ELISA (Enzyme-linked immunosorbent assay)


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 response

We 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 ELISA

Now, 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 ELISA

So 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.