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Enzyme Linked Immunosorbent Assay (ELISA) is a basic, sensitive, reliable, and efficient analytical method employed for the quantification and detection of protein biomarkers in immunoassays.1

How Does an ELISA Work?

The ELISA method is established on the strength of the interaction between an antigen and an antibody, frequently called the binding specificity and affinity of the antibody to the substrate or antigen.

An antibody-substrate complex is produced when an antibody binds to an antigen. The binding strength can then be quantified by introducing a second antibody that is bound to an enzyme to the substrate complex.

The most frequently utilized enzymes are horseradish peroxidase (HRP) or alkaline phosphatase (ALP), as they can both identify many substrates.

In some applications, β-galactosidase, acetylcholinesterase, and catalase may be employed, but these have a smaller amount of substrates, which means they are not often used.3

The strength of the interaction between the antigen and the antibody can be determined by the addition of a substrate to the enzyme, triggering a chemical reaction that results in a change of color. The intensity of the color change can then be analyzed and employed to evaluate the interaction’s strength.

When Should an ELISA Protocol be Carried Out?

An ELISA can be utilized to either identify and quantify antibodies or to analyze the antigen concentration within immunoassays. A competitive or Sandwich ELISA are the two most common types utilized in diagnostics.

A Sandwich ELISA is frequently utilized to identify and measure antigens in immunoassays as opposed to competitive ELISA, which identify and measure antibodies.5There are a range of tips and tricks available to make the most out of any ELISA to maintain efficient research.

What are the Differences Between a Sandwich ELISA and a Competitive ELISA?

A sandwich ELISA is more robust and sensitive because the antibody binds to two locations on the antigen. This enhances the binding specificity of the primary capture antibody to the antigen along with the binding specificity of the detection antibody to the antigen.

A competitive ELISA is not as sensitive to experimental mistakes because it only needs a single binding site on the antigen. It is more efficient, flexible and has optimal reproducibility.

A weakness of the Sandwich ELISA is the high chance of non-specific binding and cross-reactivity, which can be decreased by utilizing primary monoclonal antibodies raised in various species.

St John’s Laboratory provides a range of cost-effective polyclonal and monoclonal antibodies produced in various species and a range of enzyme-conjugated secondary antibodies for use in any immunoassay. The organization also offers tips on selecting the correct antibody for a particular assay.

How to Detect and Quantify the Binding Affinity of the Antibody to the Antigen?

A color change that can be quantified and plotted is generated by the reaction of an enzyme to its substrate. The color intensity is directly proportional to a high presence of antigen in a Sandwich ELISA, whereas a strong color signal in a competitive ELISA is related to a small amount of antigen.

St John’s Laboratory offers complete ELISA kits available to use immediately for Sandwich and competitive ELISA.

References and Further Reading

1. Sakamoto, S., Putalun, W., Vimolmangkang, S., Phoolcharoen, W., Shoyama, Y., Tanaka, H., & Morimoto, S. (2018). Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of natural medicines, 72(1), 32–42. doi:10.1007/s11418-017-1144-z
2. Thiha, A., & Ibrahim, F. (2015). A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc. Sensors (Basel, Switzerland), 15(5), 11431–11441. doi:10.3390/s150511431
3. Lourenço E.V., Roque-Barreira MC. (2010) Immunoenzymatic Quantitative Analysis of Antigens Expressed on the Cell Surface (Cell-ELISA). In: Oliver C., Jamur M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology (Methods and Protocols), vol 588. Humana Press
4. Konstantinou G.N. (2017) Enzyme-Linked Immunosorbent Assay (ELISA). In: Lin J., Alcocer M. (eds) Food Allergens. Methods in Molecular Biology, vol 1592. Humana Press, New York, NY
5. Aydin, S. (2015). A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides, 72, 4-15.