ELISA (Enzyme-linked immunosorbent assay)

Definitions & Key takeaways

ELISA is a biochemical assay used in immunology to detect the presence of an antigen, antibody, or another protein. It takes its name from the enzyme-linked immunosorbent assay (ELISA).

The basic principle behind ELISA is that if an antigen or antibody is present in a sample, it will bind to a specific antibody or antigen attached to a solid support. The bound antigen or antibody can then be detected using an enzyme-linked secondary antibody that recognizes the primary antibody or antigen. The presence of this enzyme can be detected using various methods depending on the assay format, such as spectrophotometry, fluorimetry, or chemiluminescence. ELISA is a very sensitive assay that can detect minute levels of antigens or antibodies in a sample. It is often used to measure the concentration of proteins in body fluids, such as serum or urine.

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Enzyme-linked immunosorbent assay - or ELISA for short - is a laboratory technique or assay where antibodies or antigens in people’s samples are immobilized in a surface, and then detected by an antibody with an enzyme attached that causes a change of color.
So it’s useful to diagnose infections, such as HIV, hepatitis B, or malaria; and autoimmune diseases, like Graves’ disease, systemic lupus erythematosus, or rheumatoid arthritis, where the body reacts to its own proteins as if they were foreign antigens.
Immune responseWe are constantly surrounded by harmful microorganisms called pathogens, that could cause a lot of damage if it wasn’t for the immune system.
These pathogens have unique parts called antigens. Now, B lymphocytes are cells of the immune system that can detect those antigens and trigger an immune response by secreting lots and lots of antibodies.
These antibodies, also called immunoglobulins or Ig, are Y-shaped proteins that have two regions: the constant fragment region, also called Fc, that determines the antibody class - IgD, IgM, IgG, IgA, or IgE; and two fragment-antigen binding, or Fab regions, that recognize and bind to the antigen in order to inactivate it, preventing the pathogen from reaching its target cell and causing damage.
Principle of ELISANow, the basic idea with ELISA is to use that specific link between the antigen and antibody to help diagnose infections or autoimmune diseases.
More specifically, ELISA can be used to look for either the pathogen’s antigens or the antibodies our body secretes against them.
More than that, it also makes them visible or measurable by using an antibody against the suspected antigen or antibody.
But this antibody comes with a +1: it has an enzyme attached, which can modify a substrate called a chromogen. Chromogen literally means that it generates color, so the change in color determines whether the test is positive or negative.So, think of an ELISA test like a cooking recipe.
We’re going to need a cooking pot, and some ingredients, or reagents. The cooking pot is a transparent plate with 96 wells, that’s made up of a special material that allows proteins to easily attach to its surface, including the walls and the bottom of the wells.
And there are seven reactants: the patient’s serum sample; some nonspecific proteins, like bovine serum albumin or casein, that don’t interact with any other reactant but can only attach to the well’s surface; the enzyme-linked antibody; the chromogenic substrate; a saline solution combined with non-ionic detergent used to wash the wells between every step of the procedure; a stop solution, like sulfuric acid; and two control samples, a positive control, that is a solution known to have the antigen or antibody you expect to find, and a negative control that doesn’t have the antigen or antibody.
Now, there are three main types of ELISA: direct, indirect, and sandwich ELISA. And depending on the type, other reactants may be needed.So, with ELISA, a positive control is put in one of the wells, a negative control in another, and as many samples as needed in the rest of the wells.
You can put one sample in each well, so a lot of patients can be tested with a single ELISA plate. But let’s rewind a little and zoom in on one of the 96 wells of the plate to see how this whole thing works.
Direct ELISASo let’s take a look at direct ELISA to get started, which is used to detect antigens. First, the serum sample is put in the well and incubated.
During this time, the proteins present in the sample, among them the expected antigen, adhere to the surface of the well.
After that, the exceeding sample solution is discarded and the well is washed a few times, so that only the attached proteins remain in the surface.
However, some empty gaps between them could remain. If these are left empty, the enzyme-linked antibody that’s added next, could bind to these sticky sites and give a false positive reaction.
So next, nonspecific proteins are added, which attach to all the remaining gaps. After an incubation period, the well is washed again.
Next, the solution with the enzyme-linked antibody specific to the searched antigen is added, incubated, and then washed again.
If the antigen is present in the sample, the enzyme-linked antibody binds to it and can’t be removed by washing. However, if the antigen isn’t in the sample, the enzyme-linked antibody has nothing to attach to, and is washed up.The next step is to add the chromogen and incubate again, this time in a dark place to let the reaction happen.
After a while, the plate is removed from the darkness and the reaction is stopped with a stop solution. If the enzyme-linked antibody found its antigen, the chromogen generates color, usually yellow, which translates as a positive result.
The more intense the color, the concentration of the antigen in the sample is greater. If the antigen wasn’t there, there’s no change in color, and this is a negative result.
For more accurate results, the intensity of the color can be measured by spectrophotometry, with a machine called an ELISA reader, to determine whether the test result is positive or negative.
Now, direct ELISA is the simplest type, but nowadays the indirect and sandwich ELISA have largely replaced it because they’re more sensitive techniques.
Indirect ELISASo, to understand the difference between indirect and sandwich ELISA, let’s say we’re trying to diagnose an HIV infection.
Both tests are appropriate for the situation, but indirect ELISA can detect IgG antibodies in the serum directed against HIV antigens, whilst sandwich ELISA can detect the HIV antigen itself.
So, basically with indirect ELISA, there’s an HIV antigen that attaches to the well’s surface; a primary antibody that binds to it, which is the IgG antibody we’re looking for in the serum; and then the secondary enzyme-linked antibody.
Then, the chromogen is added; and if the IgG antibody was present in the serum, the chromogen generates color, which translates as a positive result.
An ELISA reader can also be used and the results are interpreted the same way as direct ELISA.Sandwich ELISAFinally, with a sandwich ELISA test, we can detect HIV antigens instead of antibodies against those antigens.
Here, there’s basically an antibody-antigen sandwich: there’s a primary antibody that attaches to the well’s surface; then the HIV antigen that binds to it, which is the one we’re looking for in the serum; and finally the secondary enzyme-linked antibody.
Then, the chromogen is added; and if the HIV antigen was present in the serum sample, the chromogen generates color, which translates as a positive result.
An ELISA reader can also be used and the results are interpreted the same way as the other ELISA tests.##SummaryAll right, as a quick recap...Enzyme-linked immunosorbent assay - or ELISA for short - is a laboratory technique used to diagnose infections, and autoimmune diseases.
To do this you can look for either the antigens or the antibodies against them. With direct ELISA you can detect antigens in a sample by matching its corresponding enzyme-linked antibody to reveal its presence.
With indirect ELISA you can use an antigen attached to the well to select an antigen-specific antibody in a sample, and then match a secondary enzyme-linked antibody.
With sandwich ELISA you can use an antibody attached to the well to select an antigen in a sample, and then match a secondary enzyme-linked antibody; like with direct ELISA, but more sensitive.
Finally, by adding the chromogen, a color change indicates the presence of the antigen or antibody you’re looking for. You can use an antibody attached to the well to select an antigen in a sample and then match a secondary enzyme linked antibody like with directly but more sensitive Finally by adding the Chromagen a color change indicates the presence of the antigen or antibody you're looking for