| Source: https://pixabay.com/photos/science-lab-laboratory-research-1029385/ |
A guest post by Emma Willson.
There are four different types of ELISA, each with its benefits and drawbacks.
Direct ELISA
A direct ELISA involves immobilizing an antigen to the surface of a microplate (usually a 96-well microplate) and adding only one enzyme-labeled (conjugated) antibody to directly bind to the antigen.
With the addition of a substrate for the enzyme, the antibody creates a signal that shows the level of analytes in the sample.
Advantages:
● A straightforward and fast method
● No risk of cross-reactivity
Disadvantages:
● Not very specific results due to using only one antibody
● Risk of high background reading
Indirect ELISA
An indirect ELISA is very similar to a direct ELISA, but it includes two antibodies. After immobilizing an antigen to the well of a plate, an unconjugated primary antibody is added to bind to the antigen.
The next step involves adding a secondary conjugated antibody to bind to the primary antibody. Then, a substrate is added to trigger a reaction of the enzyme in the secondary antibody that will produce a signal for measuring antigens.
Advantages:
● Higher sensitivity due to amplifying the signal with a second antibody
● Higher specificity
● High flexibility
Disadvantages:
● The secondary antibody increases the risk of cross-reactivity.
● Potential cross-reactivity could cause high background noise.
Sandwich ELISA
A sandwich ELISA test also includes adding two antibodies to an antigen sample, to “sandwich” the antigen.
As opposed to direct and indirect ELISA tests, a sandwich ELISA doesn’t immobilize the antigen, but rather one of the two antibodies, or matched antibody pairs.
After immobilizing a capture antibody to the microplate, an antigen is added to bind to it. The next step is binding the antigen by adding a detection antibody, which can be either conjugated or unconjugated.
In case the antibody is unconjugated, a secondary enzyme-labeled detection antibody is used to link to the primary one. Finally, a substrate for the enzyme is added to produce the signal for measurement.
Advantages:
● Highest level of sensitivity
● Highest specificity due to using two antibodies
● High flexibility
Disadvantages:
● Takes more time than any other type of ELISA
● Can be more expensive
Competitive ELISA
A competitive ELISA is similar to a sandwich ELISA, except there are no detection antibodies in the test. Instead, an enzyme-labeled antigen is added to a microplate after immobilizing a capture antibody to the surface.
An enzyme-labeled or conjugated antigen then binds to the antigen in the sample. Again, the last step involves adding a substrate for the specific enzyme that will create a signal for measuring the analytes in the sample.
If the signal is weak, it means that the enzyme-labeled antigen binds less to the capture antibody, which means that a high level of analytes is present in the sample.
Advantages:
● High sensitivity and flexibility
● Ability to measure tiny molecules
● Great for measuring immune responses
Disadvantages:
● Using only one antibody means lower specificity
● Requires an enzyme-labeled antigen
● The most complex ELISA test
ELISA kits are excellent for getting quick and accurate results when measuring antigens in various biological fluids. They’re very easy-to-use and allow for great flexibility, such as adjusting sample volumes to low concentrations of antigens and meeting various other quantitative demands.
