Precipitation reactions. Agglutination reaction, precipitation reaction Immune electron microscopy

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

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

The detection of antibodies against pathogen antigens in the patient’s blood serum allows a diagnosis of the disease to be made. 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 a patient, the pathogen is identified by studying its antigenic properties using immune diagnostic sera, i.e. blood sera of hyperimmunized animals containing specific antibodies. This is the so-called serological identification 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 production technique, however, they are all basic. are based on the reaction of interaction of antigen with antibody and are used to detect both antibodies and antigens. Immune reactions are characterized by high sensitivity and specificity.

Below are the principles and diagrams of the main immunodiagnostic reactions. Detail technique formulation of reactions is given in. practical guidelines for immunodiagnostics.

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

Apply various options agglutination reactions: extensive, indicative, 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 alloantigens.

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

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

If it is necessary to determine the pathogen isolated from the patient, put indicative agglutination reaction, using diagnostic antibodies (agglutinating serum), i.e., serotyping of the pathogen is carried out. Approximate reaction carried out on a glass slide. A pure culture of the pathogen isolated from the patient is added to a drop of diagnostic agglutinating serum at a dilution of 1:10 or 1:20. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied. When a flocculent sediment appears in a drop containing serum and microbes, a 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 clearness of the liquid. The reaction is considered positive if agglutination is observed 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, the suspension of microbes in the same solution should be uniformly cloudy, without sediment.

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

Indirect (passive) hemagglutination reaction (RNGA, RPGA) is based on the use of erythrocytes with antigens or antibodies adsorbed on their surface, the interaction of which with the corresponding antibodies or antigens of the blood serum causes the erythrocytes to stick together and fall out to the bottom of the test tube or cell V in the form of scalloped sediment (Fig. 13.2). At negative reaction red blood cells settle ■ in the form of a “button”. Typically, antibodies are detected in the RNGA using an antigenic erythrocyte diagnosticum, which is erythrocytes with adsorbed on them with antigens. Sometimes antibody erythrocyte diagnostics are used, on which antibodies are adsorbed. For example, botulinum toxin can be detected by adding erythrocyte antibody botulinum toxin to it (this reaction is called reverse indirect hemagglutination reaction- RONG). RNGA is used to diagnose infectious diseases and determine gonadotropic hormone V urine when establishing pregnancy, to detect hypersensitivity To medicines, hormones and in some other cases.

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

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

Agglutination reaction for determining blood groups used to establish the ABO system (see section 10.1.4.1) using agglutination of red blood cells with immune serum antibodies against blood group antigens A (II), B (III). The control is: serum that does not contain antibodies, i.e. serum AB (GU) blood types; antigens contained in red blood cells of groups A (II), B (III). The negative control does not contain 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-Rhesus sera (at least two different series). If there is a Rh antigen on the membrane of the erythrocytes under study, agglutination of these cells occurs. Standard Rh-positive and Rh-negative erythrocytes of all blood groups serve as control.

Agglutination reaction to determine anti-Rhesus antibodies ( indirect reaction Coombs)used in patients with intravascular hemolysis. In some of these patients, anti-Rhesus antibodies are detected, which are incomplete and monovalent. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. The presence of such incomplete antibodies is determined by the indirect Coombs test. To do this, antiglobulin serum (antibodies against human immunoglobulins) is added to the system of anti-Rh antibodies + Rh-positive erythrocytes, which causes agglutination of erythrocytes (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 have passed through the placenta from a Rh-negative mother.

Precipitation reactions

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

Precipitation reactions are performed in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of precipitation reactions in semi-liquid gels of agar or agarose have become widespread: double immunodiffusion according to Ouchterlony. radial immunodiffusion, immunoelectrophoresis and etc.

Ring precipitation reaction . The reaction is carried out in narrow precipitation tubes with immune serum, onto which a soluble antigen is layered. With an optimal ratio of antigen and antibodies, an opaque ring of precipitate forms 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 organs or tissues, this reaction is called thermoprecipitation reaction (Ascoli reaction, with anthrax/

Double immunodiffusion reaction according to Ouchteruny . To set up the reaction, melted agar gel is poured into a thin layer onto a glass plate and, after it hardens, wells 2-3 mm in size are cut out. Antigens and immune sera are placed separately into these wells, which diffuse towards each other. At the meeting point, in equivalent proportions, they form a precipitate in the form of a white stripe. 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 . Immune serum with molten agar gel is poured evenly 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-shaped precipitation zones around the wells with antibodies (Fig. 13.7). The diameter of the precipitation ring is proportional to the antigen concentration. The reaction is used to determine the content of immunoglobulins of various classes, components of the complement system, etc. in the blood.

Immunoelectrophoresis- 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 groove parallel to the electrophoresis zones, the antibodies of which, diffusing into the gel, form precipitation lines at the meeting point with the antigen.

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 toxoid-antitoxin reaction. It is used to determine the activity of antitoxic serum or toxoid.

Immune electron microscopy- electron microscopy of microbes, 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 complementare based on the activation of complement by the antigen-antibody complex (complement fixation reaction, radial hemolysis, etc.).

Complement fixation reaction (RSK) is that when antigens and antibodies correspond to each other, they form an immune complex, to which, through Fc -antibody fragment is attached to complement (C), i.e., complement is bound by the antigen-antibody complex. If the antigen-antibody complex is not formed, then complement remains free (Fig. 13.8). RSK is carried out in two phases: 1st phase - incubation of a mixture containing three components 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, when the antigen-antibody complex is formed, complement binds, 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 match 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 - anti-erythrocyte antibody complex, causing hemolysis; the reaction is negative.

RSC is used to diagnose many infectious diseases, in particular syphilis (Wassermann reaction).

Radial hemolysis reaction (RRH) ) placed in the wells of an agar gel containing sheep red blood cells and complement. After introducing hemolytic serum (antibodies against sheep red blood cells) into the wells of the gel, a hemolysis zone forms around them (as a result of radial diffusion of antibodies). In this way, 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 the 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 which

Then complement components join this complex, causing hemolysis.

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

Neutralization reaction

Antibodies of immune serum are capable of neutralizing 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. 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 effects of microorganisms or their antigens or 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 (Coons method)

There are three main types of method: direct, indirect (Fig. 13.10), with complement. The Koons reaction is a rapid diagnostic method for identifying microbial antigens or determining 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 periphery of the cell in the form of a green border.

Indirect RIF method consists of identifying 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 from antimicrobial rabbit diagnostic serum. Then the antibodies that are not bound by the microbial antigens are washed, 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 of microbe + antimicrobial rabbit antibodies + antirabbit antibodies labeled with fluorochrome is formed. This complex is observed in a fluorescent microscope, as in the direct method.

Enzyme immunosorbent method, or analysis (ELISA)

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

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

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

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

Competitive ELISA option . the target antigen and the enzyme-labeled antigen compete with each other to bind a limited amount of immune serum antibodies. Another test - 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 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 intensity of the radiation is directly proportional to the number of bound antigen and antibody molecules.

At solid-phase RIA version one of the reaction components (antigen or antibodies) is sorbed on a solid support, for example, in the wells of polystyrene micropanels. Another method option is competitive RIA. the desired antigen and the radionuclide-labeled antigen compete with each other to bind 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 identify microbial antigens, 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 poses 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 electrophoresis in a polyacrylamide gel, then transferred (blotting - from English. blot, stain) from the gel onto activated paper or nitrocellulose membrane and developed using ELISA. Companies 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 and serum against human immunoglobulins labeled with an enzyme is applied. The complex antigen + patient antibody + antibody against human Ig formed on the strip is detected by adding a substrate/chromogen that changes color under the action of an enzyme (Fig. 13.12).

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

Precipitation reaction is based on the formation of a precipitate - the precipitation of antigen-antibody immune complexes. In solutions they precipitate, and in gels they are deposited in the form of stripes. To determine the concentration of antibodies in the test material, purified antigens are used, and there are several ways to carry out the precipitation reaction.

Precipitation in solution

The precipitation reaction in solutions has a peculiarity: the amount of precipitate depends on the amount of the reagent (antibodies). If the amount of antibodies is less than the amount of antigen, then an increase in the amount of the latter leads to greater formation of precipitate. Largest quantity precipitate is formed when there are relatively equal amounts of antigens and antibodies. If the antigen is in excess quantity compared to antibodies, then as its concentration increases, the amount of precipitate decreases.

Immunodiffusion

The most common way of carrying out precipitation reactions is associated with immunodiffusion - the ability of antibodies and antigens to penetrate the gel.
For simple radial immunodiffusion, a gel containing antibodies is used. Round slits are made in a thin layer of the gel and the test material with antigens is placed into them, which penetrate deep into the gel, interact with antibodies and form rings of precipitate around the wells. In this way, the content of immunoglobulins in the blood is determined.
Many reagents for biochemical tests released fixed on tablets. Each cell of the tablet contains a strictly measured amount of reagent, so you can carry out multiple analyzes at once with high accuracy of results.
With double radial immunodiffusion, two round holes are cut in a thin layer of gel at a certain distance from each other. The reagent is placed in one, and the test material is placed in the other. Antibodies and antigens penetrate the gel towards each other. A precipitate is formed at the site of their interaction.
To determine the concentration of the antigen in the test material, measure the distance from the well with it to the precipitate line.

In contrast to the agglutination reaction, the antigen for the precipitation reaction is soluble compounds, the size of whose particles approaches the size of molecules. These can be proteins, complexes of proteins with carbohydrates and lipids, bacterial extracts, various disates or filtrates of broth cultures of microbes. Antibodies involved in the precipitation reaction are called precipitins. The resulting finely dispersed antigen-antibody complex is detected using 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, and meningitis. The method is simple and accessible.

Specific immune precipitating serum is poured into narrow precipitation tubes and the antigen is very carefully layered onto it. For example, when diagnosing anthrax, pieces of skin, wool, the skin of a dead animal, etc. are taken as an antigen. They are boiled, the liquid is filtered and used as an antigen. The appearance of a ring—precipitate—at the interface of two liquids indicates the presence of the corresponding antigen.

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

The method of immunoelectrophoresis has become widespread in last years when studying the antigenic structure of microbes. The antigen complex is placed in a well, which is located in the center of an agar gel poured onto a plate. An electric current is then passed through the agar gel, causing the various antigens included in the complex to move into the field electric current depending on their electrophoretic mobility. After electrophoresis is completed, specific immune serum is added to a trench located along the edge of the plate and placed in a humid chamber. Precipitation lines appear at the sites where the antigen-antibody complex is formed.

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

In the precipitation reaction, a specific immune complex is precipitated, consisting of a soluble antigen (lysate, extract, hapten) and a specific antibody in the presence of electrolytes.

The cloudy ring or precipitate formed as a result of this reaction is called precipitate. This reaction differs mainly from the agglutination reaction in the size of the antigen particles.

The precipitation reaction is usually used to determine antigen in the diagnosis of a number of infections (anthrax, meningitis, etc.); in forensic medicine - to determine the species of blood, sperm, etc.; in sanitary and hygienic studies - when establishing falsification of products; with its help, the phylogenetic relationship of animals and plants is determined. For the reaction you need:

1. Antibodies (precipitins) - immune serum with a high titer of antibodies (not lower than 1:100,000). The titer of the precipitating serum is determined by the highest dilution of the antigen with which it reacts. The serum is usually used undiluted or in a dilution of 1:5 - 1:10.

2. Antigen - dissolved substances of protein or lipoid polysaccharide nature (full antigens and haptens).

3. Isotonic solution.

The main methods for carrying out the precipitation reaction are: ring precipitation reaction and precipitation reaction in agar (gel).

Attention! All components involved in the precipitation reaction must be completely transparent.

Ring precipitation reaction. Using a Pasteur pipette, add 0.2-0.3 ml (5-6 drops) of serum into the precipitation tube (the serum should not get on the walls of the tube). The antigen in the same volume is carefully layered onto the serum, pouring it with a thin Pasteur pipette along the wall of the test tube. The test tube is kept in an inclined position. When properly layered, there should be a clear boundary between the serum and the antigen. Carefully, so as not to mix the liquid, place the test tube in a stand. If the reaction is positive, a cloudy “ring” is formed at the interface of the antigen and antibody - a precipitate (see Fig. 48).

The reaction is accompanied by a number of controls (Table 18). The sequence of adding reaction ingredients into the test tube is very important. You cannot layer the serum on the antigen (in the control - on an isotonic solution), since relative density If there is more serum, it will sink to the bottom of the test tube, and the boundary between the liquids will not be detected.

Table 18. Scheme for setting up the ring precipitation reaction

Note. + presence of a “ring”; - absence of a “ring”.

The results are recorded after 5-30 minutes, in some cases after an hour, as always starting with the controls. The “ring” in the 2nd test tube indicates the ability of the immune serum to enter into a specific reaction with the corresponding antigen. There should be no “rings” in the 3-5 test tubes - there are no antibodies and antigens corresponding to each other. A “ring” in the 1st tube - a positive reaction result - indicates that the test antigen corresponds to the taken immune serum, the absence of a “ring” (a “ring” only in the 2nd tube) indicates their inconsistency - a negative reaction result.

Precipitation reaction in agar (gel). The peculiarity of the reaction is that the interaction of antigen and antibody occurs in a dense medium, i.e., in a gel. The resulting precipitate gives a turbid streak in the thickness of the medium. The absence of a band indicates a discrepancy between the reaction components. This reaction is widely used in biomedical research, in particular in the study of toxin formation in the causative agent of diphtheria.

Control questions

1. What is the main difference between agglutination and precipitation reactions?

2. Why can't cloudy ingredients be used in the precipitation reaction?

Exercise

1. Set up the ring precipitation reaction and sketch the result.

2. Study the nature of the interaction of antigen with antibody in the precipitation reaction in agar, sketch the result (get a cup from your teacher).

Lysis reaction (immune cytolysis)

Immune lysis is the dissolution of cells under the influence of antibodies with the obligatory participation of complement. For the reaction you need:

1. Antigen - microbes, red blood cells or other cells.

2. Antibody (lysine) - immune serum, less often patient serum. Bacteriolytic serum contains antibodies involved in the lysis of bacteria; hemolytic - hemolysins that promote the lysis of red blood cells; for the lysis of spirochetes, spirochetolysins are needed, cells - itolysins, etc.

3. Complement. Most complement in serum guinea pigs. This serum (a mixture from several animals) is usually used as a complement. Fresh (native) complement is unstable and easily destroyed by heating, shaking, or storage, so it can be used no longer than two days after receipt. To preserve complement, 2% is added to it boric acid and 3% sodium sulfate. This complement can be stored at 4°C for up to two weeks. Dry complement is most often used. Before use, it is dissolved in an isotonic solution to the original volume (indicated on the label).

4. Isotonic solution.

Hemolysis reaction(Table 19). For the reaction you need:

1. Antigen - 3% suspension of washed sheep erythrocytes at the rate of 0.3 ml of erythrocyte sediment and 9.7 ml isotonic solution.

2. Antibody - hemolytic serum (hemolysin) against sheep erythrocytes; usually prepared in production, lyophilized and the titer indicated on the label.

Hemolysin titer is the highest dilution of serum at which complete hemolysis of a 3% suspension of red blood cells occurs in the presence of complement. For the hemolysis reaction, hemolysin is taken in triple titer, i.e. diluted 3 times less than before the titer. For example, with a serum titer of 1:1200, the serum is diluted 1:400 (0.1 ml of serum * and 39.9 ml of isotonic solution). Excess hemolysin is necessary, since some of it can be adsorbed by other reaction components.

* (You should not take less than 0.1 ml of serum - the accuracy of the measurement will suffer.)

3. Complement is diluted 1:10 (0.2 ml of complement and 1.8 ml of isotonic solution).

4. Isotonic solution.

Table 19. Hemolysis reaction scheme

Accounting for results. If the reaction is carried out correctly, hemolysis will occur in the 1st test tube - its contents will become transparent. In the controls, the liquid remains cloudy: in the 2nd tube there is not enough complement for hemolysis to occur, in the 3rd tube there is no hemolysin, in the 4th tube there is neither hemolysin nor complement, in the 5th tube the antigen does not match the antibody,

If necessary, hemolytic serum is titrated according to the following scheme (Table 20).

Before titration, prepare an initial serum dilution of 1:100 (0.1 ml of serum and 9.9 ml of isotonic solution), from which the necessary dilutions are made, for example:

From these dilutions, add 0.5 ml of serum into titration test tubes, as shown in Table. 20.

Table 20. Titration scheme for hemolytic serum (hemolysin)

In the example given in table. 20, the titer of hemolytic serum is 1:1200.

When using fresh hemolytic serum, it must be inactivated to destroy the complement present in it. To do this, it is heated for 30 minutes at 56 ° C in a water bath or in an inactivator with a thermostat. The latter method is better: it eliminates the possibility of overheating the whey, i.e., its denaturation. Denatured sera are unsuitable for testing.

Bacteriolysis reaction. In this reaction, complement lyses bacteria in the presence of appropriate (homologous) serum. The reaction scheme is fundamentally similar to the hemolysis reaction scheme. The difference is that after a two-hour incubation, all test tubes are seeded onto Petri dishes with a medium favorable for the microorganism taken into the experiment to find out whether it is lysed. If the experiment is carried out correctly, cultures from 2-5 test tubes (controls) should show abundant growth. Lack of growth or weak growth in inoculation from the 1st test tube (experiment) indicates the death of microbes, i.e., that they are homologous to the antibody.

Attention! The bacteriolysis reaction must be carried out under aseptic conditions.

Control questions

1. What will happen to red blood cells if distilled water is used instead of isotonic sodium chloride solution? What is the basis of this phenomenon?

2. What reaction will occur when erythrocytes interact with homologous immune serum in the absence of complement?

Exercise

Set up the hemolysis reaction. Record and sketch the result.

In the precipitation reaction, a specific immune complex is precipitated, consisting of a soluble antigen (lysate, extract, hapten) and a specific antibody in the presence of electrolytes.

The cloudy ring or precipitate formed as a result of this reaction is called precipitate. This reaction differs mainly from the agglutination reaction in the size of the antigen particles.

2. Antigen - dissolved substances of protein or lipoid polysaccharide nature (full antigens and haptens).

3. Isotonic solution.

The main methods for carrying out the precipitation reaction are: ring precipitation reaction and precipitation reaction in agar (gel).

Attention! All components involved in the precipitation reaction must be completely transparent.

Ring precipitation reaction. Using a Pasteur pipette, add 0.2 - 0.3 ml (5-6 drops) of serum into the precipitation tube (the serum should not get on the walls of the tube). The antigen in the same volume is carefully layered onto the serum, pouring it with a thin Pasteur pipette along the wall of the test tube. The test tube is kept in an inclined position. When properly layered, there should be a clear boundary between the serum and the antigen. Carefully, so as not to mix the liquid, place the test tube in a stand. If the reaction is positive, a cloudy “ring” - a precipitate - is formed at the interface of the antigen and antibody.

Precipitation reaction in agar(gele). The peculiarity of the reaction is that the interaction of antigen and antibody occurs in a dense environment, i.e. gel. The resulting precipitate gives a turbid streak in the thickness of the medium. The absence of a band indicates a discrepancy between the reaction components. This reaction is widely used in biomedical research, in particular in the study of toxin formation in the causative agent of diphtheria.

Lysis reaction (immune cytolysis)

Immune lysis- this is the dissolution of cells under the influence of antibodies with the obligatory participation of complement. For the reaction you need:

1. Antigen- microbes, red blood cells or other cells.

2. Antibody(lysine) - immune serum, less often patient serum. Bacteriolytic serum contains antibodies involved in the lysis of bacteria; hemolytic - hemolysins that promote the lysis of red blood cells; for the lysis of spirochetes, spirochetolysins, cell-itolisins, etc. are needed.

3. Complement. The serum of guinea pigs contains the most complement. This serum (a mixture from several animals) is usually used as a complement.

4. Isotonic solution.