Enzyme-linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is an immunological method for the detection and quantification of proteins or antibodies [1]. The substance to be detected is first bound to a microtiter plate via a primary antibody, then a so-called enzyme-linked detection antibody initiates a color reaction [2]. The signal is quantified by measuring the optical density in a photometer at the appropriate wavelength [1]. The ELISA replaces the radioimmunoassay applied for a long time, in which radioactive material is used for detection [3]. A brief overview of the different ELISA methods is given below.
Overview of different ELISA methods. In direct ELISA, an enzyme (red ball)-coupled primary antibody is used that binds to the antigen (blue ball). In indirect ELISA, a labeled secondary antibody is used in addition to a primary antibody. In the case of sandwich ELISA, the antigen is surrounded by two antibodies, using a second secondary antibody to which an enzyme is coupled. In competitive ELISA, labeled antigen (orange ball) competes with unlabeled antigen from the sample for binding sites on the detection antibody, resulting in an inversely proportional relationship between signal strength and concentration of the target antigen. In each case, the coupled enzyme converts a specific substrate, producing a detectable signal (figure created with biorender.com).
Direct ELISA
In direct ELISA, a microtiter plate is coated with the antigen to be detected. An enzyme-linked primary antibody is then added, which binds to the antigen. Subsequently, a substrate is added, which is reacted by the enzyme, generating a detectable signal [4]. Direct ELISA is often used in serological diagnostic test systems to detect antibodies directed against a pathogen antigen as an indication of infection. The method is characterized by rapid performance, as only one antibody needs to be applied. On the other hand, this method is quite time-consuming and expensive in production because a suitable antibody must be labeled for each antigen to be detected [1]. This disadvantage is circumvented in indirect ELISA.
Indirect ELISA
In indirect ELISA, too, the antigen is first immobilized by direct adsorption to the microtiter plate. However, the primary antibody binding to the antigen is not labeled in this case. Instead, a capture antibody is used, which is immobilized on the microtiter plate and binds the antigen. Another, unlabeled detection antibody binds the antigen at a different site so that it is surrounded by two antibodies, the so-called sandwich. A labeled secondary antibody is now added to bind to the second primary antibody, allowing for an enzymatic color reaction [6]. By using two antibodies to capture and detect the target antigen, the sandwich ELISA shows both high sensitivity and specificity and is therefore the most commonly used variant of the assay [5].
Sandwich ELISA
In the so-called sandwich method, the antigen is not bound directly to the substrate. Instead, a capture antibody is used, which is immobilized on the microtiter plate and then binds the antigen. Another, unlabeled detection antibody binds the antigen at a different site so that it is surrounded by two layers of antibody, the so-called "sandwich". A labeled secondary antibody is now added to bind to the second primary antibody and catalyze an enzymatic color reaction. By using two antibodies to capture and detect the target antigen, the sandwich ELISA shows both high sensitivity and specificity and is therefore the most commonly used version of the assay [5].
Competitive ELISA
The competitive ELISA is often used when the antigen is very small or has only one epitope. In this method, labeled antigen is used, which competes with the unlabeled antigen from the sample for the binding sites on the detection antibody [5]. The relationship between the concentration of the antigen to be detected and the signal strength is therefore inversely proportional: the lower the signal strength, the greater the amount of the protein in the sample [6]. In our portfolio you will find, among others, Acetylcholinesterase (AChE) ELISAs from our partner Cayman Chemical, which are a well-known example of competitive ELISAs.
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Sources
[1] E. Engvall & P. Perlmann. Enzyme-linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochemistry, 8(9), 871-874 (1971).
[2] https://de.wikipedia.org/wiki/Enzyme-linked_Immunosorbent_Assay, 23.01.2023
[3] R. Yalow & S. Berson. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39(7): 1157–1175 (1960).
[5] Alhajj M, Zubair M, Farhana A. Enzyme Linked Immunosorbent Assay. StatPearls (2023) PMID: 32310382.