Enzyme Linked Immunosorbent Assay (ELISA)

by Elisakits BioSupply Manufacturer & Supplier

Enzyme linked immunosorbent assay which is most commonly referred to as ELISA for short is a routinely used qualitative and quantitative procedure in order to measure proteins present in a biological sample. There are many different variants which exists for this immunoassay method and in majority of cases the samples that are being analysed will dictate which method to be used.

What is an ELISA test?

It is essentially a test which can be used to detect and measure antibodies present in the blood. This assay can detect specific antibodies which could be related to specific infectious conditions/diseases. This is because antibodies are defined as proteins which are produced and released by the body in response to antigens (harmful substances). ELISA assays are popular as a screening tool as well as being used for more specific in-depth analysis.

The ELISA method was developed in the 1960s and 1970s and is now an established procedure which uses specific antibodies in order to analyse specific proteins that are present using a chromogenic, luminescent or fluorescent readout. Also, the absolute concentration of the protein being tested can be worked out, since accurate standards are provided with each ELISA kit, standards are solution of the target protein at known concentrations.

Different types of ELISA tests?

There are four different categories of ELISA tests: direct, indirect, sandwich and competitive ELISA.

Direct ELISA: This involves attaching an antigen to the polystyrene plate and then using an enzyme labelled antibody. The enzyme is detected in order to illustrate the signal of the antigen, there is a direct correlation between the signal generated and the amount of antigen present. This is one of the fastest ELISA methods since only one antibody is used and only a few steps are required. However, this procedure time consuming which carrying out a large number of experiments, since each primary antibody needs to be labelled individually. It is mainly useful for highly specific antibody-to-antigen reactions.
Indirect ELISA: Involves a two-step procedure, where the primary antibody is initially incubated with the antigen and this is followed by the addition of a secondary antibody. This results in the primary antibody being sandwiched between the coated antigen and the enzyme labelled secondary antibody (usually an anti-species globulin conjugate). In this method many labelled antibodies can be bound on the antigen molecules and these can be detected using a single labelled secondary antibody molecule. This makes this method highly sensitive, flexible and cost savings (since fewer labelled antibodies are needed).

Sandwich ELISA: This procedure is able to quantify the amount of antigen present using two layers of antibodies (one used for the capture and the other used for detection). This means that in this method the antigen needs to have a minimum of two antigenic epitopes that are able to bind each of the antibodies since at least two antibodies act in the sandwich process. Monoclonal antibodies are intended to recognise a single epitope and therefore are extremely useful in allowing fine detection and quantification of minor differences present in antigens whereas polyclonal antibodies can be used to capture as much as antigens as possible. The benefits of a sandwich ELISA is that sample do not need to be purified and it can offer better sensitive (between 2-5 times better when compared to direct and indirect method). The properties of a sandwich ELISA make it suitable for measuring complex samples, however, optimising this method could take longer.

Competitive ELISA: A process of competitive binding occurs between the sample antigen and an add-in antigen. In this case the greater the sample antigen concentration, then the weaker the signal that is generated (i.e. inverse correlation). One of the major benefits of this protocol is that it allows impure and crude samples to be used and still offer selective binding of any antigen present.

Final Thoughts

In comparison to many different immunoassay protocols, the ELISA test provides many advantages, it is considered as being highly specific, sensitive, flexible and generates results which are comparable with other methods (such as radioimmune assay, RIA). ELISA tests are much safer since no radioactive substances are required or expensive radiation counting equipment is needed. The many advantages of the ELISA method makes is an ideal biotechnical tool that can be used in a large number of applications in scientific research or in the diagnosis of disease conditions.

Further Reading

https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366430/

https://www.ncbi.nlm.nih.gov/pubmed/25908411

https://www.researchgate.net/publication/255958640_Enzyme_Immunoassay_and_Enzyme-Linked_Immunosorbent_Assay