Precipitation reaction. Precipitation reaction, its varieties, mechanism and application Antigen-antibody reactions and their application

Reactions of antigens with antibodies are called serological or humoral, because the specific antibodies involved are always present in the blood serum.

Reactions between antibodies and antigens that occur in a living organism can be reproduced in the laboratory for diagnostic purposes.

Serological reactions of immunity entered the practice of diagnosing infectious diseases in the late 19th and early 20th centuries.

The use of immunity reactions for diagnostic purposes is based on the specificity of the interaction of an antigen with an antibody.

Determination of the antigenic structure of microbes and their toxins made it possible to develop not only diagnosticums and therapeutic sera, but also diagnostic sera. Immune diagnostic sera are obtained by immunizing animals (for example, rabbits). These sera are used to identify microbes or exotoxins by antigenic structure using serological reactions (agglutination, precipitation, complement fixation, passive hemagglutination, etc.). Immune diagnostic sera treated with fluorochrome are used for express diagnostics of infectious diseases by the method of immune fluorescence.

With the help of known antigens (diagnosticums), it is possible to determine the presence of antibodies in the blood serum of a patient or subject (serological diagnosis of infectious diseases).

The presence of specific immune sera (diagnostic) allows you to establish the species, type of microorganism (serological identification of the microbe by antigenic structure).

The external manifestation of the results of serological reactions depends on the conditions of its setting and the physiological state of the antigen.

Corpuscular antigens give the phenomenon of agglutination, lysis, complement fixation, immobilization.

Soluble antigens give the phenomenon of precipitation, neutralization.

In laboratory practice, for diagnostic purposes, reactions of agglutination, precipitation, neutralization, complement fixation, hemagglutination inhibition, etc. are used.

Agglutination reaction (RA)

Due to its specificity, ease of setting and demonstrativeness, the agglutination reaction has become widespread in microbiological practice for the diagnosis of many infectious diseases: typhoid and paratyphoid fever (Vidal reaction), typhus (Weigl reaction), etc.

The agglutination reaction is based on the specificity of the interaction of antibodies (agglutinins) with whole microbial or other cells (agglutinogens). As a result of this interaction, particles are formed - agglomerates that precipitate (agglutinate).

Both living and dead bacteria, spirochetes, fungi, protozoa, rickettsia, as well as erythrocytes and other cells can participate in the agglutination reaction.

The reaction proceeds in two phases: the first (invisible) is specific, the connection of the antigen and antibodies, the second (visible) is nonspecific, the bonding of antigens, i.e. agglutinate formation.

Agglutinate is formed when one active center of a bivalent antibody is combined with the determinant group of the antigen.

The agglutination reaction, like any serological reaction, proceeds in the presence of electrolytes.

Externally, the manifestation of a positive agglutination reaction is twofold. In non-flagellated microbes, which have only somatic O-antigen, the microbial cells themselves stick together directly. Such agglutination is called fine-grained. It occurs within 18 - 22 hours.

Flagellated microbes have two antigens - the somatic O-antigen and the flagellated H-antigen. If the cells stick together with flagella, large loose flakes are formed and such an agglutination reaction is called coarse-grained. It comes within 2 - 4 hours.

The agglutination reaction can be set both for the purpose of qualitative and quantitative determination of specific antibodies in the patient's blood serum, and for the purpose of determining the species of the isolated pathogen.

The agglutination reaction can be set both in a detailed version, which allows working with serum diluted to a diagnostic titer, and in the variant of setting up an indicative reaction, which allows, in principle, to detect specific antibodies or determine the species of the pathogen.

When setting up a detailed agglutination reaction, in order to detect specific antibodies in the blood serum of the subject, the test serum is taken at a dilution of 1:50 or 1:100. This is due to the fact that in whole or slightly diluted serum, normal antibodies may be present in very high concentrations, and then the reaction results may be inaccurate. The test material in this variant of the reaction is the patient's blood. Blood is taken on an empty stomach or not earlier than 6 hours after a meal (otherwise, there may be droplets of fat in the blood serum, making it cloudy and unsuitable for research). The patient's blood serum is usually obtained in the second week of the disease, collecting 3–4 ml of blood sterilely from the cubital vein (by this time, the maximum amount of specific antibodies is concentrated). A diagnosticum prepared from killed but not destroyed microbial cells of a specific species with a specific antigenic structure is used as a known antigen.

When setting up a detailed agglutination reaction in order to determine the species, type of pathogen, the antigen is a live pathogen isolated from the test material. Known are the antibodies contained in the immune diagnostic serum.

Immune diagnostic serum is obtained from the blood of a vaccinated rabbit. Having determined the titer (the maximum dilution in which antibodies are detected), the diagnostic serum is poured into ampoules with the addition of a preservative. This serum is used for identification by the antigenic structure of the isolated pathogen.

When setting up an approximate agglutination reaction on a glass slide, sera with a higher concentration of antibodies are used (in dilutions of not more than 1:10 or 1:20).

With a Pasteur pipette, one drop of saline and serum is applied to the glass. Then a small amount of microbes is added to each drop in a loop and mixed thoroughly until a homogeneous suspension is obtained. A few minutes later, with a positive reaction, a noticeable clustering of microbes (granularity) appears in the drop with serum, uniform turbidity remains in the control drop.

The approximate agglutination reaction is most often used to determine the species of microbes isolated from the material under study. The result obtained allows us to roughly speed up the diagnosis of the disease. If the reaction is poorly visible to the naked eye, it can be observed under a microscope. In this case, it is called microagglutination.

An approximate agglutination reaction, which is placed with a drop of the patient's blood and a known antigen, is called blood-drip.

The reaction of indirect or passive hemagglutination (IPHA)

This reaction is more sensitive than the agglutination reaction and is used in the diagnosis of infections caused by bacteria, rickettsiae, protozoa and other microorganisms.

RPGA allows you to detect a small concentration of antibodies.

This reaction involves tannized sheep erythrocytes or human erythrocytes with group I blood, sensitized with antigens or antibodies.

If antibodies are detected in the test serum, then erythrocytes sensitized with antigens (erythrocyte diagnosticum) are used.

In some cases, if it is necessary to determine various antigens in the test material, erythrocytes sensitized with immune globulins are used.

The results of RPHA are taken into account by the nature of the erythrocyte sediment.

The result of the reaction is considered positive, in which the erythrocytes evenly cover the entire bottom of the test tube (an inverted umbrella).

With a negative reaction, erythrocytes in the form of a small disk (button) are located in the center of the bottom of the test tube.

Precipitation reaction (RP)

In contrast to the agglutination reaction, the antigen for the precipitation reaction (precipitinogen) is soluble compounds, the size of the particles of which approaches the size of the molecules.

These can be proteins, complexes of proteins with lipids and carbohydrates, microbial extracts, various lysates or filtrates of microbial cultures.

Antibodies that determine the precipitating property of the immune serum are called precipitins, and the reaction product in the form of a precipitate is called a precipitate.

Precipitating sera are obtained by artificial immunization of an animal with live or killed microbes, as well as various lysates and extracts of microbial cells.

By artificial immunization, it is possible to obtain precipitating sera to any foreign protein of plant and animal origin, as well as to haptens when an animal is immunized with a complete antigen containing this hapten.

The mechanism of the precipitation reaction is similar to that of the agglutination reaction. The action of precipitating sera on the antigen is similar to the action of agglutinating sera. In both cases, under the influence of immune serum and electrolytes, the antigen particles suspended in the liquid become larger (decrease in the degree of dispersion). However, for the agglutination reaction, the antigen is taken in the form of a homogeneous turbid microbial suspension (suspension), and for the precipitation reaction - in the form of a transparent colloidal solution.

The precipitation reaction is highly sensitive and can detect negligible amounts of antigen.

The precipitation reaction is used in laboratory practice for the diagnosis of plague, tularemia, anthrax, meningitis and other diseases, as well as in forensic medical examination.

In sanitary practice, this reaction determines the falsification of food products.

The precipitation reaction can be performed not only in test tubes, but also in a gel, and for fine immunological studies of the antigen, the immunophoresis method is used.

The agar gel precipitation reaction, or diffuse precipitation method, allows you to study in detail the composition of complex water-soluble antigenic mixtures. To set up the reaction, a gel (semi-liquid or denser agar) is used. Each component that makes up the antigen diffuses towards the corresponding antibody at a different rate. Therefore, complexes of various antigens and corresponding antibodies are located in different parts of the gel, where they form precipitation lines. Each of the lines corresponds to only one antigen-antibody complex. The precipitation reaction is usually set at room temperature.

The method of immunophoresis has become widespread in the study of the antigenic structure of a microbial cell.

The complex of antigens is placed in a well located in the center of the agar field poured onto the plate. An electric current is passed through the agar gel. Various antigens included in the complex move as a result of the action of the current, depending on their electrophoretic mobility. After the end of electrophoresis, a specific immune serum is introduced into the trench located along the edge of the plate and placed in a humid chamber. Precipitation lines appear at the sites of formation of the antigen-antibody complex.

Neutralization reaction of exotoxin with antitoxin (RN)

The reaction is based on the ability of antitoxic serum to neutralize the action of exotoxin. It is used for the titration of antitoxic sera and the determination of exotoxin.

When serum is titrated, a certain dose of the corresponding toxin is added to different dilutions of antitoxic serum. With complete neutralization of the antigen and the absence of unused antibodies, initial flocculation occurs.

The flocculation reaction can be used not only for titration of serum (for example, diphtheria), but also for titration of toxin and toxoid.

The reaction of toxin neutralization with antitoxin is of great practical importance as a method for determining the activity of antitoxic therapeutic sera. The antigen in this reaction is a true exotoxin.

The strength of the antitoxic serum is determined by conventional units of AE.

1 AU of diphtheria antitoxic serum is the amount that neutralizes 100 DLM of diphtheria exotoxin. 1 AU of botulinum serum is an amount that neutralizes 1000 DLM of botulinum toxin.

The neutralization reaction in order to determine the species or type of exotoxin (in the diagnosis of tetanus, botulism, diphtheria, etc.) can be carried out in vitro (according to Ramon), and when determining the toxigenicity of microbial cells - in the gel (according to Ouchterlony).

Lysis reaction (RL)

One of the protective properties of immune serum is its ability to dissolve microbes or cellular elements that enter the body.

Specific antibodies that cause the dissolution (lysis) of cells are called lysins. Depending on the nature of the antigen, they can be bacteriolysins, cytolysins, spirochetolizins, hemolysins, etc.

Lysines show their effect only in the presence of an additional factor - complement.

Complement, as a factor of nonspecific humoral immunity, is found in almost all body fluids, except for the cerebrospinal fluid and fluid of the anterior chamber of the eye. A fairly high and constant complement content was noted in human blood serum and a lot of it in guinea pig blood serum. In other mammals, the content of complement in the blood serum is different.

Complement is a complex system of whey proteins. It is unstable and collapses at 55 degrees for 30 minutes. At room temperature, complement is destroyed within two hours. It is very sensitive to prolonged shaking, to the action of acids and ultraviolet rays. However, the complement is stored for a long time (up to six months) in a dried state at a low temperature.

Complement promotes the lysis of microbial cells and erythrocytes.

Distinguish reaction of bacteriolysis and hemolysis.

The essence of the reaction of bacteriolysis is that when a specific immune serum is combined with its corresponding homologous living microbial cells in the presence of complement, microbes are lysed.

The hemolysis reaction consists in the fact that when erythrocytes are exposed to a specific, immune to them serum (hemolytic) in the presence of complement, erythrocytes dissolve, i.e. hemolysis.

The hemolysis reaction in laboratory practice is used to determine the complement tyre, as well as to take into account the results of the Borde-Jangu and Wassermann diagnostic complement fixation tests.

The complement titer is the smallest amount that causes the lysis of red blood cells within 30 minutes in the hemolytic system in a volume of 2.5 ml. The lysis reaction, like all serological reactions, occurs in the presence of an electrolyte.

Complement fixation reaction (CFR)

This reaction is used in laboratory studies to detect antibodies in the blood serum for various infections, as well as to identify the pathogen by antigenic structure.

The complement fixation test is a complex serological test and is characterized by high sensitivity and specificity.

A feature of this reaction is that the change in the antigen during its interaction with specific antibodies occurs only in the presence of complement. Complement is adsorbed only on the antibody-antigen complex. An antibody-antigen complex is formed only if there is an affinity between the antigen and the antibody present in the serum.

Complement adsorption on the antigen-antibody complex can affect the fate of the antigen in different ways, depending on its characteristics.

Some of the antigens undergo sharp morphological changes under these conditions, up to dissolution (hemolysis, Isaev-Pfeifer phenomenon, cytolytic action). Others change the speed of movement (treponema immobilization). Still others die without drastic destructive changes (bactericidal or cytotoxic effect). Finally, complement adsorption may not be accompanied by changes in the antigen that are easily accessible for observation (Bordet-Jangu, Wasserman reactions).

According to the mechanism, RSC proceeds in two phases:
a) The first phase is the formation of the antigen-antibody complex and adsorption on this complement complex. The result of the phase is not visually visible.
b) The second phase is a change in the antigen under the influence of specific antibodies in the presence of complement. The result of a phase may or may not be visually visible.

In the case when changes in the antigen remain inaccessible for visual observation, it is necessary to use a second system that acts as an indicator that allows you to assess the state of the complement and draw a conclusion about the result of the reaction.

This indicator system is represented by the components of the hemolysis reaction, which includes sheep erythrocytes and hemolytic serum containing specific antibodies to erythrocytes (hemolysins), but not containing complement. This indicator system is added to the test tubes one hour after setting the main CSC.

If the complement fixation reaction is positive, then an antibody-antigen complex is formed that adsorbs complement on itself. Since complement is used in the amount necessary for only one reaction, and erythrocyte lysis can occur only in the presence of complement, then when it is adsorbed on the antigen-antibody complex, erythrocyte lysis in the hemolytic (indicator) system will not occur. If the complement fixation reaction is negative, the antigen-antibody complex is not formed, the complement remains free, and when the hemolytic system is added, erythrocyte lysis occurs.

Hemagglutination reaction (RHA)

In laboratory practice, two different hemagglutination reactions are used.

In one case, the hemagglutination reaction is serological. In this reaction, erythrocytes are agglutinated when interacting with the corresponding antibodies (hemagglutinins). The reaction is widely used to determine the blood type.

In another case, the hemagglutination reaction is not serological.

In it, agglutination of red blood cells is caused not by antibodies, but by special substances (hemagglutinins) formed by viruses. For example, the influenza virus agglutinates chicken erythrocytes, the polio virus agglutinates monkeys. This reaction makes it possible to judge the presence of a particular virus in the test material.

Accounting for the results of the reaction is carried out by the location of erythrocytes. With a positive result, the erythrocytes are located loosely, lining the bottom of the test tube in the form of an "inverted umbrella". If the result is negative, the erythrocytes settle to the bottom of the test tube with a compact sediment ("button").

Haemagglutination inhibition reaction (HITA)

This is a serological reaction in which specific antiviral antibodies, interacting with the virus (antigen), neutralize it and deprive it of the ability to agglutinate red blood cells, i.e. inhibit the hemagglutination reaction.

The high specificity of the agglutination inhibition reaction makes it possible to determine the type and type of viruses or to detect specific antibodies in the test serum.

Immunofluorescence reaction (RIF)

The reaction is based on the fact that immune sera, to which fluorochromes are chemically attached, when interacting with the corresponding antigens, form a specific luminous complex visible in a fluorescent microscope. Serums treated with fluorochromes are called luminescent.

The method is highly sensitive, simple, does not require the isolation of a pure culture, because microorganisms are found directly in the test material. The result can be obtained 30 minutes after applying the luminescent serum to the preparation.

The immune fluorescence reaction is used in the accelerated diagnosis of many infections.

In laboratory practice, two variants of the immunofluorescence reaction are used: direct and indirect.

The direct method is when the antigen is immediately processed by immune fluorescent serum.

The indirect method of immune fluorescence consists in the fact that initially the drug is treated with a conventional (non-fluorescent) immune diagnostic serum specific to the desired antigen. If the preparation contains an antigen specific to this diagnostic serum, then an “antigen-antibody” complex is formed, which cannot be seen. If this preparation is additionally treated with luminescent serum containing specific antibodies to serum globulins in the “antigen-antibody” complex, luminescent antibodies will be adsorbed on diagnostic serum globulins and, as a result, glowing contours of the microbial cell can be seen in a luminescent microscope.

Immobilization reaction (RI)

The ability of the immune serum to immobilize motile microorganisms is associated with specific antibodies that act in the presence of complement. Immobilizing antibodies have been found in syphilis, cholera and some other infectious diseases.

This was the basis for the development of the treponema immobilization test, which, in its sensitivity and specificity, is superior to other serological tests used in the laboratory diagnosis of syphilis.

Virus Neutralization Test (RNV)

In the blood serum of people who have been immunized or have had a viral disease, antibodies are found that can neutralize the infectious properties of the virus. These antibodies are detected by mixing serum with the appropriate virus and then injecting the mixture into susceptible laboratory animals or infecting cell cultures. Based on the survival of the animals or the absence of the cytopathic effect of the virus, the neutralizing ability of the antibodies is judged.

This reaction is widely used in virology to determine the species or type of virus and the titer of neutralizing antibodies.

Modern methods for diagnosing infectious diseases include the immunofluorescent method for detecting antigens and antibodies, the radioimmune, enzyme immunoassay method, the method of immunoblotting, the detection of antigens and antibodies using monoclonal antibodies, the method for detecting antigens using polymerase chain reaction (PCR - diagnostics), etc.

In the form of turbidity, called precipitate. It is formed by mixing antigens and antibodies in equivalent amounts; an excess of one of them reduces the level of formation of the immune complex. The precipitation reaction is put in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of precipitation reaction in a semi-liquid gel of agar or agarose are widely used: Ouchterlony double immunodiffusion, radial immunodiffusion, immunoelectrophoresis, etc.
Ring precipitation reaction. The reaction is carried out in narrow precipitating tubes: a soluble antigen is layered on the immune serum. With an optimal ratio of antigen and antibodies, an opaque precipitate ring is formed at the border of these two solutions (Fig. 7.50). If boiled and filtered tissue extracts are used as antigens in the reaction, then such a reaction is called a thermoprecipitation reaction (Ascoli reaction, in which anthrax hapten is detected).

Rice. 7.50.

Ouchterlony double immunodiffusion reaction.

To set up the reaction, the melted agar gel is poured into a thin layer on a glass plate, and after solidification, holes are cut out in it. Antigens and immune sera are placed separately in the wells of the gel, which diffuse towards each other. At the meeting point in equivalent proportions, they form a precipitate in the form of a white band (Fig. 7.51). In multicomponent systems, several lines of precipitate appear between the wells with antigens and antibodies; in identical antigens, the precipitate lines merge; in non-identical antigens, they intersect.

Rice. 7.51

The immune serum with molten agar gel is evenly poured onto the glass. After solidification in the gel, wells are made into which the antigen (Ag) is placed in various dilutions. The antigen, diffusing into the gel, forms annular precipitation zones around the wells with antibodies. The diameter of the precipitation ring is proportional to the concentration of the antigen (Fig. 7.52). The reaction is used to determine in the blood serum immunoglobulins of various classes, components of the complement system, etc.

Rice. 7.52.

A combination of electrophoresis and immunoprecipitation: a mixture of antigens is introduced into the wells of the gel and separated in the gel using electrophoresis, then immune serum is introduced into the gel groove parallel to the electrophoresis zones. Antibodies of the immune serum diffuse into the gel and form a precipitation line at the “meeting” site with the antigen (Fig. 7.53).

Rice. 7.53.

Flocculation reaction (according to Ramon) (from lat. floccus- wool flakes) - the appearance of opalescence or flaky mass (immunoprecipitation) in a test tube during a toxin-antitoxin or anatoxin-antitoxin reaction (Fig. 7.54). It is used to determine the activity of antitoxic serum or toxoid.

Rice. 7.54.

Strains of the causative agent of diphtheria - C. diphtheriae can be toxigenic (producing exotoxin) and non-toxigenic. The formation of exotoxin depends on the presence in bacteria of a prophage carrying the tox gene encoding the formation of exotoxin. In case of illness, all isolates are tested for toxigenicity - the production of diphtheria exotoxin using a precipitation reaction in agar (Fig. 7.55).

Rice. 7.55

immunodiagnostic reactions. Antigen-antibody reactions and reactions with labeled components. Use for the identification of microorganisms and the diagnosis of infectious diseases.

Immune reactions are used in diagnostic and immunological studies in sick and healthy people. For this purpose, apply serological methods(from lat. serum - serum and logos - doctrine), 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 of hyperimmunized animals containing specific antibodies. This so-called serological identification microorganisms.

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

The principles and schemes of the main immunodiagnostic reactions are given below. A detailed technique for setting up reactions is given in. practical guidelines for immunodiagnostics.

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

Various variants of the agglutination reaction are used: expanded, approximate, indirect, etc. The agglutination reaction is manifested by the formation of flakes or sediment

RA is used for:

determination of antibodies in the blood serum of patients, for example, with brucellosis (Wright, Heddelson reactions), typhoid fever and paratyphoid fever (Vidal reaction) and other infectious diseases;

determination of the pathogen isolated from the patient;

determination of blood groups using monoclonal antibodies against erythrocyte allogenes.

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

The nature and rate of agglutination depend on the type of antigen and antibodies. An example is the interaction of diagnosticums (O- and R-antigens) with specific antibodies. Agglutination reaction with O-diagnosticum(bacteria killed by heat, retaining a thermostable O antigen) occurs in the form of fine-grained agglutination. The agglutination reaction with H-diagnosticum (bacteria killed by formalin, retaining the heat-labile flagellar H-antigen) is coarse-grained and proceeds faster.

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

Different related bacteria can be agglutinated by the same diagnostic agglutinating serum, making their identification difficult. Therefore, enjoy adsorbed agglutinating sera, from which cross-reactive antibodies have been removed by adsorption by their related bacteria. In such sera, antibodies specific only to this bacterium remain. The preparation of monoreceptor diagnostic agglutinating sera in this way was proposed by A. Castellani (1902).

The reaction of indirect (passive) hemagglutination (RNGA, RPHA) is based on the use of erythrocytes with antigens or antibodies adsorbed on the surface, the interaction of which with the corresponding antibodies or antigens of the blood serum of the ball causes the erythrocytes to stick together and fall to the bottom of the test tube or cell V the form of a scalloped sediment (Fig. 13.2). With a negative reaction, erythrocytes settle in the form of a "button". Usually, antibodies are detected in RNHA using an antigenic erythrocyte diagnosticum, which is erythrocytes with adsorbed on them antigens. Sometimes we use antibody erythrocyte diagnostics, on which antibodies are adsorbed. For example, botulinum toxin can be detected by adding erythrocyte antibody botulinum diagnosticum to it (this reaction is called reverse indirect hemagglutination reaction- RONGA). RNHA is used to diagnose infectious diseases, determine gonadotropic hormone V urine when pregnancy is established, to detect hypersensitivity to drugs, hormones, and in some other cases.

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

Hemagglutination inhibition reaction (RTGA) is based on blockade, suppression of antigens of viruses by antibodies of immune serum, as a result of which viruses lose their ability to agglutinate red blood cells (Fig. 13.3). RTHA is used to diagnose many viral diseases, the causative agents of which (influenza, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the erythrocytes of various animals.

Agglutination reaction for determining blood groups used to establish the ABO system (see Section 10.1.4.1) using agglutination of erythrocytes with immune serum antibodies against antigens of blood groups A (II), B (III). Controls are: antibody-free serum, i.e. serum AB (GU) blood groups; antigens contained in erythrocytes of groups A (II), B (III). The negative control contains no antigens, i.e. group 0 (I) erythrocytes are used.

IN agglutination reactions to determine the Rh factor(see section 10.1.4.1) use anti-Rh sera (at least two different series). In the presence of the Rh antigen on the membrane of the studied erythrocytes, agglutination of these cells occurs. Standard Rh-positive and Rh-negative erythrocytes of all blood groups serve as controls.

Agglutination reaction for the determination of anti-Rhesus antibodies (indirect Coombs reaction)used in patients with intravascular hemolysis. In some of these patients, anti-Rhesus antibodies are found, which are incomplete, monovalent. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. The presence of such incomplete antibodies is determined in the indirect Coombs reaction. To do this, antiglobulin serum (antibodies against human immunoglobulins) is added to the system of anti-Rh antibodies + Rh-positive erythrocytes, which causes agglutination of erythrocytes (Fig. 13.4). Using the Coombs reaction, pathological conditions associated with intravascular lysis of erythrocytes of immune origin are diagnosed, for example, hemolytic disease of the newborn: erythrocytes of a Rh-positive fetus combine with incomplete antibodies to the Rh factor circulating in the blood, which crossed the placenta from an Rh-negative mother.

Precipitation reactions

precipitation reaction - RP (fromlat. praeci-pito- precipitate,) is the formation and precipitation of a complex of a soluble molecular antigen with antibodies in the form of turbidity, called precipitate. It is formed by mixing antigens and antibodies in equivalent amounts; an excess of one of them reduces the level of formation of the immune complex.

Precipitation reactions put in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of the precipitation reaction in a semi-liquid gel of agar or agarose are widely used: double immunodiffusion according to Ouchterlony. radial immunodiffusion, immunoelectrophoresis and etc.

Ring precipitation reaction . The reaction is carried out in narrow precipitating tubes with immune serum, on which a soluble antigen is layered. With an optimal ratio of antigen and antibodies, an opaque precipitate ring is formed at the border of these two solutions (Fig. 13.5). An excess of antigen does not affect the result of the ring precipitation reaction due to the gradual diffusion of reagents to the liquid boundary. If boiled and filtered aqueous extracts of organs or tissues are used as antigens in the ring precipitation reaction, then such a reaction is called thermoprecipitation reaction-iii (Ascoli reaction, with anthrax /

Oukhteruni double immunodiffusion reaction . To set up the reaction, the melted agar gel is poured in a thin layer onto a glass plate, and after it hardens, holes of 2-3 mm in size are cut out in it. Antigens and immune sera are placed separately in these wells, which diffuse towards each other. At the meeting point in equivalent proportions, they form a precipitate in the form of a white band. In multicomponent systems, several lines of precipitate appear between wells with different antigens and serum antibodies; for identical antigens, the precipitate lines merge; for non-identical ones, they intersect (Fig. 13.6).

Radial immunodiffusion reaction . The immune serum with molten agar gel is evenly poured onto the glass. After solidification in the gel, wells are made into which the antigen is placed in various dilutions. The antigen, diffusing into the gel, forms ring precipitation zones around the wells with the antibodies (Fig. 13.7). The diameter of the precipitation ring is proportional to the antigen concentration. The reaction is used to determine the blood levels of immunoglobulins of various classes, components of the complement system, etc.

Immunoelectrophoresis- a combination of the method of electrophoresis and immunoprecipitation: a mixture of antigens is introduced into the wells of the gel and separated in the gel using electrophoresis. Then, parallel to the electrophoresis zones, an immune serum is introduced into the groove, the antibodies of which, diffusing into the gel, form at the meeting point with the antigen of the precipitation line.

flocculation reaction(according to Ramon) (from lat. floccus- wool flakes) - the appearance of opalescence or flocculent mass (immunoprecipitation) in a test tube during a toxin-antitoxin or anatoxin-antitoxin reaction. It is used to determine the activity of antitoxic serum or toxoid.

Immune electron microscopy- electron microscopy of microbes, more often viruses, treated with appropriate antibodies. Viruses treated with immune serum form immune aggregates (microprecipitates). A "corolla" of antibodies is formed around the virions, contrasted with phosphotungstic acid or other electron-optically dense preparations.

Reactions involving complement

Reactions involving complementbased on the activation of complement by an antigen-antibody complex (complement fixation reaction, radial hemolysis, etc.).

Complement fixation reaction (RSK) lies in the fact that, when corresponding to each other, antigens and antibodies form an immune complex, to which, through Fc -fragment of antibodies joins complement (C), i.e., complement binding occurs with an antigen-antibody complex. If the antigen-antibody complex is not formed, then the complement remains free (Fig. 13.8). RSK is carried out in two phases: 1st phase - incubation of a mixture containing three components of the antigen + antibody + complement; 2nd phase (indicator) - detection of free complement in the mixture by adding to it a hemolytic system consisting of sheep erythrocytes and hemolytic serum containing antibodies to them. In the 1st phase of the reaction, during the formation of the antigen-antibody complex, complement binding occurs, and then in the 2nd phase, hemolysis of erythrocytes sensitized by antibodies will not occur; the reaction is positive. If the antigen and antibody do not correspond to each other (there is no antigen or antibody in the test sample), the complement remains free and in the 2nd phase will join the erythrocyte-antierythrocyte antibody complex, causing hemolysis; the reaction is negative.

RSK is used to diagnose many infectious diseases, in particular syphilis (Wasserman reaction).

The reaction of radial hemolysis (RRH ) placed in wells of agar gel containing ram erythrocytes and complement. After adding hemolytic serum (antibodies against ram erythrocytes) to the wells of the gel, a hemolysis zone is formed around them (as a result of radial diffusion of antibodies). Thus, it is possible to determine the activity of complement and hemolytic serum, as well as antibodies in the blood serum of patients with influenza, rubella, tick-borne encephalitis. To do this, the corresponding antigens of the virus are adsorbed on erythrocytes, and the patient's blood serum is added to the wells of the gel containing these erythrocytes. Antiviral antibodies interact with viral antigens adsorbed on erythrocytes after

Complement components attach to this complex, causing hemolysis.

Immune adhesion reaction (RIP ) is based on the activation of the complement system by corpuscular antigens (bacteria, viruses) treated with immune serum. As a result, an activated third complement component (C3b) is formed, which attaches to the corpuscular antigen as part of the immune complex. On erythrocytes, platelets, macrophages there are receptors for C3b, due to which, when these cells are mixed with immune complexes bearing C3b, their combination and agglutination occur.

Neutralization reaction

Immune serum antibodies are able to neutralize the damaging effect of microbes or their toxins on sensitive cells and tissues, which is associated with the blockade of microbial antigens by antibodies, i.e., their neutralization. Neutralization reaction(RN) is carried out by introducing an antigen-antibody mixture into animals or into sensitive test objects (cell culture, embryos). In the absence of the damaging effect of microorganisms or their antigens, toxins in animals and test objects, they speak of the neutralizing effect of immune serum and, therefore, the specificity of the interaction of the antigen-antibody complex (Fig. 13.9).

Immunofluorescence reaction - RIF (Koons method)

There are three main varieties of the method: direct, indirect (Fig. 13.10), with a complement. The Koons reaction is a rapid diagnostic method for detecting microbial antigens or detecting antibodies.

Direct RIF method is based on the fact that tissue antigens or microbes treated with immune sera with antibodies labeled with fluorochromes are able to glow in the UV rays of a fluorescent microscope.

Bacteria in a smear, treated with such a luminescent serum, glow along the cell periphery in the form of a green border.

Indirect RIF method is to identify the antigen-antibody complex using antiglobulin (anti-antibody) serum labeled with fluorochrome. To do this, smears from a suspension of microbes are treated with antibodies of antimicrobial rabbit diagnostic serum. Then the antibodies that are not bound by microbial antigens are washed off, and the antibodies remaining on the microbes are detected by treating the smear with antiglobulin (anti-rabbit) serum labeled with fluorochromes. As a result, a complex microbe + antimicrobial rabbit antibodies + anti-rabbit antibodies labeled with fluorochrome is formed. This complex is observed in a fluorescent microscope, as in the direct method.

ELISA method, or analysis (ELISA)

ELISA -detection of antigens using their corresponding antibodies conjugated with a label enzyme (horseradish peroxidase, beta-galactosidase or alkaline phosphatase). After the antigen has been combined with the enzyme-labeled immune sera, the substrate/chromogen is added to the mixture. The substrate is cleaved by the enzyme, and the color of the reaction product changes - the color intensity is directly proportional to the number of bound antigen and antibody molecules.

Solid phase ELISA - the most common variant of the immunological test, when one of the components of the immune response (antigen or antibodies) is adsorbed on a solid carrier, for example, in the wells of polystyrene plates

When determining antibodies, the patient's blood serum, antiglobulin serum labeled with the enzyme, and the substrate (chromogen) for the enzyme are sequentially added to the wells of the plates with the adsorbed antigen.

Each time after the addition of the next component, unbound reagents are removed from the wells by thorough washing. With a positive result, the color of the chromogen solution changes. A solid-phase carrier can be sensitized not only with an antigen, but also with antibodies. Then, the desired antigen is introduced into the wells with adsorbed antibodies, the immune serum against the antigen labeled with the enzyme is added, and then the substrate for the enzyme is added.

Competitive ELISA . the target antigen and the enzyme-labeled antigen compete with each other for the binding of a limited amount of immune serum antibodies. Another test is the antibodies you are looking for

and labeled antibodies compete with each other for antigens.

Radioimmunological method, or analysis (RIA)

A highly sensitive method based on the antigen-antibody reaction using antigens or antibodies labeled with a radionuclide (125 J, 14 C, 3 H, 51 Cr, etc.). After their interaction, the resulting radioactive immune complex is separated and its radioactivity is determined in the appropriate counter (beta or gamma radiation):

the radiation intensity is directly proportional to the number of bound antigen and antibody molecules.

At solid-phase version of RIA one of the reaction components (antigen or antibodies) is adsorbed on a solid carrier, for example, in wells of polystyrene microarrays. Another version of the method is competitive RIA. the target antigen and the radionuclide-labelled antigen compete with each other for binding a limited amount of immune serum antibodies. This option is used to determine the amount of antigen in the test material.

RIA is used to detect antigens of microbes, determine hormones, enzymes, medicinal substances and immunoglobulins, as well as other substances contained in the test material in minor concentrations - 10 ~ | 0 -I0 ~ 12 g / l. The method presents a certain environmental hazard.

Immunoblotting

Immunoblotting (IB)- a highly sensitive method based on a combination of electrophoresis and ELISA or RIA.

The antigen is isolated using polyacrylamide gel electrophoresis, then it is transferred (blotting - from the English. blot, spot) from the gel onto activated paper or nitrocellulose membrane and developed by ELISA. Firms produce such strips with "blots"

antigens. The patient's serum is applied to these strips. Then, after incubation, the patient is washed from unbound antibodies of the patient and serum against human immunoglobulins, labeled with an enzyme, is applied. The complex antigen + antibody of the patient + antibody against human Ig formed on the strip is detected by adding a substrate / chromogen that changes color under the action of the enzyme (Fig. 13.12).

IB is used as a diagnostic method for HIV infection, etc.

In contrast to the agglutination reaction, the antigen for the precipitation reaction is soluble compounds, the size of the particles of which approaches the size of the molecules. These can be proteins, complexes of proteins with carbohydrates and lipids, bacterial extracts, various disates or filtrates of microbial broth cultures. Antibodies involved in the precipitation reaction are called precipitins. The resulting fine antigen-antibody complex is detected by certain methods of staging the precipitation reaction.

The ring precipitation reaction was first proposed by Ascoli. It is used in the diagnosis of anthrax, plague, tularemia, meningitis. The method is simple and accessible.

A specific immune precipitating serum is poured into narrow precipitating tubes and the antigen is very carefully layered on it. As an antigen, for example, when diagnosing anthrax, pieces of skin, wool, skins of a fallen animal, etc. are taken. They are boiled, the liquid is filtered and used as an antigen. The appearance of a ring at the boundary of two liquids - a precipitate indicates the presence of the corresponding antigen.

The agar gel precipitation reaction, or diffusion precipitation method, makes it possible to study in detail the composition of complex water-soluble antigenic mixtures. To set up the reaction, a gel (semi-liquid or thicker agar) is used. Each component that makes up the antigen diffuses towards the corresponding antibody at a different rate. Therefore, complexes of various antigens and corresponding antibodies are located in different parts of the gel, where precipitation lines are formed. Each of the lines corresponds to only one antigen-antibody complex. The precipitation reaction is usually set at room temperature.

The method of immunoelectrophoresis has become widespread in recent years in the study of the antigenic structure of microbes. The complex of antigens is placed in the well, which is located in the center of the agar gel, poured onto the plate. Then, an electric current is passed through the agar gel, as a result of which the various antigens included in the complex move in the electric current field depending on their electrophoretic mobility. After electrophoresis is completed, a specific immune serum is introduced into the trench located along the edge of the plate and placed in a humid chamber. In places where the antigen-antibody complex is formed, precipitation lines appear.

The precipitation reaction is a very sensitive method and is used in the study of various protein and polysaccharide antigens in forensic practice to determine the species of blood, semen, serum stains on linen and various objects. This reaction can also be used to identify various impurities in milk, fish and meat products, to determine the nature of the proteins that make up the paints of ancient masters of painting.

Precipitation reaction precipitation reaction

in vitro interaction reaction antigen With antibody leading to cloudiness of the medium visible to the naked eye or the formation of an immune complex precipitate (precipitate). It is applied for identification of antigens And antibodies, antigen purity control, quantitative determination of antigens and antibodies in the test material. For the setting of P. r. clear solutions of antigen and appropriate serum.

(Source: "Microbiology: glossary of terms", Firsov N.N., M: Bustard, 2006)

the test-tube reaction of the interaction of Ab with Ag in the liquid phase or in the gel, which leads to the formation of immune precipitates visible to the naked eye (turbidity). Used to identify Ag and Ab, control the purity of Ag, the quantitative content of Ab and Ag. For statement P. r. a clear solution of Ag, antiserum, saline solution or gel (1.5 - 2% agar and agarose in veronal acetate buffer, pH 6.8, ionic strength - 0.1) is required. The most commonly used methods are: 1) ring precipitation reaction. 0.2 ml of precipitating s-ki with a high titer is poured into the precipitation tube (tubes) and approximately the same amount of Ag solution (whole or diluted) is carefully layered onto it. In a positive case, after a few minutes, a mobile white ring forms at the interface of the reagents. Mandatory controls are Ag, s-ki, obviously positive, obviously negative, etc. They are used to identify Ag in extracts from various materials, incl. infectious; 2) Ouchterlony double radial immunodiffusion. In a uniform layer of 1% agar gel about 1.5 mm thick, holes are punched at a distance of 4-10 mm and filled with solutions of antiserum and Ag. The result is taken into account after a day (up to 6-7 days) of incubation at 4°, 20° and 37°C on a wet or dried and stained preparation according to the number and location of precipitation lines. In single-component systems, one line appears between the wells with Ag and Ab, in multicomponent systems, several lines appear, with identical Ag, the lines merge, with non-identical ones, they intersect. Used for qualitative analysis, determination of the purity of preparations, identification of Ab and Ag; 3) simple linear immunodiffusion according to Uden. They are used for the same purposes as the double. The gel containing antiserum is poured into test tubes or tubes. The solution of Ag is poured onto the gel in test tubes, and the tubes are placed in a glass with the solution of Ag. The results are taken into account after keeping the system at +4°C for 48 hours by the presence of a precipitate and a front, to-ry passed Ag. According to the formula, the amount of Ag is found; 4) Mancini simple radial immunodiffusion. A gel with a monospecific s-coy is poured onto a glass surface in an even layer. After solidification in the gel, holes are punched and solutions of the study are poured into them. Ag. The system is kept for 40 hours at +4° or 20°C. In positive cases, a precipitate is formed around the well, the area of ​​which reflects the amount of Ag. Similarly put the control with a series of dilutions of the standard Ag. The diameter of the precipitate zone is measured, the area is calculated, and the amount of Ag in 1 ml of the substrate is found from the calibration curve.

(Source: Glossary of Microbiology Terms)

See what "precipitation reaction" is in other dictionaries:

precipitation reaction- — [English-Russian glossary of basic terms on vaccinology and immunization. World Health Organization, 2009] Topics vaccinology, immunization EN precipitation reaction ... Technical Translator's Handbook

- (RW or EMF Express Diagnosis of Syphilis) is an outdated method for diagnosing syphilis. It has now been replaced by a precipitation microreaction (anticardiolipin test, MP, RPR Rapid Plasma Reagin). Named after the German immunologist Augustus ... ... Wikipedia

precipitation reaction- eng precipitation test eng precipitin test, precipitation test fra réaction (f) de précipitation (f) deu Präzipitintest (m), Präzipitations Reaktion (f) spa reacción (f) de precipitación, reacción (f) de precipitinas, prueba (f) de… … Occupational safety and health. Translation into English, French, German, Spanish

A method for the detection and identification of antibodies or soluble antigens based on the phenomenon of precipitation... Big Medical Dictionary

Precipitation (Latin praecipitatio rapid fall) In chemistry and biochemistry, a synonym for the word precipitation In immunology, the immunological reaction of precipitation from a solution of an insoluble antigen-antibody complex resulting from a compound ... ... Wikipedia

A method for detecting low concentrations of antibodies, monovalent haptens or polyvalent antigens, based on inhibition in their presence of reactions of precipitation, agglutination or complement fixation due to specific blocking ... ... Big Medical Dictionary

Modification of the precipitation reaction, in which the formation of a precipitate in the form of a ring is recorded at the boundary between antigen and antibody solutions ... Big Medical Dictionary

A test for the presence of syphilis, in which the determination of antibodies characteristic of this disease in the blood of the subject is carried out using a precipitation reaction. This test is less accessible than some others.