It uses red blood cells or neutral synthetic materials (for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are sorbed. Their agglutination occurs when appropriate sera or antigens are added. Red blood cells sensitized with antigens are called antigenic erythrocyte diagnosticum and are used to detect and titrate antibodies. Erythrocytes sensitized with antibodies. are called immunoglobulin erythrocyte diagnosticums and are used to identify antigens.

The passive hemagglutination reaction is used to diagnose diseases caused by bacteria (typhoid and paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (influenza, adenoviral infections, viral hepatitis B, measles, tick-borne encephalitis, Crimean hemorrhagic fever, etc.), as well as to determine certain hormones, to identify the patient’s hypersensitivity to drugs and hormones, such as penicillin and insulin.

Passive hemagglutination reaction (RPHA). The passive hemagglutination test is a sensitive method of serological diagnosis and is used for both early and retrospective diagnosis, as well as to determine the immunopogic state of vaccinated individuals. In patients with tularemia, antibodies are usually detected at the end of the 1st or 2nd week of the disease; after 1-1.5 months, RPHA titers reach maximum levels (1: 100,000-1: 20,000, less often higher), after which they decrease to the level 1:100-1:200 are stored for a long time.

In vaccinated people, antibodies are also constantly detected, however, in lower titers, not exceeding 1:2000-1:5000 1-1.5 months after vaccination, and remain for several years at a low level of 1:20-1:80.

The antigen for staging RPHA is tularemia erythrocyte diagnosticum (antigenic). The drug is formalinized sheep red blood cells, sensitized with tularemia antigen, available in liquid and dry form. Liquid preparation - a 10% suspension of red blood cells in a formaldehyde solution of 10% concentration. Dry lyophilized preparation is a vacuum-dried 10% suspension of red blood cells without a preservative. Before use, it is diluted according to the directions on the label. To set up the reaction in polystyrene plates, both drugs are used in a 2.5% concentration, and when setting up the reaction in microvolumes - at a 0.5% concentration.

Technique for setting up RPGA. The test sera are diluted with physiological solution 1:5 (1:10) and heated at 56 degrees C for 30 minutes. After this, in order to remove heterogeneous antibodies to sheep erythrocytes, the sera are treated with a 50% suspension of formalinized sheep erythrocytes. To do this, add red blood cells at the rate of 2 drops (0.05 ml) per 1 ml of serum and mix thoroughly by shaking. The serum is left until the erythrocytes have completely settled, or is centrifuged after one hour at room temperature, after which it is ready for examination.

The dilution liquid is poured in a volume of 0.5 ml into a row of wells on a polystyrene plate. During the preliminary study of sera, it is advisable to test them by setting up the reaction in a short row of the plate (6-wells). If antibodies are detected in a short series, the sera are retested in a long series of dilutions (12 wells). After spilling the dilution liquid, add 0.5 ml of test sera in a 1:5 dilution to the first well of each row (short or long). Then the same volumes of serum are titrated with twofold dilutions. Thus, serum dilutions are obtained in the short series from 1:10 to 1:320, and in the long series from 1:10 to 1:20480. After titration of the sera, one drop (0.05 ml) of a working 2.5% suspension of sensitized erythrocytes is added to each well. The contents of the plates are thoroughly shaken until a homogeneous suspension is obtained. The plates are left at room temperature on a stationary table surface. Preliminary recording of the reaction is carried out after 2-3 hours, the final determination of the titer is made after complete sedimentation of red blood cells in the wells. The following controls are provided for the reaction: 1) test serum diluted 1:10 in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 2) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 3) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of sensitized erythrocytes. All controls should give a clearly negative reaction.

Accounting and assessment of RPGA. The reaction is assessed according to the following scheme:

1) sharply positive reaction (++++) - red blood cells fall to the bottom of the hole in an even layer in the form of an “umbrella”, which often has a scalloped melting of the edges;

2) positive reaction (+++) - red blood cells cover at least 2/3 of the bottom of the well;

3) weakly positive reaction (++) - the agglutinate is small and located in the very center of the well;

4) questionable reaction (+) - around the sediment of erythrocytes in the very center of the well there are individual grains of agglutinate;

5) negative (-) - at the bottom of the hole, red blood cells settle in the form of a “button” or a small ring with smooth, sharply defined edges.

The serum titer is taken into account based on the last dilution of the serum, which gave a very clear reaction (at least three pluses). A dilution of 1:100 or higher is considered a diagnostic titer; however, just as in the case of RA, it is necessary to monitor its increase.

RPHA for tularemia is quite specific and detects some cross-reactions only with brucellosis sera. Differential diagnosis is possible by the height of titers in RPGA, which are significantly higher with the homologous antigen.

Technique for setting up RPHA in microvolumes. RPGA can be performed in microvolumes using a Takachi-type microtitrator (or round-bottomed microplates with micropipettes), which allows titration of material in volumes of 25 μl and 50 μl. The reaction technique and the sequence of all operations are the same as when studying in polystyrene plates. It should, however, be borne in mind that the sensitivity of the micromethod is usually one dilution (i.e., 2 times) lower than that of the macromethod.

To set up the reaction in a microtitrator, a dilution liquid in a volume of 50 μl is added to each well using a dropper pipette. Then, using titrators with a 50 μl head, the test serum is collected by immersing the head in it. Make sure that the liquid has filled the titrator head. The titrator with serum is transferred to the first well and, holding it in a vertical position, several rotational movements are made in both directions. Then the titrator is transferred to the next well and the manipulation is repeated. Titration can be carried out simultaneously in several rows. After titrating the entire series, the titrator is washed with distilled water (changing 2 portions) by rotating movements, the water is removed from the head using a swab and burned on a burner flame.

After titration, add 25 μl of erythrocyte diagnostic fluid to the wells. The concentration of diagnosticum for RPHA in microvolumes should be 0.5% (i.e., a 2.5% suspension of red blood cells is additionally diluted 5 times). After adding red blood cells, the plates should be shaken slightly until a homogeneous suspension is obtained. The results can be recorded within 1-1.5 hours, which is a significant advantage of RPGA in a microtiter. In addition, this technique requires small quantities of all reaction ingredients and test sera.

The reaction is taken into account according to the following scheme:

1) “+” – complete hemagglutination, in which red blood cells fall to the bottom of the well in an even layer in the form of an “umbrella”, occupying at least 2/3 of the bottom;

2) “+-“ - partial hemagglutination, in which red blood cells fall to the bottom in the form of a loose ring of small size;

3) “-“ – absence of hemagglutination, when red blood cells fall to the bottom in the form of a small button or ring with a smooth edge.

The specificity of a positive result obtained in RPHA can be tested using a three-component reaction - the passive hemagglutination inhibition reaction (PHA).

Technique for setting up RTPGA. This reaction is used to confirm the specificity of a positive RPGA result when it is in doubt or is of particular epidemiological interest. The mechanism of the reaction is the specific inhibition of hemagglutination when a suspension of killed tularemia bacteria is added to the test serum. Three components interact in the reaction: test serum, specific tularemia antigen and antigenic erythrocyte diagnosticum RTPHA is usually placed in a row of 7-8 wells. It is advisable to install a repeated RPGA in parallel with the RTPGA. 0.25 ml of dilution liquid is poured into two rows of wells, then the test serum in a volume of 0.25 ml is added to the first wells of both rows and titration is performed. Two identical rows of serum dilutions are obtained. Add 0.25 ml of dilution liquid to all wells of the second row, and 0.25 ml of a suspension of tularemia bacteria to the wells of the first row. Tularemia diagnosticum is used (containing 25 billion tularemia bacteria in 1 ml), previously diluted 50 times. This suspension contains 500 million bacteria in 1 ml or 125 million in a volume of 0.25 ml. After adding the antigen, the plate is left for 1 hour at room temperature, after which one drop (0.05 ml) of erythrocyte diagnosticum is added to all wells of both rows, the plate is shaken and left on a flat table surface. Accounting is carried out after 2-3 hours.

Accounting and assessment of RTPGA. If the test serum contains specific tularemia antibodies, they are neutralized by the added antigen and hemagglutination will not occur in the first row of wells, or, with a high serum titer, hemagglutination will be observed in a smaller (2-4) number of wells than in the row with RPHA. In this case, the specificity of the results was confirmed. If hemagglutination is noted in both rows, i.e. If the results of RTPGA and RPGA coincide, this indicates the absence of tularemia antibodies in the test serum. In this case, the primary result of RPGA is considered nonspecific.

Technique for staging RTHG in microvolumes. RTPGA, like RPGA, can be performed in microvolumes using a Takachi-type microtiter. To do this, add 0.25 μl of diluting liquid into the wells of microplates in two rows of 7-8 wells each. Then, using a titrator, 0.25 μl of the test serum is added and titrated in both rows. After this, 25 μl of tularemia antigen (the concentration of which is 500 million tularemia bacteria in 1 ml) is added to each well in the first row, and 25 μl of dilution liquid is added to the second row. The plates are left for 1 hour at room temperature, after which 25 μl of antigenic zritrocytic diagnosticum (0.5% concentration) is added to all wells of both rows. Accounting and evaluation of the results are carried out similarly to reactions in macrovolumes.

Indirect hemagglutination reaction (IRHA)

The use of sorbed diagnostic drugs is based on the principle of immunological recognition of surface structures, which is constantly realized 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 for 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 widespread as a carrier of antigens or antibodies. Based on the principle of using erythrocytes (both native and fixed) as an antigen carrier, the indirect (passive) hemagglutination reaction has become widespread.

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

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

When studying erythrocytes treated with various fixing substances, some of their common properties were established:

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

Do not hemolyze in hypotonic solutions, in water and after freezing-thawing;

Can be freeze dried;

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

The main criterion for the quality of fixed erythrocytes is the possibility of using them to obtain sensitized drugs in the absence of nonspecific gluing 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 drugs. The treatment of red blood cells with tannin has become especially widespread.

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

Along with tanning erythrocytes, chemical reagents such as bromelain, tryopsin, potassium periodate, which also modify erythrocyte membranes, are used to produce erythrocyte diagnosticums and prepare antigen carriers.

After preparing the surface of erythrocytes, the most important process occurs - the process of hemosensitization - the final stage of manufacturing erythrocyte diagnosticums.

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

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

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

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

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

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

We give an example of the production and use of erythrocyte diagnosticum for detecting antibodies to rota and coronavirus infections.

The method for preparing antibody diagnostics for identifying rota and coronavirus antigens in the RNGA is as follows: obtaining a suspension of erythrocytes; fixation of their acroleins; tanization, which allows obtaining stable sorption of the required protein on red blood cells; 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 collecting the blood of a clinically healthy sheep into a flask with glass beads. After defibrinating the blood, the red blood cells should be washed 3-5 times with isotonic (0.9%) sodium chloride solution by centrifugation for 15-20 minutes at 400 g.

In order to stabilize red blood cells, they are treated with acrolein. As is known, its effect is somewhat milder than formaldehyde, and ensures stability and higher activity of red blood cells. To do 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 pH 7.2. The resulting suspension is kept for 30 minutes at 37°C, stirring thoroughly, followed by removal of acrolein by repeated centrifugation for 5 minutes at 600-800g until the specific odor completely disappears. The advantage of red blood cells prepared in this way is that they are stored for future use and can be stored for a long time without undergoing hemolysis.

Subsequently, stabilized red blood cells 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 red blood cells, it is necessary to tannize them by mixing equal parts of a 10% suspension of washed red blood cells and a tannin solution 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 minutes, after which the red blood cells are thoroughly washed from tannin twice in PBS with a pH of 7.2 and three times with an isotonic sodium chloride solution with a pH of 7.2-7.4.

The optimal period for guaranteeing the receipt of high-quality erythrocyte diagnosticums is their sensitization within 24-48 hours after their treatment with tannin.

In the process of manufacturing diagnosticums, 0.3% phenolysed 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 minutes.

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

Sensitization is carried out by mixing equal volumes of tanned erythrocytes and immune serum preheated for 30 minutes at 560C in an optimal concentration. In addition, add 3 parts of an isotonic sodium chloride solution with a pH of 7.2 and 5 parts of a 0.1% chromium chloride solution. The mixture, stirring occasionally, is kept at room temperature for 5 minutes. After the specified time, the mixture is thoroughly washed using PBS with pH 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, provided it is stored at 4°C.

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

When performing RNGA, successive double dilutions of the test virus-containing material are prepared (20-50% suspension of fecal samples), and then an equal volume of erythrocyte diagnostic fluid is added to each well. The plates should be shaken thoroughly several times and left at room temperature until the erythrocytes in the control completely settle. 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 higher.

Indirect or passive hemagglutination reaction (IPHA)

This reaction is superior in sensitivity to the agglutination reaction and is used in the diagnosis of infections caused by bacteria, rickettsia, 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 red blood cells sensitized with antigens are used (erythrocyte diagnosticum).

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 RPGA are taken into account by the nature of the erythrocyte sediment.

The result of a reaction is considered positive when red blood cells evenly cover the entire bottom of the test tube (an inverted umbrella).

In a negative reaction, red blood cells 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 performance and demonstrativeness, the agglutination reaction has become widespread in microbiological practice for the diagnosis of many infectious diseases: typhoid fever 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).

The agglutination reaction can involve both live and killed bacteria, spirochetes, fungi, protozoa, rickettsia, as well as red blood cells and other cells.

The reaction occurs in two phases: the first (invisible) - specific, the combination of antigen and antibodies, the second (visible) - nonspecific, gluing of antigens, i.e. agglutinate formation.

An agglutinate is formed when one active center of a divalent antibody combines with the determinant group of an antigen.

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

Externally, the manifestation of a positive agglutination reaction has a twofold character. In flagellated microbes that have only somatic O-antigen, the microbial cells themselves adhere directly. This agglutination is called fine-grained. It occurs within 18 - 22 hours.

Flagellate microbes have two antigens - somatic O-antigen and flagellar H-antigen. If cells are glued together by flagella, large, loose flakes are formed and this agglutination reaction is called coarse-grained. It occurs within 2 - 4 hours.

The agglutination reaction can be performed 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 performed both in an expanded version, which allows you to work with serum diluted to a diagnostic titer, and in an indicative reaction version, which allows you, in principle, to detect specific antibodies or determine the species of the pathogen.

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

1) To detect antibodies in the patient’s blood serum, erythrocyte diagnostics are used, in which the antigen is adsorbed on the surface of tannin-treated erythrocytes. In relation to this reaction, the term RPHA 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 along the walls of the hole in the form of a “lace umbrella”; with a negative reaction, a dense sediment of red blood cells in the form of a “button” appears.

2) To detect toxins and bacterial antigens in the test material, antibody erythrocyte diagnostics 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 diagnostics, plague bacillus antigen, diphtheria exotoxin, and botulinum exotoxin are detected.

Coombs test (aptiglobulin test)

The reaction is used to detect incomplete antibodies, for example, antibodies to the Rh factor. Test serum is added to Rh + erythrocytes, in which the presence of incomplete antibodies to the Rh factor is assumed. Having attached to red blood cells, incomplete antibodies do not cause agglutination, since they have only one active center. Then antiglobulin serum containing antibodies to human globulins is added. Combining with incomplete antibodies, antiglobulin serum causes agglutination of red blood cells.

Precipitation reaction

The essence of the reaction is the sedimentation (precipitation) of the antigen under the influence of specific antibodies. To obtain a visible reaction, the presence of an electrolyte is necessary. The antigen in the precipitation reaction is molecularly dispersed substances.

Ring precipitation reaction placed in narrow precipitation tubes. Immune serum is poured into a test tube, and the test material is carefully layered onto it. If there is an antigen in it, an opaque ring of precipitate forms at the border of the two liquids.

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

Agar gel precipitation reaction carried out using several methods. These are the double immunodiffusion reaction, the radial immunodiffusion reaction, and the immunoelectrophoresis reaction.

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

Radial immunodiffusion reaction(according to Mancini). The immune serum is added to the melted agar gel and poured into a cup. After the agar hardens, wells are cut out and antigens are placed in them, which, diffusing into the agar, form ring-shaped precipitation zones around the wells. The higher the antigen concentration, the larger the diameter of the ring. The reaction is used, for example, to determine immunoglobulins of various classes 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 an agar gel. 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 where they meet.

Immune lysis reactions

An antigen (erythrocytes or bacteria), combining with specific antibodies, forms an immune complex to which complement (C1) attaches, and complement is activated 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 red blood cells, and complement is activated, which lyses red blood cells. The cloudy suspension of red blood cells 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 the gel(Erne 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 red blood cells, and after the agar has solidified, complement is added. A hemolysis zone forms around each hemolysin-producing cell. The number of hemolysin-producing cells is determined by the number of such zones.

Laboratory diagnosis of almost all infectious diseases is based on identifying antibodies in the patient’s blood that are produced against pathogen antigens using serological reactions. They entered medical practice from the late nineteenth to early twentieth centuries.

The development of science has helped determine the antigenic structure of microbes and the chemical formulas of their toxins. This made it possible to create not only therapeutic, but also diagnostic sera. They are obtained by injecting weakened pathogens into laboratory animals. After several days of incubation, preparations are prepared from the blood of rabbits or mice that are used to identify microbes or their toxins using serological tests.

The external manifestation of such a reaction depends on the conditions of its production and on the state of antigens in the patient’s blood. If microbial particles are insoluble, they precipitate, lyse, bind or immobilize in the serum. If antigens are soluble, then the phenomenon of neutralization or precipitation occurs.

Agglutination reaction (RA)

The serological agglutination reaction is highly specific. It is simple to perform and visual enough to quickly determine the presence of antigens in the patient’s blood serum. It is used to perform the Widal reaction (diagnosis of typhoid fever and paratyphoid fever) and Weigl reaction (typhus fever).

It is based on the specific interaction between human antibodies (or agglutinins) and microbial cells (agglutenogens). After their interaction, particles are formed that precipitate. This is a positive sign. Live or killed microbial agents, fungi, protozoa, and somatic cells can be used to stage the reaction.

Chemically, the reaction is divided into two stages:

1. Specific combination of antibodies (AT) with antigens (AG).
2. Nonspecific - precipitation of AG-AT conglomerates, that is, the formation of an agglutinate.

Indirect agglutination reaction (IAR)

To perform it, purified sheep erythrocytes and human red blood cells, pre-treated with antibodies or antigens (this depends on what exactly the laboratory technician wants to find), are used. In some cases, human red blood cells are treated with immunoglobulins. Serological reactions of erythrocytes are considered successful if they settle to the bottom of the tube. We can speak of a positive reaction when the cells are arranged in the form of an inverted umbrella, occupying the entire bottom. A negative reaction is counted if the red blood cells settle in a column or in the form of a button in the center of the bottom.

Precipitation reaction (RP)

Serological tests of this type are used to detect extremely small particles of antigens. These can be, for example, proteins (or parts thereof), compounds of proteins with lipids or carbohydrates, parts of bacteria, and their toxins.

Sera for the reaction are obtained by artificially infecting animals, usually rabbits. Using this method, you can obtain absolutely any precipitating serum. The production of serological precipitation reactions is similar in mechanism of action to agglutination reactions. Antibodies contained in the serum combine with antigens to form large protein molecules that are deposited at the bottom of the tube or onto the substrate (gel). This method is considered highly specific and can detect even minute amounts of a substance.

Used to diagnose plague, tularemia, anthrax, meningitis and other diseases. In addition, he is involved in forensic medical examination.

in gel

Serological reactions can be carried out not only in a liquid medium, but also in an agar gel. This is called the diffuse precipitation method. With its help, the composition of complex antigenic mixtures is studied. This method is based on the chemotaxis of antigens to antibodies and vice versa. In the gel they move towards each other at different speeds and, meeting, form precipitation lines. Each line is one set of AG-AT.

Exotoxin neutralization reaction with antitoxin (RN)

Antitoxic serums are able to neutralize the effect of exotoxin produced by microorganisms. These serological reactions are based on this. Microbiology uses this method to titrate serums, toxins and toxoids, as well as determine their therapeutic activity. The strength of toxin neutralization is determined by conventional units - AE.

In addition, thanks to this reaction, it is possible to determine the species or type of exotoxin. This is used for diphtheria, botulism. The study can be carried out both “on glass” and in gel.

Lysis reaction (RL)

The immune serum that enters the patient’s body has, in addition to its main function of passive immunity, also lysing properties. It is capable of dissolving microbial agents, foreign cellular elements and viruses entering the patient’s body. Depending on the specificity of the antibodies contained in the serum, bacteriolysins, cytolysins, spirochetolysins, hemolysins and others are isolated.

These specific antibodies are called "complement". It is found in almost all fluids of the human body, has a complex protein structure and is extremely sensitive to increased temperature, shaking, acids and direct sunlight. But when dried, it can retain its lysing properties for up to six months.

There are the following types of serological reactions of this type:

Bacteriolysis;

Hemolysis.

Bacteriolysis is carried out using the patient's blood serum and specific immune serum with live microbes. If a sufficient amount of complement is present in the blood, the researcher will see lysis of bacteria, and the reaction will be considered positive.

The second serological blood reaction is that a suspension of the patient’s red blood cells is treated with serum containing hemolysins, which are activated only in the presence of a certain compliment. If there is one, the laboratory assistant observes the dissolution of red blood cells. This reaction is widely used in modern medicine to determine the titer of complement (that is, its smallest amount that provokes the lysis of red blood cells) in blood serum and to perform a complement fixation test. It is in this way that a serological reaction to syphilis is carried out -

Complement fixation reaction (CFR)

This reaction is used to detect antibodies to an infectious agent in the patient’s blood serum, as well as to identify the pathogen by its antigenic structure.

Up to this point we have described simple serological reactions. RSC is considered a complex reaction, since not two, but three elements interact in it: antibody, antigen and complement. Its essence lies in the fact that the interaction between antibody and antigen occurs only in the presence of complement proteins, which are adsorbed on the surface of the resulting AG-AT complex.

The antigens themselves, after the addition of complement, undergo significant changes, which indicate the quality of the reaction performed. This may be lysis, hemolysis, immobilization, bactericidal or bacteriostatic effect.

The reaction itself occurs in two phases:

1. Formation of an antigen-antibody complex that is not visually noticeable to the researcher.
2. Change in antigen under the influence of complement. This phase can most often be observed with the naked eye. If the reaction is not visually visible, then an additional indicator system is used to identify changes.

Indicator system

This reaction is based on complement fixation. Purified sheep erythrocytes and hemolytic serum that does not contain complement are added to the test tube an hour after RSC placement. If there is unbound complement left in the test tube, it will join the AG-AT complex formed between sheep blood cells and hemolysin and cause their dissolution. This will mean that the RSC is negative. If the red blood cells remain intact, then the reaction is positive.

Hemagglutination reaction (HRA)

There are two fundamentally different hemagglutination reactions. One of them is serological, it is used to determine blood groups. In this case, red blood cells interact with antibodies.

And the second reaction is not serological, since red blood cells react with hemagglutinins produced by viruses. Since each pathogen acts only on specific red blood cells (chicken, lamb, monkey), this reaction can be considered highly specific.

You can tell whether a reaction is positive or negative by the location of the blood cells at the bottom of the test tube. If their pattern resembles an inverted umbrella, it means that the desired virus is present in the patient’s blood. And if all the red blood cells are formed like a column of coins, then the desired pathogens do not exist.

Hemagglutination inhibition reaction (HAI)

This is a highly specific reaction that allows you to determine the type, type of viruses or the presence of specific antibodies in the patient’s blood serum.

Its essence lies in the fact that antibodies added to a test tube with the test material prevent the deposition of antigens on red blood cells, thereby stopping hemagglutination. This is a qualitative sign of the presence in the blood of specific antigens for the specific virus being sought.

Immunofluorescence reaction (RIF)

The reaction is based on the ability to detect AG-AT complexes after they are treated with fluorochrome dyes. This method is easy to use, does not require isolation of pure culture and takes little time. It is indispensable for the rapid diagnosis of infectious diseases.

In practice, these serological reactions are divided into two types: direct and indirect.

Direct RIF is produced with an antigen that has been pre-treated with fluorescent serum. And the indirect one is that the drug is first treated with a conventional diagnosticum containing antigens to the desired antibodies, and then a luminescent serum is applied again, which is specific to the proteins of the AG-AT complex, and microbial cells become noticeable under microscopy.