General principle and reaction mechanism
In enzyme-linked immunosorbent assay (ELISA), a key role is played by the conjugate — antibodies chemically linked to an enzyme. Horseradish peroxidase, $\beta$-galactosidase, or alkaline phosphatase are most commonly used as labels.
The reaction proceeds in several stages:
- Formation of the antigen-antibody immune complex.
- Addition of a specific substrate or chromogen.
- The enzyme in the conjugate cleaves the substrate.
- As a result of cleavage, the color of the solution changes.
The intensity of the resulting color directly depends on the number of bound molecules. This allows for quantitative evaluation of the analyte. To stop the reaction at the desired moment, an acid is added to the mixture, which denatures the enzyme.
Solid-phase ELISA
Solid-phase ELISA (ELISA) is the most widely used. Its main feature is that one of the components (antigen or antibody) is pre-attached (sorbed) to a solid support. Typically, the wells of polystyrene microplates serve as the support.
A crucial condition for the successful performance of solid-phase ELISA is thorough washing of the plate wells after each addition of a new reagent. This is necessary to completely remove all unbound molecules and prevent false-positive results.
Main types of ELISA
Depending on the diagnostic goals, different assay schemes are used:
- Antibody detection (serodiagnosis): The goal is to find specific immunoglobulins against the pathogen in the patient's serum. A known antigen is immobilized on the bottom of the well. When the patient's serum is added, their antibodies (if present) bind to the antigen. Then a conjugate (antiglobulin serum with an enzyme) is added, which attaches to the Fc fragments of the patient's antibodies. The addition of the substrate causes coloration.
- Antigen detection ("sandwich method"): Used to search for the pathogen itself or its particles. Diagnostic antibodies are immobilized in the well. They "capture" the target antigen from the patient's serum. After this, labeled antibodies against the same antigen are introduced, completing the complex ("sandwich"). The presence of the enzyme is confirmed by a color reaction.
- Competitive ELISA: The method is based on the competition of molecules for a limited number of binding sites. For example, when searching for an antigen, the patient's target antigen and the added labeled standard antigen compete for binding to the antibodies.
Clinical applications
Due to its ultra-high sensitivity (detecting concentrations down to $10^{-10}$ — $10^{-12}$ g/L), ELISA is widely used in modern medicine.
Main areas of application:
- Diagnosis of infectious diseases: viral (including hepatitis B and HIV), bacterial, and parasitic.
- Quantitative determination of biologically active molecules such as hormones and enzymes.
- Monitoring the concentration of drugs in the body.
Briefly: answers to topical questions
Which classes of antibodies (immunoglobulins) are differentially determined by ELISA to assess the stage of the infectious process?
Class M and G immunoglobulins are differentially determined by ELISA.
- Class M immunoglobulins (IgM) are a marker of recent infection; in herpesvirus infection, a 4-fold or greater increase in IgM titer indicates a primary infection.
- Class G immunoglobulins (IgG) — in herpesvirus infection, an increasing titer indicates a recurrent form of the disease; in rotavirus infection, post-infection immunity is mediated by IgG antibodies.
To remember the essence of the sandwich method, imagine a sandwich: the bottom piece of bread represents the immobilized antibodies at the bottom of the well, the filling (meat) is the target antigen from the patient's blood, and the top piece of bread is the added labeled antibodies that complete the structure.
Class and exam questions
What is a conjugate in ELISA?
A conjugate is an antibody firmly bound (conjugated) to an enzyme molecule, which serves as a label for subsequent visualization of the reaction.
What is the difference between direct and indirect detection methods?
The scheme for detecting antibodies in serum (serodiagnosis) is an indirect method because the enzyme label is located on the secondary (antiglobulin) antibodies, whereas the sandwich method is aimed at detecting antigens.
What else to study on this topic
- Detailed mechanism of horseradish peroxidase action
- Causes of false-positive and false-negative ELISA results
- Spectrophotometry: how instruments read color intensity
- Features of ELISA in the diagnosis of HIV infection
- Comparison of ELISA with PCR diagnostics
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