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What is ELISA Protocol?

By H.R. Childress
Updated May 17, 2024
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An enzyme-linked immunosorbent assay (ELISA) is a common test used in immunology to detect antigens or antibodies. Antigens provoke an immune system reaction — in other words, they cause illnesses. ELISA is used to detect many bacterial and viral antigens, including human immunodeficiency virus (HIV), malaria, cholera, measles, and mumps. A slightly different ELISA protocol can be used to detect antibodies to these and many other pathogens. An ELISA protocol is the step-by step procedure for performing a specific ELISA.

Though the ELISA protocol varies slightly, depending on the type of ELISA being performed, the basic concept is the same for all of them. ELISA depends on enzyme-linked antibodies, also called antiglobulins. A signal molecule attached to these antiglobulins causes a substrate added during the assay to change color, if the desired antigen or antibody is present. The amount of antigen or antibody present is proportional to the amount of color change, which can be measured with an electronic plate reader.

There are three main types of ELISA. A direct ELISA is usually used to detect antigens rather than antibodies. This is the simplest ELISA protocol, as the enzyme-linked antibody binds directly to the desired antigen. Substrate is then added to determine the quantity of antigen present.

An indirect ELISA is used to detect antibodies, while another type of indirect ELISA — called a sandwich ELISA — can be used to detect antigens. A sandwich ELISA protocol requires two antibodies, called the capture and detection antibodies, to bind to the desired antigen. An antiglobulin is added, which binds to the detection antibody, and the substrate is added. The indirect ELISA follows a similar protocol, but uses a capture antigen rather than a capture antibody in order to detect the desired antibody.

Competitive ELISA is often used for detecting antigens that are present in low concentrations, because it is a very sensitive assay. In contrast to the other ELISA protocols, competitive ELISA uses an enzyme-linked antigen instead of an antibody, and a slightly different quantification method. In it, the sample containing the antigen to be detected and the enzyme-linked antigen are both added to an antibody, and the two antigens compete for antibody-binding sites. When the substrate is added, the color change is greater, with a higher ratio of bound enzyme-linked antigens to bound sample antigens. This means that higher color change is detected at lower concentrations of the sample antigen, which is what makes this method so sensitive.

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Discussion Comments
By ysmina — On Mar 26, 2012

@simrin-- Yes, an ELISA protocol can give a false positive. Of course it's something that is being worked on but it hasn't been resolved as of yet.

A false positive can happen when there are different antibodies that are really similar to one another in structure. So the ELISA protocol can react to a wrong antibody confusing it with a different one because it can't tell the difference.

I do think you can get an accurate result with the competitive protocol for ELISA since it tests for antigens and not antibodies. If there was a case of a false positive, it should be eliminated there.

I'm not sure what other test the hospital suggested to you is. But ELISA is one of the most affordable diagnostic tests available. So I don't think there is any harm in having it done again.

By SteamLouis — On Mar 26, 2012

@burcidi-- Do you know why the ELISA gives false positives sometimes?

I think my ELISA test had a false positive because the result came back as inconclusive. I was tested for malaria. They want me to take another test now to see what the results will be for that one.

I think the ELISA came back positive because the nurse said that I might have malaria but they're not entirely sure. So it's probably a false positive. I'm not sure what this means though.

Doesn't the false-positive issue get resolved at the lab? Is it possible to get false-positives during direct ELISA protocol? Do you think I should ask for a competitive ELISA test?

I'm kind of worried, I would appreciate any suggestions.

By burcidi — On Mar 26, 2012

My brother works in a lab where they develop ELISA assay protocol for different viruses and antigens. From what he tells me about it, it's hard work.

As far as I understand, he tries to make various combinations of antibodies, proteins and enzymes so that the ELISA protocol he's working with correctly identifies the virus. Sometimes it takes a lot longer than expected, he can get false positives and might have to start all over.

What this lab and similar labs are doing is really important though. ELISA is used for diagnosis of so many different diseases. The more developed and accurate ELISA tests are, the better it will be for patients.

So I have a lot of respect for his work. When I didn't understand any of this, I thought he was playing around with viruses in the lab. But it's actually a really big deal.

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