Basic principles and types of reactions
The essence of the serological method lies in searching for specific antigen–antibody complexes in the patient's blood serum. Depending on the research goals, a physician may look either for traces of the pathogen itself (antigens) or the patient's immune system reaction (antibodies).
To visualize and assess this interaction, various classes of reactions are used in microbiology:
- Classical tests: agglutination reaction (including modifications such as indirect hemagglutination assay (IHA) and hemagglutination inhibition assay (HI)), complement fixation test (CFT), neutralization test (NT), and precipitation reaction (PR).
- High-sensitivity methods: radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The latter possess maximum accuracy in detecting minimal concentrations of target molecules.
Antibody detection diagnostics: the paired sera method
The classical method for confirming an acute infection is the paired sera method. Its algorithm requires strict adherence to time intervals and storage conditions of the biomaterial.
- The first blood portion is collected at the very beginning of the illness (in the acute period). The obtained serum must be stored in a refrigerator at a temperature of 4–8 °C.
- The second portion is taken 10–14 days later.
- The main rule: both sera must be tested simultaneously in a single laboratory assay. This eliminates the influence of external factors and errors from different reagent lots.
The criterion for a positive result (confirmation of a current infection) is seroconversion — an increase in antibody titer in the second sample by at least 4 times compared to the first.
It is important to understand that this approach provides only retrospective diagnostics. The laboratory report arrives two weeks after the onset of the disease, when the patient is often already in the recovery stage. The only exceptions are certain infections where a single detection of extremely high antibody titers is sufficient for diagnosis.
Determination of immunoglobulin classes
Differentiation of synthesized antibodies by class (IgM, IgG, IgA) provides the physician with valuable information about the nature of immunity and the phase of the pathological process. This is also an excellent tool to distinguish a real infectious disease from post-vaccination immunity.
- Acute infection: IgM class antibodies predominate in the blood. Early antibodies of other classes (e.g., low-avidity IgG specific to early pathogen proteins) may also be detected during this phase.
- Chronic process, secondary infection, or carriage: characterized by the stable presence of IgG and IgA.
- Past infection (immunological history): stably low titers of IgG are detected, with no tendency to increase.
A serious limitation of the serological antibody search is the state of marked immunosuppression in the patient. In suppressed immunity, the synthesis of protective proteins is sharply reduced, which creates a high risk of obtaining a false-negative result.
Antigen detection and evaluation of method reliability
Unlike the search for antibodies, detecting pathogen antigens allows for rapid diagnostics. Antigens can be detected at the earliest stages of infection, when the immune system has not yet had time to produce its own antibodies. Quantitative determination of antigens is actively used to monitor disease dynamics: a systematic decrease in their concentration serves as a reliable criterion for the effectiveness of the therapy.
As for the reliability of serological methods, their specificity varies within 70–90%. Analysis errors are divided into two categories:
- False-positive results: occur due to non-specific binding of molecules in the sample or as a result of cross-reactivity (when different species of microorganisms share evolutionarily similar antigens).
- False-negative results: are most often associated with the fact that the amount of target antigens or antibodies in the patient's blood drops below the sensitivity threshold of the test system used.
Briefly: answers to topical questions
In which infections is a single detection of high antibody titers sufficient for a diagnosis?
For diagnosis, a single detection of high antibody titers is sufficient in acute rheumatic fever (streptococcal infection).
- Acute rheumatic fever — the determination of antistreptolysin O (ASO) in titers $\ge$ 250 Todd units in adults and $\ge$ 333 Todd units in children older than 5 years is diagnostically significant in a single test.
In most other infectious diseases (e.g., shigellosis), a single test has no diagnostic significance, and the "paired sera" method is required.
What is the difference between low-avidity and high-avidity IgG in serological diagnostics?
In serological diagnostics, low-avidity and high-avidity IgG differ in their time of appearance and diagnostic significance, reflecting different stages of the infectious process.
| Characteristic | Low-avidity IgG | High-avidity IgG |
|---|---|---|
| Time of appearance | Produced on days 10–14 of the illness, disappear in 1–3 months | Appear later, circulate in the blood for life |
| Diagnostic significance | Indicate recent primary infection or active antibody synthesis by a newborn | Indicate past infection, carriage, or maternal origin of antibodies in the fetus |
What is the mechanism of the complement fixation test (CFT)?
The mechanism of the complement fixation test (CFT) is based on the ability of the complement system to be activated and fixed only upon the formation of an immune complex. The reaction includes two systems:
- Primary system — antigen, test serum, and complement.
- Indicator system — sheep red blood cells and hemolytic serum.
If specific antibodies are present in the serum, an antigen–antibody complex is formed, which fixes the added complement. As a result, no free complement remains for the indicator system, and hemolysis of red blood cells does not occur (positive reaction). If there are no antibodies, the complex is not formed, and free complement destroys the red blood cells of the indicator system, causing visible hemolysis.
What modifications of the agglutination reaction exist besides IHA and HI?
Besides IHA and HI, the following variants and reactions exist:
- Standard tube agglutination reaction — performed in test tubes.
- Slide agglutination test — performed on a glass slide.
- Latex agglutination test — used to detect antigens or antibodies.
- Wright agglutination test — used during the febrile period of an acute form of infection.
- Huddleson plate agglutination test — a rapid method for mass screening examinations.
- Coombs test — allows the detection of incomplete blocking IgA and IgG antibodies in subacute, latent courses, or remission.
Rule of classes: "M" means "Momental" (acute phase, IgM). "G" means "Global/Years" (past infection, stable IgG).
Class and exam questions
Why is the paired sera method considered retrospective?
To assess seroconversion (titers rise), it is necessary to wait 10–14 days before taking a second sample. By the time the results are ready, the acute phase has passed, and the patient has usually already recovered.
Is it possible to test the first serum sample immediately, and the second two weeks later?
No, the algorithm requires simultaneous testing of both samples in a single assay run. The first serum is stored at 4–8 °C to be tested simultaneously with the second and eliminate test system errors.
What can trigger a false-negative result when searching for antibodies?
The main reasons are pronounced immunosuppression (when the body is physically unable to synthesize antibodies) or a very low antibody concentration below the sensitivity threshold of a specific test.
What is the main advantage of antigen-based diagnosis?
This is a rapid diagnostic method that works at the very early stages of the disease. Antigens appear in the blood before the immune system has time to produce specific immunoglobulins.
What else to study on this topic
- Mechanisms of classical reactions: agglutination, precipitation, complement fixation.
- Principles of high-sensitivity tests: radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).
- The concept of antibody avidity: why low-avidity IgG indicates an early stage of the disease.
- The nature of cross-reactivity and molecular mimicry of pathogens.
- Features of serological diagnosis in immunodeficient states.
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