Background to ELISA

What is ELISA?

Enzyme-linked immunosorbent assay (ELISA) is a laboratory technique, via which antigens can be detected in biological samples. Similar to the rest of the immunoassays, this technique also relies on specific antibodies to detect the antigen of interest. This detection consists of specific antigen-antibody interactions.

ELISA principles:

During the ELISA assay, the antigen is fixed to a solid surface, either directly or with the help of a capture antibody. Following the antigen immobilization, a detection antibody, which has been conjugated with a suitable molecule for a detection is added. This molecule can be a fluorophore or an enzyme.

The greatest advantage of this assay is its high specificity and sensitivity. ELISAs can easily detect antigens at the picogram level under the right conditions. Carrying out the experiment is relatively simple, thanks to the protocols that are supplied with the kits.

The main disadvantage of this assay is that the readout must be obtained in a short time, because the detection is based on the enzyme and substrate reactions. The information that can be obtained about the antigen is quite limited, because it solely relies on the amount of it present in the sample.

Types of ELISA detection:

ELISA uses the enzymatic label for production of a detectable signal, which is directly correlating to the antibody-antigen binding. Several types of enzymes and substrates can be used in this assay, leading to different methods being incorporated.

The major difference is that in direct ELISA only one antibody is used, compared to two that are needed for indirect ELISA.

Direct detection:

When performing direct detection, the antibody is conjugated to the detection enzyme, which is both cost and time effective. The downside to this method is that the antibody reactivity can be altered, due to the attached enzyme. Also, the enzyme labelled antibody has to be different for every target antigen.

Indirect detection:

In this type of detection, the antibodies are coupled with biotin and then a streptavidin-conjugated step is performed. Using unlabelled primary antibodies combined with either enzyme or biotinylated secondaries is also possible.