The reaction of indirect or passive hemagglutination (RNHA or RPHA) is more sensitive and specific than the agglutination reaction. This reaction is also used in two directions.

1) To detect antibodies in the patient's blood serum, erythrocyte diagnosticums are used, in which the antigen is adsorbed on the surface of erythrocytes treated with tannin. In relation to this reaction, the term RPGA is more often used.

The test serum is diluted in the wells of plastic plates and erythrocyte diagnosticum is added. With a positive reaction, a thin film appears on the walls of the well in the form of a "lace umbrella", with a negative reaction, a dense sediment of erythrocytes in the form of a "button" appears.

2) To detect toxins and bacterial antigens in the test material, antibody erythrocyte diagnosticums are used, obtained by adsorption of antibodies on erythrocytes. In relation to this reaction, the term RNGA is more often used. For example, with the help of antibody diagnosticums, the antigen of the plague bacillus, diphtheria exotoxin, botulinum exotoxin are detected.

Coombs reaction (aptiglobulin test)

The reaction is used to detect incomplete antibodies, for example, antibodies to the Rh factor. The test serum is added to Rh + erythrocytes, in which the presence of incomplete antibodies to the Rh factor is expected. Attached to erythrocytes, incomplete antibodies do not cause agglutination, since they have only one active center. Then add antiglobulin serum containing antibodies to human globulins. When combined with incomplete antibodies, antiglobulin serum causes erythrocyte agglutination.

precipitation reaction

The essence of the reaction is the precipitation (precipitation) of the antigen under the action of specific antibodies. The presence of an electrolyte is necessary to obtain a visible reaction. The antigen in the precipitation reaction are molecularly dispersed substances.

Ring precipitation reaction placed in narrow precipitating tubes. The immune serum is poured into the test tube, and the test material is carefully layered on it. In the presence of an antigen in it, an opaque ring of precipitate is formed at the boundary of two liquids.

The reaction is used in forensic medicine to determine the species of proteins in blood spots, in semen, etc.; to determine the antigen in the diagnosis of anthrax (Ascoli reaction), meningitis and other infections; in sanitary and hygienic studies - to establish falsification of food products. Immune precipitating sera are obtained by immunizing animals with the appropriate antigen. For example, serum precipitating human protein is obtained by immunizing a rabbit with human protein. The titer of precipitating serum is the highest dilution of the antigen with which it reacts. Serum is usually used undiluted or diluted 1:5.

Agar gel precipitation reaction carried out in several ways. This is a double immunodiffusion reaction, a radial immunodiffusion reaction, an immunoelectrophoresis reaction.

Double immunodiffusion reaction(according to Ouchterlony). The melted agar gel is poured into a Petri dish and, after solidification, wells are cut out in it. Antigen is placed in some wells, immune sera are placed in others, which diffuse into the agar, form a precipitate in the form of white stripes at the meeting point.

Radial immunodiffusion reaction(according to Mancini). Immune serum is added to the melted agar gel, poured into a dish. After the agar solidifies, wells are cut out in it and antigens are placed in them, which, diffusing into the agar, form annular precipitation zones around the wells. The higher the antigen concentration, the larger the ring diameter. The reaction is used, for example, to determine various classes of immunoglobulins in the blood. Immunoglobulins of the classes IgG, IgM, IgA act as antigens in this reaction, and antibodies against them are contained in specific monoreceptor sera.

Immunoelectrophoresis. Electrophoresis of protein antigens is carried out in agar gel. The precipitating serum is introduced into the groove, which runs parallel to the direction of movement of the proteins. Antigens and antibodies diffuse into the agar, and precipitation lines form at their meeting point.

Immune lysis reactions

An antigen (erythrocytes or bacteria), having combined with specific antibodies, forms an immune complex, to which complement (C1) is attached, and complement activation occurs along the classical pathway. Activated complement lyses red blood cells (hemolysis) or bacteria (bacteriolysis). The bacteriolysis reaction is used to identify Vibrio cholerae.

hemolysis reaction. The antigen in the reaction is erythrocytes, antibodies (hemolysins) are contained in hemolytic serum. Hemolysins attach to erythrocytes, complement activation occurs, which lyses erythrocytes. A cloudy suspension of erythrocytes turns into a transparent bright red liquid - “lacquer blood”. Since the hemolysis reaction occurs only in the presence of complement, it is used as an indicator for the detection of complement.

Local hemolysis reaction in gel(Jerne reaction) - a variant of the hemolysis reaction. Serves to determine the number of antibody-forming cells (AFC) in the spleen and lymph nodes.

The melted agar gel is mixed with a suspension of spleen cells and erythrocytes, and after the agar has solidified, complement is added. Around each cell that produces hemolysins, a zone of hemolysis is formed. The number of such zones determines the number of cells producing hemolysins.

The reaction of indirect hemagglutination (RIHA)

The use of adsorbed diagnostic preparations is based on the principle of immunological recognition of surface structures that is constantly implemented in nature. From this point of view, there are no fundamental differences between the use of, for example, a microbial cell or an artificial carrier loaded with antigen (antibodies). Various carriers are used as the basis of sorbed preparations - microbial cells, erythrocytes, latex, bentonin, cholesterol, Sephadex, dermatol, activated carbon, fungal spores, etc.

After the work of A.T. Kravchenko (1945), erythrocytes deprived of viability and fixed with various chemical reagents became most widely used as a carrier of antigen or antibodies. On the principle of using erythrocytes (both native and fixed) as a carrier of the antigen, the reaction of indirect (passive) hemagglutination has become widespread.

The suggestion to use the indirect hemagglutination reaction (IHA) for diagnostic purposes arose on the basis of previously developed knowledge about the high sorption activity of erythrocytes. In comparison with many other carriers of antigens and antibodies, erythrocytes have certain advantages in the reaction of indirect hemagglutination. First, in isotonic saline solutions, they form a fairly stable cohesion and do not settle in a short time. Secondly, the red blood cells of the same animal species are the same in size. And, finally, erythrocytes relatively easily directly or as a result of special processing attach various serologically active components.

The creation of erythrocyte diagnosticums from stabilized erythrocytes makes it possible to overcome the difficulties that arise when using native erythrocytes.

In the study of erythrocytes treated with various fixatives, some of their common properties were established:

In morphology, they practically do not differ from fresh ones;

Not hemolyzed in hypotonic solutions, in water and after freezing-thawing;

Can be freeze dried;

Their surface structures retain the ability to be chemically modified (eg, tannin, diazo compounds) and react with antigens and antibodies.

The main criterion for the quality of fixed erythrocytes is the possibility of their use for obtaining sensitized preparations in the absence of nonspecific agglutination in stabilizing liquids.

Preparation of the surface of erythrocytes in order to increase the sorption capacity for antigens is important in the design of diagnostic preparations. Especially widespread is the treatment of erythrocytes with tannin.

Under its influence, the antigenic properties of erythrocytes (permeability, sedimentation rate) change, their resistance to the hemolytic effects of certain fatty acids increases. However, the main thing is achieved - tanized erythrocytes have a much higher sorption capacity of proteins, which is currently widely used in the manufacture of high-quality antigenic and antibody diagnosticums.

Along with the tanization of erythrocytes, for the manufacture of erythrocyte diagnosticums and the preparation of an antigen carrier, chemical reagents such as bromelain, triopsin, and potassium periodate are used, which also modify erythrocyte membranes.

After preparing the surface of erythrocytes, the most important process takes place - the process of hemosensitization - the final stage in the manufacture of erythrocyte diagnosticums.

To strengthen the connection between the carrier and the antigen, various conjugating substances are used - bisdiazobenzidine, difluorodinitrobenzine, cyanide chloride, cadmium chloride and acetic acid, bromine chloride, formaldehyde, glutaraldehyde, cyanogen bromide, rivanol, amidol, etc.

The use of conjugating substances helps to eliminate the shortcomings that tanized erythrocytes used for sensitization without conjugating substances have.

The most widely used conjugating substances are chromium chloride, glutaraldehyde, diazole black C, and amidol. The process of sensitization with the help of chromium chloride occurs very quickly - from 4 to 10 minutes, which attracts the attention of researchers. In this case, concentrations of chromium chloride from 0.05 to 2% are used, pH is not lower than 5.0 at a temperature of the reaction mixture of 20-250 C. In the process of sensitization with chromium chloride, carboxyl groups of erythrocyte membrane proteins are activated. As a result, a covalent bond is formed between the erythrocyte membrane and sensitin.

The reaction of indirect hemagglutination has a high specificity, since the agglutination of erythrocytes occurs as a result of the interaction of the antigen with antibodies adsorbed on erythrocytes. Its distinctive feature is high sensitivity: 0.02-0.05 μg of antibody protein is detected in it. In terms of sensitivity, it significantly exceeds the reactions of immunodiffusion, immunofluorescence, radial immunodiffusion, complement fixation, and neutralization. RNGA is less sensitive than the methods of radioimmunoelectrophoresis, determination of the amount of protein of radioactive antibodies in the immunosorbent, enzyme immunoassay.

The advantages of RNGA include sufficiently high reproducibility, ease of implementation, and the need for minimal amounts of ingredients, especially when using the micromethod.

In recent years, RNHA has found wide application in the diagnosis of infectious rhinotracheitis, diarrhea, adenovirus infection, rota- and coronavirus infection, parainfluenza-3 and respiratory syncytial infection.

We give an example of the manufacture and use of an erythrocyte diagnosticum for the detection of antibodies to rota- and coronavirus infections.

The method of preparation of antibody diagnosticums for the detection of rota- and coronavirus antigens in RNGA is as follows: obtaining a suspension of erythrocytes; fixing their acroleins; tanization, which allows obtaining stable sorption of the required protein on erythrocytes; sensitization of tanized erythrocytes with immunoglobulins; determination of the specificity of the prepared diagnosticum. The preparation of a suspension of erythrocytes is carried out by taking the blood of a clinically healthy ram into a flask with glass beads. After blood defibrination, erythrocytes should be washed 3-5 times with isotonic (0.9%) sodium chloride solution by centrifugation for 15-20 minutes at 400g.

In order to stabilize red blood cells, they are treated with acrolein. As you know, its action is somewhat milder than formaldehyde, and provides stability and higher activity of erythrocytes. For this, equal volumes of a 10% suspension of erythrocytes are mixed with a 0.2% solution of acrolein prepared in phosphate buffer solution (PBS) with a pH of 7.2. The resulting suspension is kept for 30 min at 37°C, thoroughly mixing, followed by the release of acrolein by repeated centrifugation for 5 min at 600-800g until the specific odor disappears completely. The advantage of erythrocytes prepared in this way is that they are harvested for the future, they can be stored for a long time without being subjected to hemolysis.

Subsequently, stabilized erythrocytes in a 10% concentration are kept at 2-4 °C in PBS with a pH of 7.2 for two months.

To increase the sorption capacity and sedimentation of erythrocytes, it is necessary to tanize them by mixing equal parts of a 10% suspension of washed erythrocytes and a solution of tannin in PBS and pH 7.2 at a dilution of 1:30,000. The mixture is thoroughly mixed and kept at 370 C for 15 min, after which the erythrocytes are thoroughly washed from tannin twice in PBS with pH 7.2 and three times with isotonic sodium chloride solution with pH 7.2-7.4.

The optimal time to guarantee the receipt of high-quality erythrocyte diagnosticums is their sensitization within 24-48 hours after their treatment with tannin.

In the process of making diagnosticums, 0.3% phenolized isotonic sodium chloride solution with 0.1% normal rabbit or horse serum, previously inactivated at 56°C for 30 minutes and adsorbed by normal sheep erythrocytes, is used as a solvent and preservative. 37°C for 30 min.

Sensitization of tanized erythrocytes should be carried out with hyperimmune serum, respectively, against bovine rota- and coronaviruses (produced at the Ya.R. Kovalenko All-Russian Research Institute of Experimental Veterinary Medicine). At the same time, the sensitizing dose (protein concentration) of anti-rotavirus immune serum is 280 μg/ml, and that of the immune serum to coronavirus is 260 μg/ml.

Sensitization is carried out by mixing equal volumes of tanized erythrocytes and immune serum preheated for 30 minutes at 560C in optimal concentration. In addition, 3 parts of an isotonic sodium chloride solution with a pH of 7.2 and 5 parts of a 0.1% chromium chloride solution are added. The mixture, stirring occasionally, is kept at room temperature for 5 minutes. After the specified time, the mixture is thoroughly washed using PBS with a pH of 7.2, and then the prepared erythrocyte diagnosticums are resuspended in a preservative to a 1% concentration. The prepared diagnosticum retains its basic qualities for 8 months, if stored at 4°C.

At all stages of the manufacture of diagnosticums, self-agglutination is monitored. The specificity of the prepared diagnosticums is determined by staging RNGA, where standard diagnosticums of RTI viruses, PG-3, adenoviruses, viral diarrhea, as well as rota- and coronavirus antigens were used as antigens.

When setting up RNHA, serial double dilutions of the studied virus-containing material are prepared (20-50% suspension of fecal samples), and then an equal volume of erythrocyte diagnosticum is added to each well. The plates should be thoroughly shaken several times, left at room temperature until the erythrocytes are completely settled in the control. An indicator of a positive reaction is agglutination of the erythrocyte diagnosticum with an agglutination intensity of 2+ in a titer of 1:4 and above.

The reaction of indirect or passive hemagglutination (IPHA)

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

RPGA allows you to detect a small concentration of antibodies.

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

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

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

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

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

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

Agglutination reaction (RA)

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

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

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

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

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

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

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

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

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

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

The reaction is carried out to detect the antigens of pathogens in the test material by adding immune serum to it. In the presence of a homologous antigen, antibodies bind, therefore, after the addition of antigen-sensitized erythrocytes, their agglutination does not occur, which is assessed as a positive result. For greater sensitivity of the reaction, specific immune serum is added to the test material in a minimum amount (2 hemagglutination units). The titer of immune serum is determined previously using RNGA. For one hemagglutinating unit, take the limiting dilution of serum, which causes agglutination of erythrocytes.

Methodology. 0.025 ml of a 1% solution of normal rabbit serum is added to the wells of polystyrene plates or agglutination tubes and serial 2-fold dilutions of the test material are made. Then, 0.025 ml of immune serum is added to all wells, the plate is shaken and left for 30 minutes at a temperature of 37ºС or for 1 hour at room temperature. Further, 0.025 ml of antigenic diagnosticum is added to all wells, shaken and left for 2 hours, after which the results are taken into account.

When carrying out this reaction, an immune serum specificity control is added to the control for RNGA, consisting of a specific antigen, immune serum (2, 1, 0.5 hemagglutinating units) and a suspension of sensitized erythrocytes.

RTNGA is also used to detect specific antibodies (complete and blocking), for which a dosed amount of antigen is added to the test serum. If it contains antibodies, then they are bound by an antigen, therefore, after the addition of erythrocytes sensitized with antibodies (2nd stage of RTNHA), erythrocytes do not stick together.

Due to the use of a minimum amount of antigen (2 neutralizing doses), the reaction is highly sensitive. The number of neutralizing doses in the antigenic preparation is determined using RNGA, while serial 2-fold dilutions of the antigen are made in a 1% solution of normal rabbit serum and an antibody erythrocyte diagnosticum is added to the wells. The neutralizing dose of the antigen is its maximum dilution, which gives complete adhesion of sensitized erythrocytes.

Before the reaction, the test sera are diluted 1:5-1:10 and inactivated at a temperature of 56°C for 30 minutes. For adsorption of hemagglutinins, a 50% suspension of formalized or freshly washed sheep erythrocytes is added to the sera at the rate of 0.1 ml of the suspension per 1 ml of diluted serum. The mixture is shaken and incubated for 30 minutes at 37°C or 1 hour at room temperature, after which the erythrocytes are precipitated by centrifugation. You can leave the serum in the refrigerator until the next day for the precipitation of erythrocytes.

During the experiment, the test material is diluted in 1% solution of normal rabbit serum in a volume of 0.025 ml in the wells of the microtiter panel. Then, 2 neutralizing doses of antigen in a volume of 0.025 ml are added to each well. The plates are shaken and left for 30 minutes at 37°C or 1 hour at room temperature. Then, 0.025 ml of antibody erythrocyte diagnosticum is added to all wells. The plates are shaken and incubated for 1.5-2 hours at room temperature, after which the reaction results are taken into account. Accounting can also be done the next day. The titer of the studied serum is considered to be its maximum dilution, in which erythrocytes do not stick together.

Experience is accompanied by the following types of control:

1) stability of sensitized erythrocytes (erythrocytes + 1% solution of normal rabbit serum);

2) completeness of depletion of hemagglutinins in each test serum (test serum in the smallest dilution + formalinized erythrocytes);

3) the correctness of determining the minimum neutralizing dose of the antigen (2, 1 and 0.5 of the neutralizing dose of the antigen + antibody erythrocyte diagnosticum).

Reverse indirect hemagglutination (rong) is used to indicate bacterial and viral antigens in test materials, as well as for express diagnostics of a number of infections.

In contrast to RNHA, in this reaction, erythrocytes are not sensitized by an antigen, but by antibodies, the agglutination of which occurs when the antigen is added.

Erythrocytes are preliminarily fixed with formalin or glutaraldehyde, and then they are bound to gamma globulin, which is isolated from immune sera and purified from other serum proteins. The binding of gamma globulin to the surface of erythrocytes occurs with the help of chromium chloride. To do this, 1 volume of immunoglobulins isolated from immune serum, 1 volume of 50% suspension of formalized erythrocytes and 1 volume of 0.1-0.2% chromium chloride solution are added to 8 volumes of distilled water. The mixture is left for 10-15 minutes at room temperature, then the erythrocytes are treated as in a passive hemagglutination reaction.

The specificity of the antibody diagnosticum is checked in the reaction of inhibition of passive hemagglutination with a homologous antigen. The reaction must be inhibited by a homologous antigen by at least 16 times and not inhibited by a heterologous one. Use control for the absence of spontaneous hemagglutination.

With the help of this reaction, pathogens are indicated in the material taken from the organs of dead people and animals, for example, from the brain, spleen, liver of the lungs. Prepare a 10% suspension of these organs in isotonic sodium chloride solution, centrifuge them for 30-60 minutes at 10,000 rpm and use the supernatant as an antigen.

Methodology. Prepare 2-fold dilutions of the test material (antigen) in a stabilizing solution. Make 1 drop of each antigen dilution into 3 adjacent wells of the microarray (the reaction takes 3 parallel rows of wells). 1 drop of stabilizing solution is added to each well of the first row, 1 drop of homologous immune serum in a dilution of 1: 10 is added to the wells of the second row, 1 drop of heterologous immune serum is added to the wells of the third row. The second and third rows serve as controls for the specificity of the reaction. The mixture is left for 20 minutes at room temperature.

Add 1 drop of 1% suspension of sensitized erythrocytes (erythrocyte antibody diagnosticum) to all wells and shake the plates thoroughly. The results of the reaction are taken into account after 30-40 minutes. In the presence of a specific antigen, hemagglutination is noted in the first and third row (with heterologous serum) and is absent in the second row, where the antigen is previously neutralized with homologous serum.

The reaction is accompanied by controls of sensitized erythrocytes for spontaneous agglutination.

Reverse indirect hemagglutination inhibition reaction (RTONGA) allows you to determine the presence of antibodies in the sera of humans and animals.

Methodology. Serums are diluted 10 times with isotonic sodium chloride solution, heated for 20 minutes at a temperature of 65 ° C to destroy non-specific inhibitors, and then 2-fold dilutions of sera are prepared in a stabilizing solution and a working dose of antigen containing 4 agglutinating units. Make 1 drop of each serum dilution into the wells of the micropanel and add 1 drop of antigen to them, the dilution of which corresponds to the working dose. After the components of the mixture have been in contact for 20 minutes at room temperature, 1 drop of erythrocyte antibody diagnosticum is added to all wells and shaken thoroughly. After 1.5-2 hours of incubation at room temperature, the results of the reaction are taken into account by hemagglutination. Serum titer is its highest dilution, which completely inhibits the hemagglutination reaction with four antigen agglutinating units.

The reaction is accompanied by controls of sensitized erythrocytes for spontaneous agglutination in the presence of: a) a stabilizing solution; b) normal antigen (from a material that does not contain a virus); c) the test serum. The advantage of the reaction lies in its versatility and the possibility of using it to detect various antigens.

Evaluation of the results of hemagglutination. The results of RNGA, RONGA and RTONGA are taken into account according to the degree of erythrocyte agglutination: (++++) - complete agglutination; (+++) - less complete agglutination; (++) - partial agglutination; (+) - traces of agglutination; (–) – absence of agglutination.

The reaction is considered positive if the agglutination is complete (++++) or almost complete (+++), the diagnosticum does not spontaneously agglutinate in the presence of each component of the reaction, and the antigen or antibody specificity control is positive.

These reactions called indirect (passive), since they use Ag (or AT) artificially sorbed on the surface of various corpuscular particles.

The reaction of indirect, or passive, hemagglutination (RNGA, RPGA) is one of the most sensitive serological reactions. It is based on the ability of AT to interact with Ag fixed on various erythrocytes, which at the same time agglutinate. For greater stability of diagnosticums, erythrocytes are formalized.

Reverse RNGA used to detect antigen in blood serum; for this, not Ag, but AT are fixed on erythrocytes. Reactions of this type are widely used to diagnose infectious diseases, establish pregnancy, detect hypersensitivity to drugs, etc.

Passive hemagglutination inhibition reaction (RTPGA) - further development RNGA; in some sense controls its specificity. Unlike RNGA, includes three components; Ag, AT and Ag (AT) adsorbed on erythrocytes. Initially, Ag reacts with AT (standard antiserum), then erythrocytes sensitized with the same Ag (or AT) are added to the mixture. If, during the interaction of Ag with AT, free AT (or Ag) does not remain in the system, then agglutination of the erythrocyte diagnosticum is not observed.