ELISA Tests and Their Mechanisms Explained – ELISA, short for enzyme-linked immunosorbent assay, is a test often used for detecting and quantifying antibodies, antigens, peptides, or proteins. The key driver of the ELISA immunoassay is the highly specific binding of an antibody to a specific antigen. Antibodies interact with antigens in a similar way to a lock and key — for each lock, only one key can open it. In the same way, only one kind of antibody can bind to its respective antigen.
ELISA Assay Principle
In a typical ELISA setup, the antigen is immobilized on a solid surface, like a 96- or 384-well plate. The antigen can either be directly immobilized onto the surface of the plate or held on by a capture antibody, which itself is immobilized on the surface of the plate. A detecting antibody with an enzyme conjugated to it is then added to the plate. Common reporter enzymes used for ELISA are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
The detecting antibody binds specifically to the antigen, and the enzyme conjugated to it produces a fluorescent or colored product when an appropriate substrate is added. The products can be measured, which help quantify the amount of antigen in the sample. The higher the quantity of the product detected, the more antigen is present in the sample.
The ELISA immunoassay comes in four types: direct, indirect, sandwich, and competitive ELISA. Though each one is different, all of them rely on these four basic steps:
- Coating: Antigens are immobilized directly onto the wells of the multiwell plates or “captured” through capture antibodies.
- Blocking: The multiwell plates are flooded with bovine serum albumin, nonfat dry milk, or whole serum to cover all binding sites that have no antigen. This reduces the chances of false positives.
- Probing: Antibodies specific to the antigens in the sample are added, which bind specifically and with high affinity to the antigens.
- Signal measurement: Detecting and quantifying the absorbance, luminescence, or fluorescence produced by the enzyme-conjugated detecting antibodies.
Direct ELISA Principle
In a direct ELISA, the mechanism behind it is straightforward. First, the target antigen is immobilized onto the wells of a multiwell plate through direct adsorption. Then, an antibody specific to that antigen, and conjugated to HRP, AP, or other enzyme, is later added. If there is more of the antigen present in the sample, the signal will be greater.
Direct ELISA has the quickest and easiest protocols of all four types. However, it also has a few disadvantages. First of all, the signal produced by direct ELISA is not as strong compared to the other types. This may also create difficulty in distinguishing background signal from that of the actual antigen.
Indirect ELISA Principle
To address the weak signal problem of direct ELISA, indirect ELISA can be used. Here, the target antigen is still first immobilized onto the well plate. The difference is two antibodies are involved instead of just one. A primary antibody binds specifically to the antigen, while a secondary antibody is used to target the primary antibody. Only the secondary antibody is conjugated to an enzyme. Also, the secondary antibody has no affinity for the antigen.
The biggest advantage of indirect ELISA is signal amplification. Multiple secondary antibodies can bind to each primary antibody. In turn, with more reporter enzymes that can be detected, the signal intensifies.
In effect, indirect ELISA is more specific and generates less background signal compared to direct ELISA. It is also more flexible, as one secondary antibody can be used for different primary antibodies.
One issue with indirect ELISA is the potential for cross-reactivity of the secondary antibody. If the secondary antibody also has an affinity for the antigen, this interaction will interfere with the resulting signal.
Sandwich ELISA Principle
Sandwich ELISA requires a pair of antibodies specific to two different regions (epitopes) of the target antigen. Here, the antigen is not directly immobilized onto the well plate. Instead, a capture antibody is immobilized on the well plate, and this holds the antigen on one side. The primary antibody targets another epitope, and a secondary antibody conjugated to a reporter enzyme binds to the primary antibody. The sandwich ELISA method relies on a similar mechanism to that of indirect ELISA.
Using two antibodies to target the same antigen adds a layer of specificity to this method. That means fewer false positives and less background signal as well.
However, there is one huge challenge with sandwich ELISA, and that is to find a suitable antibody pair. The antibodies must also have very little to no cross-reactivity, otherwise this will interfere with the signal.
Competitive ELISA Principle
In competitive ELISA, a known, purified antigen, called reference antigen, is labeled with a reporter enzyme instead of the antibody. The well plates are coated with capture antibodies, similar to sandwich ELISA. The sample with the target antigen is added along with the reference antigen, and the two compete for binding to the capture antibodies immobilized on the well plate.
Unlike the other three methods of ELISA, competitive ELISA depends on the weakening of the signal to determine the amount of the target antigen in the sample. The lower the signal, the more target antigen is in the sample.
Competitive ELISA is useful if the antigen of interest is small and has only one or a limited number of possible epitopes. Any other ELISA method can be adapted into a competitive method as well.
Different instruments can be used to detect signals from ELISA reporter enzymes. Researchers may use a fluorometer to measure fluorescence, a spectrophotometer to determine optical absorbance, or a luminometer to measure luminescence.
Usually, a standard curve is first prepared from serial dilutions of a known antigen. Through the standard curve, the concentration of the target antigen in various samples can then be calculated.
Some ELISAs may also be qualitative, determining either the presence or absence of the target antigen in samples. The samples are compared to blank wells that contain no antigen or control wells containing a reference antigen.
To learn more about the ELISA mechanism, check out Boster Bio’s ELISA technical resources where you’ll find information about the ELISA assay principle, protocol, troubleshooting tips, and more. Boster Bio is a biotechnology company that has been providing high quality antibodies and ELISA kits to scientists since 1993.