Serological reactions. Precipitation reaction

This article will focus on the phenomenon of precipitation reaction. Here we will look at the features of this phenomenon, the phenomenon of diffusion, general characteristics, role in human life and much more.

Introduction to the phenomenon

Precipitation is a phenomenon of the serological type, during which soluble antigens interact with antibodies and, as a result, a precipitate forms.
The general characteristic of the precipitation reaction is a form of concerted influence of antigen and antibody. These types of interactions make it possible to determine the presence of unknown antigens in the test substance by adding known antibodies and antigens. The precipitation process without the presence of salts will proceed worse, and the best optimum lies within the range of 7.0-7.4 pH.

Components of a reaction

Among the components of the precipitation reaction, three main elements are distinguished:

1. An antigen that is molecular in nature. It is in a finely divided state, in other words, it is soluble. And also such an antigen is called precipitogen, which is a lysate or extract of tissue, etc. Precipitogen has a characteristic difference from an agglutinogen, which lies in the size of the particles of which it consists. Agglutinogen has an inherent cell size, while precipitogens are proportional to the size of the molecule. The antigen solution is characterized by transparency.
2. An antibody found in human blood serum, as well as in immune diagnostic serum, which contains the studied antibodies.
3. Electrolytes are a solution of sodium chloride, which is characterized by an isotonic state.

Obtaining precipitogen

The precipitation reaction is impossible without precipitogen, which is obtained by grinding materials and extracting protein antigens from them. Extraction occurs by boiling or other methods.
A striking example of precipitogens are lysates, as well as tissue and organ extracts, blood serum, various types of filtrates based on broth cultures of microbes, as well as saline extracts of microorganisms and autolysate substances.

Staging in precipitation

Now let's look at the method of setting up the precipitation reaction.
A ring precipitation reaction is carried out, which occurs in specially prepared test tubes. The serum is introduced into the cavity of the dish, pouring it along the wall using the nose of a pipette. Next, the appropriate amount of precipitogen is carefully layered on top, and then the test tube is brought into a vertical position from a horizontal one. Setting up and taking into account the precipitation reaction is a very scrupulous operation. The result is taken into account after the appearance of a white ring at the border between the antigen and antibody. If the reacting elements of the reaction correspond to each other, then they communicate, but this becomes noticeable after a long period of time of their interaction.
The precipitation reaction is also carried out in a Petri dish or on a glass slide, where the agar gel is transferred, applying it in a small layer. After it hardens, a small number of wells are cut out in the gel into which antigens and antibodies will be placed. There are two ways to perform this action: the method of radial immunodiffusion and double immunodiffusion.

General information

The mechanics of precipitation are similar to the agglutination device. When exposed to the influence of immune-type serum, the antigen, which has already reacted, reduces its. An important condition is the transparency of both the serum and the antigen.
The registration of a reaction can be improved by layering antigens onto antibodies. As a result, the appearance of ring-shaped precipitates can be observed. This phenomenon is called ring precipitation and is carried out in special tubes with a diameter of 2.5 to 3.5 mm. One of the most widespread examples of the precipitation reaction is the diagnosis of anthrax.
Precipitation makes it possible to determine the level of toxigenicity of a diphtheria culture in agar.
During the reaction under consideration, the precipitation of antigenic complexes and antibodies occurs. Precipitation is an immunological phenomenon that allows one to determine the amount of antibody content in the blood serum of sick or vaccinated humans and animals.

Consequence of titration

It is important to know that the data obtained by titration of the above method is not quantitative. To create and analyze a quantitative estimate of the contained number of antibodies, a special reaction technique was developed by M. Heidelberger and E. Kabat, which is based on the search and identification of the equivalence zone. Mixing the age-specific number of antigens with a constant volume of antiserum leads to an increase in the initially formed precipitate, and then it decreases again due to an increase in the ability to dissolve antigen complexes. By determining the amount of antibodies in the supernatants contained in each tube, you can find that in a certain number of dishes with antibodies there will be no liquid. Here, in comparison with other test tubes, the largest precipitate will be formed. Thanks to this and subtraction of the antigenic protein precipitate from the total value of proteins, it is possible to obtain the exact value of the antibodies contained in the volume of the specifically studied serum. Next, the amount of protein molecules in the precipitate is determined by the amount of nitrogen or using colorimetric methods.

Estimation of values

An assessment of precipitation values ​​in diagnostic methodology must take into account the likelihood of the presence in the immune serum of an antibody that does not have the property of precipitin, which means that the precipitate itself may not be formed after reacting with antigens. The list of such molecules includes partial antibodies and some species from the group of gamma A-globulins.

The precipitation reaction in laboratory conditions finds its application in various types of modifications. For example, the thermoprecipitation reaction is used to detect bacterial antigens of botulism, anthrax, etc., which are not subject to thermal denaturation. Unlike ring precipitation, this type of reaction uses filtrates of the material in question in a boiled state.
Precipitation in a complex mixture does not allow characterizing the properties of individual elements of the mixture. In such cases, a person resorts to the method of precipitation in agar, and also uses immunoelectropheresis.

Diffuse precipitation

In this area of ​​research, there is the concept of diffuse precipitation reaction (DPR). It is based on the ability of antibodies and soluble antigens to diffuse in a gel. Diffusion is the ability of a molecule of a certain substance to penetrate the molecules of another, which is caused by thermal movement.
A gel is a dispersed type system in which the liquid phase is distributed uniformly in the solid phase. Most often, an agar gel is used for this reaction.
After giving parameters under which the molecules can diffuse towards each other, their meeting will be accompanied by the formation of an antigen + antibody complex. Such a neoplasm can diffuse while in the gel itself, and it will precipitate, taking the form of a strip that can be detected with the naked eye. If the antigen and antibody are homologous, a band will not form.
Creating conditions under which diffusion will take place while in the agar layer involves pouring the components, but the total number of wells and their relative position is determined by the type of problem that needs to be solved. RPD gives a person the ability to detect and identify unknown isolated viruses by testing using a known antibody serum.

Application

Precipitation is widely used not only in the diagnosis of diseases, but also finds its application in forensic medicine. It is difficult to imagine an analysis in which it is possible to determine the species of blood, part of an organ or tissue found on a crime weapon that does not use the precipitation reaction. During this process, precipitating sera are used, which are obtained by immunizing various animals and birds. It is important that the serum titer level is at least 1:10,000, and it must also have sufficient specificity. From the detected blood stain or its crust, an extract is made for physical examination. solution, which will subsequently be exposed to precipitating serum. Using this reaction, it is possible to determine the types of tissue and organ proteins of both humans and animals. Obtaining cloudy extracts forces one to resort to precipitation on agar.

conclusions

Analyzing the information we have read, we can conclude that precipitation reactions are extremely important for humans, as they allow one to diagnose various antigens using antibodies; this phenomenon is also widely used in forensic medicine and allows one to identify the type of blood, tissue or organ in relation to a specific subject. There are several types and methods of precipitation that are used in accordance with the emerging needs of the problem being solved.

Precipitation reaction (RP) is the formation and precipitation of a complex of soluble molecular antigen with antibodies in the form of a cloud 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.

RP is placed in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of RP in semi-liquid agar or agarose gel are widespread: double immunodiffusion according to Ouchterlony, radial immunodiffusion, immunoelectrophoresis, etc.

Mechanism. It is carried out with transparent colloidal soluble antigens extracted from pathological material, environmental objects or pure bacterial cultures. The reaction uses clear diagnostic precipitating sera with high antibody titers. The titer of the precipitating serum is taken to be the highest dilution of the antigen, which, when interacting with the immune serum, causes the formation of a visible precipitate - turbidity.

The ring precipitation reaction is carried out in narrow test tubes (diameter 0.5 cm), into which 0.2-0.3 ml of precipitating serum is added. Then, using a Pasteur pipette, 0.1-0.2 ml of antigen solution is slowly layered. The tubes are carefully transferred to a vertical position. The reaction is recorded after 1-2 minutes. In the case of a positive reaction, a precipitate appears in the form of a white ring at the border between the serum and the test antigen. In the control tubes, no precipitate is formed.

15. Reaction involving complement: hemolysis reaction, complement fixation reaction. Mechanism, components, application.

The complement fixation reaction (CFR) is that when antigens and antibodies correspond to each other, they form an immune complex to which complement (C) is attached through the Fc fragment of antibodies, i.e., complement is bound by the antigen-antibody complex. If the antigen-antibody complex is not formed, then complement remains free.

The specific interaction of AG and AT is accompanied by adsorption (binding) of complement. Since the process of complement fixation is not visually apparent, J. Bordet and O. Zhang suggested using the hemolytic system (sheep red blood cells + hemolytic serum) as an indicator, which shows whether complement is fixed

AG-AT complex. If AG and AT correspond to each other, i.e. an immune complex has formed, then complement is bound by this complex and hemolysis does not occur. If AT does not correspond to AG, then the complex is not formed and complement, remaining free, combines with the second system and causes hemolysis.

Components. The complement fixation reaction (CFR) is a complex serological reaction. To carry it out, 5 ingredients are needed, namely: AG, AT and complement (first system), sheep erythrocytes and hemolytic serum (second system).

The antigen for CSC can be cultures of various killed microorganisms, their lysates, components of bacteria, pathologically altered and normal organs, tissue lipids, viruses and virus-containing materials.

Fresh or dried guinea pig serum is used as a complement.

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

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 sick 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. A detailed technique for setting up 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.

Various options for the agglutination reaction are used: 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 reaction), 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. An indicative reaction is carried out on a glass slide. A pure culture of the pathogen isolated from the patient is added to a drop of diagnostic agglutinating serum at a dilution of 1:10 or 1:20. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied. When a flocculent sediment appears in a drop containing serum and microbes, a 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 red blood cells with antigens or antibodies adsorbed on their surface, the interaction of which with the corresponding antibodies or antigens of the blood serum causes sticking and precipitation of red blood cells to the bottom of the test tube or cell V in the form of scalloped sediment (Fig. 13.2). In case of a 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 identify hypersensitivity to medications, 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 for determining anti-Rhesus antibodies (indirect Coombs test)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 of organs or tissues are used as antigens in the ring precipitation reaction, then 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, and 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. If the result is positive, 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, drugs 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.

13.1. Antigen-antibody reactions and their applications

When an antigen is introduced, antibodies are formed in the body. Antibodies are complementary to the antigen that caused their synthesis and are able to bind to it. The binding of antigens to antibodies consists of two phases. The first phase is specific, in which rapid binding of the antigenic determinant to the active center of the Fab fragment of antibodies occurs. It should be noted that the binding is due to van der Waals forces, hydrogen and hydrophobic interactions. The strength of the bond is determined by the degree of spatial correspondence between the active site of the antibody and the epitope of the antigen. After the specific phase, a slower phase begins - nonspecific, which is manifested by a visible physical phenomenon (for example, the formation of flakes during agglutination, etc.).

Immune reactions are interactions between antibodies and antigens, and these reactions are specific and highly sensitive. They are widely used in medical practice. With the help of immune reactions, the following problems can be solved:

Determination of unknown antibodies by known antigens (antigenic diagnosticum). This task occurs when it is necessary to determine antibodies to a pathogen in the patient’s blood serum (serodiagnosis). Finding antibodies allows you to confirm the diagnosis;

Determination of unknown antigens using known antibodies (diagnostic serum). This study is carried out when identifying a pathogen culture isolated from a patient’s material (serotyping), as well as when detecting

microbial antigens and their toxins in blood and other biological fluids. There are many types of immune reactions, differing in the technique of staging and the recorded effect. These are agglutination reactions (RA), precipitation reactions (RP), reactions involving complement (RSC), reactions using labeled components (RIF, ELISA, RIA).

13.2. Agglutination reaction

An agglutination reaction (RA) is an immune reaction of the interaction of an antigen with antibodies in the presence of electrolytes, and the antigen is in a corpuscular state (erythrocytes, bacteria, latex particles with adsorbed antigens). During agglutination, corpuscular antigens are glued together by antibodies, which is manifested by the formation of a flocculent precipitate. The formation of flakes occurs due to the fact that antibodies have two active centers, and antigens are polyvalent, i.e. have several antigenic determinants. RA is used to identify the pathogen isolated from the patient’s material, as well as to detect antibodies to the pathogen in the patient’s blood serum (for example, the Wright and Heddleson reactions for brucellosis, the Widal reaction for typhoid fever and paratyphoid fever).

The simplest way to diagnose RA is the reaction on glass; this is an approximate RA, which is used to determine the pathogen isolated from the patient. When a reaction is established, diagnostic agglutinating serum is applied to a glass slide (at a dilution of 1:10 or 1:20), then a culture from the patient is added. The reaction is positive if a flocculent sediment appears in the drop. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied. If the diagnostic agglutinating serum is not adsorbed 1, then it is diluted (to the titer - the dilution to which agglutination should occur), i.e. put expanded RA in test tubes with increasing

1 Unadsorbed agglutinating serum can agglutinate related bacteria that have common (cross-reacting) antigens. Therefore they useadsorbed 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.

dilutions of agglutinating serum, to which 2-3 drops of a suspension of the pathogen isolated from the patient are added. Agglutination is taken into account by the amount of sediment and the degree of clearing of the liquid in the test tubes. The reaction is considered positive if agglutination is observed in a dilution close to the titer of the diagnostic serum. The reaction is accompanied by controls: the serum diluted with isotonic sodium chloride solution should be transparent, the suspension of microbes in the same solution should be uniformly cloudy, without sediment.

To determine antibodies to the pathogen in the patient's blood serum, full-scale RA is used. When setting it up, the patient’s blood serum is diluted in test tubes and an equal amount of diagnosticum suspension (suspension of killed microbes) is added to the test tubes. After incubation, the highest serum dilution at which agglutination occurred is determined, i.e. a precipitate (serum titer) has formed. In this case, the agglutination reaction with O-diagnosticum (bacteria killed by heating, retaining the thermostable 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.

Indirect (passive) hemagglutination reaction(RNGA or RPGA) is a type of RA. This method is highly sensitive. With the help of RNGA, two problems can be solved: to determine antibodies in the patient’s blood serum, to which is added an antigenic erythrocyte diagnosticum, which is erythrocytes on which known antigens are adsorbed; determine the presence of antigens in the test material. In this case, the reaction is sometimes called the reverse indirect hemagglutination reaction (RONHA). During the procedure, an antibody erythrocyte diagnosticum (erythrocytes with antibodies adsorbed on their surface) is added to the test material. In this reaction, red blood cells act as carriers and are passively involved in the formation of immune aggregates. With a positive reaction, passively glued red blood cells cover the bottom of the hole in an even layer with scalloped edges (“umbrella”); in the absence of agglutination, red blood cells accumulate in the central recess of the hole, forming a compact “button” with sharply defined edges.

Coagglutination reaction used to determine pathogen cells (antigens) using antibodies adsorbed on Staphylococcus aureus, containing protein A. Protein A has an affinity for the Fc fragment of immunoglobulins. Thanks to this, antibodies bind to staphylococcus indirectly through the Fc fragment, and Fab fragments are oriented outward and are able to interact with the corresponding microbes isolated from patients. In this case, flakes are formed.

Hemagglutination inhibition reaction (HAI) used in the diagnosis of viral infections, and only infections caused by hemagglutinating viruses. These viruses contain a protein on their surface - hemagglutinin, which is responsible for the hemagglutination reaction (HRA) when red blood cells are added to the viruses. RTGA involves blocking viral antigens with antibodies, as a result of which viruses lose their ability to agglutinate red blood cells.

Coombs reaction - RA for determination of incomplete antibodies. In some infectious diseases, such as brucellosis, incomplete antibodies to the pathogen circulate in the patient’s blood serum. Incomplete antibodies are called blocking antibodies because they have one antigen-binding site, and not two, like complete antibodies. Therefore, when an antigenic diagnosticum is added, incomplete antibodies bind to antigens, but do not glue them together. To manifest the reaction, antiglobulin serum (antibodies to human immunoglobulins) is added, which will lead to agglutination of immune complexes (antigenic diagnosticum + incomplete antibodies) formed in the first stage of the reaction.

The indirect Coombs reaction is used in patients with intravascular hemolysis. In some of these patients, incomplete monovalent anti-Rhesus antibodies are detected. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. Therefore, antiglobulin serum is added to the system of anti-Rh antibodies + Rh-positive erythrocytes, which causes agglutination of erythrocytes. Using the Coombs reaction, pathological conditions associated with intravascular lysis of erythrocytes of immune origin are diagnosed, for example, hemolytic disease of the newborn caused by Rh conflict.

RA for determining blood groups is based on the agglutination of erythrocytes by immune serum antibodies to blood group antigens A(II), B(III). The control is serum that does not contain antibodies, i.e. serum AB(IV) blood group, and erythrocyte antigens of groups A(P) and B(III). Group 0(I) red blood cells are used as a negative control because they do not have antigens.

To determine the Rh factor, anti-Rh sera are used (at least two different series). If there is a Rh antigen on the membrane of the erythrocytes under study, agglutination of these cells occurs.

13.3. Precipitation reaction

RP is an immune reaction of the interaction of antibodies with antigens in the presence of electrolytes, and the antigen is in a soluble state. During precipitation, soluble antigens are precipitated by antibodies, which is manifested by cloudiness in the form of precipitation bands. The formation of a visible precipitate is observed when both reagents are mixed in equivalent ratios. An excess of one of them reduces the number of precipitated immune complexes. There are various ways to perform the precipitation reaction.

Ring precipitation reaction placed in precipitation tubes with a small diameter. The immune serum is added to the test tube and the soluble antigen is carefully layered. If the result is positive, a milky ring forms at the interface of the two solutions. The ring precipitation reaction, which is used to determine the presence of antigens in organs and tissues, the extracts of which are boiled and filtered, is called the thermoprecipitation reaction (Ascoli reaction for determining thermostable anthrax antigen).

Ouchterlony double immunodiffusion reaction. This reaction is carried out in an agar gel. In a layer of gel of uniform thickness, wells are cut out at a certain distance from each other and filled with antigen and immune serum, respectively. After this, antigens and antibodies diffuse into the gel, meet each other and form immune complexes, which precipitate in the gel and become visible as precision lines.

nutrition. This reaction can be used to identify unknown antigens or antibodies, and also to test the similarity between different antigens: if the antigens are identical, the precipitation lines merge, if the antigens are not identical, the precipitation lines intersect, if the antigens are partially identical, a spur is formed.

Radial immunodiffusion reaction. Antibodies are added to the melted agar gel and the gel is applied in an even layer to the glass. Wells are cut out in the gel and a standard volume of antigen solutions of different concentrations is added to them. During incubation, antigens diffuse radially from the well and, meeting antibodies, form a precipitation ring. As long as excess antigen remains in the well, a gradual increase in the diameter of the precipitation ring occurs. This method is used to determine antigens or antibodies in the test solution (for example, to determine the concentration of immunoglobulins of different classes in blood serum).

Immunoelectrophoresis. The antigen mixture is first electrophoretically separated, then precipitating antiserum is added to the groove running along the direction of protein movement. Antigens and antibodies diffuse into the gel towards each other; interacting, they form arcuate precipitation lines.

Flocculation reaction(according to Ramon) - a type of precipitation reaction that is used to determine the activity of antitoxic serum or toxoid. The reaction is carried out in test tubes. In a test tube where the toxoid and antitoxin are in an equivalent ratio, turbidity is observed.

13.4. Complement fixation reaction

Antibodies, interacting with the corresponding antigen, bind added complement (1st system). An indicator of complement fixation is erythrocytes sensitized with hemolytic serum, i.e. antibodies to red blood cells (2nd system). If complement is not fixed in the 1st system, i.e. If the antigen-antibody reaction does not occur, the sensitized red blood cells are completely lysed (negative reaction). When complement is fixed by immune complexes of the 1st system after adding sensitized erythrocytes, hemolysis from

absent (positive reaction). The complement fixation reaction is used to diagnose infectious diseases (gonorrhea, syphilis, influenza, etc.).

13.5. Neutralization reaction

Microbes and their toxins have a damaging effect on the organs and tissues of the human body. Antibodies are able to bind to these damaging agents and block them, i.e. neutralize. The diagnostic neutralization reaction is based on this feature of antibodies. It is carried out by introducing an antigen-antibody mixture into animals or into sensitive test objects (cell culture, embryos). For example, to detect toxins in the material of a patient, animals of the 1st group are injected with material from the patient. Animals of the 2nd group are injected with similar material, pre-treated with the appropriate antiserum. Animals of the 1st group die if there is a toxin in the material. The second group of animals survives; the damaging effect of the toxin does not manifest itself, as it is neutralized.

13.6. Reactions using labeled antibodies or antigens

13.6.1. Immunofluorescence reaction (RIF, Koons method)

This method is used for express diagnostics. It can be used to detect both microbial antigens and antibodies.

Direct RIF method- an immune reaction of the interaction of antibodies with antigens, and the antibodies are labeled with a fluorochrome - a substance capable of emitting light quanta of a certain wavelength when exposed to light of a certain wavelength. The peculiarity of this method is the need to remove unreacted components in order to exclude the detection of nonspecific luminescence. To do this, wash off unreacted antibodies. The results are assessed using a fluorescent microscope. Bacteria in a smear treated with such a luminescent serum glow against a dark background along the periphery of the cell.

Indirect RIF method is used more often than the previous one. This reaction is carried out in two stages. At the first stage, antigens mutually

interact with the corresponding antibodies, forming immune complexes. All components that have not reacted (i.e., are not part of immune complexes) must be removed by washing. At the second stage, the resulting antigen-antibody complex is detected using fluorochromized antiglobulin serum. As a result, a complex of microbe + antimicrobial rabbit antibodies + antibodies to rabbit immunoglobulins, labeled with fluorochrome, is formed. The results are assessed using a fluorescent microscope.

13.6.2. Enzyme immunosorbent method or assay

ELISA is the most common modern method used for the diagnosis of viral, bacterial, protozoal infections, in particular for the diagnosis of HIV infection, viral hepatitis, etc.

There are a lot of ELISA modifications. Solid-phase non-competitive ELISA is widely used. It is carried out in 96-well polystyrene plates (solid phase). When carrying out a reaction, it is necessary to wash off unreacted components at each stage. When determining antibodies, the test blood serum is added to the wells on which antigens are sorbed, then antiglobulin serum labeled with an enzyme. The reaction is carried out by adding a substrate for the enzyme. In the presence of an enzyme, the substrate changes, and the enzyme-substrate complex is selected so that the product formed in the reaction is colored. Thus, with a positive reaction, a change in the color of the solution is observed. To determine antigens, the solid-phase carrier is sensitized with antibodies, then the test material (antigens) and enzyme-labeled serum to the antigens are sequentially added. For the reaction to occur, a substrate for the enzyme is added. A change in the color of the solution occurs with a positive reaction.

13.6.3. Immunoblotting

This method is based on a combination of electrophoresis and ELISA. When performing immunoblotting (blotting from English. blot- spot) a complex mixture of antigens is first subjected to electrophoresis in a polyacrylamide gel. The resulting fractionated anti-

gene peptides are transferred to a nitrocellulose membrane. The blots are then treated with enzyme-labeled antibodies to a specific antigen, i.e. carry out ELISA blot. Immunoblotting is used in the diagnosis of infections such as HIV.

13.6.4. Immune electron microscopy

The method involves microscopying viruses (less commonly other microbes) in an electron microscope, pre-treated with the appropriate immune serum labeled with electron-optically dense preparations, for example ferritin, an iron-containing protein.

13.7. Flow cytometry

Blood cells are differentiated based on laser cytofluorometry. To do this, the desired cells are stained with fluorescent monoclonal antibodies to CD antigens. The blood sample, after being treated with labeled antibodies, is passed through a thin tube and a laser beam is passed through it, which excites the fluorochrome to glow. Fluorescence intensity correlates with the density of antigens on the cell surface and can be quantitatively measured using a photomultiplier tube. The results obtained are converted into a histogram.

Flow cytometry is used to determine the immune status (content of the main populations of lymphocytes, content of intracellular and extracellular cytokines, functional activity of NK cells, phagocytosis activity, etc.).

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. The precipitation reaction is carried out in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of the precipitation reaction in a semi-liquid agar or agarose gel are widespread: double immunodiffusion according to Ouchterlony, radial immunodiffusion, immunoelectrophoresis, etc.
Ring precipitation reaction. The reaction is carried out in narrow precipitation tubes: soluble antigen is layered onto the immune serum. With an optimal ratio of antigen and antibodies, an opaque ring of precipitate forms at the boundary of these two solutions (Fig. 7.50). If boiled and filtered tissue extracts are used as antigens in the reaction, then this 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, a thin layer of melted agar gel is poured onto a glass plate and after hardening, wells are cut out in it. Antigens and immune sera are placed separately into 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 stripe (Fig. 7.51). In multicomponent systems, several lines of precipitate appear between the wells with antigens and antibodies; For identical antigens, the precipitate lines merge, and for non-identical antigens, they intersect.

Rice. 7.51

Immune serum with molten agar gel is poured evenly onto the glass. After hardening in the gel, wells are made into which the antigen (Ag) is placed in various dilutions. The antigen, diffusing into the gel, forms ring 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 immunoglobulins of various classes, components of the complement system, etc. in blood serum.

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 added into the gel groove parallel to the electrophoresis zones. Antibodies from immune serum diffuse into the gel and form precipitation lines at the site of “meeting” with the antigen (Fig. 7.53).

Rice. 7.53.

Flocculation reaction (according to Ramon) (from lat. floccus- wool flakes) - the appearance of opalescence or flocculent mass (immunoprecipitation) in a test tube during the toxin-antitoxin or toxoid-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 an exotoxin depends on the presence in bacteria of a prophage carrying a tox gene encoding the formation of an exotoxin. In case of disease, all isolates are tested for toxigenicity - production of diphtheria exotoxin using the agar precipitation reaction (Fig. 7.55).

Rice. 7.55