaction and produces the color change. The product, or end result of the ELISA, is generally a color change that will detect/quantify the compound of interest. This technique is usually performed in a plastic well plate, since antibodies and antigens have an affinity to adhere to the plastic wells. 

Direct ELISA

What distinguishes the Direct ELISA from other types? That only one antibody (a primary antibody) is used to detect an antigen. The enzyme is attached to the primary antibody and produces the colorimetric product in the presence of a substrate. The main steps include:

  1. Coat well with antigen of interest
  2. Wash wells
  3. Add primary antibody with conjugated enzyme
  4. Wash wells
  5. Add substrate for enzyme
  6. Observe colorimetric change

Indirect ELISA

What distinguishes the Indirect ELISA from other types? It uses a secondary antibody with a conjugated enzyme to detect the antigen indirectly through a primary antibody. The main steps include:

  1. Coat well with antigen of interest
  2. Wash wells
  3. Add primary antibody
  4. Wash wells
  5. Add secondary antibody with conjugated enzyme 
  6. Wash wells
  7. Add substrate for enzyme
  8. Observe colorimetric product development

Competitive ELISA

What distinguishes the Competitive ELISA from other types? The sample of antibodies or antigens will compete with a reagent that is mixed with it (hence the competition terminology). The amount of color produced at the end of the reaction is inversely proportional to the amount of protein of interest present in the sample. In the steps below we will be measuring the amount of protein in the sample. The main steps include:

  1. Coat wells with capture antibody
  2. Block plate with BSA or detergent
  3. Mix protein of interest sample with an enzyme conjugate (like Horseradish Peroxidase)
  4. Add mixture to wells
  5. Protein of interest and enzyme conjugate will compete for the limited binding sites on capture antibodies
  6. Wash wells
  7. Add colorless chemical substrate (TMB substrate)
  8. Substrate will cause any enzyme conjugate that managed to bind to the capture antibodies to produce a color. The more color present in a sample, the more enzyme conjugate was able to bind to capture antibodies. The less color present in a sample, the more protein of interest was able to bind to capture antibodies.
  9. Add stop solution and observe color change

Sandwich ELISA

What distinguishes the Sandwich ELISA from other types? It is for the detection of antigens. This procedure coats the well with capture antibody first, followed by the addition of the antigen. Then, antibody conjugated with enzyme is added, which forms a sandwich with antigen in between two types of antibodies. The main steps include:

  1. Coat wells with capture antibody
  2. Wash wells
  3. Add antigen of interest 
  4. Wash wells
  5. Add secondary antibody conjugated to an enzyme 
  6. Wash wells
  7. Add the substrate
  8. Observe colorimetric change

Interested in learning more about ELISA? Check out the following EDVO-Kits to learn more!

Simulation of HIV Detection by ELISA

Expanding Our Testing: Using ELISA to Detect COVID-19

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