ELISA was originally conceived independently by Eva Engvall and Peter Perlman of Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen of the Netherlands. They sought an immune assay that could determine the presence of antigens or antibodies instead of radioimmunoassays because radioimmunoassays used potentially dangerous radiolabeled antigens or antibodies, so they designed an enzyme-based alternative.
There are now four main ELISA types, direct method, indirect method, sandwich method and competition method. The following pictures illustrate the determination of antigens; however, the same principle applies to the determination of antibodies, except that the antigen and primary antibodies do the opposite.
ELISA Direct MethodIn the ELISA Direct Method, antigens or antibodies in the sample are directly adsorbed onto the coated plate in a nonspecific manner. Then, the specific assay antibody or antigen is ligated into the well. The assay substrate is then used to generate measurable color changes and quantified on the plate reader. Because this detection method has fewer steps, faster speeds, and has fewer chances of introducing errors than other ELISA methods. However, because the adsorption step is nonspecific, the background noise can be high. The absence of secondary antibody steps means no signal amplification, reducing detection sensitivity. It also requires the creation of conjugated assay antibodies/antigens for each desired target.
ELISA indirect methodELISA indirect method, or iELISA, was originally developed in 1978 to detect human serum albumin. It works very similarly to the ELISA direct method, except that it adds a secondary antibody step. This amplifies the test signal and overcomes the limitations of the ELISA direct method. It also negates the need for target-specific conjugation assays of antibodies/antigens, since the conjugated secondary antibodies only need to be species specific to primary antibodies. If the total sample antigen is bound to the coated plate, background noise remains a problem, just like the ELISA direct method. However, if the assay method is used to detect sample antibodies, the purified target antigen is coated on the plate and the primary antibody is derived from the sample. This greatly reduces background noise, so these assays are most popular in determining antibody titers in samples. Disadvantages of the ELISA indirect method include longer regimen time, more opportunities for error, and the possibility of cross-reacting with secondary antibodies.
ELISA Sandwich MethodAs the name suggests, the ELISA sandwich method sandwichs the antigen between antibodies. The technology was developed in 1977 and can be used in the form of ELISA direct or indirect methods (the ELISA sandwich method described above is based on the ELISA indirect method). In addition to the nonspecific binding of the antigen to the detection plate, the capture of the antibody makes it a specific process. This combination further improves the sensitivity and specificity of the detection. However, they do require determining compatible capture and assay antibody pairs to function effectively, and there may be problems with cross-reactions. This detection method also usually has the most steps, providing a greater chance of error. Due to the selectivity of the antigen binding step, the ELISA sandwich method is particularly useful when the antigen is in a complex mixed state, since there is no need to purify the antigen.
ELISA Competition LawELISA Competition Law, also known as ELISA inhibition method or ELISA blocking method, may be the most complex one of ELISA technology. Originally developed in 1976 to detect human chorionic gonadotropin, the detection method creates an inverse relationship by detecting interference to the expected output signal levels. The more targets are in the sample applied, the lower the measured output signal. Other ELISA methods can also be changed to competition methods. There are two general principles here: the sample antigen or antibody competes with the reference, respectively, and binds to a limited number of labeled antibodies or antigens; or, the sample antigen or antibody can compete with the labeled reference, respectively, and binds to a limited number of antibodies or antigens. The ELISA competition law, like the form it takes, has some of the same advantages and disadvantages. However, its effect is highlighted when the antigen is smaller, limiting the ability of two antibodies to bind simultaneously (as required by the ELISA sandwich method), or when only one antibody is available.
The types of ELISA tests have their own characteristics, and choosing the right test method is the key to ensuring the accuracy of the experimental results. Whether it is direct ELISA, indirect ELISA, sandwich ELISA or competitive ELISA, they all have their own unique application scenarios and advantages. In practice, researchers should make choices based on factors such as sample type, experimental objectives, and required sensitivity. With the advancement of biotechnology, ELISA technology is also constantly improving, providing strong support for the fields of medical care, scientific research and public safety.
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