Choosing Between a Sandwich and Competitive ELISA
1. What is an ELISA?
Enzyme Linked Immunosorbent Assay (ELISA) is a simple, quick, sensitive and reliable analytical technique used for detecting and quantifying protein biomarkers in immunoassays1. ELISA’s have been used for decades as a diagnostic tool and remains a gold standard for pharmacological and clinical diagnostics2.
2. How does an ELISA work?
The ELISA technique is based on the strength of interaction between an antibody and an antigen, commonly referred to as the binding specificity and affinity of the antibody to the antigen or substrate. When the antibody binds to the antigen, it forms an antibody-substrate complex. The binding strength can then be measured by attaching a second antibody which is bound to an enzyme to the substrate complex. Common enzymes used are alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as these can recognise a lot of substrates. In some instances,β-galactosidase, acetylcholinesterase and catalase may be used although these have limited substrates and are therefore not widely used3.
The strength of interaction between the antibody and the antigen can be measured by adding a substrate to the enzyme, which triggers a chemical reaction leading to a colour change. The intensity of the colour change can then be measured and used to assess the strength of interaction.
3. When would you carry out an ELISA protocol?
An ELISA can be used to either detect and quantify antibodies or measure the concentration of antigens in an immunoassays. Sandwich and competitive ELISA are the two main types used in diagnostics. A Sandwich ELISA is commonly used to detect and quantify antigens in immunoassays whereas competitive ELISA detects and quantifies antibodies5. There are various tips and tricks to get the most out of your ELISA and keep your research on track.
4. What is the differences between a Sandwich ELISA and a Competitive ELISA?
A sandwich ELISA is more sensitive and robust as the antibody binds to two sites on the antigen. This increases the binding specificity of the primary capture antibody to the antigen as well as the binding specificity of the detection antibody to the antigen.
A competitive ELISA on the other hand is less sensitive to experimental errors as it only requires one binding site on the antigen. Nonetheless, It is quicker, more flexible and has good reproducibility.
A disadvantage of the sandwich ELISA is the risk of cross reactivity and non-specific binding, which can be reduced by using primary monoclonal antibodies raised in different species.
St John’s laboratory has a range of affordable monoclonal and polyclonal antibodies raised in different species and a variety of enzyme conjugated secondary antibodies for use in your immunoassay. We also have tips on choosing the right antibody for your assay.
5. How to detect and quantify the binding affinity of the antibody to the antigen?
The reaction of an enzyme to its substrate generates a colour change which can be quantified and plotted. With a sandwich ELISA, the colour intensity is directly proportional to the amount of antigen present whereas a strong colour signal in a competitive ELISA correlates to a low amount of antigen.
St John’s laboratory provides complete ELISA kits ready to use for competitive and sandwich ELISA.
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.