Mechanism of antigen-antibody interaction
Any in vitro immunodiagnostic reaction proceeds in two successive phases:
- Specific phase. It occurs rapidly and is invisible to the naked eye. The mechanism consists in the binding of the active center (Fab fragment) of the antibody to the determinant (epitope) of the antigen. The interaction is provided by physicochemical forces: hydrogen bonds, van der Waals forces, and hydrophobic interactions. Efficiency depends on affinity (the affinity of the antibody for the epitope) and avidity (the total bond strength).
- Nonspecific phase. A slower stage that requires the presence of electrolytes and an optimal pH level. It is in this phase that the result of the reaction becomes physically visible — for example, in the form of turbidity or flock formation (the agglutination phenomenon).
Strategy of the serological method
The serological method is aimed at making a diagnosis by detecting antibodies or antigens in the patient's blood serum. Since one component in the reaction is always known and the second is being searched for, two options are possible:
- Serodiagnosis (search for antibodies). Known antigens — diagnostic agents — are used as "bait". If the patient's serum contains specific antibodies, they will bind to the diagnostic agent.
- Identification (search for antigen). To determine the species or type affiliation of a microorganism isolated from a patient, immune diagnostic sera are used. They are obtained by hyperimmunization of laboratory animals (e.g., rabbits), and therefore they are guaranteed to contain the necessary antibodies.
Agglutination reaction: stages and variants
Agglutination is used for clumping particulate antigens (bacteria, erythrocytes).
Stage A: Preliminary reaction. Performed on a glass slide to preliminarily determine the type of bacteria. 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. Control is mandatory: saline plus culture (to exclude spontaneous agglutination).
Stage B: Tube agglutination test. Performed in test tubes to confirm specificity and determine the titer. The serum is sequentially diluted (titrated) and a suspension of the pathogen is added. A reaction is considered positive if the amount of precipitate and clearing of the liquid are observed at a dilution close to the passport value (titer) of the serum.
Indirect hemagglutination assay (IHA). Sheep erythrocytes (or latex) are used as carriers.
- Classical IHA: antigens are adsorbed onto erythrocytes (antigenic diagnostic agent). Used to search for antibodies in infections.
- Reverse IHA (RIHA): antibodies are adsorbed onto erythrocytes. Searches for antigens (e.g., botulinum toxin) or hormones (human chorionic gonadotropin in pregnancy tests).
Reading IHA results: a positive reaction looks like an "umbrella" (erythrocytes coat the bottom of the well, scalloped edge). Negative — like a compact "button" in the center.
Complement fixation test (CFT)
A complex two-phase reaction. Before testing, the patient's serum is necessarily heated at 56 °C to inactivate its own complement, otherwise it will distort the results.
- Main phase. Three components are incubated: antigen, antibody, and a strictly dosed exogenous complement. If the Ag and Ab match each other, an immune complex is formed that binds (consumes) all the added complement.
- Indicator phase. A hemolytic system (sheep erythrocytes + hemolytic serum with 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, the complex did not form, the complement remained free. In the second phase, it attacks the erythrocytes, forming a membrane attack complex. Hemolysis occurs — the liquid turns transparent red ("lakelike blood"). A historical example of such a method is the diagnosis of syphilis (Wassermann test).
Solid-phase methods and immunodiffusion
In addition to classical reactions, methods using enzyme labels and gel diffusion are actively used.
- Determination of antibodies (serodiagnosis): A known antigen is sorbed in the wells on a solid phase. The test serum is added, followed by an enzyme-labeled antiglobulin serum. After introducing the chromogenic substrate, the presence of antibodies is confirmed by a color change.
- Determination of antigens: Antibodies are fixed on the carrier. After adding the material, the target structure is detected with an immune serum with an enzyme and substrate.
- Radial diffusion: 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.
Briefly: answers to topical questions
What enzymes are used as labels in enzyme-linked immunosorbent assay (ELISA)?
Three main substances are used as enzyme labels in ELISA. These include:
- Horseradish peroxidase — used to cleave the substrate and change the color of the reaction product.
- Beta-galactosidase ($\beta$-galactosidase) — acts as a specific enzyme label.
- Alkaline phosphatase — used as part of the conjugate to detect the antigen-antibody complex.
These enzymes chemically bind to antibodies to form a key reagent (conjugate) which, after the addition of a chromogen, makes it possible to visualize and quantify the test results.
Which classes of immunoglobulins have the highest avidity and agglutination capacity?
According to sources, immunoglobulin class M (IgM) has the greatest agglutination capacity. IgM has a pentameric structure and 10 antigen-binding sites, so it can bind several bacteria or cells, forming large conglomerates. IgG has a monomeric structure and also participates in the formation of a lattice between neighboring cells. Sources do not establish that IgM has the highest avidity compared to other classes of immunoglobulins.
Which eponymic reactions are used in the clinic as a tube agglutination test?
The Widal reaction is a tube agglutination test mentioned as a historically used method in the serological diagnosis of infections associated with Enterobacteriaceae.
Which particulate particles can act as antigen carriers in indirect agglutination reactions?
In indirect agglutination reactions, the following particulate particles can act as carriers of antigens or antibodies:
- Erythrocytes — used in the indirect hemagglutination assay. Soluble antigens are adsorbed on their surface, which makes it possible to visualize the formation of immune complexes in the form of cell clumping.
- Latex particles — used in latex agglutination. These can be colored polystyrene or superparamagnetic particles sensitized with specific antibodies or antigens.
How to remember the CFT result: "If the antigen and antibody match, they 'eat up' the complement. The erythrocytes do not get complement, and they remain intact (no hemolysis, positive reaction)."
Class and exam questions
Why is the serum heated to 56 °C when performing the complement fixation test (CFT)?
This is a critical step to inactivate the patient's own complement. Failure to do so will result in an uncontrolled amount of endogenous complement leading to false hemolysis in the second phase.
How to visually distinguish between a positive and negative IHA reaction?
In a positive reaction, erythrocytes clump and settle in the form of an "umbrella" with scalloped edges. In a negative one, they slide down to the center of the well, forming a compact "button".
What is antibody avidity?
Avidity is an indicator characterizing the overall bond strength between an antibody and an antigen. It depends on affinity (affinity for a specific epitope) and the number of formed bonds.
Why are controls set up in slide agglutination reactions?
The "bacteria + saline" control is necessary to ensure there is no spontaneous agglutination of the pathogen culture. Normally, a uniform turbidity without sediment should be observed.
What else to study on this topic
- Structure and functions of the antibody Fab fragment
- Nature of physicochemical interactions (van der Waals forces, hydrophobic bonds) in the specific phase
- Preparation and standardization of erythrocyte diagnostic reagents for IHA and HIHA
- Mechanism of membrane attack complex formation in hemolysis
- Evaluation of complement activity and hemolytiс serum
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