General Principle and Reaction Mechanism
In an enzyme-linked immunosorbent assay (ELISA), the conjugate plays a key role—these are 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 within the conjugate cleaves the substrate.
- The cleavage results in a change in solution color.
It directly depends on the number of bound molecules, allowing 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 is the most widely used variant. 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 this support.
A critical condition for a successful 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 ELISA Formats
Depending on diagnostic goals, different assay schemes are used:
- Antibody Detection (Serodiagnosis): The goal is to find specific immunoglobulins against a pathogen in the patient's serum. A known antigen is fixed to the bottom of the well. When the patient's serum is added, their antibodies (if present) bind to the antigen. A conjugate (antiglobulin serum with an enzyme) is then added, which attaches to the Fc fragments of the patient's antibodies. The addition of a substrate causes color development.
- Antigen Detection ("Sandwich Assay"): Used to search for the pathogen itself or its structural components. Diagnostic antibodies are immobilized in the well. They "capture" the target antigen from the patient's serum. Next, labeled antibodies against the same antigen are introduced, completing the complex ("sandwich"). The presence of the enzyme is confirmed by a colorimetric reaction.
- Competitive ELISA: This 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 a 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 fields 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 drug concentrations in the body.