Elisa Test

Elisa Test

What is an Elisa Test ?

The enzyme-linked immunosorbent assay, or ELISA, is a widely used laboratory method for quality and quantity analysis of substances present in various samples. Since the method has been around for over 50 years, there has been developed various modifications, but the general principle remains the same. The ELISA method uses antibodies immobilized on the surface of the wells of microwell plates. Into those wells the samples are pipetted. The antibodies immobilized in the wells capture the antigen of interest during the incubation. From here come the “immunosorbent” part of the name. Then a washing step is performed, and the sample is removed from the well, leaving only the antigen of interest captured by the antibodies. Then a second antibody is added to each well that also recognizes the same antigen, the one already captured on the immobilized antibodies. Another incubation follows and the another washing to remove the unbound antibodies. The second antibodies are conjugated with the enzyme Horseradish peroxidase (HRP), hence “enzyme-linked immuno assay”. After this a substrate for the HRP is added. Several such substrates are used but what they all have in common is the color reaction. The HRP oxidases the substrate and as a result the substrate precipitates into colored product. Other substrates become luminescent after oxidation and emit light. The choice of substrate depends on the specific purpose of the assay. The amount of the protein or other antigen of interest from the analyzed samples correlates to the intensity of the color or the emitted light.

Why is this correlation available?

Because the more antigen is present in the sample, the more antigen molecules will get caught on the immobilized antibodies. More caught antigens mean that more of the second antibodies (the ones linked to HRP) will remain in the wells after the second washing step. And the more HRP is present in the sample, the more of the substrate will be converted to a colored product for given time. The reaction is stopped by using a stop solution – an acidic buffer that makes the HRP inactive. The period in which the HRP is allowed to work on the substrate is strictly fixed and the instructions should be followed closely, otherwise the quantitative results will be inaccurate.

Why will this happen?

Let’s say that you have samples in two wells. In the first well the antigen was in such amount that in the end there are 100 units of HRP present in this well. In the second well the antigen was twice less than in the first well and only 50 units of HRP are present after the second wash. Let’s say that each unit of HRP oxidases 1 molecule of substrate per 1 second. You add to both wells the same amount of substrate at the same time. You let the reaction go on for 100 seconds in the first well. During this time the 100 units of HRP oxidase 100 molecules of the substrate. When you stop the reaction in the first well, you leave the reaction in the second well for another 100 seconds (200 seconds in total). During this time the 50 units of HRP will convert 100 molecules of the substrate, same as in the first well. When you compare the intensity of the color if the two wells, they will be the same (both wells will have 100 molecules of oxidized substrate). This will lead to the inaccurate conclusion that the antigen of interest is present in the same amount in the two samples. That is why stopping all the reactions after the same time is very important for the accuracy of your results.
The above described principle is called “sandwich ELISA”. There are a few other modifications such as:

Direct ELISA

in this case the sample of interest in incubated in a plate. The proteins from the sample adhere on the plate due to forces generated by electrical charges. Amongst all the immobilized antigens the protein of interest will also be present. The wells are washed to remove the antigens from the samples that didn’t adhere on the plate. Then a labeled with HRP antibody is added in the wells. The antibody will bind to the specific antigen. Another washing is performed to remove the unbound antibody. Then the HRP substrate is added and everything continues as with the sandwich ELISA.

Competitive ELISA

the difference here is that before adding the sample in the wells (in this case covered with the antigen), the sample is incubated with unlabeled antibodies. Then the complex of antigen-antibody is added to the wells and incubated. The more antigen I present in the sample, the more antibody-antigen complexes will be form and the less unbound antibodies will remain. The unbound antibodies will attach to the antigen that is covered on the well. Then a wash follows. Afterwards a secondary antibody is added that recognizes the primary antibody. The secondary antibody is conjugated with HRP. After another wash an HRP substrate is added. The more free antibodies were available in the sample as a result of less antigen presence (less antigen-antibody complexes formed), the more antibodies will remain free to bind to the antigen on the well and remain there. In turn, this means that more secondary antibodies (conjugated with HRP) will bind to the primary antibodies and remain in the wells. This will lead to higher HRP activity and more intense color. In this case the higher the intensity of the color, the less antigen was present in the analyzed sample.
In conclusion, despite the various modifications, all ELISA tests, in their essence, are a clever utilization of antibodies’ ability to recognize antigens with high specificity. This property coupled with an appropriate color/fluorescence signaling molecules conjugated to the antibodies allow us not only to prove the presence or absence of a molecule in a sample but also to calculate with great accuracy its concentration.

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