Serological reactions in the diagnosis of infectious diseases. Serological method for the diagnosis of viral infections Serological tests used to diagnose viral infections

Antigens- genetically alien substances that, when introduced into the body of an animal or human, cause a specific immune response - the synthesis of antibodies, the formation of sensitized T-lymphocytes, immunological memory or tolerance. Foreign substances are chemical structures that are not present in the body. Foreign to the human body are viruses, microorganisms, as well as cells, tissues, organs of animals and other people. Antigens have several receptors for binding to antibodies and are able to react with them both in the animal or human body (in vivo) and outside the body - in vitro (in vitro).

Antibodies- high molecular weight proteins of the globulin fraction of blood serum. Antibodies are synthesized under the influence of an antigen and are able to specifically react (combine) with the corresponding antigen. All antibodies have a characteristic immunoglobulin structure; differ in immunological, biological and physical properties; and are divided into 5 classes - IgG, IgA, IgM, IgD and IgE.

Serological reactions

In laboratory practice, they use serological reactions- laboratory reactions between antigens and antibodies, which lead to registered changes in the system under study. These reactions are called serological, since serum (serum) containing antibodies is used for their production.

Serological studies performed to detect specific antibodies and pathogen antigen in infectious diseases are more accessible methods laboratory diagnostics than bacteriological detection of the pathogen. In some cases, serological studies remain the only method for diagnosing infectious diseases.

Some methods for the detection of antibodies used in laboratory practice

All serological reactions are based on the interaction of an antigen and an antibody with the formation of immune complexes that can be detected in in vitro tests (i.e. "in vitro" - outside a living organism). Antigen-antibody reactions in the in vitro system may be accompanied by several phenomena - agglutination, precipitation, lysis, and others. The external manifestations of the reaction depend on the physicochemical properties of the antigen (particle sizes, physical state), class and type of antibodies, as well as experimental conditions (consistency of the medium, salt concentration, pH, temperature).

1. Complement binding reaction

Complement is a system of blood plasma proteins, which includes 9 components indicated by the letter C (C1, C2, C3, ... C9), factor B, factor D and a number of regulatory proteins. Some of these components consist of 2-3 proteins, for example C1 is a complex of three proteins. These proteins circulate in the bloodstream and are present on cell membranes. Complement is the most important system of both innate and acquired immunity. This system is designed to protect the body from the action of foreign agents and is involved in the implementation of the body's immune response. The complement was discovered at the end of the 19th century by the Belgian scientist J. Borde.

Complement fixation reaction (CFR)- a serological test used to quantify complement-fixing antibodies and antigens. First described by Bordet and Zhangu (Bordet - Gengou) in 1901. RSK is based on the fact that the antigen-antibody complex is able to absorb the complement that is added to the reaction mixture. When antigens and antibodies correspond to each other, they form an immune complex, to which complement is attached. The specific immune complex adsorbs the complement added to the system, i.e. binding of complement by the antigen-antibody complex. The more antibodies, the more complement is fixed. If the antigen-antibody complex is not formed, then the complement remains free.

The complexity of CSC is that the reaction of formation of the "antigen - antibody - complement" complex is invisible. An additional indicator hemolytic system is used to identify the components of the reaction. Using the hemolysis reaction, a quantitative determination of the complement residue is carried out after the end of the reaction of the antigen with antiserum.

Complement fixation test (RCT) is used to detect antibodies to a specific antigen or determine the type of antigen by a known antibody. This complex serological reaction involves two systems and complement. The first system - bacteriological (main), consists of an antigen and an antibody. The second system is hemolytic (indicator). It includes sheep erythrocytes (antigen) and their corresponding hemolytic serum (antibody).

RSK is put in two steps: first, the antigen is combined with the test blood serum, in which antibodies are searched for, and then complement is added. If the antigen and antibody match, then an immune complex is formed that binds complement. In the absence of antibodies in the serum, the immune complex is not formed and the complement remains free. Since the process of complement adsorption by the complex is visually invisible, a heme system is added to reveal this process.

Due to its high sensitivity, the complement fixation reaction (CFR) is used both for serological diagnosis bacterial and viral infections, allergic conditions, and for the identification of antigens (isolated bacterial culture).

Precipitation reaction (RP)(from Latin praecipitatio - precipitation, falling down) is based on the precipitation of a specific immune complex consisting of a soluble antigen and a specific antibody in the presence of an electrolyte. As a result of the reaction, a cloudy ring or a loose precipitate is formed - a precipitate. A precipitation reaction occurs between a water-soluble antigen and an antibody, resulting in large complexes that precipitate

3. Flocculation reaction

Flocculation reaction (according to Ramon)(from Latin floccus - wool flakes, flocculi - shreds, flakes; flocculation - the formation of loose flocculent aggregates (flocculi) from small particles of the dispersed phase) - the appearance of opalescence or flocculent mass (immunoprecipitation) in a test tube during the reaction of toxin - antitoxin or anatoxin - antitoxin. It is used to determine the activity of antitoxic serum or toxoid.

The flocculation reaction is based on the detection of "initial" flocculation - turbidity during the formation of a complex of exotoxin (anatoxin) + antitoxin in the optimal quantitative ratios of ingredients.

4. Agglutination reaction

Agglutination(from Latin agglutinatio - bonding) is the reaction of the interaction of an antigen with a specific antibody, which manifests itself in the form of bonding. In this case, antigens in the form of particles-corpuscles (microbial cells, erythrocytes, etc.) are glued together by antibodies and precipitate (agglutinate) in the form of flakes. Agglutinates are usually visible to the naked eye. For the reaction to occur, the presence of electrolytes (for example, isotonic sodium chloride solution) is necessary, accelerating the agglutination process.

Using the agglutination test (RA), reactio agglutinationis (English agglutination test), antibodies or corpuscular antigens are detected. Depending on the type of immunodiagnosticum used, there are reactions of microbial agglutination, hemagglutination, latex agglutination, coagglutination, etc.

5. Name of the antibodies involved in the precipitation reactions

Antibodies involved in sedimentary reactions received the traditional name for their interaction with the antigen:

agglutinins - cause agglutination of the corpuscular antigen - agglutinogen and precipitation of the antigen-antibody complex (agglutinate);

precipitins - form a precipitate with a soluble antigen - precipitinogen.

Bacteriolysins (cause lysis of bacteria) and hemolysins (cause lysis of red blood cells) are involved in lytic reactions.

Serological diagnostics based on the antigen-antibody reaction can be used to determine both, and plays a role in determining the etiology of a viral infection even with negative results of virus isolation.

The success of serological diagnosis depends on the specificity of the reaction and compliance with the temporary conditions for taking blood necessary for the synthesis of antibodies by the body.

In most cases, paired blood sera are used, taken at intervals of 2-3 weeks. A positive reaction is considered at least a 4-fold increase in antibody titer. It is known that most specific antibodies belong to the IgG and IgM classes, which are synthesized at different times of the infectious process. Wherein IgM antibodies are early, and the tests used to determine them are used for early diagnosis (it is enough to examine one serum). Antibodies of the IgG class are synthesized later and stored for a long time.

For virus typing, pH is used, for group-specific diagnostics, for example, of adenovirus infection, they use complement fixation reaction(RSK). The most used are hemagglutination inhibition reaction(RTGA), RSK, RIF, passive reactions and reverse passive hemagglutination(RPGA, ROPGA), various variants of ELISA, which almost everywhere replaced RIA, equal in sensitivity to it.

RTGA used to diagnose diseases caused by hemagglutinating viruses. It is based on the binding of antibodies to the patient's serum of the added standard virus. The reaction indicator is erythrocytes agglutinated by the virus (formation of a characteristic "umbrella") in the absence of specific antibodies and settling to the bottom, non-agglutinated if they are present.

RSK is one of the traditional serological tests and is used to diagnose many viral infections. Two systems take part in the reaction: antibodies of the patient's serum + standard virus and sheep erythrocytes + antibodies to them, as well as a titrated complement. If the antibodies and the virus match, this complex binds complement and lysis of sheep erythrocytes does not occur ( positive reaction). With a negative RSC, complement contributes to the lysis of erythrocytes. The disadvantage of the method is its insufficiently high sensitivity and the difficulty of standardizing the reagents.

To take into account the significance of RSK, as well as RTGA, it is necessary to titrate paired sera, that is, taken at the onset of the disease and during convalescence.

RPGA- agglutination of erythrocytes (or polystyrene beads) sensitized by viral antigens in the presence of antibodies. Any viruses can be sorbed on erythrocytes, regardless of the presence or absence of hemagglutinating activity in them. Due to the presence of non-specific reactions, sera are tested at a dilution of 1:10 or more.

RNGA- agglutination of erythrocytes sensitized by specific antibodies in the presence of viral antigens. ROPHA received the greatest distribution in the detection of the HBs antigen both in patients and in blood donors.

IF method as well ELISA used to detect antibodies in serum. ELISA for diagnostic purposes is becoming increasingly important and widespread. The viral antigen is sorbed onto the solid phase (the bottom of the wells of polystyrene plates or polystyrene beads). When the corresponding antibodies in the serum are added, they bind to the adsorbed antigens. The presence of the desired antibodies is detected using anti-antibodies (for example, human) conjugated with an enzyme (peroxidase). Substrate addition and substrate-enzyme reaction give color. ELISA can also be used to determine antigens. In this case, antibodies are adsorbed onto the solid phase.

monoclonal antibodies. Great progress in the diagnosis of viral infections has been made in the last decade, when, with the development of genetic engineering research, a system for obtaining monoclonal antibodies was developed. This dramatically increased the specificity and sensitivity diagnostic methods determination of viral antigens. The narrow specificity of monoclones, representing a small proportion of viral proteins that may not be present in clinical material, is successfully overcome by using several monoclonal antibodies to various viral determinants.

No. 1 Serological tests used for diagnosis viral infections.

immune reactions used in diagnostic and immunological studies in patients and healthy people. For this purpose, apply serological methods, i.e., methods for studying antibodies and antigens using antigen-antibody reactions determined in blood serum and other fluids, as well as body tissues.

The detection of antibodies against the antigens of the pathogen in the patient's blood serum makes it possible to diagnose the disease. Serological studies are also used to identify microbial antigens, various biologically active substances, blood groups, tissue and tumor antigens, immune complexes, cell receptors, etc.

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

In microbiology and immunology, agglutination, precipitation, neutralization reactions, reactions involving complement, using labeled antibodies and antigens (radioimmunological, enzyme immunoassay, immunofluorescent methods) are widely used. The listed reactions differ in the registered effect and staging technique, however, they are all based on the reaction of the interaction of the antigen with the antibody and are used to detect both antibodies and antigens. Immunity reactions are characterized by high sensitivity and specificity.

Features of the interaction of an antibody with an antigen are the basis of diagnostic reactions in laboratories. The in vitro reaction between an antigen and an antibody consists of a specific and a non-specific phase. In the specific phase, there is a rapid specific binding of the active site of the antibody to the determinant of the antigen. Then comes the non-specific phase - slower, which is manifested by visible physical phenomena, such as the formation of flakes (agglutination phenomenon) or precipitate in the form of turbidity. This phase requires certain conditions (electrolytes, optimal pH of the medium).

The binding of an antigen determinant (epitope) to the active site of an antibody Fab fragment is due to van der Waals forces, hydrogen bonds, and hydrophobic interactions. The strength and amount of antigen bound by antibodies depend on the affinity, avidity of antibodies and their valency.

No. 2 The causative agents of leishmaniasis. Taxonomy. Feature. Microbiological diagnostics. Treatment.

Taxonomy: type Sarcomastigophorae, subtype Mastigophora - flagella, class Zoomastigophora, order Kinetoplastida, genus Leishmania.

cultivation: NNN culture medium containing defibrinated rabbit blood agar. Leishmania also grows on the chorion-allantoic membrane of the chick embryo and in cell cultures.

Epidemiology: in countries with a warm climate. The transmission mechanism of pathogens is transmissible, through the bite of vectors - mosquitoes. The main sources of pathogens: in cutaneous anthroponotic leishmaniasis - people; with cutaneous zoonotic leishmaniasis - rodents; with visceral leishmaniasis - people; with mucocutaneous leishmaniasis - rodents, wild and domestic animals.

Pathogenesis and clinic. There are two causative agents of cutaneous leishmaniasis: L. tropica, the causative agent of anthroponotic leishmaniasis, and L. major, the causative agent of zoonotic cutaneous leishmaniasis.

Anthroponotic cutaneous leishmaniasis is characterized by a long incubation period - several months. At the site of a mosquito bite, a tubercle appears, which increases and ulcerates after 3 months. Ulcers are most often located on the face and upper limbs, cicatrize by the end of the year. Zoonotic cutaneous leishmaniasis (early ulcerative leishmaniasis, Pendinsky ulcer, rural form) is more acute. Incubation period is 2-4 weeks. Weeping ulcers are more often localized on lower limbs. Mucocutaneous leishmaniasis is caused by the leishmania of the L. braziliensis complex; develops granulomatous and ulcerative lesions of the skin of the nose, mucous membranes of the mouth and larynx. Antraponous visceral leishmaniasis is caused by the leishmania of the L. donovani complex; in patients, the liver, spleen, lymph nodes, bone marrow and digestive tract are affected.

Immunity: persistent lifelong

In smears (from tubercles, contents of ulcers, punctates from organs), stained according to Romanovsky-Giemsa, small, oval-shaped leishmania (amastigotes) are found intracellularly located. To isolate a pure culture of the pathogen, inoculation is done on NNN medium: incubation for 3 weeks. Serological methods are not specific enough. It is possible to use RIF, ELISA.

The skin allergy test for HRT to leishmanin is used in epidemiological studies of leishmaniasis.

Treatment: At visceral leishmaniasis antimony preparations and diamidines (pentamidine) are used. With cutaneous leishmaniasis - quinacrine, amphotericin.

Prevention: destroy sick animals, carry out the fight against rodents and mosquitoes. Immunoprophylaxis of cutaneous leishmaniasis is carried out by inoculation of a live culture of L. major.

TICKET#28

№ 1Immunoglobulins, structure and functions.

nature of immunoglobulins. In response to the introduction of an antigen, the immune system produces antibodies - proteins that can specifically combine with the antigen that caused their formation, and thus participate in immunological reactions. Antibodies belong to?-globulins, i.e., the fraction of blood serum proteins that is the least mobile in an electric field. In the body,?-globulins are produced by special cells - plasma cells. α-globulins that carry the functions of antibodies are called immunoglobulins and are denoted by the symbol Ig. Therefore, antibodies are immunoglobulins produced in response to the introduction of an antigen and capable of specifically interacting with the same antigen.

Functions. The primary function is the interaction of their active centers with complementary determinants of antigens. A secondary function is their ability to:

Bind the antigen in order to neutralize it and eliminate it from the body, i.e., take part in the formation of protection against the antigen;

Participate in the recognition of a "foreign" antigen;

Ensure cooperation of immunocompetent cells (macrophages, T- and B-lymphocytes);

Participate in various forms immune response (phagocytosis, killer function, GNT, HRT, immunological tolerance, immunological memory).

Structure of antibodies. Proteins of immunoglobulins chemical composition belong to glycoproteins, as they consist of protein and sugars; built from 18 amino acids. They have species differences associated mainly with a set of amino acids. Their molecules have a cylindrical shape, they are visible in an electron microscope. Up to 80% of immunoglobulins have a sedimentation constant of 7S; resistant to weak acids, alkalis, heating up to 60 °C. It is possible to isolate immunoglobulins from blood serum by physical and chemical methods (electrophoresis, isoelectric precipitation with alcohol and acids, salting out, affinity chromatography, etc.). These methods are used in production in the preparation of immunobiological preparations.

Immunoglobulins are divided into five classes according to their structure, antigenic and immunobiological properties: IgM, IgG, IgA, IgE, IgD. Immunoglobulins M, G, A have subclasses. For example, IgG has four subclasses (IgG, IgG 2 , IgG 3 , IgG 4). All classes and subclasses differ in amino acid sequence.

Molecules of immunoglobulins of all five classes consist of polypeptide chains: two identical heavy chains H and two identical light chains - L, connected by disulfide bridges. According to each class of immunoglobulins, i.e. M, G, A, E, D, distinguish five types of heavy chains: ? (mu), ? (gamma), ? (alpha), ? (epsilon) and? (delta), differing in antigenicity. Light chains of all five classes are common and come in two types: ? (kappa) and? (lambda); L-chains of immunoglobulins of various classes can join (recombine) with both homologous and heterologous H-chains. However, in the same molecule there can only be identical L-chains (? or?). Both H- and L-chains have a variable - V region, in which the amino acid sequence is unstable, and a constant - C region with a constant set of amino acids. In light and heavy chains, NH 2 - and COOH-terminal groups are distinguished.

During processing? -globulin mercaptoethanol destroys disulfide bonds and the immunoglobulin molecule decomposes into separate chains of polypeptides. When exposed to the proteolytic enzyme papain, immunoglobulin is cleaved into three fragments: two non-crystallizing fragments containing determinant groups to the antigen and called Fab fragments I and II, and one crystallizing Fc fragment. FabI and FabII fragments are similar in properties and amino acid composition and differ from the Fc fragment; Fab- and Fc-fragments are compact formations interconnected by flexible sections of the H-chain, due to which immunoglobulin molecules have a flexible structure.

Both H-chains and L-chains have separate, linearly connected compact regions called domains; there are 4 of them in the H-chain, and 2 in the L-chain.

Active sites, or determinants, that form in the V-regions occupy approximately 2% of the surface of the immunoglobulin molecule. Each molecule has two determinants related to hypervariable sections H-and L-chains, that is, each immunoglobulin molecule can bind two antigen molecules. Therefore, antibodies are divalent.

The typical structure of an immunoglobulin molecule is IgG. The remaining classes of immunoglobulins differ from IgG in additional elements of the organization of their molecules.

In response to the introduction of any antigen, antibodies of all five classes can be produced. Usually, IgM is produced first, then IgG, the rest - a little later.

No. 2 The causative agent of chlamydia. Taxonomy. Feature. Microbiological diagnostics. Treatment.

Taxonomy: order Chlamydiales, family Chlamydaceae, genus Chlamydia. The genus is represented by the species C.trachomatis, C.psittaci, C.pneumoniae.

Diseases caused by chlamydia are called chlamydia. Diseases caused by C. trachomatis and C. pneumoniae are anthroponoses. Ornithosis, the causative agent of which is C. psittaci, is a zooanthroponotic infection.

Morphology of chlamydia: small, gram "-" bacteria, spherical shape. Do not form spores, no flagella and capsules. Cell wall: 2-layer membrane. They have glycolipids. Gram is red. The main staining method is according to Romanovsky-Giemsa.

2 forms of existence: elementary bodies (inactive infectious particles, outside the cell); reticular bodies (inside cells, vegetative form).

Cultivation: Can only be propagated in living cells. AT yolk sac developing chick embryos, susceptible animals and in cell culture

Enzymatic activity: small. They ferment pyruvic acid and synthesize lipids. Not capable of synthesizing high-energy compounds.

Antigenic structure: Antigens of three types: genus-specific thermostable lipopolysaccharide (in the cell wall). Identified with the help of RSK; species-specific antigen of a protein nature (in the outer membrane). Detect using RIF; variant-specific antigen of a protein nature.

pathogenicity factors. The proteins of the outer membrane of chlamydia are associated with their adhesive properties. These adhesins are found only in elementary bodies. Chlamydia produce endotoxin. Some chlamydia have been found to have a heat shock protein that can cause autoimmune reactions.

resistance. High to various environmental factors. Resistant to low temperatures, drying. Sensitive to heat.

C. trachomatis is the causative agent of diseases of the genitourinary system, eyes and respiratory tract in humans.

Trachoma is a chronic infectious disease characterized by damage to the conjunctiva and cornea of ​​the eyes. Anthroponosis. Transmitted by contact-household way.

Pathogenesis: affects the mucous membrane of the eyes. It penetrates the epithelium of the conjunctiva and cornea, where it multiplies, destroying cells. Follicular keratoconjunctivitis develops.

Diagnostics: examination of scrapings from the conjunctiva. In the affected cells, when stained according to Romanovsky-Giemsa, cytoplasmic inclusions of purple color are found, located near the nucleus - Provachek's body. RIF and ELISA are also used to detect a specific chlamydial antigen in affected cells. Sometimes they resort to the cultivation of chlamydia trachoma on chicken embryos or cell culture.

Treatment: antibiotics (tetracycline) and immunostimulants (interferon).

Prevention: Nonspecific.

Urogenital chlamydia is a sexually transmitted disease. This is an acute / chronic infectious disease, which is characterized by a predominant lesion of the genitourinary tract.

Human infection occurs through the mucous membranes of the genital tract. The main mechanism of infection is contact, the mode of transmission is sexual.

Immunity: cellular, with the serum of infected - specific antibodies. After the transferred disease - it is not formed.

Diagnostics: In diseases of the eyes, a bacterioscopic method is used - intracellular inclusions are detected in scrapings from the epithelium of the conjunctiva. RIF is used to detect chlamydia antigen in affected cells. In case of damage to the genitourinary tract, a biological method can be applied, based on infection with the test material (scrapings of the epithelium from the urethra, vagina) of cell culture.

Statement RIF, ELISA allow you to detect chlamydia antigens in the test material. Serological method - for the detection of IgM against C. trachomatis in the diagnosis of pneumonia in newborns.

Treatment. antibiotics (azithromycin from the macrolide group), immunomodulators, eubiotics.

Prevention. Only non-specific (treatment of patients), personal hygiene.

Venereal lymphogranuloma is a sexually transmitted disease characterized by lesions of the genital organs and regional lymph nodes. The mechanism of infection is contact, the route of transmission is sexual.

Immunity: persistent, cellular and humoral immunity.

Diagnostics: The material for the study is pus, biopsy from the affected lymph nodes, blood serum. Bacterioscopic method, biological (cultivation in the yolk sac of a chicken embryo), serological (RCC with paired sera is positive) and allergological (intradermal test with chlamydia allergen) methods.

Treatment. Antibiotics - macrolides and tetracyclines.

Prevention: Nonspecific.

C. pneumoniae - the causative agent of respiratory chlamydia, causes acute and chronic bronchitis and pneumonia. Anthroponosis. Infection is by airborne droplets. They enter the lungs through the upper respiratory tract. Cause inflammation.

Diagnostics: setting RSK for the detection of specific antibodies (serological method). In primary infection, IgM detection is taken into account. RIF is also used to detect chlamydial antigen and PCR.

Treatment: Carried out with the help of antibiotics (tetracyclines and macrolides).

Prevention: Nonspecific.

C. psittaci - the causative agent of ornithosis - acute infectious disease characterized by damage to the lungs nervous system and parenchymal organs (liver, spleen) and intoxication.

Zooanthroponosis. Sources of infection - birds. The mechanism of infection is aerogenic, the route of transmission is airborne. The causative agent is through the mucus. shells breathe. pathways, into the epithelium of the bronchi, alveoli, multiplies, inflammation.

Diagnostics: The material for the study is blood, sputum of the patient, blood serum for serological testing.

A biological method is used - the cultivation of chlamydia in the yolk sac of a chicken embryo, in cell culture. Serological method. Apply RSK, RPHA, ELISA, using paired blood serum of the patient. Intradermal allergy test with ornithine.

Treatment: antibiotics (tetracyclines, macrolides).

TICKET#29

No. 1 The causative agent of diphtheria. Taxonomy and characteristics. Conditionally pathogenic corynebacteria. Microbiological diagnostics. Detection of anatoxic immunity. Specific prevention and treatment.

Diphtheria - acute infectious disease, characterized by fibrinous inflammation in the pharynx, larynx, less often in other organs and intoxication phenomena. Its causative agent is Corynebacterium diphtheriae.

Taxonomy. Corynebacterium belongs to the Firmicutes division, the genus Corynebacterium.

Morphological and tinctorial properties. The causative agent of diphtheria is characterized by polymorphism: thin, slightly curved rods (most common), coccoid and branching forms are found. Bacteria are often located at an angle to each other. They do not form spores, do not have flagella, many strains have a microcapsule. A characteristic feature is the presence of volutin grains at the ends of the stick (causes the club-shaped shape). The causative agent of diphtheria according to Gram stains positively.

cultural properties. Facultative anaerobe, opt. temperature. The microbe grows on special nutrient media, for example, on Clauberg's medium (blood-tellurite agar), on which the diphtheria bacillus produces colonies of 3 types: a) large, gray, with uneven edges, radial striation, resembling daisies; b) small, black, convex, with smooth edges; c) similar to the first and second.

Depending on the cultural and enzymatic properties, 3 biological variants of C. diphtheriae are distinguished: gravis, mitis and intermediate intermedius.

enzymatic activity. High. They ferment glucose and maltose in the formation of acid, do not decompose sucrose, lactose and mannitol. They do not produce urease and do not form indole. Produces the enzyme cystinase, which cleaves cysteine ​​to H 2 S. Forms catalase, succinate dehydrogenase.

antigenic properties. O-antigens are thermostable polysaccharides located deep in the cell wall. K-antigens - superficial, thermolabile, grayish-specific. With the help of sera to the K-antigen C. diph. divided into serovars (58).

pathogenicity factors. An exotoxin that disrupts protein synthesis and, as a result, affects the cells of the myocardium, adrenal glands, kidneys, and nerve ganglia. The ability to produce exotoxin is due to the presence in the cell of a prophage carrying the tox gene responsible for the formation of the toxin. Enzymes of aggression - hyaluronidase, neuraminidase. The microcapsule also belongs to the pathogenicity factors.

resistance. Resistant to drying, action low temperatures, therefore, for several days it can be stored on objects in water.

Epidemiology. The source of diphtheria - sick people Infection occurs more often through the respiratory tract. The main route of transmission is airborne, and the contact route is also possible - through linen, dishes.

Pathogenesis. The entrance gate of infection is the mucous membranes of the pharynx, nose, respiratory tract, eyes, genital organs, wound surface. At the site of the entrance gate, fibrinous inflammation is observed, a characteristic film is formed, which is hardly separated from the underlying tissues. Bacteria secrete exotoxin that enters the blood - toxinemia develops. The toxin affects the myocardium, kidneys, adrenal glands, and the nervous system.

Clinic. There are different localization forms of diphtheria: diphtheria of the pharynx, which is observed in 85-90% of cases, diphtheria of the nose, larynx, eyes, vulva, skin, wounds. The incubation period is from 2 to 10 days. The disease begins with an increase in body temperature, pain when swallowing, the appearance of a film on the tonsils, an increase lymph nodes. Swelling of the larynx, diphtheria croup develops, which can lead to asphyxia and death. Other severe complications that can also cause death are toxic myocarditis, paralysis of the respiratory muscles.

Immunity. After the disease - persistent, intense antitoxic immunity. Of particular importance is the formation of antibodies to fragment B. They neutralize diphtheria histotoxin, preventing the attachment of the latter to the cell. Antibacterial immunity - unstressed, grayish-specific

Microbiological diagnostics. With the help of a tampon, a film and mucus from the throat and nose are taken from the patient. To make a preliminary diagnosis, it is possible to use a bacterioscopic method. The main diagnostic method is bacteriological: inoculation on Klauber II medium (blood-tellurite agar), on a dense serum medium to detect cystinase production, on Hiss medium, on a medium to determine the toxigenicity of the pathogen. Intraspecific identification consists in determining the bio- and serovar. For accelerated detection of diphtheria toxin, the following are used: RIHA (indirect hemagglutination reaction) with an antibody erythrocyte diagnosticum, an antibody neutralization reaction (the presence of a toxin is judged by the effect of preventing hemagglutination); RIA (radioimmune) and ELISA (enzymatic immunoassay).

Treatment. The main method of therapy is the immediate administration of a specific antitoxic antidiphtheria equine liquid serum. Human immunoglobulin antidiphtheria for intravenous administration.

Associated vaccines: DTP (absorbed pertussis-tetanus vaccine), DTP (absorbed diphtheria-tetanus toxoid).

№ 2 Classes of immunoglobulins, their characteristics.

Immunoglobulins are divided into five classes according to their structure, antigenic and immunobiological properties: IgM, IgG, IgA, IgE, IgD.

Immunoglobulin class G. Isotype G is the bulk of the Ig serum. It accounts for 70-80% of all serum Ig, while 50% is found in tissue fluid. The average content of IgG in the blood serum of a healthy adult is 12 g/l. The half-life of IgG is 21 days.

IgG is a monomer that has 2 antigen-binding centers (it can simultaneously bind 2 antigen molecules, therefore, its valence is 2), a molecular weight of about 160 kDa, and a sedimentation constant of 7S. There are subtypes Gl, G2, G3 and G4. Synthesized by mature B-lymphocytes and plasma cells. It is well defined in blood serum at the peak of the primary and secondary immune response.

Has high affinity. IgGl and IgG3 bind complement, and G3 is more active than Gl. IgG4, like IgE, has cytophilicity (tropism, or affinity, to mast cells and basophils) and is involved in the development allergic reaction I type. In immunodiagnostic reactions, IgG can manifest itself as an incomplete antibody.

Easily passes through the placental barrier and provides humoral immunity to the newborn in the first 3-4 months of life. It can also be secreted into the secret of mucous membranes, including milk by diffusion.

IgG provides neutralization, opsonization and labeling of the antigen, triggers complement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity.

Immunoglobulin class M. The largest molecule of all Ig. This is a pentamer that has 10 antigen-binding centers, i.e. its valency is 10. Its molecular weight is about 900 kDa, the sedimentation constant is 19S. There are subtypes Ml and M2. The heavy chains of the IgM molecule, unlike other isotypes, are built from 5 domains. The half-life of IgM is 5 days.

It accounts for about 5-10% of all serum Ig. The average content of IgM in the blood serum of a healthy adult is about 1 g/l. This level in humans is reached by the age of 2-4 years.

IgM is phylogenetically the most ancient immunoglobulin. Synthesized by precursors and mature B-lymphocytes. It is formed at the beginning of the primary immune response, it is also the first to be synthesized in the body of a newborn - it is determined already at the 20th week of intrauterine development.

It has high avidity and is the most effective complement activator in the classical pathway. Participates in the formation of serum and secretory humoral immunity. Being a polymeric molecule containing a J-chain, it can form a secretory form and be secreted into the secretion of mucous membranes, including milk. Most of the normal antibodies and isoagglutinins are IgM.

Does not pass through the placenta. The detection of specific isotype M antibodies in the blood serum of a newborn indicates a former intrauterine infection or placental defect.

IgM provides neutralization, opsonization and labeling of the antigen, triggers complement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity.

Immunoglobulin class A. Exists in serum and secretory forms. About 60% of all IgA is found in mucosal secretions.

Serum IgA: It accounts for about 10-15% of all serum Ig. The blood serum of a healthy adult contains about 2.5 g / l of IgA, the maximum is reached by the age of 10. The half-life of IgA is 6 days.

IgA is a monomer, has 2 antigen-binding centers (i.e., 2-valent), a molecular weight of about 170 kDa, and a sedimentation constant of 7S. There are subtypes A1 and A2. Synthesized by mature B-lymphocytes and plasma cells. It is well defined in blood serum at the peak of the primary and secondary immune response.

Has high affinity. May be an incomplete antibody. Does not bind complement. Does not pass through the placental barrier.

IgA provides neutralization, opsonization and labeling of the antigen, triggers antibody-dependent cell-mediated cytotoxicity.

Secretory IgA: Unlike serum, secretory sIgA exists in polymeric form as a di- or trimer (4- or 6-valent) and contains J- and S-peptides. Molecular weight 350 kDa and above, sedimentation constant 13S and above.

It is synthesized by mature B-lymphocytes and their descendants - plasma cells of the corresponding specialization only within the mucous membranes and is released into their secrets. The volume of production can reach 5 g per day. The slgA pool is considered the most numerous in the body - its number exceeds the total content of IgM and IgG. It is not found in blood serum.

The secretory form of IgA is the main factor in the specific humoral local immunity of the mucous membranes of the gastrointestinal tract, genitourinary system and respiratory tract. Due to the S-chain, it is resistant to proteases. slgA does not activate complement but effectively binds to antigens and neutralizes them. It prevents the adhesion of microbes to epithelial cells and generalization of infection within the mucous membranes.

Immunoglobulin class E. Also called reagin. The content in the blood serum is extremely low - approximately 0.00025 g / l. Detection requires the use of special highly sensitive diagnostic methods. Molecular weight - about 190 kDa, sedimentation constant - about 8S, monomer. It accounts for about 0.002% of all circulating Ig. This level is reached by 10-15 years of age.

It is synthesized by mature B-lymphocytes and plasma cells mainly in the lymphoid tissue of the bronchopulmonary tree and the gastrointestinal tract.

Does not bind complement. Does not pass through the placental barrier. It has a pronounced cytophilicity - tropism for mast cells and basophils. Involved in the development of hypersensitivity immediate type- type I reaction.

Immunoglobulin class D. There is not much information about Ig of this isotype. Almost completely contained in the blood serum at a concentration of about 0.03 g / l (about 0.2% of the total number of circulating Ig). IgD has a molecular weight of 160 kDa and a sedimentation constant of 7S, a monomer.

Does not bind complement. Does not pass through the placental barrier. It is a receptor for precursors of B-lymphocytes.

TICKET#30

No. 1 The causative agent of amoebiasis. Taxonomy. Characteristic. Microbiological diagnostics. specific treatment.

Taxonomy: phylum Sarcomastigophorae, subphylum Sarcodina, class Lobosia, order Amoebida.

Morphology: There are two stages of pathogen development: vegetative and cystic. The vegetative stage has several forms: large vegetative (tissue), small vegetative; precystic form, similar to the translucent, forming cysts.

The cyst (resting stage) has an oval shape. A mature cyst contains 4 nuclei. The translucent form is inactive, lives in the lumen of the upper colon as a harmless commensal, feeding on bacteria and detritus.

A large vegetative form is formed, under certain conditions, from a small vegetative form. It is the largest, forms pseudopodia and has movement. Can phagocytose erythrocytes. Found in fresh feces in amoebiasis.

cultivation: on nutrient-rich media.

Resistance: Outside the body, the vegetative forms of the pathogen quickly (within 30 minutes) die. Cysts are resistant to environment are preserved in faeces and water. In foodstuffs, on vegetables and fruits, cysts persist for several days. They die when boiled.

Epidemiology: Amebiasis - anthroponotic disease; the source of the invasion is man. The transmission mechanism is fecal-oral. Infection occurs when cysts are introduced with food, water, through household items.

Pathogenesis and clinic: Cysts that have entered the intestine, and then formed from them, luminal forms of amoebae can live in the large intestine without causing disease. With a decrease in the body's resistance, amoeba penetrate into the intestinal wall and multiply. Intestinal amebiasis develops.

Trophozoites of the tissue form are mobile due to the formation of pseudopodia. They penetrate the wall of the colon, causing necrosis; able to phagocytose erythrocytes; can be found in human feces. With necrosis, ulcers form. Clinically, intestinal amebiasis manifests itself in the form of frequent liquid stools with blood, accompanied by fever and dehydration. In the feces, pus and mucus are found, sometimes with blood.

Amoeba with blood flow can enter the liver, lungs, brain, resulting in the development of extraintestinal amoebiasis.

Immunity: Unstable, mainly the cellular link is activated.

Microbiological diagnostics. The main method is microscopic examination of the patient's feces, as well as the contents of abscesses. internal organs. Smears are stained with Lugol's solution or hematoxylin. Serological studies (RNGA, ELISA, RSK): the highest titer of antibodies in the blood serum is detected with extraintestinal amoebiasis.

Treatment: Apply metronidazole, furamid.

Prevention: identification and treatment of cystic excretors and amoeba carriers, general sanitary measures.

No. 2Interferons. Nature, methods of obtaining. Application.

Interferons are glycoproteins produced by cells in response to viral infection and other stimuli. Block the reproduction of the virus in other cells and participate in the interaction of cells immune system. There are two serological groups of interferons: type I - IFN-? and IFN -?; II type - IFN-.? Type I interferons have antiviral and antitumor effects, while type II interferons regulate the specific immune response and nonspecific resistance.

Interferon (leukocytic) is produced by leukocytes treated with viruses and other agents. α-interferon (fibroblast) is produced by virus-treated fibroblasts.

Type I IFN binds to healthy cells and protects them from viruses. The antiviral effect of type I IFN may also be due to the fact that it is able to inhibit cell proliferation by interfering with the synthesis of amino acids.

IFN-? produced by T-lymphocytes and NK. Stimulates the activity of T- and B-lymphocytes, monocytes / macrophages and neutrophils. Induces apoptosis of activated macrophages, keratinocytes, hepatocytes, cells bone marrow, endotheliocytes and suppresses apoptosis of peripheral monocytes and herpes-infected neurons.

Genetically engineered leukocyte interferon is produced in prokaryotic systems (E. coli). Biotechnology obtaining leukocyte interferon includes the following steps: 1) treatment of leukocyte mass with interferon inducers; 2) isolation of the mRNA mixture from the treated cells; 3) obtaining total complementary DNA using reverse transcriptase; 4) insertion of cDNA into the plasmid coli and its cloning; 5) selection of clones containing interferon genes; 6) inclusion in the plasmid of a strong promoter for successful transcription of the gene; 7) expression of the interferon gene, i.e. synthesis of the corresponding protein; 8) destruction of prokaryotic cells and purification of interferon using affinity chromatography.

Interferons apply for the prevention and treatment of a number of viral infections. Their effect is determined by the dose of the drug, but high doses of interferon have a toxic effect. Interferons are widely used for influenza and other acute respiratory diseases. The drug is effective on early stages disease, applied topically. Interferons have a therapeutic effect in hepatitis B, herpes, and also in malignant neoplasms.

Detection in blood serum the patient's antibodies against the antigens of the pathogen allows you to make a diagnosis of the disease. Serological studies are also used to identify microbial antigens, various biologically active substances, blood groups, tissue and tumor antigens, immune complexes, cell receptors, etc.

When isolating a microbe from the 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 so-called serological identification microorganisms.

Widely used in microbiology and immunology agglutination, precipitation, neutralization reactions, reactions involving complement, using labeled antibodies and antigens (radioimmunological, enzyme immunoassay, immunofluorescent methods). The listed reactions differ in the registered effect and staging technique, however, they are all based on the reaction of the interaction of the antigen with the antibody and are used to detect both antibodies and antigens. Immunity reactions are characterized by high sensitivity and specificity.

Features of the interaction of an antibody with an antigen are the basis of diagnostic reactions in laboratories. Reaction in vitro between antigen and antibody consists of a specific and non-specific phase. AT specific phase there is a rapid specific binding of the active site of the antibody to the determinant of the antigen. Then comes non-specific phase - slower, which is manifested by visible physical phenomena, for example, the formation of flakes (agglutination phenomenon) or precipitate in the form of turbidity. This phase requires certain conditions (electrolytes, optimal pH of the medium).

The binding of an antigen determinant (epitope) to the active site of an antibody Fab fragment is due to van der Waals forces, hydrogen bonds, and hydrophobic interactions. The strength and amount of antigen bound by antibodies depend on the affinity, avidity of antibodies and their valency.

To the question about express diagnostics:

1. A culture isolated in its pure form can be diagnosed.
2. In Specially equipped laboratories (must have permission)
3. Compliance with strict rules such as: an isolated room, the necessary special protective suits, mandatory complete sanitization of the premises after working with the pathogen, sanitization of researchers after work.

Methods of express diagnostics.
1. Bacteriology - combined polytropic nutrient media for rapid study of morphs, tinctor, biochem. properties. The use of an enzyme indicator tape, the electrophysical method, the method of paper discs impregnated with various substances (glucaso, lactose, etc.)
2. Phage diagnostics.
3. Serodiagnosis - Mancini's method, precipitation reaction in the gel according to Ascoli, RA, RPGA.
4. Bacterioscopy - direct and indirect RIF.

Express diagnostic methods for:
Cholera - M. Z. Ermolyeva, immobilization district with cholera diagnostic serum, RIF.
Tularemia - RA on glass, RPGA
Chume - phage typing, the method of carbohydrate paper disks, RPHA.
Anthrax - Ascoli method, RIF, RPGA.

The nature of growth: there are three diffuse (facultative anaerobes), near-bottom (obligate anaerobes) and surface (obligate aerobes.)

Isolation of pure culture anaerobic bacteria

In laboratory practice, it is often necessary to work with anaerobic microorganisms. They are more fastidious to nutrient media than aerobes, they often need special growth supplements, they require the cessation of oxygen supply during their cultivation, their growth period is longer. Therefore, working with them is more complicated and requires considerable attention of bacteriologists and laboratory assistants.

It is important to protect the material that contains anaerobic pathogens from the toxic effects of atmospheric oxygen. Therefore, it is recommended to take the material from the foci of purulent infection during their puncture with a syringe, the time between taking the material and sowing it on a nutrient medium should be as short as possible.

Since special nutrient media are used for the cultivation of anaerobic bacteria, which should not contain oxygen and have a low redox potential (-20 -150 mV), indicators are introduced into their composition - resazurin, methylene blue and the like, which react to a change in this potential. With its growth, the colorless forms of the indicators are resumed and change their color: resazurin stains the medium pink, and methylene blue - blue. Such changes indicate the impossibility of using media for the cultivation of anaerobic microbes.

It helps to reduce the redox potential of introducing into the medium at least 0.05% agar, which, by increasing its viscosity, helps to reduce the supply of oxygen. This, in turn, is also achieved by using fresh (not later than two hours after production) and reduced culture media.

It should be noted that due to the peculiarities of the fermentative type of metabolism of anaerobic bacteria, they require media richer in nutrients and vitamins. The most commonly used are cardio-brain and liver infusions, soy and yeast extracts, casein hydrolytic digest, peptone, tryptone. It is mandatory to add growth factors such as tween-80, hemin, menadione, whole or hemolyzed blood.

The isolation of a pure culture of aerobic microorganisms consists of a number of steps.
On the first day (stage 1 of the study), pathological material is taken into a sterile container (test tube, flask, vial). It is studied for appearance, consistency, color, smell and other signs, a smear is prepared, stained and examined under a microscope. In some cases (acute gonorrhea, plague), at this stage it is possible to make a previous diagnosis, and in addition, select the media on which the material will be sown. I took it with a bacteriological loop (used most often), with a spatula following the Drygalsky method, with a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimum temperature (37 ° C) for 18-48 years. The purpose of the stage is to obtain isolated colonies of microorganisms.
However, sometimes in order to pile up the material, it is sown on liquid nutrient media.

Smears are prepared from suspicious colonies, stained by the Gram method to study the morphological and tinctorial properties of pathogens, and mobile bacteria are examined in a “hanging” or “crushed” drop. These signs are of extremely great diagnostic value in the characterization of certain types of microorganisms.
The remains of the studied colonies are carefully removed from the surface of the medium without touching the others and inoculated on a slant agar or on sectors of a Petri dish with a nutrient medium to obtain a pure culture. Test tubes or dishes with crops are placed in a thermostat at the optimum temperature for 18-24 hours.

On liquid nutrient media, bacteria can also grow differently, although the features of growth manifestations are poorer than on solid ones.

Bacteria are capable of causing diffuse turbidity of the medium, while its color may not change or acquire the color of the pigment. This growth pattern is most often observed in most facultative anaerobic microorganisms.

Sometimes a precipitate forms at the bottom of the tube. It can be crumbly, homogeneous, viscous, slimy, etc. The medium above it can remain transparent or become cloudy. If the microbes do not form a pigment, the precipitate has a livid or yellowish color. As a rule, anaerobic bacteria grow in a similar rank.

Wall growth is manifested by the formation of flakes, grains attached to the inner walls of the test tube. The medium remains transparent.

Aerobic bacteria tend to grow on the surface. Often a delicate colorless or bluish film is formed in the form of a barely noticeable coating on the surface, which disappears when the medium is shaken off or agitated. The film can be moisture, thick, have a knitted, slimy consistency and stick to the loop, stretching for it. However, there is also a dense, dry, brittle film, the color of which depends on the pigment, which is produced by microorganisms.

If necessary, a smear is made, stained, examined under a microscope, and the microorganisms are seeded with a loop on the surface of a dense nutrient medium to obtain isolated colonies.

On the third day (stage 3 of the study), the nature of the growth of a pure culture of microorganisms is studied and its identification is carried out.

First, attention is paid to the characteristics of the growth of microorganisms on the medium and a smear is made, staining it using the Gram method, in order to check the culture for purity. If bacteria of the same type of morphology, size and tinctorial (ability to dye) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, already by the appearance and characteristics of their growth, one can draw a conclusion about the type of isolated pathogens. Determination of the type of bacteria for their morphological features called morphological identification. Determining the type of pathogens by their cultural characteristics is called cultural identification.

However, these studies are not enough to make a final conclusion about the type of isolated microbes. Therefore, they study the biochemical properties of bacteria. They are quite varied.

Most often, saccharolytic, proteolytic, peptolytic, hemolytic properties, the formation of decarboxylase, oxidase, catalase, plasmacoagulase, DNase, fibrinolysin enzymes, the reduction of nitrates to nitrites, and the like are studied. For this, there are special nutrient media that are inoculated with microorganisms (variegated Hiss series, MPB, curdled whey, milk, etc.).

Determining the type of pathogen by its biochemical properties is called biochemical identification.

CULTURING METHODS
AND ISOLATION OF PURE BACTERIA CULTURE

For successful cultivation, in addition to properly selected media and properly seeded, optimal conditions are necessary: ​​temperature, humidity, aeration (air supply). Cultivation of anaerobes is more difficult than aerobes; various methods are used to remove air from the nutrient medium.
Isolation of certain types of bacteria (pure culture) from the test material, which usually contains a mixture of various microorganisms, is one of the stages of any bacteriological study. A pure microbial culture is obtained from an isolated microbial colony.
When isolating a pure culture from blood (hemoculture), it is preliminarily “growth” in a liquid medium: 10–15 ml of sterile blood is inoculated into 100–150 ml of a liquid medium. The ratio of sown blood and nutrient medium 1:10 is not accidental - this is how blood dilution is achieved (undiluted blood has a detrimental effect on microorganisms).
Steps for isolating a pure culture of bacteria
Stage I (native material)
Microscopy (rough idea of ​​the microflora).
Sowing on dense nutrient media (obtaining colonies).
Stage II (isolated colonies)
The study of colonies (cultural properties of bacteria).
Microscopic study of microbes in a stained smear
(morphological properties of bacteria).
Inoculation on nutrient agar slant to isolate a pure culture.
Stage III (pure culture)
Determination of cultural, morphological, biochemical
and other properties for bacterial culture identification
IDENTIFICATION OF BACTERIA

Identification of isolated bacterial cultures is carried out by studying the morphology of bacteria, their cultural, biochemical and other characteristics inherent in each species.

Similar information.

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Serological reactions are designated in accordance with the phenomena accompanying the formation of an antigen-antibody complex during the interaction of components with different properties. There are reactions of agglutination, precipitation and lysis.

Agglutination reaction (RA)

The agglutination reaction (RA) is based on the use of a corpuscular antigen (suspension of bacteria, sensitized erythrocytes, latex particles, etc.) interacting with specific antibodies, as a result of which the resulting antigen-antibody complex precipitates. This reaction is widely used in laboratory practice for the serological diagnosis of bacterial infections and for the identification of isolated microorganisms.

RA is used to diagnose many infectious diseases: brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis (RAL - leptospira agglutination and lysis reaction), listeriosis, typhus(PAP - rickettsia agglutination reaction), shigellosis, yersiniosis, pseudotuberculosis, etc.

The reaction of indirect, or passive, agglutination (RIGA or RPGA).

To set up this reaction, erythrocytes of animals (sheep, monkeys, guinea pigs, some birds), sensitized by antibodies or antigen, which is achieved by incubation of a suspension of erythrocytes and a solution of antigen or immune serum.

Diagnosticums obtained on the basis of erythrocytes sensitized with antigens are called antigenic erythrocyte diagnosticums. They are designed to detect antibodies in serial dilutions of blood sera, such as erythrocyte shigellosis diagnosticums, erythrocyte salmonella O-diagnosticums.

Accordingly, diagnosticums based on erythrocytes sensitized with specific immunoglobulins are called antibody(immunoglobulin) diagnosticums and they serve to detect antigens in different material, for example, an erythrocyte immunoglobulin diphtheria diagnosticum for RIGA, used to detect diphtheria exotoxin of corynebacteria in a liquid nutrient medium when material from the nose and oropharynx is inoculated into it.

The hemagglutination reaction is used to diagnose both bacterial (typhoid fever, paratyphoid, dysentery, brucellosis, plague, cholera, etc.) and viral (influenza, adenovirus infections, measles, etc.) infections. RIGA is superior in sensitivity and specificity to RA.

Haemagglutination inhibition reaction (HITA)

The hemagglutination inhibition test (HITA) is used to titrate antiviral antibodies in blood sera, as well as to establish the type of affiliation of isolated viral cultures. RTHA can be used to diagnose those viral infections whose pathogens have hemagglutinating properties.

The principle of the method is that serum containing antibodies to a specific type of virus suppresses its hemagglutinating activity and erythrocytes remain non-agglutinated.

Reaction of inhibition (delay) of passive hemagglutination (RTPGA).

Three components are involved in RTGA: immune serum, antigen (test material) and sensitized erythrocytes.

If the test material contains an antigen that specifically reacts with antibodies of the immune standard serum, then it binds them, and with the subsequent addition of erythrocytes sensitized with an antigen homologous to serum, hemagglutination does not occur.

RTPHA is used to detect microbial antigens, to quantify them, and also to control the specificity of TPHA.

Latex agglutination reaction (RLA)

Latex particles are used as carriers of antibodies (immunoglobulins). RLA is an express method for diagnosing infectious diseases, given the time it takes (up to 10 minutes) and the ability to detect an antigen in a small amount of the test material.

RLA is used to indicate antigens of Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis in cerebrospinal fluid, to detect group A streptococci in throat swabs, to diagnose salmonellosis, yersiniosis and other diseases. The sensitivity of the method is 1-10 ng/ml, or 10³ -10⁶ bacterial cells in 1 µl.

Coagglutination reaction (RKoA)

The coagglutination reaction (RKoA) is based on the ability of protein A of staphylococci to attach specific immunoglobulins. RKA - a method of express diagnostics - serves to identify soluble thermostable antigens in human secrets and in the composition of circulating immune complexes (CIC). The detection of specific antigens in the composition of the CEC requires their preliminary precipitation from the blood serum.

precipitation reaction

In the precipitation reaction (RP), as a result of the interaction of antibodies with highly dispersed soluble antigens (proteins, polysaccharides), complexes are formed with the participation of complement - precipitates. It is a sensitive test used to detect and characterize a variety of antigens and antibodies. The simplest example of high-quality RP is the formation of an opaque precipitation band in a test tube at the border of antigen layering on immune serum - a ring precipitation reaction. Various types of RP are widely used in semi-liquid agar or agarose gels (double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis).

Complement fixation reaction (CFR)

The complement fixation reaction (CFR) is based on the phenomenon of hemolysis involving complement, i.e. can only detect complement-fixing antibodies.

RSC is widely used to diagnose many bacterial and viral infections, rickettsiosis, chlamydia, infectious mononucleosis, protozoal infections, helminthiases. RSK is a complex serological reaction in which two systems are involved: the test (blood serum), represented by the antigen-antibody and complement system, and the hemolytic (sheep erythrocytes + hemolytic serum). Hemolytic serum is a heat-inactivated blood serum of a rabbit immunized with ram erythrocytes. It contains antibodies against sheep erythrocytes.

A positive result of RSK - the absence of hemolysis - is observed if the test serum contains antibodies homologous to the antigen. In this case, the resulting antigen-antibody complex binds complement, and in the absence of free complement, the addition of the hemolytic system is not accompanied by hemolysis. In the absence of antibodies corresponding to the antigen in the serum, the formation of the antigen-antibody complex does not occur, the complement remains free and the serum causes hemolysis of erythrocytes, i.e. the presence of hemolysis is a negative result of the reaction.

Yushchuk N.D., Vengerov Yu.Ya.