The analysis for syphilis is one of the most common laboratory tests. Tests for syphilis are widely used in preventive examinations. With the help of microscopy, the causative agent of syphilis is revealed -. With the help of serological reactions, the diagnosis of syphilis is confirmed, the diagnosis of latent syphilis is established, 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, the detection of the causative agents of syphilis in the 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 detected by doctors of different specialties. Differential diagnosis of primary syphilis is carried out with a number of diseases.

Fig. 1. In the photo, the primary manifestation of syphilis is a hard chancre.

Antibodies to Treponema Pallidum and Serologic Diagnosis

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

Antibodies to treponema pallidum IgM

IgM antibodies are the first to develop 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 has not been treated, then the antibodies disappear after six months. IgM antibodies disappear after 1 to 2 months. after, after 3 - 6 months. - after treatment of late syphilis. If their growth is recorded, then this serves or speaks of re-infection. IgM molecules are large and do not cross the placenta to the fetus.

Antibodies to treponema pallidum IgG

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

Non-specific antibodies

There are many serological reactions. This is due to the antigenic plurality of pale treponemas. In the blood serum of a sick person at different stages of syphilis, in addition to specific ones, certain nonspecific antibodies are formed - agglutinins, complement-binding, immobilisins, antibodies that cause immune fluorescence, precipitins, etc. Serological reactions for the detection of nonspecific antibodies have relative specificity, therefore, to avoid diagnostic errors you should use not one, but a complex of serological reactions (CSR).

False positive tests for syphilis

A distinctive feature of non-treponemal tests is the receipt of false positive reactions. Antibodies-reagins, which are produced in human blood against the cardiolipin antigen, are recorded not only in syphilis, but also in other diseases: collagenoses, hepatitis, kidney diseases, thyrotoxicosis, oncological diseases, and infectious diseases (leprosy, tuberculosis, brucellosis, malaria , scarlet fever), during pregnancy and menstruation, when eating fatty foods and alcohol. It is noted that the number of false positive reactions increases with age.

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

Laboratory diagnosis of syphilis using serological tests

Serologic tests for syphilis are classified as treponemal and non-treponemal.

1. Non-treponemal tests

The cardiolipin antigen is used as an antigen in this group of tests. Lipid antigens of the causative agents of syphilis are the most numerous. They make up 1/3 of the dry mass of the cell. Non-treponemal tests detect reagin antibodies that are produced against the cardiolipin antigen. This group includes the complement fixation reaction (RSCcard), the microprecipitation reaction (RMP), the rapid determination of plasma reagins (RPR), etc. Using non-treponemal tests, primary screening for syphilis (examination of population groups) is carried out, and the possibility of obtaining results in a quantitative version allows you to use these tests to monitor the effectiveness of treatment. Positive non-treponemal test results should 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 treponemal pallidus. With their help, the positive results of non-treponemal tests are confirmed. The group includes: RSKtrep - the reaction of complement binding, RIF - the reaction of immunofluorescence and its modification, RIT, RIBT - the reaction of immobilization of pale treponemas, RPHA - the reaction of passive hemagglutination, ELISA - enzyme immunoassay.

3. Tests for syphilis using recombinant antigens

Antigens for this group of tests are genetically engineered and used in reactions - RPHA and ELISA, in immunoblotting (IB) analyzes and immunochromatographic analysis.

Fig. 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 tests (CSR) are used. Serological diagnostics are applied from the 5th week from the moment of infection or from 2-3 weeks after the appearance. Antibodies are detected in almost all patients with fresh primary,. Serological reactions are positive in 70 - 80% of patients with, in 50 - 60% of cases in patients with tertiary latent syphilis.

Serologic tests using non-treponemal tests can give false positive results.

Fig. 4. Blood sampling for testing for syphilis.

Complement binding reaction (CSC card, CSC with CA, Wasserman reaction)

The Wasserman reaction (RW, RV), invented by A. Wasserman more than 100 years ago, has undergone many changes today, however, as a tribute to tradition, it has retained its name to the present. The complement binding reaction using a cardiolipin antigen is intended not only for the detection of antibodies, but is also performed in a quantitative form - with different dilutions of serum, which makes it possible to use it to monitor the effectiveness of treatment. Low sensitivity and specificity, obtaining false positive results are the negative aspects of this type of study.

The essence of the Wasserman reaction is as follows: the antigens that are used when staging 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 (little sediment or +), slightly positive (++), positive (+++) and sharply positive (++++).

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

In case of sharply positive reactions, a quantitative determination of reagins is carried out, for which serum is used in dilutions from 1:10 to 1: 320, which makes it possible to use this type of study to monitor the effectiveness of treatment. For example, a decrease in the titer of antibodies and their subsequent seronegativeness (obtaining negative results) indicates a successful cure of the disease.

Fig. 5. Blood test for syphilis - Wasserman's reaction.

Precipitation microreaction (MPR)

The microreaction of precipitation is used for mass examinations of certain groups of the population, for the diagnosis of syphilis and control over the effectiveness of treatment. To carry out this type of research, a small amount of the test material is required. The microreaction of precipitation is based on the immunological antigen-antibody reaction. In the case of the presence of antibodies in the blood serum of the subject, the antigen-antibody complex precipitates with the formation of flakes. The reaction is carried out in the wells of a special glass plate. It is estimated by the intensity of the precipitate and the size of the flakes in (+) as Wasserman's reaction. When examining pregnant women, donors and to monitor the effectiveness of treatment is not used. VDRL and RPR are types of microreactions.

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

Fig. 7. Blood test for syphilis - a microprecipitation reaction.

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

All positive tests obtained during non-specific 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 them to control the effectiveness of the treatment, obtaining positive results in spirochetosis and non-venereal treponematosis and obtaining false positive results in cancer, leprosy, and some endocrine pathology. Tests such as RPHA, ELISA and RIF remain positive for many years after syphilis is cured, and in some cases for life.

RIBT and RIF are the more specific of all serological tests used to diagnose syphilis. They make it possible to distinguish between false positive reactions, 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 the child's infection.

The reaction of immobilization of pale treponemas (RIBT, RIT)

The essence of the reaction is that the antibodies in the patient's blood serum immobilize pale treponema. A negative reaction is considered when immobilizing up to 20% of pathogens, weakly positive - 21 - 50%, positive - 50 - 100%. RIBT sometimes gives false positive results. The test is complex and time-consuming, however, it is indispensable for 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 in secondary, early and late syphilis, in 94 - 100% of cases - in other forms of syphilis.

Immunofluorescence reaction (RIF)

The essence of the reaction lies in the fact that pale treponemes (antigens), combined with antibodies labeled with fluorochromes, emit a yellow-green glow in a luminescent microscope. The result is evaluated with a (+) sign. With the help of 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 (RIF modification) is more sensitive, RIF-200 and RIF-abs are more specific.

Fig. 9. Blood test for syphilis - the reaction of immunofluorescence (RIF).

Treponema pallidum immune adherence reaction (PALRT)

The essence of the reaction is that pale treponemas sensitized by the patient's serum in the presence of complement adhere to the surface of erythrocytes. The resulting complexes are precipitated upon cetrofugation. The sensitivity and specificity of this test are close to that of RIF and RIBT.

Immunoassay for syphilis (ELISA)

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

Fig. 10. Immunoassay analyzer.

Passive (indirect) hemagglutination reaction (RPHA)

RPHA is based on the ability of erythrocytes, on which antigens of treponema pallidum are adsorbed, to stick together in the presence of patient's serum (hemagglutination). RPGA is used to diagnose all forms of syphilis, including latent. When a high quality antigen is used, this type of serological reaction exceeds all other tests in terms of specificity and sensitivity.

Fig. 11. RPHA is used to diagnose all forms of syphilis.

Fig. 12. Analysis for syphilis - a reaction of passive (indirect) hemagglutination (scheme).

Fig. 13. The sight of an inverted umbrella occupying the entire bottom of the tube indicates a positive reaction. In the case when erythrocytes settle in a column ("button") in the center of the bottom of the test tube, a negative reaction is indicated.

Fig. 14. RPGA test in laboratory conditions.

Microbiological diagnostics

Along with serological diagnostics, the method of detecting pale treponemas (microbiological diagnostics) plays an important role, especially during the period of seronegative syphilis, when there are no antibodies in the blood, but there are already the first manifestations of fresh primary syphilis (chancre).

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

The best technique for detecting the causative agents of syphilis is the examination of biological material in the dark field of a microscope. This technique allows you to see pale treponema in a live state, to study its structural features and movements, to distinguish pathogenic pathogens from saprophytes.

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

Fig. 16. When studying dry smears, staining according to Romanovsky-Giemsa is used. Pale treponemas turn pink, all other types of spirochetes turn purple.

The detection of pale treponema with dark field microscopy is an absolute criterion for the final diagnosis of syphilis.

Fig. 17. To detect 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 luminescent microscope, gives a greenish glow of bacteria.

Fig. 18. The causative agents of syphilis are clearly visible in smears prepared according to the Levaditi method (silver impregnation). Pale treponemas of a dark color against a yellow background of cells of infected tissues.

Fig. 20. Photo of a colony of pale treponema. Getting bacteria cultured is hard. 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

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

Fig. 21. PCR for syphilis allows detecting DNA or its fragments of pale treponema.

The sensitivity of this research method depends on the presence of pale treponemas in the biological material and reaches 98.6%. The specificity of this test largely depends on the correct choice of a target for amplification during diagnostics and reaches 100%.

At the same time, due to the insufficiently studied comparative characteristics of the sensitivity and specificity of direct methods for diagnosing syphilis and PCR, this method of examination in the Russian Federation for diagnosing the disease is not yet allowed.

PCR for syphilis is allowed to be carried out only in some cases, as an additional method for diagnosing congenital syphilis, neurosyphilis, when it is difficult to diagnose syphilis using serological research methods in HIV patients.

Fig. 22. The detection of treponema palette DNA using PCR indicates either the presence of viable bacteria, or the remains of the dead, but containing additional copies of individual sections of chromosomal DNA capable of forming additional copies.

Serological method is a set of reactions based on antigen-antibody interaction (Ag-Ab) and aimed at detecting antibodies to antigens of pathogens of infectious diseases, or actually microbial antigens in blood serum and other body fluids. The serological method is characterized by high sensitivity and specificity. Most of the reactions of this method are easy to carry out and record, available to a wide range of laboratories, as a rule, are safe, economical, and amenable to standardization. TO disadvantages the serological method can be attributed: 1) the indirect nature of the result, when the etiology of the disease is judged not by the isolation of the pathogen, but by the response of the (immune) organism to the pathogen; 2) the need for parenteral intervention in the patient's body; 3) in most cases, late diagnosis, which is explained by the natural dynamics of the humoral immune response; 4) the ability to take anamnestic Ab (as a result of a previous illness or vaccination) for Ab to the current infection. When determining microbial antigens of the 3rd and 4th there are no drawbacks, but it is necessary to take into account the peculiarities of the circulation of antigens of different microbes and correlate these features with the possibility of taking material for research.

Stages of the serological method:

1) taking research materials... In most cases, the material is blood serum... It is obtained after the formation of a blood clot, the blood should be collected under strictly aseptic conditions according to the standard method,

2) the choice of a serological reaction for a given case depends on the purpose of the study, the alleged disease, the phase of the disease, the material for the study, the sensitivity of the reaction, the capabilities of a particular laboratory. To detect Ab, as well as Ar, agglutination reactions are used ( RA), passive hemagglutination ( RPGA)), immunofluorescence (REEF), inhibition of hemagglutination ( RTGA), precipitation, flocculation, complement fixation reaction (CSC), etc.

3) staging a serological reaction,

4) registration of a serological reaction in order to determine the presence of serological markers of infection.

Various serological reactions are characterized by several general characteristics:

1) since any serological reactions are reactions of the interaction of Ab and Ar, in all cases, to establish the presence of Ab in the studied substrate, a set of known standard corpuscular or soluble Ar is required, called diagnosticums... In turn, to establish the presence of Ar, a set immune diagnostic sera,

2) the interaction of Ar and Ab is carried out only in the presence of an electrolyte, which is usually used as an isotonic solution of sodium chloride or buffer mixtures, the pH of the system should be about 7,

3) for the formation of the Ag-Ab complex, an incubation period is required under special temperature conditions (from +4 ° C to 37 ° C). The formation of a specific immune complex occurs quickly; a phenomenon visible to the naked eye (agglutination, lysis, etc.) - slowly, after a few hours or even days,

4) both components of the serological reaction (antigen and antibodies) must be present in an equivalent ratio. An excess of any of the components blocks the formation of the Ag-Ab complex and contributes to false negative results.

Serological reactions are recorded visually, sometimes using a magnifying glass. The essence of accounting for a serological reaction is reduced to the determination of the phenomenon of binding of Ar and Ab by the formation of the Ag-Ab complex. Visually, the formation of the Ag-Ab complex is accompanied by two main phenomena - agglutination and precipitation. The differences between them are determined by the characteristics of antigens and antibodies specific to them. At the same time, among the microbial antigens there are also those that induce the synthesis of non-precipitating ones, the formation of the Ag-Ab complex in this case is not accompanied by either the phenomenon of agglutination or the phenomenon of precipitation, and the identification of the fact of the formation of the Ag-Ab complex requires marking the diagnostic component of the reaction with special marks , or transfer of the diagnostic antigen to another state of aggregation.

When assessing the reaction, 3 main criteria are used: 1) the presence and intensity of the reaction (in pluses, etc.); 2) diagnostic titer, 3) increase in Ab titer during the course of the disease by 4 times or more. The presence of a reaction is established by visual phenomena or by the binding of an immunochemical marker. To assess the intensity of the serological reaction, the 4 "+" principle is used (Table 7).

Table 7.

System for evaluating serological reactions by 4 "+" for agglutination and precipitation reactions

For the quantitative presentation of the results of a serological reaction, the concept of an antibody or antigen titer is used. To determine the type of Ab (or titer of Ar), it is necessary to put a serological reaction by preparing a series of dilutions of blood serum or other material (titrate). When preparing dilutions of blood serum, an electrolyte solution is used (most often isotonic sodium chloride solution). The dilution (titration) step is set by the ratio of the volume of the electrolyte solution and the volume of blood serum. For example, with successive dilutions with a step of 2 times, equal volumes of electrolyte solution and blood serum are mixed in the 1st test tube, at a step of 5 times, 1 volume of serum is added to 4 volume parts of the electrolyte solution, at a step of 10 times - to 9 volumes of electrolyte solution add 1 volume of blood serum (Table 8).

The serological diagnostic method provides for the use of 2 areas of research: serodiagnostics (detection of specific antibodies in the blood serum of the subjects) and sero-identification (determination of the antigenic properties of an isolated pathogen in order to establish its type and type).

In serodiagnostics, agglutination, precipitation, lysis, CSC reactions, reactions using labeled antigens or antibodies are used. The components of these reactions are: blood serum of the examined patients and standard antigenic preparations. The purpose of the research is to determine the titers of antibodies to pathogens of infectious diseases in the studied sera.

Detection of high titers of specific antibodies, especially if they reach the level of the so-called "diagnostic" titers, confirms the presumptive diagnosis. Great importance is attached to the study of paired sera, when sera taken at the very beginning of the disease (3-4 days) and sera obtained on the 7th - 10th day of the disease are simultaneously titrated. A 4-fold increase in antibody titers is of diagnostic value.

In sero-identification, the agglutination reaction on glass is most often used. Reaction components: grown pure culture of the pathogen (antigen) and standard diagnostic immune rabbit sera (antibodies).

Dignity of the serological method - simplicity, availability, high sensitivity, specificity, rapidity.

Disadvantages: the need for expensive standard drugs and equipment.

Allergic method

This method provides for the detection of body sensitization to a particular infectious agent, which is of great diagnostic value. It is known that the course of some infectious diseases is accompanied by the formation of delayed-type hypersensitivity. In this regard, the intradermal administration of small doses of an allergen to such patients causes a corresponding reaction from the body, caused by a repeated encounter with the allergen. The reaction proceeds in a delayed manner, and redness, swelling, and sometimes infiltration (positive reaction) appear at the injection site of the allergen after 48-72 hours. If the body is not sensitized, intradermal administration will not cause any reaction from the body (negative reaction). The industry produces special preparations for skin-allergic tests: tuberculin, dysenterin, brucellin, tularin, etc.

The allergic method is especially important in cases where identification of the causative agent of an infectious disease is difficult or impossible (for example, with leprosy) or when it takes a long time (for example, with brucellosis). The test with tuberculin is used in practice not only for diagnostic purposes, but also to determine the timing of revaccination of children with BCG vaccine, as well as to study collective anti-tuberculosis immunity.

Advantages of the allergy method - its simplicity, reliability, expressiveness.

Disadvantage - limited use, since not all infectious diseases develop delayed-type hypersensitivity.

SEROLOGICAL REACTIONS IN DIAGNOSTICS

INFECTIOUS DISEASES

Currently, immunological research methods are widely used for laboratory diagnosis of infectious and non-infectious diseases.

Interaction reactions between antigens and antibodies are called serological (serum - serum, logos - learning).

The essence of all serological reactions consists in the specific combination of antigens and their corresponding antibodies to form a complex. A serological reaction is possible if there is an immunological correspondence (homology) - the main components - antigens and antibodies. The basis of immunological specificity is the structural complementarity of antigen and antibody.

The process of interaction of antigens and antibodies proceeds in two phases - specific and non-specific. The first phase is developing rapidly. It consists of a specific connection of the active center of the antibody with the determinant groups of the corresponding (homologous) antigen. The subsequent phase develops more slowly, nonspecific - this is an external manifestation of the antigen-antibody reaction (flocculation, turbidity, etc.)

Serological tests are used for two purposes:

1) to detect antibodies in the studied serum using known antigens (serodiagnostics);

2) to establish an unknown antigen using known sera (sero-identification).

The determination of an unknown antigen in microbiological studies is carried out in order to establish the generic, species, type of pathogens isolated from the subject. In such cases, of the two main components of the reaction (antibody, antigen), the antigen is unknown; the reaction must be carried out with known antibodies. In this case, the antigen is a pure culture of microorganisms isolated from the test material. Immune diagnostic sera are used as known antibodies. The latter are obtained from the blood of animals (most often rabbits), previously immunized with the corresponding bacterial antigens. Immune sera should contain high levels of specific antibodies.

In addition, serological tests can be used to determine various non-bacterial antigens: to establish blood groups, tissue antigens, tumors, to select donor-recipient pairs for organ and tissue transplantation, etc.

Serological tests can also be used to detect antibodies in serum. In such cases, a known antigen (diagnosticum) is required. Suspensions of live or killed microbes, extracts or isolated chemical fractions from them can be used as antigens.

The numerous serological tests currently used in healthcare practice differ in the phenomenon of antigen-antibody association. The apparent manifestation of the reaction will be different depending on which technique is used, in what physical state the antigen or immune serum is used, whether complement is involved in the reaction. For example, if a corpuscular antigen is used, the phenomenon of agglutination occurs - the adhesion of particles (agglutination reaction). Since the particulate antigens are composed of relatively large particles, a visible flocculent precipitate forms. If a soluble antigen is used in the reaction, the precipitation of a finely granular precipitate is observed (precipitation reaction). If complement is introduced into the reaction of interaction of bacterial antigens and immune serum, bacteriolysis (dissolution of bacterial antigens) or lysis of erythrocytes (when they are used as an antigen) occurs. The use in serological reactions of immune sera, labeled with fluorochromes, enzymes or radioisotopes, makes it possible to establish the specific binding of antigens to antibodies by the corresponding phenomenon (luminescence, change in color or activity of a radioisotope label).

Criteria such as specificity and sensitivity are used to assess serological responses. Specificity is the ability of antigens to react only with homologous antibodies. Sensitivity is the ability to interact specifically with minimal amounts of antigens and antibodies.

Agglutination reaction

Agglutination reaction (RA) is a specific interaction of antigens with antibodies, expressed in the adhesion of antigens under the influence of serum antibodies and their precipitation in the presence of an electrolyte. Was the first of the proposed serological reactions.

The peculiarity of the agglutination reaction is that cells or other corpuscular particles are used as antigens during its setting. The reaction is based on the ability of corpuscular antigens to specifically bind with homologous antibodies of immune sera to form a precipitate in the form of grains, flakes, lumps. The reaction takes place only in the presence of electrolytes. The antigens used in the reaction are called agglutinogens, antibodies are called agglutinins, and the resulting precipitate is called agglutinate.

As an antigen in the agglutination reaction, a suspension of live cultures in isotonic sodium chloride solution or microbes killed by formalin, alcohol or heating, as well as erythrocytes, leukocytes or other cells, can be used.

The source of antibodies is a known agglutinating serum obtained by immunizing animals with appropriate antigens, or the serum of the patient being examined.

There are different ways of staging the agglutination reaction. Of these, the most commonly used are: orientation reaction on glass, expanded agglutination in test tubes, hemagglutination and indirect hemagglutination reactions (RNGA).

Similar information.

Serological research (tests) - laboratory research methods based on the detection of antibodies or antigens in the patient's biological material. Most often, blood is used for analysis, less often urine, saliva, purulent discharge, or tissue samples taken during a biopsy.

Application area

• Determination of the blood group.
• Identification of specific tumor proteins - tumor markers (for example, in case of suspected cancer of the ovaries, prostate, bladder, stomach, etc.).
• Diagnostics of viral, bacterial, fungal, protozoal infections (HIV, syphilis, toxoplasmosis, chlamydia, rubella, herpes, helminthiasis, tick-borne encephalitis, etc.).
• Determination of hormones, enzymes and drugs contained in the test biomaterial in minor concentrations (less than 10-10 g / l).

The essence of the method is serological tests

Serological tests differ in their technique, but they all result from the interaction of antigens (foreign compounds) with the corresponding antibodies. The study consists of two successive phases. The first phase is characterized by the interaction between antigens and antibodies with the formation of immune complexes (positive reaction). In the second phase, external signs appear, confirming the presence of these very complexes (depending on the type of reaction, this may be a turbidity of the test solution, a change in its color, loss of flakes, etc.). The absence of visible physical phenomena is regarded as a negative test result.

Preparation for serological tests

Depends on the type of study. A medical specialist should tell about the peculiarities of passing a specific test when registering for the procedure.

You can take the required serological test at the Spectra clinic. We order analyzes in the best metropolitan laboratories that work according to European standards, which guarantees fast and reliable results. Our doctors will help to decipher the conclusion and give recommendations for further diagnostics.

SEROLOGICAL RESEARCH (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.

The beginning of S. and. put at the end of the last century, after it was found that the combination of an antigen with an antibody (see. Antigen - antibody reaction) is accompanied by a number of phenomena available to visual observation - agglutination (see), precipitation (see) or lysis. There was a 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's reaction) can be used for lab. diagnostics of typhoid fever. In the same year, it was shown that the filtrates of the cultures of plague, typhoid and cholera bacteria, when combined with the corresponding immune sera, form flakes, or precipitate.

The precipitation reaction proved to be suitable for the detection of any protein antigens. In 1900-1901. K. Landsteiner found that in erythrocytes of people 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 reaction to determine the blood group and Rh factor is used in obstetric practice, in blood transfusions and tissue transplantation. Antibodies against the Rh factor (see) are incomplete antibodies, they are not capable of a direct reaction with Rh-positive erythrocytes, therefore, to detect them, use the Coombs reaction (see Coombs reaction), based on the detection of incomplete antibodies using antiglobulin sera. The test blood serum is added to erythrocytes of known specificity, followed by antiglobulin serum against IgG (indirect Coombs reaction). Fab-fragments of incomplete antibodies of the studied blood serum bind to erythrocytes, and anti-IgG antibodies bind to the free Fc-fragments of these antibodies, and erythrocytes agglutinate. To diagnose hemolytic anemia, a direct Coombs reaction is used.In the body of such patients, erythrocytes are combined with antibodies against the Rh factor circulating in the blood. To identify them, antibodies against IgG are added to the erythrocytes taken from the patient. The appearance of erythrocyte agglutination confirms the diagnosis of the disease.

The reaction of inhibition of hemagglutination - RTGA (see. Hemagglutination) - is based on the phenomenon of prevention (inhibition) by the immune serum of hemagglutination of erythrocytes by viruses. The phenomenon of viral hemagglutination is not serol. reaction and occurs as a result of the connection of the virus with erythrocyte receptors, however, RTGA is a serological reaction used to detect and titrate antiviral antibodies. RTGA is the main method of 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 erythrocytes or neutral synthetic chemistry (for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are sorbed (see Boyden's reaction). Their agglutination occurs when the appropriate sera or antigens are added. Erythrocytes sensitized with antigens are called antigenic erythrocyte diagnosticum and are used to detect and titrate antibodies. Erythrocytes 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, adenovirus infections, tick-borne encephalitis, Crimean hemorrhagic fever, etc.) ... The reaction of passive hemagglutination in sensitivity is not inferior to the method of isolating the virus in arenovirus diseases (see), in particular in 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 in the reaction of passive hemagglutination, bacteria are determined 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 diagnostic is made, which is chicken erythrocytes sensitized with goat immunoglobulin against the HBs antigen. A drop of the diagnosticum is combined with an equal volume of blood serum of the examined people, and if the HBs antigen is present in it, agglutination occurs. The reaction is able to capture up to 1.5 ng / ml of HBs antigen. To detect HBs antibodies, erythrocytes are used with HBs antigen, isolated from the blood of patients, sorbed on them. The passive hemagglutination reaction is also used to identify the patient's hypersensitivity to drugs and hormones, for example, penicillin or insulin. In this case, the erythrocytes of the 0 blood group of a person are sensitized with a drug and then used to detect agglutinins 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). For this, a standard serum for this hormone is incubated with the urine to be examined. With the subsequent addition of erythrocytes 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 detect a wide variety of antigens and antibodies. The simplest example of a qualitative reaction is the formation of an opaque precipitation band at the border of antigen-antibody deposition in a test tube. Various types of precipitation reactions in semi-liquid agar or agarose gels are widely used (Ouchterlon's double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis), to-rye are both qualitative and quantitative (see Immunodiffusion, Immunoelectrophoresis).

To set up double immunodiffusion, a layer of melted gel is poured onto a glass plate and, after solidification, holes with a diameter of 1.5-3 mm are cut out. The test antigens are placed in the wells located in a circle, and the 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 (by the Mancini method), the 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 antigen concentration. 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 agar, and blood serum is added to the wells. Then the plates are treated with immune sera against IgM or IgG antibodies, which helps to identify the reaction of the corresponding antibodies with antigens. The method allows you to simultaneously determine the titers of antibodies and their belonging to a certain class of immunoglobulins.

A type of immunoelectrophoresis is radioimmunophoresis. In this case, after electrophoretic separation of antigens into the groove cut parallel to the movement of antigens in the gel, first poured radioactive iodine-labeled immune serum against the antigens to be determined, and then immune serum against IgG antibodies, the edges of the formed antibody-antigen complexes precipitate. All unbound reagents are washed out, 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 to titrate antigens or antibodies 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 tested blood serum (test system) and the interaction of hemolytic serum with sheep erythrocytes (indicator system). If the reaction is positive, complement is bound in the test system, and then, when adding antibodies sensitized erythrocytes, no hemolysis is observed (see Complement Binding Reaction). The reaction is widely used for serodiagnosis of visceral syphilis (see Wasserman's reaction) and viral infections (see Virological studies).

Cytolysis. Antibodies against cellular structures can, with the participation of complement, dissolve the cells that bear these structures. Erythrocyte lysis can be easily assessed by the degree and intensity of hemoglobin release. Lysis of nuclear cells is assessed by calculating the percentage of dead cells that are not stained with methylene blue. Radioactive chromium is also often used, which is preliminarily chemically bound to 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 proceed in a gel. A suspension of ram erythrocytes is placed in an agarose gel, adding complement; holes are made in the layer frozen on the glass and hemolytic serum is introduced into them. As a result of radial diffusion of antibodies, a hemolysis zone will form around the wells. The radius of the hemolysis zone is directly proportional to the serum titer. If you sorb any antigen on erythrocytes, for example, the glycoprotein hemagglutinin of the influenza virus, rubella or tick-borne encephalitis, you can reproduce the phenomenon of hemolysis by immune sera to these viruses. The reaction of radial hemolysis in gel has found application in the diagnosis of viral infections due to its simplicity of setting, insensitivity to serum inhibitors, and the ability to titrate blood serum along the diameter of the hemolysis zone without resorting to serial dilutions.

Immune adhesion. Erythrocytes, platelets and other blood cells have receptors on the surface for the third component of complement (C3). If the corresponding immune serum and complement are added to the antigen (bacteria, viruses, etc.), then an antigen-antibody complex is formed, covered with the C3 component of the complement. When mixed with platelets, thanks to the C3 component of the complement, the antigen-antibody complex will settle on the cells and cause them to agglutinate (see Immune adhesion). This reaction is used to determine the antigens of the HLA system (see. Transplant immunity) and when studying a number of viral infections (tick-borne encephalitis, dengue fever), to-rye are accompanied by immunopathol. processes and circulation of viral antigens in the blood 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, pathogenicity of viruses. In bacteriology, this reaction is used to detect antistreptolysins, antistreptokinase, and antistaphylolysins. The reaction of neutralization of toxins can be assessed by biol. effect, for example, titrate antitetanus and antibotulinic sera (see Toxin - antitoxin reaction). The mixture of toxin and antiserum administered to the animals prevents their death. Various variants of the neutralization reaction are used in virology. By mixing viruses with an appropriate antiserum and introducing this mixture to animals or into cell cultures, the pathogenicity of the viruses is neutralized.

Reactions using chemical and physical tags

Immunofluorescence, developed by Koons (A. N. Coons) in 1942, is used for serol. reactions labeled with fluorochrome of sera (see. Immunofluorescence). The serum labeled with fluorochrome forms an antigen-antibody complex with the antigen, to-ry becomes available for observation under a microscope in ultraviolet rays that excite the fluorochrome glow. Direct immunofluorescence is used to study cellular antigens, detect the virus in infected cells, and detect bacteria and rickettsia in smears. So, for the diagnosis of rabies, the prints of pieces of the brain of animals suspected of carrying a virus are treated with luminescent rabies serum. With a positive result, lumps of bright green color are observed in the protoplasm of nerve cells. Express diagnostics of influenza, parainfluenza and adenovirus infection is based on the detection of viral antigens in the cells of imprints from the nasal mucosa.

The method of indirect immunofluorescence is more widely used, based on the detection of an antigen-antibody complex using luminescent immune serum against IgG antibodies and is used to detect not only antigens, but also titrate antibodies. The method has found application in serodiagnostics of herpes, cytomegalip, and Lassa fever. In the laboratory, a stock of antigen-containing cell preparations should be stored at -20 ° C, for example, VERO cells grown on thin glass pieces and infected with a virus or chicken fibroblasts fixed with acetone. The test blood serum is layered on the preparations, the preparation is placed in a thermostat at f 37 ° for the formation of immune complexes, and then, after washing the unbound reagents, these complexes are detected with labeled luminescent serum against human globulins. Using labeled immune sera against IgM or IgG antibodies, it is possible to differentiate the type of antibodies and detect an 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 compound of the labeled serum with the antigen, a substrate is added, which is decomposed by an enzyme attached to the serum with the appearance of a yellow-brown (peroxidase) or yellow-green (phosphatase) color. They also use enzymes that decompose not only the chromogenic, but also the lumogenic substrate. In this case, with a positive reaction, a glow appears. Like immunofluorescence, the enzyme immunoassay is used to detect antigens in cells or to 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, they sorb the antigen. Most often, the carrier is the surface of the micropanel wells. The test blood serum is added to the wells with the sorbed antigen, then the enzyme-labeled antiserum and the substrate. Positive results are taken into account by the change in the color of the liquid medium. To detect antigens, antibodies are sorbed onto the carrier, then the test material is introduced into the wells and the reaction is developed with an enzyme-labeled antimicrobial serum.

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

Comparative characteristics and use of serological research methods in medical practice

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

Serodiagnostics inf. 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. Investigate pairs of blood serum taken at the beginning of the disease and 2-3 weeks. later. An increase in antibodies in the second blood serum by at least 4 times compared to the first is diagnostically significant. It also matters what class of immunoglobulins antibodies are represented. IgM antibodies are detected at the end of the acute period of the disease and in the early stage of convalescence. IgG antibodies appear at a later stage of convalescence and circulate for a long time. If a woman in the first trimester of pregnancy is found to have IgM antibodies to the rubella virus, then this serves as the basis for terminating the pregnancy, since during this period the fetus is especially sensitive to the virus. With different inf. diseases, they selectively use the most specific and convenient methods.

S. and. widely used in epidemiology. The systematic collection and study of blood samples from various groups of the population makes it possible to understand the contacts of the population with the source of pathogens inf. diseases. The study of the level of herd immunity makes it possible to identify high-risk groups and plan vaccination measures, to study the geographical spread of infections. S. and. different age groups of the population made it possible, for example, to retrospectively identify the circulation of different variants of the 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 immunodiagnostics of liver cancer is based on the determination of alpha-fetoprotein and other embryonic antigens in the blood serum of patients by immunodiffusion and radioimmunological methods.

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

Bibliography: Research Methods in Immunology, ed. I. Lefkovits and B. Pernis, trans. from English., M., 1981; Guide to Immunology, ed. OE Vyazov and Sh. X. Khodzhaeva, M., 1973; Guidelines for Clinical Laboratory Diagnostics, ed. V.V. Menshikov, M., 1982; Immunology, ed. by J.-F. Bach, N. Y., 1978.

S. Ya. Gaidamovich.