Tuesday, 19 May 2020

4 Types of ELISA with Their Advantages and Disadvantages

Source: https://pixabay.com/photos/science-lab-laboratory-research-1029385/
ELISA (Enzyme-linked Immunosorbent Assay) is a technique used in labs to measure the levels of antigens or antibodies in biological fluids. ELISA kits are commonly used to analyze plasma, saliva, urine, serum, cell culture supernatants, and lysates of broken-down cells and tissues due to enzymes and viruses.

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.


A straightforward and fast method
No risk of cross-reactivity


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.


Higher sensitivity due to amplifying the signal with a second antibody
Higher specificity
High flexibility


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.


Highest level of sensitivity
Highest specificity due to using two antibodies
High flexibility


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.


High sensitivity and flexibility
Ability to measure tiny molecules
Great for measuring immune responses


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.

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)


Pharmaceutical Microbiology Resources

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