Beginners Guide to Understanding ELISA Assay

What Are ELISA Assays?

ELISA is an acronym for Enzyme-Linked Immunosorbent Assay. Like all bioassays, these are tests used to determine the presence and concentration of a certain substance. ELISA assays are typically used to identify and measure hormones, antibodies, proteins, and peptides. These are a kind of biochemical assay first described by Engvall and Perlmann in 1971.

Since ELISA assays are essentially immunoassays, they function in much the same way. An antibody is used to identify a target antigen through specific reactions between the antibody and the antigen. We mostly know ELISA assays as the tests that are used to detect HIV or HBV.

What Is The Basic Principle Underlying ELISA Assays?

The first step is to immobilize the antigen on the surface of a multi-well plate. It can be done directly or by immobilizing an antibody on the surface and using it to capture the antigen. This antigen is then connected to an antibody that is itself conjugated to a molecule that can be detected. This detectable molecule can either be a fluorophore or an enzyme.

ELISA assays are normally done on multi-well plates that have anywhere between 96 to 384 wells. If the analytes can be immobilized, the antigen can be separated from the other constituents of the sample. This is what allows scientists to perform ELISA assays on different samples at the same time.

Four Types of ELISA Assays

1. Direct ELISA- The antigen can directly be immobilized in this process. After which, it is detected by an antibody specific to that antigen. This antibody is conjugated with a detectable molecule like HRP.

2. Indirect ELISA- A two-step process has to be followed for detection. Here the detectable molecule carrying antibody cannot directly conjugate with the antigen. A primary antibody that is specific to the antigen first has to bind with the antigen.

Then a secondary antibody – which is opposed to the host species of the main antibody – must become bound to that antibody. Since the secondary antibody carries the detectable molecule, only after it attaches to the combination of the primary antibody and the antigen, does the antigen become detectable.

The first step however remains the same. Similar to Direct ELISA, the immobilization of the antigen occurs on the surface of the multi-well plate. If an antibody needs to be detected in serum, this method of Indirect ELISA can be followed. The primary antibody just has to be replaced with the serum.

3. Sandwich ELISA- This is quite similar to the Indirect ELISA in principle, but there is a slight difference. The two antibodies trap the antigen between them in a sandwich structure. One of the antibodies is meant to capture the antigen, while the other is meant to make it detectable.

The two antibodies are used as specific to two different epitopes of the antigen. Therefore these antibodies are also called matched antibody pairs. The first antibody is coated on the multi-well plate.

The presence of this on the surface immobilizes and captures the antigen. This is when the second antibody, carrying the detectable molecule, is added and completes the sandwich structure, making the whole combination detectable.

This is the most commonly used type of ELISA assay.

4. Competitive ELISA- These are also known as Inhibition ELISA. This kind of assay not only detects the antigen but also measures the concentration of the antigen by detecting signal interference.

A specific quantity of antibodies is taken for this purpose. The antigen whose concentration is to be tested is called the sample antigen. Another antigen is taken as the baseline. It is called the reference antigen. Whether or not the sample antigen can beat the reference antigen at binding with the antibody determines how strong or weak it is.

The reference antigen is coated on the surface of the multi-well plate and the sample antigen is added to the wells. The sample antigen must be pre-incubated with the predetermined antibody. Then the antibody is introduced into this combination.

If the concentration of the sample antigen is higher, then the antibodies are supposed to fuse with them more than the reference antigen. And that’s why if a lower quantity of reference antigen is detected, it means the concentration of the sample antigen is high.

To put it simply- the weaker the signal by the reference antigens, the stronger the antigen concentration is in the sample.

All the above ELISA assays can be modified to be performed competitively. Experienced scientists working for a reputed biochemistry lab will be able to help you choose the appropriate ELISA assay for your purpose.

Which Type of ELISA Assay Should You Use?

That depends on your priorities. If you’re afraid of cross-reactivity from another antibody and want to save time, go for Direct ELISA. If flexibility is your priority, then opt for Indirect ELISA. You can use the same secondary antibody for the detection of several primary antibodies.

If you have a complex sample on your hands, you should opt for Sandwich ELISA. And as mentioned before, Competitive ELISA is simply a modification of the other types to test the concentration of the antigen.

There are some drawbacks to all the types as well. Direct ELISA might be quick and easy, but it does not allow any flexibility of signal amplification. Indirect ELISA takes a long time, and there is a risk of the two antibodies producing cross-reactions. Sandwich ELISA is difficult to perform simply because finding two matching antibodies that suit the epitopes of the same antigen is not easy.

In Conclusion

There are a variety of ELISA kits available on the market. But not all research kits can be used as diagnostic kits. It must be kept in mind because ELISA assays are used extensively to test for HIV, HBV, Lyme Disease, influenza, and even food allergies. Finding the right type and the kit is the key.