Mechanism of Antigen-Antibody Interaction
Any immunodiagnostic reaction in vitro proceeds in two sequential phases:
- Specific Phase. This occurs rapidly and is invisible to the naked eye. The mechanism involves the binding of the active center (Fab fragment) of the antibody to the determinant (epitope) of the antigen. The interaction is driven by physicochemical forces: hydrogen bonds, Van der Waals forces, and hydrophobic interactions. The efficacy depends on affinity (the binding strength of an antibody to a single epitope) and avidity (the total combined binding strength).
- Non-specific Phase. A slower stage that requires electrolytes and an optimal pH level. It is during this phase that the reaction result becomes physically visible—for example, as turbidity or flake formation (the agglutination phenomenon).
Strategy of the Serological Method
The serological method aims to establish a diagnosis by detecting antibodies or antigens in the patient's blood serum. Since one component in the reaction is always known while the second is being sought, two approaches are possible:
- Serodiagnostics (searching for antibodies). Known antigens—diagnostic agents—are used as "bait". If specific antibodies are present in the patient's serum, they will bind to the diagnostic agent.
- Identification (searching for an antigen). To determine the species or type affiliation of a microorganism isolated from a patient, immune diagnostic sera are used. These are obtained by hyperimmunizing laboratory animals (e.g., rabbits) and therefore guaranteed to contain the necessary antibodies.
Agglutination Reaction: Stages and Variants
Agglutination is used to clump particulate antigens (bacteria, erythrocytes).
Stage A: Preliminary (Slide) Test. Performed on a glass slide to make a preliminary determination of the bacterial species. A drop of diagnostic serum at a low dilution (1:10 or 1:20) is mixed with a pure culture of the pathogen. The appearance of a flaky precipitate indicates a positive result. A control (saline plus culture) is mandatory to rule out spontaneous agglutination.
Stage B: Tube Agglutination Test. Performed in test tubes to confirm specificity and determine the titer. The serum is serially diluted (titrated) and a suspension of the pathogen is added. The reaction is considered positive when the amount of precipitate and clearing of the liquid are observed at a dilution close to the passport value (titer) of the serum.
Indirect (Passive) Hemagglutination Assay (IHA / HIHA). Sheep erythrocytes (or latex particles) are used as carriers.
- Classical IHA: antigens are adsorbed onto erythrocytes (antigenic diagnostic agent). Used to screen for antibodies in infections.
- Reverse IHA: antibodies are adsorbed onto erythrocytes. Used to detect antigens (e.g., botulinum toxin) or hormones (human chorionic gonadotropin in pregnancy tests).
- Interpretation of IHA results: A positive reaction appears as an "umbrella" (erythrocytes coat the bottom of the well with a scalloped edge). A negative reaction appears as a compact "button" in the center.
Complement Fixation Test (CFT)
A complex two-phase reaction. Before setting up the assay, the patient's serum must be heated at 56 °C to inactivate its own complement; otherwise, it will distort the results.
- Primary Phase. Three components are incubated: antigen, antibody, and a strictly dosed exogenous complement. If the Ag and Ab match, an immune complex forms, which binds (consumes) all the added complement.
- Indicator Phase. A hemolytic system (sheep erythrocytes + hemolytic serum containing antibodies against them) is added to the system. The goal is to visualize the presence of free complement.
Interpretation:
- Positive (+): In the first phase, the complement bound to the immune complex. None is left for the second phase. There is no hemolysis; erythrocytes settle to the bottom.
- Negative (–): In the first phase, no complex formed, leaving the complement free. In the second phase, it attacks the erythrocytes, forming the membrane attack complex. Hemolysis occurs, turning the fluid transparent red ("laconic blood"). A historical example of this method is the diagnosis of syphilis (Wassermann reaction).
Solid-Phase Assays and Immunodiffusion
In addition to classical reactions, enzyme-labeled and gel diffusion methods are widely used.
- Antibody Detection (Serodiagnostics): A known antigen is sorbed into the wells of a solid phase. The test serum is added, followed by an enzyme-labeled antiglobulin serum. After adding a chromogenic substrate, the presence of antibodies is confirmed by a color change.
- Antigen Detection: Antibodies are fixed to a solid support. After adding the sample, the target structure is identified using an enzyme-labeled immune serum and substrate.
- Radial Immunodiffusion: Performed in an agar gel containing sheep erythrocytes and complement. Hemolytic serum is introduced into the wells. Antibodies diffuse radially, forming a visible zone of hemolysis around the well.