5. “SENSORY-MOTOR REACTION. MOTOR RESPONSE OF A BOXER TO THE APPEARANCE OF EXTERNAL IRRITIVE In the speed performance of a punch, an important role is played by how the boxer reacts to the appearance of an external stimulus (sound, signal, light bulb on the dynamometer before

RZGA (RTGA)- highly specific serological reaction, which allows to solve the following tasks: to determine the titer of antibodies to the hemagglutinating virus in serum; identify an unknown hemagglutinating virus by known serum; establish the degree of antigenic relationship of two hemagglutinating viruses. RZGA is widely used for the retrospective diagnosis of certain viral infections (influenza, parainfluenza of cattle, viral abortion of sheep, etc.). For this, paired sera samples are used, taken at intervals of 2-3 weeks, and the disease is determined by an increase in antibody titer by 4 or more times.

Advantages of RZGA: simplicity of setting technique, quick response, sterile work is not required, specificity, low cost. Disadvantage - RZGA is possible only with hemagglutinating viruses.

The essence of the RZGA lies in the fact that when the antiviral immune serum interacts with the corresponding strains of the virus, a delay (inhibition) of the hemagglutinating ability of the virus occurs. The result of RHA is affected by non-specific inhibitors of hemagglutination contained in blood sera. They have the ability to bind with viruses and inhibit hemagglutination. In order to eliminate the negative effect of inhibitors on the course of the reaction, they must be destroyed (removed). For this, several methods are used.

ComponentsRZGA : tested virus-containing material; diagnostic immune serum; 1% suspension of erythrocytes; 2nd option - the studied blood serum; standard antigen (viral diagnosticum); 1% suspension of erythrocytes.

When staging reactions, it should be borne in mind that some body fluids (blood serum, urine, saliva, milk, etc.) may contain inhibitors that can inhibit the reaction and delay hemagglutination. In these cases, normal sera, i.e. sera from healthy animals, may give a delay in hemagglutination, leading to erroneous results. In the sera of some healthy people and animals revealed two types of inhibitors - thermostable and thermolabile. Thermolabile inhibitors are found in (beta-fractions of globulin and they have the properties of beta-globulin, inhibit hemagglutination, neutralize the virus and are an important factor in antiviral immunity. They interfere with the production of RHA and get rid of them by inactivating the serum at 56-65 ° C in a water bath for 30 minutes Thermostable inhibitors (Francis inhibitors) are contained in a-fractions of globulin, they are resistant to high temperatures(even to boiling) and the action of alkali, they suppress hemagglutination, but do not possess virus-neutralizing properties. Heat-stable inhibitors are removed from blood serum by treatment of sera with carbon dioxide, vibrio cholera culture filtrate or potassium periodate.
For setting RHA, it is necessary to preliminarily titrate the virus in the RHA to determine 1 AU. When staging RHA, the virus is taken in a titer of 4 AU, that is, the dilution is 4 times less than the titer of the virus in the RHA. For example, the titer of a virus containing 1 AU is 1:64, which means that 4 AU will be contained in a 1:16 dilution of the virus.
All sera are heated to remove heat-labile inhibitors for 30 minutes at a temperature of 56-65°C, depending on the type of animal, before setting the RPHA.
To set up the reaction in one row of test tubes or wells, dilutions of serum in saline are prepared. Usually, a dilution of 1:10 is prepared in the first test tube, and then 2-fold dilutions up to 1:1280 and sometimes higher are prepared from it. Then, from this row, 0.25 ml of the serum corresponding to the dilution is transferred to the second row of test tubes and 0.25 ml of the virus containing 4 AU is added there. The liquid is mixed by shaking the tubes and the mixture is kept in contact for 30-60 minutes at room temperature or in a thermostat, after which 0.5 ml of 1% suspension of erythrocytes is added to each tube, the tubes are then shaken and the mixture is kept again in a room of 30-60 minutes.

Hemadsorption delay reaction (RZGAd).

It is used to detect, identify and type viruses in infected cell culture.

Its essence is in that the antibodies of the immune serum, interacting with the antigen of hemagglutinating viruses that multiply in cell culture, screen them, and as a result, the adsorption of erythrocytes on the surface of infected cells is not observed.

Reaction components: 1) infected and non-infected with hemagglutinating virus cell culture in test tubes; 2) Diagnostic antiviral serum; 3) 0.4% suspension of chicken erythrocytes.

Setting technique. The nutrient medium is drained from the test tubes with cell culture, the cells are washed from its protein components by adding 2-3 ml of Hank's solution to the test tubes. After 1-2 minutes of exposure, the Hank's solution is removed. Then, 0.2 ml of diagnostic serum is added to each experimental and control tubes. The test tubes are left in an inclined position, with the strip up, and kept at room temperature for 15–20 min. Then add 0.2 ml of 0.4% suspension of erythrocytes and again leave for 3...5 min in an inclined position at room temperature. Subsequently, the tubes are gently rotated 5-10 times in order to resuspend the settled, but not adsorbed, erythrocytes.

The hemagglutination inhibition test (HITA) is a method for identifying a virus or detecting antiviral antibodies in the patient's blood serum, based on the phenomenon of the absence of erythrocyte agglutination by a drug containing a virus in the presence of blood serum immune to it.

The hemagglutination inhibition reaction (RTHA) is based on the blockade, suppression of antigens of viruses by antibodies of the immune serum, as a result of which the viruses lose their ability to agglutinate red blood cells.

RTHA is used to diagnose many viral diseases, the causative agents of which (influenza, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the erythrocytes of various animals.

Mechanism. Virus typing is carried out in the hemagglutination inhibition test (HITA) with a set of type-specific sera. The results of the reaction are taken into account by the absence of hemagglutination.

Subtypes of virus A with antigens H0N1, H1N1, H2N2, H3N2, etc. can be differentiated in RTGA with a set of homologous type-specific sera.

Complement binding reaction. Mechanism. Components. Application

Complement fixation reaction (RCC) consists in the fact 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 the complement remains free.

The specific interaction of AG and AT is accompanied by the adsorption (binding) of complement. Since the process of complement fixation does not appear visually, J. Bordet and O. Zhangu proposed to use the hemolytic system (sheep erythrocytes + hemolytic serum) as an indicator, which shows whether the complement is fixed by the AG-AT complex. If AG and AT correspond to each other, i.e., an immune complex has formed, then the complement binds to this complex and hemolysis does not occur. If AT does not correspond to AG, then the complex is not formed and the complement, remaining free, connects with the second system and causes hemolysis.

Components. The complement fixation reaction (CFR) is a complex serological reactions. For its implementation, 5 ingredients are needed, namely: AG, AT and complement (the first system), sheep erythrocytes and hemolytic serum (the 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.

As a complement, fresh or dry guinea pig serum is used.

Mechanism. RSK is carried out in two phases: 1st phase - incubation of a mixture containing three components of the antigen + antibody + complement; 2nd phase (indicator) - detection of free complement in the mixture by adding to it a hemolytic system, consisting of sheep erythrocytes, and hemolytic serum containing antibodies to them. In the 1st phase of the reaction, during the formation of the antigen-antibody complex, complement binding occurs, and then in the 2nd phase, hemolysis of erythrocytes sensitized by antibodies will not occur; the reaction is positive. If the antigen and antibody do not correspond to each other (there is no antigen or antibody in the test sample), the complement remains free and in the 2nd phase it will join the erythrocyte-antierythrocyte antibody complex, causing hemolysis; the reaction is negative.

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

Hemagglutination inhibition reaction. Mechanism. Components. Application

(RTGA) - a method for identifying a virus or detecting antiviral antibodies in the patient's blood serum, based on the phenomenon of the absence of agglutination of erythrocytes by a drug containing a virus in the presence of blood serum immune to it.

Hemagglutination inhibition reaction (RTGA) based on the blockade, suppression of antigens of viruses by antibodies of the immune serum, as a result of which the viruses lose their ability to agglutinate red blood cells.

RTGA is used for the diagnosis of many viral diseases, the causative agents of which (influenza, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the erythrocytes of various animals.

Mechanism. Virus typing is carried out in the hemagglutination inhibition test (HITA) with a set of type-specific sera. The results of the reaction are taken into account by the absence of hemagglutination. Subtypes of virus A with antigens H 0 N 1 , H 1 N 1 , H 2 N 2 , H 3 N 2 and others can be differentiated in RTGA with a set of homologous type-specific sera.

precipitation reaction. Mechanism. Components. Ways of setting. Application

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

RP put in test tubes (ring precipitation reaction), in gels, nutrient media, etc. Varieties of RP in a semi-liquid gel of agar or agarose are widely used: Ouchterlony double immunodiffusion, 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 transparent diagnostic precipitating sera with high antibody titers. The titer of the precipitating serum is taken as the highest dilution of the antigen, which, when interacting with the immune serum, causes the formation of a visible precipitate - turbidity.

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

Neutralization reaction (PH)

This universal reaction serves as a benchmark in the evaluation of other serological reactions.

Its principle is that when an antigen (virus) interacts with homologous antibodies, an antigen + antibody complex is formed, as a result, the infectivity of the virus is neutralized. An indicator of a free (not bound to antibodies) virus is a living system sensitive to the virus: animals, chicken embryos or cell culture.

When setting up the reaction in a test tube, equal volumes of the virus and serum are mixed, the mixture is kept at the appropriate temperature (4, 22 or 37 ° C) for one hour or 16-18 hours (depending on the virus and the conditions of the experiment). Then, this mixture is used to infect a virus-sensitive living system, monitor the state of living objects, and take into account the result of neutralization of the virus by absence after an appropriate period:

1) death, development clinical picture illness and pathological changes in organs and tissues of laboratory animals;

2) death, pathological changes in the membranes, tissues of the embryo and the absence of hemagglutinins in the fluids of the cavities of chicken embryos;

3) cytopathic action (CPE) or plaque formation in cell culture.

Since a certain amount of antibodies is required to neutralize a certain amount of the virus, and one of these components is still unknown, the PH can be set in two ways:

1) to different doses (dilutions) of serum (usually in the form of serial 2-fold dilutions) add the same doses of the virus (usually 100-1000 ED50). In this variant, the titer of virus-neutralizing antibodies in the serum is determined, the indicator of which is the dilution of the serum that protects 50% of the infected from the action of the virus. biological systems;

2) to different doses (dilutions) of the virus (usually in the form of serial 10-fold dilutions) add the same doses of serum (usually in dilutions of 1:10 or 1:20). When setting this variant of PH, in addition to the studied (or specific) serum, normal (negative) serum is also used. In this case, the neutralization index (IN) is determined, which shows how many times the immune (specific) or test serum reduces the virus titer compared to normal serum.

The advantages of PH are its versatility and high specificity; disadvantages - high labor intensity; the need to strictly observe the sterility of materials, utensils and tools; high cost of living biological systems; the relative duration of the bioassay and the need for mathematical calculations.

Haemagglutination inhibition reaction (HITA)

Widely used in the study of hemagglutinating viruses.

It is based on the fact that antibodies, upon encountering a homologous virus (antigen), neutralize not only its infectious, but also hemagglutinating activity, since they block the virion receptors responsible for hemagglutination, forming an antigen + antibody complex with them.

The principle of RTGA is that equal volumes of blood serum and virus are mixed in a test tube (well), and after exposure (30-40 minutes), erythrocytes of the corresponding animal species are added.

Red blood cells are an indicator of the presence of the virus in the mixture. Agglutination of erythrocytes indicates the presence of the virus in the mixture, and the absence of hemagglutination indicates its absence, since the antibodies completely neutralized the hemagglutinating activity of the virus.

RTGA can be installed in two versions:

1) to different dilutions of serum (usually 2-fold) add the same dose of virus (4-8 HAU);

2) to different dilutions of the virus (usually 2-fold) add the same dose of serum.

The setting of RTGA according to the first option is carried out according to the following steps:

The virus is titrated in RGA and its hemagglutination titer is determined;

Prepare and control the working dose of the virus (4 or 8 HAU);

They put the main experience of RTGA;

The results are taken into account.

Assess hemagglutination in each mixture in crosses and determine the antibody titer. Serum antibody titer is taken as the highest serum dilution that still completely inhibits hemagglutination.

RGTA allows you to solve the following diagnostic tasks:

Detect and determine the antibody titer in blood serum using a known virus;

Identify an unknown virus by examining it with various known sera (antibodies).

The advantages of RTGA are the simplicity of the setting technique and the quick result. However, this reaction can only be used for hemagglutinating viruses.