What does a serological blood test show? Diagnostic measures are the most important stage in the treatment of any disease. The success of treatment depends not only on the prescribed drugs, but also largely on how correctly the diagnosis was made.

In addition, diagnosis helps prevent complications and accompanying illnesses. Using a serological test of the patient's blood, the presence of antibodies and antigens is detected. The study helps to find many diseases, determine their phase and monitor the progress of treatment.

What is serology?

Serology is the branch of immunology that studies the reactions of antigens to antibodies. This branch of medicine deals with the study of blood plasma and its immunological characteristics.

Today, a serological blood test for antibodies is a reliable way to detect human immunodeficiency virus, hepatitis, brucellosis, STDs and other life-threatening diseases. Let's figure out in what cases it is prescribed.

Indications for use

A serological blood test is necessary to identify the causative agent of the disease if it is difficult to make a diagnosis.

To carry out this reaction, antigens of pathogens are introduced into the plasma, and then the ongoing process is studied by a laboratory assistant. Or they carry out the reverse reaction: antibodies are injected into the infected blood to determine the specific identity of the pathogen.

Scope of application

This research is used in various branches of medicine. This reaction identifies specific cells and antibodies produced by the body to fight infections and viruses.

In addition, a person’s blood type is determined using the serological method.

A similar serological blood test is used in gynecology to diagnose sexually transmitted diseases. This method is also used for comprehensive examinations of pregnant women (detection of toxoplasmosis, HIV, syphilis, etc.). Passing this test is mandatory when registering with a antenatal clinic.

In children, a serological reaction is used to confirm the diagnosis of so-called “childhood” diseases (chickenpox, measles, rubella, etc.) if the symptoms do not have pronounced manifestations and it is impossible to identify the disease by analyzing clinical indications.

Detection of sexually transmitted diseases

For venereologists, this testing is truly irreplaceable and allows you to make a diagnosis very accurately.

With a blurred clinical picture, a serological blood test for syphilis, giardiasis, ureaplasmosis, chlamydia, herpes and other diseases can quickly detect the presence of antibodies.

Viral and infectious diseases

Serological analysis is actively used by gastroenterologists, hepatologists and infectious disease specialists for diagnosis viral hepatitis.

Deciphering a serological blood test makes it possible to determine the stage of the disease and answer the question of how necessary hospitalization is. this moment. How to prepare properly?

Preparing for the test

Serological blood tests are done in both public and commercial clinics. Preference should be given to a laboratory with modern equipment and qualified personnel.

Biological samples for testing can be saliva and feces, but in most cases the patient's venous blood is used. Blood for a serological test is taken from the cubital vein in a laboratory. Before taking the test, you should consult with your doctor about preparing for this procedure.

To prepare for a serological test, you need to follow a few simple rules.

Blood is donated in a calm state before meals, that is, on an empty stomach. Before this, you should not undergo other tests, such as x-rays, ultrasound, etc.

It is necessary to avoid taking antibacterial and some other drugs several weeks before donating blood. Certain recommendations in this case depend on the disease for which the test is being done. For example, a test for hepatitis involves eliminating fatty foods and alcohol 48 hours before the procedure.

Fluorescence reaction

Among the types of serological reactions there is a fluorescence reaction. This technique uses a reagent that illuminates antibodies in the blood serum.

Setting up a direct serological reaction involves marking specific antibodies with a fluorescent substance. This reaction is the fastest and is carried out in one stage.

Another option for conducting such an analysis is called indirect, or RNIF. It is carried out in two stages. In the first step, antibodies are not labeled with fluorescent tags, and in the second, appropriately labeled antibodies are used to identify antigens and antibodies. The glow occurs only after binding to a specific antibody occurs.

What does a serological blood test show? The result of the entire procedure is assessed by a special device that analyzes the strength of the radiation and reveals the shape and size of the object under study. The causative agents of infectious diseases are detected with a result whose reliability is 90-95%, depending on the type and stage of the pathology.

Linked immunosorbent assay

These types of serological testing use unique, stable reagents. The marked substances seem to stick to the desired antibodies. As a result, we get a qualitative or quantitative result.

If no pronounced markers are found, the result will be considered negative. If the presence of antibodies in biological samples is detected during a qualitative study, then the test result is considered positive. By quantifying cells, the analysis gives a more accurate result.

By analyzing the analysis indicators (for example, the sum of detected cells), the specialist determines whether the disease is at initial stage, in the acute stage, or it has worsened chronic form pathology. In order to make a diagnosis, the doctor takes into account not only the data of a serological study, but also the clinical picture of the disease.

Features of this test

Carrying out this analysis is not always able to provide 100% confidence that a certain disease has been detected. It happens that the results may be ambiguous and other procedures are required.

For example, during a test for brucellosis, blood serum is controlled for self-retention without antigen. This significantly increases the reliability of testing. The test for brucellosis may be positive or negative meaning, and also raise doubts.

If you receive questionable results that do not have an unambiguous interpretation, it is recommended to take the test again. In addition, brucellosis can be detected by blood cultures, testing Bone marrow and cerebrospinal fluid.

Pros of serological blood test

Diagnostic techniques using serological reactions are widely used in modern medical practice. This is especially often done when determining viral and infectious pathologies.

The same tests are used when conducting geographic screening and medical examination in order to prevent the epidemiological spread of infection.

The advantages of the method include:

• High level of confidence.
• Fast reaction and results. The results of the RSC are known within 24 hours. In a special situation, in a hospital setting, the analysis will be ready in a few hours.
• Monitoring the development of the disease and the effectiveness of the therapy.
• Low cost and accessibility for patients.

Disadvantages of the method

However, serological studies also have their drawbacks.

These include the fact that when conducting an analysis, the incubation period of the disease should be taken into account in order to obtain a more reliable picture.

For example, the definition herpes simplex the first or second type is possible only 14 days after infection. An analysis for the presence of the immunodeficiency virus is carried out 30 days, 90 days and six months after contact with an infected person.

Of course, the reliability of the results can also be influenced by the human factor: neglect of the rules for preparing for blood sampling or an error made by the laboratory assistant when carrying out the reaction.

According to statistics, an erroneous result can be obtained in 5% of cases. An experienced doctor, when examining a patient, having studied the clinical picture, in most cases can calculate the mistake made.

Testing for syphilis is one of the most common laboratory tests. Tests for syphilis are widely used during preventive examinations. Using microscopy, the causative agent of syphilis is identified. Using serological reactions, the diagnosis of syphilis is confirmed, the diagnosis is established latent syphilis, the effectiveness of treatment is monitored, and the cure of patients is determined.

The diagnosis of syphilis is established on the basis of clinical data, detection of syphilis pathogens in material samples and confirmation of the diagnosis by serological research methods. The manifestations of syphilis are numerous and varied, which is why the disease is diagnosed by doctors of different specialties. Differential diagnosis of primary syphilis is carried out with a number of diseases.

Rice. 1. In the photo, the primary manifestation of syphilis is chancre.

Antibodies to Treponema pallidum and serological diagnosis

When infected with syphilis, antibodies are formed in the patient's body. Serological diagnostics helps the doctor to study the dynamics of antibody formation in the body of a patient with syphilis in initial stages diseases, during the treatment period and after its completion, resolve the issue of relapse of the disease in the patient or re-infection (reinfection), make a diagnosis of syphilis in mass medical conditions.

Antibodies to Treponema pallidum IgM

IgM antibodies are the first to be produced after infection. They begin to be detected using serological reactions from the second week after infection. At 6–9 weeks of illness, their number becomes maximum. If the patient is not treated, the antibodies disappear after six months. IgM antibodies disappear after 1 - 2 months. after, after 3 - 6 months. - after treatment of late syphilis. If their growth is recorded, then this serves or indicates re-infection. IgM molecules are large and do not pass through the placenta to the fetus.

Antibodies to Treponema pallidum IgG

Antibodies IgG immunoglobulins appear at the end of the first month (4th week) from the moment of infection. Their titer is higher than the IgM titer. IgG persists for quite a long time after treatment.

Nonspecific antibodies

There are many serological reactions. This is explained by the antigenic multiplicity of Treponema pallidum. In the blood serum of a sick person different stages syphilis, in addition to specific ones, certain nonspecific antibodies are formed - agglutinins, complement-fixing antibodies, immobilins, antibodies that cause immune fluorescence, precipitins, etc. Serological reactions to detect nonspecific antibodies have relative specificity; therefore, in order to avoid diagnostic errors, you should use not one, but a complex of serological reactions (RAC).

False positive tests for syphilis

Distinctive feature non-treponemal tests result in false-positive reactions. Antibodies-reagins, which are produced in human blood against the cardiolipin antigen, are registered not only with syphilis, but also with other diseases: collagenosis, hepatitis, kidney diseases, thyrotoxicosis, cancer, infectious diseases(leprosy, tuberculosis, brucellosis, malaria, typhus, scarlet fever), during pregnancy and menstrual cycles, when taking fatty foods and alcohol. It has been noted that the number of false-positive reactions increases with age.

Rice. 2. The photo shows primary syphilis in women.

Laboratory diagnosis of syphilis using serological tests

Serological tests for syphilis are divided into treponemal and nontreponemal.

1. Non-treponemal tests

The antigen used in this group of tests is cardiolipin antigen. Lipid antigens of syphilis pathogens are the most numerous. They make up 1/3 of the dry mass of the cell. Using non-treponemal tests, reagin antibodies are detected that are produced against the cardiolipin antigen. This group includes the complement fixation test (FFR), microprecipitation test (MPR), rapid plasma reagin test (RPR), etc. Using non-treponemal tests, primary screening for syphilis is carried out (survey of population groups), and the possibility of obtaining results in a quantitative form allows these tests to be used to monitor the effectiveness of treatment. Positive results from nontreponemal tests must be confirmed by treponemal tests. A distinctive feature of non-treponemal tests is the receipt of false-positive reactions.

2. Treponemal tests

Treponemal tests use antigens of treponemal origin isolated from a culture of Treponema pallidum. With their help, positive results of non-treponemal tests are confirmed. The group includes: RSKtrep - complement fixation reaction, RIF - immunofluorescence reaction and its modifications, RIT, RIBT - immobilization reaction of Treponema pallidum, RPHA - passive hemagglutination reaction, ELISA - enzyme-linked immunosorbent assay.

3. Tests for syphilis using recombinant antigens

Antigens for this group of tests are obtained by genetic engineering and are used in reactions - RPGA and ELISA, in immunoblotting (IB) analyzes and immunochromatographic analysis.

Rice. 3. A set of serological tests is used to diagnose syphilis.

Diagnosis of syphilis using non-treponemal tests

To detect syphilis, non-treponemal tests or a complex of serological reactions (SSR) are used. Serological diagnosis is used from the 5th week from the moment of infection or from 2-3 weeks after the onset. Antibodies are detected in almost all patients with fresh primary. Serological reactions are positive in 70 - 80% of patients with syphilis, in 50 - 60% of cases in patients with tertiary latent syphilis.

Serological tests using nontreponemal tests may give false-positive results.

Rice. 4. Blood sampling for testing for syphilis.

Complement fixation reaction (RSK card, KSK with KA, Wasserman reaction)

The Wasserman reaction (RW, РВ), invented by A. Wasserman more than 100 years ago, has today undergone many changes, however, as a tribute to tradition, it has retained its name to the present day. The complement fixation reaction using cardiolipin antigen is intended not only to detect antibodies, but is also performed quantitatively - with different serum dilutions, which allows it to be used to monitor the effectiveness of treatment. Low sensitivity and specificity, obtaining false positive results— negative aspects of this type of research.

The essence of the Wasserman reaction is as follows: the antigens that are used to stage the Wasserman reaction, in the case of the presence of antibodies to the causative agents of syphilis in the human blood, bind to them through a compliment and precipitate. The intensity of the reaction is indicated by a sign (+). The reaction can be negative (-) - no sediment, doubtful (small sediment or +), weakly positive (++), positive (+++) and strongly positive (++++).

The modified Wasserman reaction, the Kolmer reaction, is more sensitive. With its help, antibodies are detected in sera where the Wasserman reaction gave a negative result.

In case of strongly positive reactions, it is carried out quantitation reagins, for which serum is used in dilutions from 1:10 to 1:320, which allows this type of research to be used to monitor the effectiveness of treatment. For example, a decrease in antibody titer and their subsequent seronegativity (obtaining negative results) indicates a successful cure of the disease.

Rice. 5. Blood test for syphilis - Wasserman reaction.

Microprecipitation reaction (MPR)

Precipitation microreaction is used for mass examinations of certain population groups, diagnosis of syphilis and monitoring the effectiveness of treatment. To conduct this type of research it is necessary small amount the material being studied. The microreaction of precipitation is based on the immunological antigen-antibody reaction. If antibodies are present in the blood serum of the subject, the antigen-antibody complex precipitates to form flakes. The reaction is carried out in the wells of a special glass plate. It is assessed by the intensity of the precipitate and the size of the flakes in (+) as the Wasserman reaction. It is not used when examining pregnant women, donors and to monitor the effectiveness of treatment. VDRL and RPR are types of micro-reactions.

Rice. 6. Type of precipitation reaction in a drop on glass.

Rice. 7. Blood test for syphilis - microprecipitation reaction.

Rice. 8. Kit for rapid determination of plasma reagins (RPR test for syphilis).

All positive tests obtained during nonspecific serological reactions require confirmation by specific reactions - treponemal tests.

Diagnosis of syphilis using treponemal tests

When conducting treponemal tests, antigens of treponemal origin are used. Their negative side is the impossibility of using to monitor the effectiveness of treatment, obtaining positive results for spirochetosis and non-venereal treponematoses and obtaining false positive results for oncological diseases, leprosy, some endocrine pathology. Tests such as RPGA, ELISA and RIF remain positive for many years after syphilis has been cured, and in some cases for life.

RIBT and RIF are the most specific of all serological reactions used to diagnose syphilis. They make it possible to distinguish between false-positive reactions and to identify late forms of syphilis that occur with negative reactions. With the help of RIBT, false-positive reactions in pregnant women are recognized when it is necessary to resolve the issue of infection of the child.

Treponema pallidum immobilization reaction (RIBT, RIT)

The essence of the reaction is that the antibodies in the patient’s blood serum immobilize Treponema pallidum. A reaction is considered negative when up to 20% of pathogens are immobilized, weakly positive - 21 - 50%, positive - 50 - 100%. RIBT sometimes gives false positive results. The test is complex and labor-intensive, however, it is indispensable in the differential diagnosis of latent forms of the disease and false-positive results of serological reactions, including in pregnant women. RIBT gives a 100% positive result for secondary, early and late syphilis, in 94 - 100% of cases - for other forms of syphilis.

Immunofluorescence reaction (RIF)

The essence of the reaction is that treponema pallidum (antigens), combined with antibodies labeled with fluorochromes, emit a yellow-green glow in a fluorescent microscope. The result is evaluated with a (+) sign. Using RIF, class A immunoglobulins are detected. The immunofluorescence reaction becomes positive earlier than the Wasserman reaction. It is always positive in secondary and latent syphilis, in 95 - 100% of cases it is positive in tertiary and congenital syphilis. The technique for conducting this type of research is simpler than that of RIBT, but it is impossible to replace RIF with RIBT, since this reaction is inferior to RIBT in specificity. RIF-10 (modification of RIF) is more sensitive, RIF-200 and RIF-abs are more specific.

Rice. 9. Blood test for syphilis - immunofluorescence reaction (RIF).

Treponema pallidum immune adhesion reaction (IPAT)

The essence of the reaction is that treponema pallidum, sensitized by the patient’s serum, in the presence of complement, adheres to the surface of red blood cells. The resulting complexes precipitate during centrifugation. Sensitivity this test and specificity are close to RIF and RIBT.

Enzyme immunoassay for syphilis (ELISA)

Using ELISA, immunoglobulins of class M and G are determined. The IgM - ELISA technique can be used as a screening and confirmatory test. The sensitivity of ELISA and its specificity are similar to RIF. For syphilis, ELISA gives positive results from the third month of infection and remains positive for quite a long time (sometimes throughout life).

Rice. 10. Enzyme immunoassay analyzer.

Passive (indirect) hemagglutination reaction (RPHA)

RPHA is based on the ability of red blood cells on which treponema pallidum antigens are adsorbed to stick together (hemagglutination) in the presence of the patient’s serum. RPGA is used to diagnose all forms of syphilis, including latent. When using High Quality antigen, this type of serological reaction exceeds all other tests in specificity and sensitivity.

Rice. 11. RPGA is used to diagnose all forms of syphilis.

Rice. 12. Test for syphilis - passive (indirect) hemagglutination reaction (scheme).

Rice. 13. The appearance of an inverted umbrella occupying the entire bottom of the test tube indicates a positive reaction. In the case when red blood cells settle in a column (“button”) in the center of the bottom of the test tube, a negative reaction is indicated.

Rice. 14. RPGA test in laboratory conditions.

Microbiological diagnostics

Along with serological diagnostics, the method of detecting Treponema pallidum (microbiological diagnostics) plays a role important role, especially during the period of seronegative syphilis, when there are still no antibodies in the blood, but there are already the first manifestations of fresh primary syphilis (chancroid).

The biological material for the study is discharge from the surface of hard ulcers (chancres), the contents of pustular syphilides, weeping and erosive papules, punctures of infected lymph nodes, cerebrospinal fluid and amniotic fluid, for PCR - blood.

The best method for detecting syphilis pathogens is to examine biological material in a dark field microscope. This technique allows you to see treponema pallidum in a living state, study its structural features and movement, and distinguish pathogenic pathogens from saprophytes.

Rice. 15. Analysis for syphilis - dark-field microscopy.

Rice. 16. When studying dry smears, Romanovsky-Giemsa staining is used. Pale treponemas are stained with pink color, all other types of spirochetes are in purple.

Detection of Treponema pallidum by dark-field microscopy - absolute criterion final diagnosis of syphilis.

Rice. 17. To identify bacteria, an immunofluorescence reaction (RIF) is used - a treponemal test. A specific antigen-antibody complex, when combined with a specific serum labeled with a fluorochrome, in the light of a fluorescent microscope gives the bacteria a greenish glow.

Rice. 18. The causative agents of syphilis are clearly visible in smears prepared using the Levaditi method (silver impregnation). Pale treponema is dark in color against the background of the yellow color of the cells of infected tissues.

Rice. 20. The photo shows a colony of Treponema pallidum. It is difficult to obtain a bacterial culture. They practically do not grow on artificial nutrient media. On media containing horse and rabbit serum, colonies appear on days 3–9.

PCR for syphilis

An effective and promising technique today is the polymerase chain reaction. PCR for syphilis allows you to get results within a few hours, and at least several pathogens may be present in the material collected for diagnosis.

Rice. 21. PCR for syphilis can detect DNA or its fragments from Treponema pallidum.

The sensitivity of this research method depends on the presence of Treponema pallidum in the biological material and reaches 98.6%. The specificity of this test largely depends on the right choice targets for amplification during diagnostics and reaches 100%.

However, due to insufficiently studied comparative characteristics sensitivity and specificity of direct methods for diagnosing syphilis and PCR, this examination method in the Russian Federation for diagnosing the disease has not yet been approved.

PCR for syphilis is allowed to be carried out only in certain cases, such as additional method diagnostics congenital syphilis, neurosyphilis, if there are difficulties in diagnosing syphilis using serological research methods in HIV patients.

Rice. 22. Detection of Treponema pallidum DNA using PCR indicates either the presence of viable bacteria or the remains of dead bacteria, but containing individual sections of chromosomal DNA capable of forming additional copies.

Serological research, or, in other words, serological analysis, is the study of biological materials in the laboratory. This analysis allows you to determine the presence of pathogenic bacteria in the organism under study, or in products that undergo control testing.

Serological diagnostic methods

The study of sera or other biological objects (milk, bile, saliva, intestinal mucosal washings, as well as copromaterials) provides a fairly reliable idea of ​​the body’s response to the introduction of an infectious agent. It should be noted that the use of serological research methods has not only diagnostic value, but also provides reliable information about the level of body protection, the state of population immunity, circulation various types rotaviruses in the surveyed region. The results of serological surveys can also provide information to optimize the antigenic composition of prophylactic drugs and be used to assess the immunological effectiveness of vaccines.

However, it should be noted that the need to study paired blood sera taken at intervals of 1.5-2 weeks and the availability of rapid diagnostic methods reduce the diagnostic value of serological reactions.

Therefore, traditional serological tests - complement fixation test (FFR) and hemagglutination inhibition test (HAI) - are currently only of limited use. Nevertheless, the simplicity of these methods, the availability and low cost of reagents make them still applicable in laboratory practice. The setting of RSC and RTGA is carried out according to generally accepted methods.

An example of the use of RSCs to study post-vaccination immunity is the work of K. Midhan (1989). When examining sera from 116 children aged 5 months, reliable seroconversions, according to the RSC, were noted in 44% of cases, and according to the results of the neutralization test (PH) and enzyme-linked immunosorbent assay (ELISA) - in 83 and 96%, respectively.

Immunity research

In order to study population immunity using RSC, we examined 1,246 sera from patients with ACC aged from several months to 80 years and older. It was shown that the geometric mean titers of complement-fixing antibodies were highest in the age groups 2 - 4 and over 60 years, which confirms our previously obtained data on the highest prevalence rotavirus infection among persons of the specified age groups.

Rotavirus analysis

RTGA is used in the study of rotavirus infection more often than RSC, and, as a rule, in combination with other laboratory tests. Thus, Andrade J. R. et al. used RTGA, immunoblot, ELISA block to study antibodies to the structural proteins of rotavirus VP2, VP4, VP6 and VP7. According to the authors, antihemagglutinating antibodies to rotaviruses were detected in 70-80% of children aged 2 to 4 years.

Depending on the level of antibodies to certain proteins, according to RTGA data, the authors identified 4 groups of individuals. Groups I and II (60%) included children with high level antibodies to VP4 and VP7 and were classified as “immune”. Group III (4%) includes persons with low level antibodies to VP7 and VP6, the so-called “partially immune”. Children of group IV (36%), in whom, according to RTGA, no antibodies were detected, were designated as “non-immune” and constituted a risk group, since among them the development of severe rotavirus infection is possible. It should be noted that in this observation, the sensitivity indicators of RTGA and the ELISA block were quite comparable.

RTGA has been repeatedly used to study the structural protein VP4. Studies have shown that its hemagglutinating activity is specifically inhibited by the antiserum, thereby confirming that VP4 is the hemagglutinin of rotaviruses. Similar data were previously published by M. Ezekiel (1995).

Thus, the presented works show that RSC and RTGA are still used in the study of rotavirus infection, however, these methods are of an auxiliary nature and should be duplicated by other tests.

Neutralization tests

The neutralization reaction is based on the ability of human or animal immune sera to neutralize virus reproduction and thereby prevent manifestations associated with this phenomenon. Depending on the biological model, these manifestations can be: the development of a disease with a specific clinic, reproduction and release of the virus, the production of specific antibodies, as well as the appearance of a cytopathic effect or the formation of spots when using cell culture.

Macrobiological models are currently used mainly in veterinary medicine; the detection of viruses in humans is in most cases carried out on cell cultures, both primary and transfused. PH methods are divided into two groups according to the basic design scheme. In one group of methods, undiluted serum is combined with serial dilutions of the virus, in the other, serum dilutions are tested with a constant dose of virus. To set up a neutralization reaction, a pathogen that causes a pronounced cytopathic effect or intensively reproduces in a biological model is preferable.

However, rotaviruses, unfortunately, have a weak cytopathic effect, as convincingly demonstrated by researchers led by B. Weber (1992). A study of 121 stool samples from children with acute respiratory disease using the classical method of virus isolation on MA-104 cells under the control of CPD revealed only 4 positive cases (3.3%), while the use modern methods detection of rotaviruses (ELISA, PCR, EF in PAGE) allowed to increase this figure to 54.4%. Consequently, to increase the sensitivity of PH, additional methodological techniques are required to facilitate visualization of virus reduction, which include: radioactive or immunofluorescent labels, spot formation, immunoperoxidase staining, etc. It should be noted that the very principle of determining the level of virus-neutralizing antibodies is identical classical method PH, differing from it only in the method of resolution, that is, visualization of the virus-neutralizing activity of the serum. In this case, the activity of the serum is determined by its ability to suppress the manifestations of the infectious properties of the virus.

Currently, most studies use two methods to measure the amount of virus-neutralizing antibodies to rotavirus. One of them is based on counting the number of individual virus-infected cells (the plyak method) that specifically bind to fluorescent-conjugated antibodies. In another test, the level of virus neutralization is judged by the decrease in the production of viral antigen using the enzyme immunoassay method. A comparative study of both methods showed a linear relationship between ELISA indicators and the number of ply-forming units for each prototype strain of the rotavirus serotype: Wa, DS-1, P, VA-70. The data obtained make it possible to easily determine the dilution of serum that ensures the neutralization of 60% of the infectious virus (neutralizing antibody titer). It turned out that antibody titers when testing materials by both methods are the same and both tests do not differ in reproducibility of results. Some advantage of the method using antibodies labeled with fluorescein, according to the authors, is greater objectivity in recording the results (automated registration) and less labor intensity.

To measure titers of virus-neutralizing antibodies, modified PH is also used by blocking ELISA with monoclonal antibodies.

• studying the intensity of the production of virus neutralizing antibodies (VNA) during natural infection and vaccination;
• formation of VNA to various structural proteins of rotaviruses;
• assessing the role of VNA in blood serum and secretory antibodies of saliva and intestinal mucosa in protection against natural infection (Ward R. et al., 1990, 1992, 1993, 1995, 1997).

In conclusion, it must be emphasized that the neutralization reaction in its various modifications is still widely used both experimentally and clinical studies and, having high sensitivity and specificity, serves as a standard for all other serological methods.

Precipitation analysis

Precipitation tests are based on the interaction of serum with viral antigens through osmotic processes or under the influence electric field in a gel medium or other type of carrier. One of these methods is the reaction of counter immunoelectrophoresis (CIEF). The authors examined the blood sera of healthy and sick adults and children with rotavirus diarrhea, as well as specific immunoglobulin preparations for the presence of antibodies to human rotavirus using the VIEF method in 7% agarose with the addition of 4% polyethylene glycol. It turned out that antibodies to RV circulated quite widely and were found in sick adults and children in 90.2-87.7%, respectively, and also in 78.4% healthy children under 1 year of age. All 32 series of immunoglobulin contained antibodies to RV in titers of 1:4-1:128. According to the authors, the method is suitable for studying population immunity.

Radioimmunoprecipitation

Another precipitation technique is radioimmunoprecipitation. The authors used this method to study the immune response to structural and nonstructural proteins during primary rotavirus infection and showed that the immune response was more pronounced to VP4 than to VP7.

When studying the serum and secretory immunological response to the use of a tetravalent reassortant vaccine in newborns, radioimmunological research techniques were used along with ELISA and PH. It has been shown that the immune response in the serum depends on the dose of the administered antigen; As for the detection of antibodies in saliva, the authors explain their appearance with the consumption of breast milk.

The radioimmunoprecipitation reaction is also used in advanced scientific research. Thus, J. Tosser used this method to study the topology of the VP6 protein in the genome structure and suggested that VP 6 is involved in the formation of internal capsid channels.

Other researchers in 1994 also used the radioimmunoprecipitation method to study immunological changes during rotavirus infection. The authors showed that in the acute period, mainly IgA to VP2 and VP6 are recorded, while during the convalescence period the intensity of the production of secretory antibodies (IgA) not only to VP2, but also to other structural and non-structural proteins decreased. Later, similar data were obtained using the radioimmunoprecipitation method. The authors showed that in addition to VP4 and VP7, VP2, VP6 and NSP2 are also involved in the immunological process.

Thus, immunoprecipitation has been used for diagnostic purposes to study the immune response during natural rotavirus infection.

In recent years, immunoprecipitation has been replaced by the radioimmunoprecipitation method, which is used to assess post-vaccination and post-infectious immunity during RV infection and for fine research developments.

Hybridization methods

The immunoblot method is widely used to determine antibodies to rotaviruses. An example of the use of Western blot in the diagnosis of rotavirus infection is the work of H. Ushijima (1989), who, using immunoblotting, characterized the specificity of antibodies to the structural proteins of RV in 21 children with ACC, as well as the level of coproantibodies of the IgA and IgG classes to these proteins in uninfected children. The authors suggested that immunoblotting may make it possible to establish a diagnosis of the disease using one sample of coproantibodies without examining paired blood sera. The possibility of using Western blot to determine the level of antibodies to VP1, VP2, VP4, VP6 and VP7 was demonstrated by Pavlov I. et al. (1991). The authors examined the blood sera of humans and animals for the presence of antibodies to rotavirus strains SA-11, DS-1, Wan Ito and came to the conclusion that Western blot can be successfully used to assess immunity in clinical practice.

It is believed that immunoblotting can be used to confirm the diagnosis of rotavirus infection without the use of paired sera from one coproantibody sample.

Along with RTGA and ELISA, immunoblot was used to study population immunity. A group of individuals who are non-immune to VP2, VP4, VP6 and VP7 have been found, which represent a risk group that primarily requires protection by means of active and passive immunization (Andrade J. P. et al., 1996).

The use of immunoblotting in the study of serological changes in the process of natural RV infection is also noted by Begue R. et al. (1998). The authors confirmed that antibodies to VP2 and VP6 most often participate in the immune response to infection, and antibodies to VP7 and VP4 less often.

Immunofluorescence analysis

The indirect immunofluorescent method using anti-species sera labeled with fluorochrome is used to titrate test sera on cells infected with rotaviruses. The reaction is based on the specific interaction of labeled antibodies and a homologous antigen, and the antigen-antibody complex is easily detected using a fluorescent microscope.

Using this method, the authors conducted a serological survey of two groups of South American Indians and found high percent seropositive individuals in both groups: 67.8 and 77.4% according to ELISA and 45.5 and 56.7% according to IFM results, respectively.

Another study examining the level of antibodies to RV group C in umbilical cord blood using IFM showed that 30% of women of childbearing age had these antibodies, indicating previous infection.

Enzyme immunoassay is currently used, along with PH, in the serological diagnosis of rotavirus infection extremely widely. This method, based on the use of enzyme-labeled antibodies or antigen, is, due to its ease of implementation and cost-effectiveness, the most acceptable and promising for the serological diagnosis of rotavirus infection. The method makes it possible to conduct mass seroepidemic surveys of the population, evaluate the immunological and epidemiological effectiveness of vaccines, and study the protective role of antibodies of various classes in various biological fluids human body, as well as carry out serological diagnosis of rotavirus infection.

Numerous studies using ELISA were conducted by R. Azeredo (1989), which showed that the number of patients with acute respiratory disease of established rotavirus etiology was significantly lower than the level of their infection according to the results of a serological examination. These data revealed that many clinical cases of rotavirus infection are not diagnosed by detection of RV in feces. Further research to study the prevalence of rotavirus infection confirmed this assumption. When conducting seroepidemiological surveys, it was found that 50-70% of the population had high levels of antibodies, which indicates widespread circulation of RV in the human population.

ELISA opens up even greater opportunities for determining the dynamics of the level of antibodies belonging to different classes of immunoglobulins. Thus, according to the authors of the method in 1989, they claim that during the vaccination process, a significant increase in antibodies to RV in the blood was observed in 83-96%. Anti-rotavirus antibodies of the IgA and IgG classes were equally good at detecting ELISA and PH by reducing spots - 67.6 and 70.0%, respectively. Seroconversion of IgM class antibodies was detected in 53 and 44% of children by ELISA and RSK methods, respectively. Based on the results of an analysis of the intensity of production of antibodies of different classes, the authors concluded that the most effective, simple and fast method for detecting seroconversions after vaccination is the method of detecting IgA antibodies using ELISA.

This conclusion was confirmed in the work of R. Bishop (1996), which indicated that according to the results of a survey of 68 mother-child pairs with diagnosed RV infection, it was shown that the detection of IgA antibodies using ELISA in copromaterials serves as the most sensitive marker both clinically significant and asymptomatic infections. Similar results when examining children with severe RV diarrhea were obtained by J. Kolomina in 1998.

However, to study the intensity of seroconversions, it is necessary to study paired sera, which significantly lengthens the diagnostic process. At the same time, it is well known that the appearance of IgM class antibodies is evidence of the onset of an infectious process. According to our data obtained during the examination of patients with rotavirus infection using ELISA and RSK methods, it turned out that, according to the results of ELISA, all examined persons contained in blood IgM antibodies to RV, while according to RSC data - only in 77% (R

In recent years, with the realization of the possibility of determining gene- and serotype-specific antibodies, ELISA has become a truly universal method for studying rotavirus infection, which is used:

• when studying the production of antibodies of classes IgA, M, G to individual structural and non-structural proteins of RV;
• in assessing immunological effectiveness various types vaccines: attenuated, cold-adapted, DNA, reassortant;
• when studying the production of antibodies in various biological fluids of the body under conditions of natural infection and during immunization.

Thus, as follows from the presented data, the immunological aspects of rotavirus infection are studied using a wide range of laboratory methods. As a result, the choice optimal method research, taking into account its resolution, economic and time costs, is quite complex and depends on the tasks facing the researchers, as well as the equipment of the laboratory. And yet, from the variety of methods, in our opinion, two should be highlighted - the neutralization reaction in cell culture and enzyme-linked immunosorbent assay - which provide the study of antibodies to various classes of immunoglobulins. The particular promise of these methods for use in widespread practice is determined by the realized possibility of rapid diagnosis of rotavirus infection.

SEROLOGICAL STUDIES(Latin serum serum + Greek logos doctrine) - methods of immunology that study the specific properties of human or animal blood in order to identify antigens or antibodies using serological reactions.

Beginning of S. and. laid down at the end of the last century, after it was established that the combination of an antigen with an antibody (see Antigen - antibody reaction) is accompanied by a number of phenomena accessible to visual observation - agglutination (see), precipitation (see) or lysis. There is now the possibility of specific recognition of antigens (see) or antibodies (see), if one of these components is known.

In 1897, F. Vidal reported that the blood serum of patients with typhoid fever selectively agglutinates typhoid bacteria and therefore this reaction (see Vidal reaction) can be used in the laboratory. diagnosis of typhoid fever. In the same year, it was shown that filtrates of cultures of plague, typhoid and cholera bacteria, when combined with the corresponding immune sera, form flakes, or precipitates.

The precipitation reaction turned out to be suitable for the detection of any protein antigens. In 1900-1901 K. Landsteiner found that in human erythrocytes there are two different antigens (A and B), and in blood serum there are two agglutinins (a and P), which contributed to the use of the hemagglutination reaction to determine blood groups (see).

The agglutination test to determine blood group and Rh factor is used in obstetric practice, for blood transfusions and tissue transplantation. Antibodies against the Rh factor (see) are incomplete antibodies; they are not capable of direct reaction with Rh-positive erythrocytes, therefore, to detect them, the Coombs reaction is used (see Coombs reaction), based on the detection of incomplete antibodies using antiglobulin sera. The test blood serum is added to red blood cells of known specificity, followed by antiglobulin serum against IgG ( indirect reaction Coombs). Fab fragments of incomplete antibodies of the blood serum under study attach to erythrocytes, and anti-IgG antibodies attach to the free Fc fragments of these antibodies, and agglutination of erythrocytes occurs. To diagnose hemolytic anemia, the direct Coombs reaction is used. In the body of such patients, red blood cells combine with antibodies circulating in the blood against the Rh factor. To identify them, anti-IgG antibodies are added to red blood cells taken from the patient. The appearance of red blood cell agglutination confirms the diagnosis of the disease.

The hemagglutination inhibition reaction - HRI (see Hemagglutination) - is based on the phenomenon of prevention (inhibition) by immune serum of hemagglutination of erythrocytes by viruses. The phenomenon of viral hemagglutination is not serol. reaction and occurs as a result of the combination of the virus with red blood cell receptors, however, HAI is a serological reaction used to detect and titrate antiviral antibodies. RTGA is the main method for serodiagnosis of influenza, measles, rubella, mumps, tick-borne encephalitis and other viral infections, the causative agents of which have hemagglutinating properties.

The reaction of passive, or indirect, hemagglutination. It uses red blood cells or neutral synthetic materials (for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are sorbed (see Boyden reaction). Their agglutination occurs with the addition of appropriate sera or antigens. Red blood cells sensitized with antigens are called antigenic erythrocyte diagnosticum and are used to detect and titrate antibodies. Red blood cells sensitized with antibodies are called immunoglobulin erythrocyte diagnosticums (see) and are used to detect antigens:

The passive hemagglutination reaction is used to diagnose diseases caused by bacteria (typhoid and paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (influenza, adenoviral infections, tick-borne encephalitis, Crimean hemorrhagic fever, etc.). The sensitivity of the passive hemagglutination reaction is not inferior to the method of virus isolation for arenoviral diseases (see), in particular for lymphocytic choriomeningitis. The viral antigen of lymphocytic choriomeningitis is detected in virus carriers (house mice) in a passive hemagglutination reaction with suspensions of extracted organs diluted tens of thousands of times. In case of salmonellosis, the passive hemagglutination reaction detects bacteria at a concentration of up to several hundred microbial bodies in 1 g of feces; dysentery bacteria in food products are detected when there are at least 500 microbial bodies in 1 g of material.

The passive hemagglutination reaction is used in the diagnosis and prevention of viral hepatitis B. In the Soviet Union, to detect the HBs antigen (see Australian antigen) in the blood of patients with acute hepatitis B, a diagnosticum is produced, which is chicken erythrocytes sensitized with goat immunoglobulin against the HBs antigen. A drop of diagnosticum is combined with an equal volume of blood serum from the people being examined, and if HBs antigen is present in it, agglutination occurs. The reaction is capable of capturing up to 1.5 ng/ml of HBs antigen. To detect HBs antibodies, red blood cells with the HBs antigen adsorbed on them, isolated from the blood of patients, are used. The passive hemagglutination reaction is also used to detect hypersensitivity the patient to medications and hormones, for example, penicillin or insulin. In this case, red blood cells of human blood group 0 are sensitized with a drug and then used to detect agglutinins to it in the patient’s blood serum.

The passive hemagglutination reaction is used to detect gonadotropic hormone in urine in order to establish pregnancy (see Chorionic gonadotropin). To do this, standard serum for this hormone is incubated with the urine being tested. With the subsequent addition of red blood cells with the hormone sorbed on them, agglutination does not occur (positive response), since the hormone contained in the urine neutralized the agglutinating antibodies.

Reactions based on the phenomenon of precipitation

They are used to determine a wide variety of antigens and antibodies. The simplest example of a qualitative reaction is the formation of an opaque precipitation band at the boundary of the layering of antigen on antibody in a test tube. Various types of precipitation reactions in semi-liquid agar or agarose gels are widely used (double immunodiffusion method according to Ouchterlohn, radial immunodiffusion method, immunoelectrophoresis), which are both qualitative and quantitative in nature (see Immunodiffusion, Immunoelectrophoresis).

To perform double immunodiffusion, a layer of melted gel is poured onto a glass plate and, after hardening, wells with a diameter of 1.5-3 mm are cut out. The test antigens are placed in wells located in a circle, and immune serum of known specificity is placed in the central well. Diffusing towards each other, homologous sera and antigens form a precipitate. With radial immunodiffusion (according to the Mancini method), immune serum is added to agar. The antigen placed in the wells diffuses through the agar, and as a result of precipitation with immune serum, opaque rings are formed around the wells, the outer diameter of which is proportional to the concentration of the antigen. A modification of this reaction is used in the diagnosis of influenza to recognize IgM and IgG antibodies (see Immunoglobulins). Influenza antigen is added to the agar, and blood serum is added to the wells. The plates are then treated with immune sera against IgM or IgG antibodies, which helps to detect the reaction of the corresponding antibodies with antigens. The method allows you to simultaneously determine antibody titers and their belonging to a specific class of immunoglobulins.

A type of immunoelectrophoresis is radioimmunophoresis. In this case, after electrophoretic separation of antigens, a labeled radioactive iodine immune serum against the identified antigens, and then immune serum against IgG antibodies, which precipitates the resulting complexes of the antibody with the antigen. All unbound reagents are washed away, and the antigen-antibody complex is detected by autoradiography (see).

Reactions involving complement. Reactions involving complement (see) are based on the ability of the complement subcomponent Cl(Clq) and then other complement components to attach to immune complexes.

The complement fixation reaction allows antigens or antibodies to be titrated according to the degree of complement fixation by the antigen-antibody complex. This reaction consists of two phases: the interaction of the antigen with the test blood serum (test system) and the interaction of hemolytic serum with sheep red blood cells (indicator system). If the reaction is positive, complement fixation occurs in the test system, and then when erythrocytes sensitized with antibodies are added, hemolysis is not observed (see Complement fixation reaction). The reaction is widely used for the serodiagnosis of visceral syphilis (see Wasserman reaction) and viral infections (see Virological studies).

Cytolysis. Antibodies against cellular structures can, with the participation of complement, dissolve the cells bearing these structures. Lysis of red blood cells is easily assessed by the degree and intensity of hemoglobin release. Nuclear cell lysis is assessed by calculating the percentage of dead cells that are not stained with methylene blue. Radioactive chromium is also often used, which is previously chemically bonded to the cells. The number of destroyed cells is determined by the amount of unbound chromium released during cell lysis.

The reaction of radial hemolysis of erythrocytes can occur in the gel. A suspension of sheep red blood cells is placed in an agarose gel, adding complement; Wells are made in the layer frozen on the glass and hemolytic serum is added to them. A hemolysis zone will form around the wells as a result of radial diffusion of antibodies. The radius of the hemolysis zone is directly proportional to the serum titer. If you adsorb any antigen on erythrocytes, for example, the glycoprotein hemagglutinin of influenza virus, rubella or tick-borne encephalitis, then you can reproduce the phenomenon of hemolysis with immune sera to these viruses. The radial hemolysis reaction in a gel has found application in the diagnosis of viral infections due to its ease of production, insensitivity to serum inhibitors, and the ability to titrate blood serum according to the diameter of the hemolysis zone without resorting to serial dilutions.

Immune adhesion. Red blood cells, platelets and other blood cells have receptors for the third component of complement (C3) on their surface. If the appropriate immune serum and complement are added to an antigen (bacteria, viruses, etc.), an antigen-antibody complex coated with the C3 component of complement is formed. When mixed with platelets, due to the C3 component of complement, the antigen-antibody complex will settle on the cells and cause their agglutination (see Immune adhesion). This reaction is used to determine antigens of the HLA system (see Transplantation immunity) and in the study of a number of viral infections(tick-borne encephalitis, dengue fever), which are accompanied by immunopathol. processes and circulation in the blood of viral antigens in combination with antibodies.

The neutralization reaction is based on the ability of antibodies to neutralize certain specific functions of macromolecular or soluble antigens, for example, enzyme activity, bacterial toxins, and pathogenicity of viruses. In bacteriology, this reaction is used to detect antistreptolysins, antistreptokinase and antistaphylolysins. The neutralization reaction of toxins can be assessed by biol. effect, for example, antitetanus and antibotulinum serums are titrated (see Toxin - antitoxin reaction). A mixture of toxin and antiserum administered to animals prevents their death. Various options Neutralization reactions are used in virology. By mixing viruses with the appropriate antiserum and injecting this mixture into animals or cell cultures, the pathogenicity of the viruses is neutralized.

Reactions using chemical and physical labels

Immunofluorescence, developed by A. N. Coons in 1942, is used for serol. reactions of fluorochrome-labeled sera (see Immunofluorescence). Fluorochrome-labeled serum forms an antigen-antibody complex with the antigen, which becomes accessible to observation under a microscope in ultraviolet rays, which excite the fluorochrome. The direct immunofluorescence reaction is used to study cellular antigens, detect virus in infected cells, and detect bacteria and rickettsiae in smears. Thus, to diagnose rabies, prints of pieces of the brain of animals suspected of carrying the virus are treated with luminescent anti-rabies serum. If the result is positive, lumps of bright green color are observed in the protoplasm of nerve cells. Express diagnostics of influenza, parainfluenza and adenoviral infection is based on the detection of viral antigens in fingerprint cells from the nasal mucosa.

The more widely used method is indirect immunofluorescence, based on the detection of the antigen-antibody complex using luminescent immune serum against IgG antibodies and used to detect not only antigens, but also titration of antibodies. The method has found application in the serodiagnosis of herpes, cytomegalips, and Lassa fever. In the laboratory, a stock of preparations of antigen-containing cells, for example, grown on pieces, should be stored at -20° thin glass and virus-infected VERO cells or chicken fibroblasts fixed with acetone. The test blood serum is layered onto the preparations, the preparation is placed in a thermostat at f 37° to form immune complexes, and then, after washing off unbound reagents, these complexes are detected with labeled luminescent serum against human globulins. By using labeled immune sera against IgM or IgG antibodies, it is possible to differentiate the type of antibodies and detect the early immune response by the presence of IgM antibodies.

In the enzyme-immunological method, antibodies conjugated with enzymes are used, ch. arr. horseradish peroxidase or alkaline phosphatase. To detect the combination of labeled serum with antigen, a substrate is added that is decomposed by the enzyme attached to the serum, producing a yellow-brown (peroxidase) or yellow-green (phosphatase) color. Enzymes are also used that decompose not only chromogenic, but also lumogenic substrates. In this case, with a positive reaction, a glow appears. Like immunofluorescence, the enzymatic immunological method is used to detect antigens in cells or titrate antibodies on antigen-containing cells.

The most popular type of enzyme-immunological method is immunosorption. On a solid carrier, which can be cellulose, polyacrylamide, dextran and various plastics, the antigen is absorbed. Most often, the surface of the micropanel wells serves as the carrier. The test blood serum is added to the wells with the sorbed antigen, then the enzyme-labeled antiserum and substrate. Positive results are taken into account by changes in the color of the liquid medium. To detect antigens, antibodies are sorbed onto the carrier, then the test material is added to the wells and the reaction is carried out with an enzyme-labeled antimicrobial serum.

The radioimmunological method is based on the use of radioisotope labels of antigens or antibodies. It was originally developed as a specific method for measuring the levels of hormones circulating in the blood. The test system was an isotope-labeled hormone (antigen) and antiserum to it. If a material containing the desired hormone is added to such an antiserum, it will bind part of the antibodies; upon subsequent addition of a labeled titrated hormone, a reduced amount of it will bind to the antibodies compared to the control. The result is assessed by comparing the curves of the bound and unbound radioactive tracer. This type of method is called a competitive reaction. There are other modifications of the radioimmunological method. Radioimmunological method is the most sensitive method for determining antigens and antibodies, used to determine hormones, medicinal substances and antibiotics, for the diagnosis of bacterial, viral, rickettsial, protozoal diseases, the study of blood proteins, tissue antigens.

Comparative characteristics and use of serological research methods in medical practice

Methods S. and. are constantly being improved to increase sensitivity and versatility of use. Initially serol. diagnosis was based on the detection of antibodies. With the advent of the mid-20th century. immunofluorescence and passive hemagglutination reactions, which are more sensitive, made it possible to detect not only antibodies, but also antigen directly in material from patients. Enzyme-immunological and radioimmunological methods, which are 2-3 orders of magnitude more sensitive than immunofluorescence and passive hemagglutination, are close to biological methods. detection of bacteria and viruses. The scope of their application for the detection of both antigens and antibodies is theoretically unlimited.

Serodiagnosis info. diseases is based on the appearance of antibodies to an isolated or suspected pathogen, regardless of whether the pathogen was detected in the acute stage of the disease. Pairs of blood serum taken at the onset of the disease and 2-3 weeks later are examined. later. An increase in antibodies in the second blood serum of at least 4 times compared to the first is diagnostically significant. It also matters which class of immunoglobulins the antibodies are represented by. IgM antibodies are detected at the end of the acute period of the disease and in early stage convalescence. IgG antibodies appear in more late dates convalescence and circulate for a long time. If IgM antibodies to the rubella virus are detected in a woman in the first trimester of pregnancy, this serves as a basis for terminating the pregnancy, since during this period the fetus is especially sensitive to the virus. With different inf. diseases, the most specific and convenient methods are selectively used.

S. and. widely used in epidemiology. Systematic collection and examination of blood samples from various population groups makes it possible to understand the contacts of the population with the source of infectious agents. diseases. Studying the level of collective immunity allows us to identify high-risk groups and plan vaccination activities, and study the geographic spread of infections. S. and. different age groups of the population made it possible, for example, to retrospectively identify circulation different options influenza virus at certain periods of time.

S. and. are of great importance in the study of hereditary diseases (see) and autoimmune diseases, accompanied by the appearance of tissue- and organ-specific antibodies that destroy the corresponding target cells, as well as in oncology for the detection of tumor antigens. Thus, the immunodiagnosis of liver cancer is based on the determination of alpha-fetoprotein and other embryonic antigens in the blood serum of patients using the immunodiffusion method and the radioimmunological method.

Significant scientific progress in the study of the fine antigenic structure of cellular antigens, antigens of bacteria and viruses is achieved through the use of serol. reactions of monoclonal antibodies, which can be obtained to individual antigen determinants.

Bibliography: Research methods in immunology, ed. I. Lefkovits and B. Pernis, trans. from English, M., 1981; Guide to immunology, ed. O. E. Vyazova and Sh. X. Khodzhaeva, M., 1973; Guide to Clinical Laboratory Diagnostics, ed. V.V. Menshikova, M., 1982; Immunology, ed. by J.-F. Bach, N. Y., 1978.

S. Ya. Gaidamovich.

Serological research method.

Antigen-antibody reactions

Features of the interaction of antibodies with antigens are the basis of diagnostic reactions in laboratories.

Reaction in vitro between antigen and antibody comprises:

specific

nonspecific phase.

IN spicesphysical phase rapid specific binding of the active center of the antibody to the antigen determinant occurs.

Then comes nonspecific phase - slower, which manifests itself visible physical phenomena, for example, the formation of flakes (agglutination phenomenon) or precipitate in the form of turbidity. This phase requires the presence of certain conditions (electrolytes, optimal pH of the environment).

For this purpose they use serological methods (from lat. serum - whey and logos - teaching), i.e. methods for studying antibodies and antigens using antigen-antibody reactions determined in blood serum and other fluids, as well as body tissues.

When isolating a microbe from a patient, the pathogen is identified by studying its antigenic properties using immune diagnostic sera, i.e. blood sera of hyperimmunized animals containing specific antibodies. This is the so-called serologicalidentification microorganisms.

Agglutination reactions

Agglutination reaction- RA(from lat. agglutinatio - gluing) is a simple reaction in which antibodies bind corpuscular antigens (bacteria, erythrocytes or other cells, insoluble particles with antigens adsorbed on them, as well as macromolecular aggregates). It occurs in the presence of electrolytes, for example, when an isotonic sodium chloride solution is added.

Various response optionsagglutination:

expanded,

approximate,

indirect, etc.

Agglutination reaction manifested by the formation of flakes or sediment (cells “glued together” by antibodies that have two or more antigen-binding centers).

Ra is used for:

1) antibody determinations pain in blood serumnykh, for example, with brucellosis (Wright, Heddelson reaction), typhoid fever and paratyphoid fever (Vidal reaction) and other infectious diseases;

determining the pathogen , isolated from a patient;

determination of blood groups using monoclonal antibodies against erythrocyte allo-antigens.

To determine antibodies in a patient putdetailed agglutination reaction: add to dilutions of the patient's blood serum diagnosticum(suspension of killed microbes) and after several hours of incubation at 37 °C, the highest serum dilution (serum titer) is noted, at which agglutination occurred, i.e., a precipitate formed.

The nature and speed of agglutination depend on the type of antigen and antibodies. An example is the peculiarities of the interaction of diagnosticums (O- and K-antigens) with specific antibodies. Agglutination reaction with O-diagnosticum(bacteria killed by heat, retaining heat-stable O-antigen) occurs in the form of fine-grained agglutination. The agglutination reaction with H-diagnosticum (bacteria killed by formaldehyde, retaining the thermolabile flagellar H-antigen) is coarse and proceeds faster.

If it is necessary to determine the pathogen isolated from the patient, put approximately immediate agglutination reaction, using diagnostic antibodies (agglutinating serum), i.e., serotyping of the pathogen is carried out. An indicative reaction is carried out on a glass slide. A pure culture of the pathogen isolated from the patient is added to a drop of diagnostic agglutinating serum at a dilution of 1:10 or 1:20. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied. When a flocculent sediment appears in a drop containing serum and microbes, a expanded agglutination reaction in test tubes with increasing dilutions of agglutinating serum, to which 2-3 drops of the pathogen suspension are added. Agglutination is taken into account by the amount of sediment and the degree of clearness of the liquid. The reaction is consideredpositive, if agglutination is observed at a dilution close to the titer of the diagnostic serum. At the same time, controls are taken into account: serum diluted with isotonic sodium chloride solution should be transparent, the suspension of microbes in the same solution should be uniformly cloudy, without sediment.

Different related bacteria can be agglutinated by the same diagnostic agglutinating serum, which makes their identification difficult. Therefore they use adsorbed agglutinating serummouths, from which cross-reacting antibodies have been removed by adsorption to related bacteria. Such sera retain antibodies that are specific only to a given bacterium. The production of monoreceptor diagnostic agglutinating sera in this way was proposed by A. Castellani (1902).

Indirect (passive) hemagglutin reactionnation (RNGA, RPGA) based on usedResearch Institute of Red Blood Cells(or latex) with adsorbirobathed on their surface with antigens orantibodies, the interaction of which with the corresponding antibodies or antigens in the blood serum of patients causes gluingdepletion and loss of red blood cells to the bottom of the test tube or cells in the form of scalloped sediment.

If there is a negative reaction red blood cells settle in the form of a “button”. Typically, the RNGA detects antibodies using an antigen erythrocyte diagnosticum, which is erythrocytes with antigens adsorbed on them. Sometimes antibody erythrocyte diagnostic fluids are used, on which antibodies are adsorbed. For example, botulinum toxin can be detected by adding erythrocyte antibody botulinum diagnosticum to it (this reaction is called reverse reactionindirect hemagglutination -RONG). RNGA is used to diagnose infectious diseases, determine gonadotropic hormone in urine when establishing pregnancy, to identify hypersensitivity to drugs, hormones, and in some other cases.

Coagglutination reaction. The pathogen cells are determined using staphylococci pre-treated with immune diagnostic serum. Staphylococci containing protein A, having an affinity for the Fc fragment of immunoglobulins, nonspecifically adsorb antimicrobial antibodies, which then interact with active centers with the corresponding microbes isolated from patients. As a result of coagglutination, flakes are formed consisting of staphylococci, diagnostic serum antibodies and the detected microbe.

Hemagglutination inhibition reaction(RTGA) based on blockade suppressionvirus tigens antibodies of immune serum, as a result of which viruses lose their ability to agglutinate red blood cells. RTGA is used to diagnose many viral diseases, the causative agents of which (influenza viruses, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the red blood cells of various animals.

Agglutination reaction to determine anti-Rhesus antibodies (indirect reactionCoombs) used in patients with intravascular hemolysis. In some of these patients, anti-Rhesus antibodies are detected, which are incomplete and monovalent. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. The presence of such incomplete antibodies is determined by the indirect Coombs test. To do this, antiglobulin serum (antibodies against human immunoglobulins) is added to the system of anti-Rh antibodies + Rh-positive erythrocytes, which causes agglutination of erythrocytes. Using the Coombs reaction, pathological conditions associated with intravascular lysis of erythrocytes of immune origin are diagnosed, for example, hemolytic disease of the newborn: erythrocytes of a Rh-positive fetus combine with incomplete antibodies to the Rh factor circulating in the blood, which have passed through the placenta from a Rh-negative mother.