General characteristics and types of reactions
All serological reactions in this group are based on a strict rule: complement molecules come into play only when a specific antibody finds its antigen and binds to it. Until then, complement remains inactive.
In laboratory practice, three main types of such reactions are distinguished:
- Complement fixation test (CFT). This is the most complex technique, which requires extremely rigorous preparation of all components prior to the assay.
- Radial hemolysis reaction (RHR). This method is technically based on the diffusion of components in a special gel.
- Immune adherence reaction (IAR). Aimed at working with large particulate antigens and evaluating their interaction with cells.
Mechanism of the complement fixation test (CFT)
CFT is a multi-step test. Performing it requires two independent component systems that are introduced into the reaction sequentially.
The first (primary) system includes:
- Known antigen (Ag);
- The patient's test serum (which may or may not contain the target antibodies);
- Exogenous complement (C).
The second (indicator or hemolytic) system is necessary for visualizing the results. It consists of:
- Sheep red blood cells (erythrocytes);
- Hemolytic serum (specific antibodies produced against sheep red blood cells).
The essence of the method lies in the competition for complement between the primary and indicator systems.
Interpretation of CFT results
The result of the reaction depends on whether the complement was bound in the first stage to the complexes from the patient's serum. Two scenarios are possible:
Option A: Positive reaction (patient is sick)
- At the first stage, the patient's serum contains specific antibodies.
- They combine with the antigen, forming a stable immune complex.
- The added complement recognizes this complex and firmly binds to it (is consumed).
- When the hemolytic system is added at the second stage, there is no free complement left in the solution.
- Result: Hemolysis is absent. Sheep red blood cells remain intact and simply settle to the bottom of the test tube. The diagnosis is confirmed.
Option B: Negative reaction (patient is healthy)
- The serum of a healthy person lacks the necessary antibodies.
- The immune complex is not formed at the first stage.
- All added complement remains completely free.
- After adding the indicator system, the free complement attacks sensitized sheep red blood cells.
- Result: Total destruction of red blood cells occurs. Pronounced hemolysis is observed, and the liquid takes on the appearance of "lakelike blood" (hemolyzed blood). The diagnosis is not confirmed.
Immune adherence reaction (IAR)
This reaction allows the visualization of the interaction of large objects. Its essence lies in the activation of complement by particulate antigens, which include whole bacterial cells or viruses pre-treated with immune serum.
Step-by-step mechanism of IAR:
- A primary complex forms between the particulate antigen and the antibody.
- This complex activates the complement system, leading to the massive production of the C3b component.
- C3b molecules deposit on the surface of the complex and begin to function as a biological "glue".
Result: The particulate antigen, coated with C3b molecules, adheres to the membranes of target cells that have the corresponding receptors. Such cells include human red blood cells, platelets, and macrophages. Visually, when mixing such cells with immune complexes, their union is observed, which looks like a classic agglutination reaction.
Briefly: answers to topical questions
Which classes of immunoglobulins are capable of binding complement in serological reactions?
Immunoglobulin classes IgM and IgG have the ability to bind and activate the complement system. Among class G immunoglobulins, this effector function is predominantly expressed in the IgG1 and IgG3 subclasses. Activation occurs via the classical pathway when the initiating component C1q binds to the Fc fragment of these antibodies, which are part of the "antigen-antibody" immune complex.
For the diagnosis of which infectious diseases is the complement fixation test used?
The complement fixation test is used in the serological diagnosis of a number of infections.
In the cited sources, CFT is indicated for the following diseases and situations:
- North Asian tick-borne rickettsiosis — detection of complement-fixing antibodies; diagnostic titer 1:40; the test can also be performed on paired sera.
- Astrakhan spotted fever — serological diagnosis using CFT; diagnostic titer of 1:40 and higher.
- Infection caused by R. prowazekii — serological diagnosis using CFT, IHA, and ELISA.
- Q fever / coxiellosis — serological diagnosis using CFT, ELISA, and indirect IFA.
- Syphilis — historically, CFT (Wassermann reaction) was used.
- Dengue fever — serological method with the study of paired sera, including CFT.
- Rotavirus infection — CFT is used as a serological method for retrospective diagnosis.
- Tick-borne encephalitis — CFT is listed among serological research methods.
In which specific gel is the radial hemolysis reaction performed?
The radial hemolysis reaction is performed in an agar gel. Sheep red blood cells and complement are preliminarily introduced into the composition of this medium. When hemolytic serum is added to the wells, radial diffusion of antibodies occurs, leading to the destruction of red blood cells and the formation of a visually detectable zone of hemolysis.
The CFT paradox: if there is NO hemolysis (red blood cells are intact, sedimented) — the reaction is POSITIVE (antibodies are present). If hemolysis IS present ("lakelike blood") — the reaction is NEGATIVE (no antibodies).
Class and exam questions
Under what circumstances does the complement system begin to function in serological reactions?
Activation occurs exclusively at the moment when the specific antigen-antibody complex is formed.
Why are sheep erythrocytes needed in the CFT?
They act as part of the indicator system. If the complement remains free, it will destroy the sheep red blood cells, and we will observe hemolysis.
Which cells have receptors for the C3b component?
Specific receptors for C3b are present on the surface of human erythrocytes, platelets, and macrophages.
What is the "lake blood" phenomenon in the CFT?
This is a visual manifestation of sheep erythrocyte hemolysis, which is observed in a negative test result when there are no antibodies in the serum.
What else to study on this topic
- Precise rules for preparation and titration of components for the Complement Fixation Test
- The mechanics of antigen and antibody diffusion in gel during the radial hemolysis assay
- The role of macrophages and platelets in the clearance of immune complexes from the body via C3b receptors
- Specifics of treating particulate antigens with immune sera
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