Complement binding reaction. Description, application. Microbiology with the technique of microbiological research - complement fixation reaction

(RSK) - serol. reaction used for quantification complement-fixing Ab and Ag. The principle of CSC is that a specific immune complex completely or partially adsorbs C added to the system. C is taken as a quantitative indicator of C binding hemolytic system, hemolysis a swarm indicates the absence of an immune complex in the diagnostic system (negative RSK), hemolysis delay - for the presence of the immune complex (positive RSK). In the RSC, the following are used: 1) heated at 57 ° C for 30 min. s-ku, it should be fresh, without suspended particles, cytotoxins, C. In the experiment, they usually take several multiples of 2 dilutions of s-ki, depending on the diagnostic titer of RSK in the suspected disease; 2) a diagnosticum for sale or specially prepared for RSK; 3) C in a working dose, which, when titrated with Ag, is equal to C titer, and when titrated without Ag, it is 20–25% higher than C titer; 4) sheep erythrocytes are washed several times with saline and a 3% suspension is prepared; 5) hemolytic acid is diluted 3 times less than the titer indicated on the label. All 5 components are taken in the same volume (0.5 ml or 0.25 ml). Research is introduced into the experimental (diagnostic) system. s-ku, diagnosticum, S. Controls are prepared: s-ki - s-ku, C and saline solution (instead of Ag); diagnosticum - diagnosticum, C and saline solution (instead of s-ki); C-C and double the volume of saline; obviously positive - standard immune s-ka, Ag, C; obviously negative - normal s-ka, Ar, C. Shake the tubes and put in a thermostat at 37 ° C for 45 minutes. At the same time, a hemolytic system is prepared by mixing equal volumes of a 3% suspension of sheep erythrocytes and hemolytic s-ki, and incubated in a thermostat for the same time. After sensitization, a double volume of the hemolytic system (1 ml or 0.5 ml) is added to all experimental and control tubes. All tubes are kept in a thermostat at 37°C until complete hemolysis occurs in the control tubes (about 30 - 40 min). With a quantitative method of setting the reaction, a dilution is found, in Krom there is a hemolysis delay of at least 2+ (At titer of research. S-ki) and compare it with the diagnostic titer of the disease being recognized. A semi-quantitative method with one dilution of s-ki (usually 1:5) makes it possible to determine the degree of delay in hemolysis by 4+, 3+, 2+ and, based on this, evaluate the intensity of the reaction. More accurate results are the finding of the s-ki titer not by 100%, but by 50% hemolysis (see. complement definition). A variant of the quantitative setting of RSK is proposed, in which one dilution of s-ki is taken, but several doses of C (1; 1.5; 2; 2.5; 3; 4). All other components are used in the same quantities. When evaluating have in mind the fact that the more doses C binds the study. s-ka, the more At in it.

(Source: Glossary of Microbiology Terms)

See what the "Complement fixation reaction" is in other dictionaries:

complement fixation reaction- RSK laboratory method. [English-Russian glossary of basic terms on vaccinology and immunization. World Organization health care, 2009] Topics vaccinology, immunization Synonyms RSK EN complement fixation testCFTCF test ... Technical Translator's Handbook

COMPLEMENT BONDING REACTION- complement fixation reaction, RSK, Bordet-Gangu reaction [named Belg. bacteriologists J. Bordet and O. Zhangu (O. Gengou), 1901], a highly specific and very sensitive serological reaction based on the property of the complex ... ... Veterinary encyclopedic Dictionary

I Complement fixation test a serological test used to determine an antigen or antibody, based on an assessment of the consumption (binding) of complement by antigen-antibody complexes, see Immunological methods of research. II… … Medical Encyclopedia

complement fixation reaction

complement fixation reaction- (RSK), a highly sensitive and specific serological test used to diagnose many infectious and parasitic animal diseases. Consists of two consecutive phases. In the first phase, the antigen is mixed with the test serum and ... ... Agriculture. Big encyclopedic dictionary

- (RSK; synonym: Borde Zhangu reaction, complement rejection reaction obsolete, alexin fixation reaction obsolete.) a serological test method based on the ability of the resulting antigen-antibody complex to bind complement, which is detected ... ... Big medical dictionary

COMPLEMENT BONDING REACTION (CFR)- highly sensitive and specific. serological reaction used for diagnosis. infectious and invasive diseases. Consists of two consecutive phases. In the first phase, the antigen is mixed with the test serum and complement (a complex of proteins ... ... Agricultural Encyclopedic Dictionary

- (obsolete) see Complement fixation reaction ... Big Medical Dictionary

complement rejection reaction- rus reaction (g) of complement fixation, reaction (g) of Borde Zhang; reaction (g) complement deviation; alexin fixation reaction (g) eng complement fixation test fra réaction (f) de fixation du complément deu Komplementbindungsreaktion (f) spa… … Occupational safety and health. Translation into English, French, German, Spanish

See Complement fixation reaction. (

The complement fixation test is one of the traditional serological tests used to diagnose many viral diseases, and also forms the basis of some other methods for the detection of antiviral antibodies. Although the reaction of neutralization, inhibition of hemagglutination, indirect hemagglutination, enzyme immunoassay is superior to the complement fixation reaction in sensitivity, however, the simplicity of the technique, the early detection of antibodies, and the detection of viral antigens that do not have hemagglutinating properties explain it. wide application in virological research.

It does not require such high degree concentration and purity of antigenic preparations, which are necessary for a number of other immunological reactions. In addition, the group specificity of the complement-fixing antigens of many viruses makes it possible to use the complement fixation reaction in mass diagnostics: this reaction does not reveal strain differences and is of particular value in the study of antigenic relationships between viruses.

The name itself largely reflects the essence of the method, which consists of two separate reactions. At the first stage, antigens and antibodies participate in the reaction (one of these components is known in advance), as well as a complement in a titrated amount. If the antigen and antibodies match, their complex binds complement, which is detected at the second stage using an indicator system (a mixture of sheep erythrocytes and antiserum to them). If the complement is bound during the interaction of the antigen and antibodies, then lysis of sheep erythrocytes does not occur (positive complement fixation reaction). In a negative complement fixation reaction, unbound complement contributes to hemolysis, which is used to judge the results of the reaction.

The main components of the complement fixation reaction are antigens (known or detectable), antibodies (known antisera or test sera), complement, hemolytic serum and ram erythrocytes; isotonic saline is used as a diluent sodium chloride(pH 7.2-7.4) or various buffer solutions. Antigens and sera may have anti-complementarity, i.e. the ability to adsorb complement, which delays hemolysis and distorts the result of the reaction.

Antigens for CSC are prepared from the organs of infected animals, allantoic or amniotic fluid of infected chicken embryos, as well as culture fluid after culturing viruses in tissue cell cultures. Antigenic preparations always contain a lot of ballast substances from animal cells or tissue cultures, which can also distort the results of the reaction.

The resulting viral antigens are inactivated, the working dose is determined, as well as anti-complementary and hemolytic properties. The antigen is then titrated in the presence of complement.

The second main component in the formulation of the reaction is complement. This term refers to a complex system of proteins and factors present in the blood of animals and humans. Typically, guinea pig serum is used in the complement fixation reaction. The activity (titer) of complement is its smallest amount, in the presence of which hemolysin causes complete hemolysis of the hemolytic system used.

To detect complement-binding antibodies to viruses, mandatory inactivation of the serum is carried out to eliminate complement at 560C for 30 minutes. Sera from immunized animals are usually used as reference sera. Moreover, for immunization, viral antigens are used, carefully purified from tissue impurities, since otherwise a pronounced non-specific reaction occurs.

An obligatory component of the complement fixation reaction is the hemolytic system, which includes ram erythrocytes and hemolytic serum obtained by hyperimmunization of rabbits with ram erythrocytes.

To set up the complement fixation reaction, the following modifications are used: the micromethod of the complement fixation reaction in the microtiter of the Takachi system, the long-term complement fixation reaction, the indirect complement fixation reaction, the complement fixation reaction in the gel, the complement fixation reaction by the microdroplet method, the complement fixation reaction on a solid basis.

Although the sensitivity of the complement fixation reaction is low, it has a high specificity. In this regard, it is widely used for the diagnosis of viral pneumoenteritis in young cattle.

Complement fixation test (CFR) is a serological test that is comparable in sensitivity to precipitation, agglutination and neutralization methods. RSK is a method where two antigen-antibody systems are used:

the first is specific;

the second is indicator (hemolytic).

5 components are required for RSC: diagnostic antigen, diagnostic antibodies, indicator antigen (sheep erythrocytes), indicator antibodies (rabbit hemolysins), complement.

The specific interaction of antigen and antibody is accompanied by complement fixation. The resulting complex is not visually manifested. The hemolytic system is used as an indicator. Hemolytic serum sensitizes erythrocytes to the action of complement, in the presence of which lysis of erythrocytes (hemolosis) occurs. If there is no hemolysis, then the complement is bound by the first system, and, therefore, the antigen in it corresponds to the antibody - a positive answer. If the antibody does not match the antigen, the complex is not formed and the complement, remaining free, combines with the second system, causing hemolysis - the reaction is negative.

To set up RSC, all reaction ingredients are prepared and titrated before the main experiment.

Serum (patient or diagnostic) on the eve of the reaction is heated in a water bath at a temperature of 56ºС for 30 minutes to inactivate its own complement. Some sera, especially from immunized animals, have anti-complement properties, i. the ability to bind complement in the absence of a homologous antigen. Anticomplementarity is eliminated by treating them with carbon dioxide, heating at a temperature of 57-58ºС, single freezing at a temperature of -20ºС or -70ºС and removing sediment by centrifugation, adding complement serum in a volume of 1:10 serum and heating it after incubation in the cold for 18- 20 hours. To prevent anticomplementarity of sera, they are stored in lyophilized form or frozen at low temperatures.

Cultures of various killed microorganisms, their lysates, components of bacteria, pathologically altered and normal organs, tissue lipids, viruses, and virus-containing materials can serve as an antigen for CSC. Many microbial antigens are produced industrially. The anticomplementarity of antigens is eliminated using methods such as thermolysis (multiple freezing and thawing), treatment with fat solvents (ether, chloroform, acetone), alcohols (methanol, ethanol), etc.

As a complement, guinea pig serum taken immediately before the experiment is used; dry complement may also be used. To obtain a basic solution for subsequent titration, the complement is diluted 1:10 with isotonic sodium chloride solution.

Sheep erythrocytes are used in the form of 3 suspensions in isotonic sodium chloride solution. Blood (100-150 ml) is taken from jugular vein, placed in a sterile jar with glass beads, defibrinated by shaking for 10-15 minutes and filtered through 3-4 layers of sterile gauze to remove fibrin. Erythrocytes are washed 3 times with isotonic sodium chloride solution, adding it to the erythrocyte sediment to the initial blood volume. Erythrocytes can be stored for 5-6 days at 4-6ºС. The shelf life of erythrocytes increases when they are preserved with formalin.

Hemolytic serum for CSC is obtained as follows Rabbits are immunized by injecting them into the ear vein with 50% suspension of washed ram erythrocytes (1 ml 4-6 times every other day). Sample serum is obtained 7 days after the last injection. If the serum titer is not lower than 1:1200, then bloodletting is done. The serum is heated for 30 minutes at a temperature of 56ºС. To prevent bacterial growth, a preservative is added to the hemolytic serum (merthiolate 1:10,000 or 1 boric acid).

The hemolytic system consists of hemolytic serum mixed in equal volumes (taken in a triple titer) and 3 suspensions of ram erythrocytes. To sensitize erythrocytes with hemolysins, the mixture is kept in a thermostat at a temperature of 37ºС for 30 minutes.

Titration of hemolytic serum. Serum is titrated by combining 0.5 ml of it (in dilutions of 1:600, 1:1200, 1:1600, 1:3200, etc.) with 0.5 ml of 3 erythrocyte suspensions and 0.5 ml of fresh complement in dilution 1:10. The volume of the mixture for the reaction in the control test tubes was adjusted to 1.5 ml by adding isotonic sodium chloride solution. The results of the reaction are taken into account after 1 hour of incubation at a temperature of 37ºС. The titer of hemolytic serum is its highest dilution that causes complete hemolysis. Serum is stored in lyophilized form.

Titration scheme for hemolytic serum

Complement titration. Before the experiment, the basic complement solution (1:10) is poured into a number of test tubes from 0.05 to 0.5 ml and added to each isotonic sodium chloride solution, bringing the volume of liquid to 1.5 ml. The tubes are placed in a thermostat at a temperature of 37ºC for 45 minutes, then a hemolytic system is added to them and kept in a thermostat for 30 minutes, after which the complement titer is determined.

For the experiment, a working dose of complement (contained in a volume of 0.5 ml) is taken, which is 20-30% higher than the titer.

Complement titration scheme

Thermostat at 37ºС for 45 min

Thermostat at 37ºС for 30 min

Titration of the antigen. Antigens used for CSC may adsorb some amount of complement, i.e. have anticomplementary properties. Therefore, before the experiment, the antigens are titrated in the presence of a working dose of complement. To a lesser extent, anticomplementary properties are expressed in specific antigens produced by production. Their titer is determined not before each experiment, but once a month, taking into account the possibility of its decrease during storage.

To determine the titer of the antigen, it is poured into a number of test tubes in a decreasing amount from 0.5 to 0.05 ml, bringing the volume in them to 1 ml by adding sodium chloride solution. Then, 0.5 ml of the working dose of complement is added to each test tube and placed in a thermostat for 1 hour at a temperature of 37ºС. After that, 1 ml of the hemolytic system is added to all test tubes, they are again incubated for 1 h in a thermostat and the reaction results are taken into account.

Antigen titer is considered to be the smallest amount at which complete hemolysis occurs. For CSC, a working dose of antigen is used, which is approximately 1/2-1/3 titer. Antigens in the presence of which the complement titer decreases by more than 30% are unsuitable for the reaction.

Antigen titration scheme

Thermostat at 37ºС for 1 hour

Thermostat at 37ºС for 1 hour

Carrying out the main experience of the RSC. The total volume of the reaction ingredients is 2.5 ml, the volume of the working dose of each of them is 0.5 ml. Serum in the appropriate dilution, antigen and complement are introduced into the first tube, serum in the appropriate dilution, complement and isotonic sodium chloride solution (serum control) into the second tube, antigen, complement and isotonic sodium chloride solution (antigen control) into the third tube. At the same time, a hemolytic system is prepared by mixing 2 ml of hemolytic serum in a triple titer (in relation to that indicated in the instructions) and 3% suspension of sheep erythrocytes (in relation to the initial blood volume). The tubes are kept in a thermostat at a temperature of 37ºС for 1 hour, then 1 ml of the hemolytic system (second system) is added to the first 3 tubes (first system). After thoroughly mixing the ingredients, the test tubes are again placed in a thermostat for 1 hour at a temperature of 37ºС.

Scheme of conducting the main experiment of the RSC

Thermostat at 37ºС for 1 hour

Thermostat at 37ºС for 1 hour

The results of the reaction are taken into account preliminary - after removing the test tubes from the thermostat and finally - after they have been kept for 15-18 hours in the refrigerator or at room temperature.

In the final count, the intensity of the reaction is expressed in pluses: (++++) - a sharply positive reaction, characterized by a complete delay in hemolysis (the liquid in the test tube is colorless, all erythrocytes settle to the bottom); (+++, ++) - a positive reaction, manifested by an increase in the color of the liquid due to hemolysis and a decrease in the number of erythrocytes in the sediment; (+) - weakly positive reaction (the liquid is intensely colored, there is a small amount of erythrocytes at the bottom of the tube). At backlash(–) complete hemolysis is observed, the liquid in the test tube has an intense pink color (lacquer blood).

A number of RSC modifications have been proposed, which are characterized by increased sensitivity and a smaller amount of ingredients used. So, for example, in virological studies, the volume of ingredients for CSC in the cold is 1 ml. For drip RSC, take 1 drop of serum + 1 drop of antigen + 1 drop of complement + 2 drops of the hemolytic system.

The compliment binding reaction has found the widest distribution in microbiological and serological diagnostics.

With the help of RSK, complement-fixing antibodies are detected in the blood serum of patients with syphilis, glanders, chronic gonorrhea, rickettsiosis, viral diseases, etc.

RSC is of particular interest for clinical immunology. The reaction is used to isolate various subpopulations of lymphocytes, to detect HLA antigens on platelets, transplanted lymphoblasts, fibroblasts, tumor cells and platelet-specific antigens, as well as the corresponding antibodies.

complement fixation reaction , RSK, Bordet-Gangu reaction [named Belg. bacteriologists J. Bordet and O. Zhangu (O. Gengou), 1901], a highly specific and very sensitive serological reaction based on the property of the antigen-antibody complex to fix free complement (), used in the diagnosis of many bacterial and viral and some protozoal and helminthic diseases, as well as to study processes accompanied by a change in the amount of antigen or antibodies. CSC proceeds in 2 phases: 1) the interaction of antibodies, antigen and complement, as a result of which the free complement is bound by the formed antigen-antibody complex (specific phase); 2) indication of the reaction sensitized by erythrocytes (non-specific phase). In RSK, 2 systems are used: a specific bacteriological system, consisting of an antibody (test serum), antigen and complement, as well as a non-specific "indicator" system containing hemolysin (hemolytic serum) and a suspension of ram erythrocytes. An antigen binds to an antibody only in the presence of complement. If the test serum contains antibodies homologous to the antigen taken, then the complement present in the reacting mixture is adsorbed by the resulting antigen-antibody complex and loses the ability to lyse sensitized erythrocytes, that is, without complement, hemolysin (hemolytic antibody) does not destroy erythrocytes (positive reaction). In cases where there is no specific relationship between the antigen and antibodies of the test serum, the complex is not formed and the complement remains in a free state. When adding a hemolytic system in this case, unbound complement causes hemolysis of sensitized red blood cells (negative reaction). There are various options for staging RSK: the classical method of staging in the form of macro- and microvariants, the long-term complement fixation reaction (RDSK) in the cold, the method of quantitative RSK according to 50% hemolysis of sensitized erythrocytes, etc. In veterinary diagnostic practice, the classical method of RSK is more often used in in the form of a macro-variant, in which each component is taken in 0.5 or 0.25 ml in a certain working titer. Complement is titrated in a bacteriological system on the sera of the same animal species whose blood is to be examined. The test sera are usually examined in dilutions of 1:5 and 1:10 with antigen and 1:5 without antigen (control). Complement binding time in the specific phase 20 min, hemolysis reaction time in the nonspecific phase 20 min at t 37-38ºC. For the diagnosis of a number of diseases, a more sensitive method is used - RDSC. In this case, the first phase of the reaction is carried out at t 4-6ºC for 16-18 hours, which leads to increased adsorption of complement by small amounts of antigen and antibodies. The hemolytic system is then added and the reaction proceeds at t 37-38ºC for 20 min. The results of RSK and RDSK are taken into account by the degree of hemolysis delay, which is indicated by crosses or minus, corresponding to the following percentages of erythrocyte hemolysis: ++++ from 0 to 10%; +++ from 10 to 40%; ++ from 40 to 70%; + 70 to 90%; – from 90 to 100%. The veterinary legislation provides for special instructions for setting, recording and evaluating RSK and RDSK for each disease separately.

Literature:
Laboratory researches in veterinary medicine, M., 1971;
Guide to Immunology, ed. O. E. Vyazova and Sh. Kh. Khodzhaeva. Moscow, 1973.

Veterinary encyclopedic dictionary. - M.: "Soviet Encyclopedia". Chief editor V.P. Shishkov. 1981 .

See what "" is in other dictionaries:

Complement fixation reaction- (RSK) serol. a reaction used to quantify complement-fixing Ab and Ag. The principle of RSK is that a specific immune complex completely or partially adsorbs C added to the system. As ... ... Dictionary of microbiology

complement fixation reaction- RSK Laboratory method. [English-Russian glossary of basic terms on vaccinology and immunization. World Health Organization, 2009] Topics vaccinology, immunization Synonyms RSK EN complement fixation testCFTCF test ... Technical Translator's Handbook

Complement fixation reaction- I Complement fixation test a serological test used to determine an antigen or antibody, based on an assessment of the consumption (binding) of complement by antigen-antibody complexes, see Immunological methods of research. II… … Medical Encyclopedia

complement fixation reaction- rus reaction (g) of complement fixation, reaction (g) of Borde Zhang; reaction (g) complement deviation; alexin fixation reaction (g) eng complement fixation test fra réaction (f) de fixation du complément deu Komplementbindungsreaktion (f) spa… …

complement fixation reaction- (RSK), a highly sensitive and specific serological test used to diagnose many infectious and parasitic animal diseases. Consists of two consecutive phases. In the first phase, the antigen is mixed with the test serum and ... ... Agriculture. Big encyclopedic dictionary

complement fixation reaction- (RSK; synonym: Borde Zhangu reaction, complement rejection reaction obsolete, alexin fixation reaction obsolete.) a serological test method based on the ability of the resulting antigen-antibody complex to bind complement, which is detected ... ... Big Medical Dictionary- rus reaction (g) of complement fixation, reaction (g) of Borde Zhang; reaction (g) complement deviation; alexin fixation reaction (g) eng complement fixation test fra réaction (f) de fixation du complément deu Komplementbindungsreaktion (f) spa… … Occupational safety and health. Translation into English, French, German, Spanish

Complement binding reaction- see Complement fixation reaction. (

Complement fixation test (CFR) and long-term complement fixation reaction (RDSC)

COMPLEMENT BONDING REACTION (RSK) AND LONG-TERM COMPLEMENT BONDING REACTION (RDSK)

COMPLEMENT BONDING REACTION, RSK, Bordet-Jangu reaction [named after bacteriologists J. Bordet and O. Gengou, 1901], highly specific and very sensitive serological. a reaction based on the property of the antigen-antibody complex to fix free complement.

Complement (complement system) (from lat.complementum - addition), a group of globular proteins in the blood serum of animals and humans, which are part of immune system organism. When bacteria or viruses infecting it enter the body, some toxins or the appearance of its own transformed cells, complement is activated, as a result of which target cells are lysed (destroyed), and toxins and viruses are neutralized. Therefore, the complement system is considered, along with macrophages, as the frontier of the body's immune defense.

This reaction is widely used in the identification of antigens and in the serodiagnosis of infections, especially diseases caused by spirochetes (Wassermann reaction), rickettsia and viruses.

RSK is a complex serological reaction. It involves complement and two antigen-antibody systems. Essentially, these are two serological reactions.

The first system - the main one - consists of an antigen and an antibody (one is known, the other is not). A certain amount of complement is added to it. When the antigen and antibody of this system match, they will connect and bind the complement. The resulting complex is finely dispersed and is not visible.

complement fixation reaction serodiagnosis

The formation of this complex is known with the help of a second hemolytic or indicator system. It includes sheep erythrocytes (antigen) and their corresponding hemolytic serum (antibody), i.e. ready-made immune complex. In this system, erythrocyte lysis can only occur in the presence of complement. If the complement is bound by the first system (if the antigen and antibody correspond in it), then there will be no hemolysis in the second system - since there is no free complement. The absence of hemolysis (the contents of the tube are cloudy or there is a sediment of erythrocytes at the bottom of the tube) is recorded as positive result RSK.

If in the first system the antigen does not match the antibody, then the immune complex is not formed and the complement remains free. Remaining free, the complement is involved in the second system, causing hemolysis - the result of RSC is negative (the contents of the tubes are transparent - "lacquer blood").

Components of the complement fixation reaction:

1. Antigen - usually a lysate, extract, hapten; less often a suspension of microorganisms

2. Antibody - patient's serum

3. Complement - guinea pig serum

4. Antigen - sheep erythrocytes Hemolytic system

5. Antibody - hemolysin to sheep erythrocytes

6. Isotonic solution

Preparation of ingredients

1. Serum is diluted 3 times less than its titer. Prepare a total serum dilution for the entire experiment, the volume of which is determined by multiplying the volume of serum in one tube (for example, 0.5 ml) by the number of tubes slightly greater than the number of them in the experiment. An excess of liquid is necessary in the preparation of all components of the reaction: part of it remains on the walls of test tubes, flasks, pipettes. Volumes of diluted hemolysin and suspension of erythrocytes, adding serum to erythrocytes, mix thoroughly and incubate for 30 min at 37°C (sensitize).

2. Sheep erythrocytes. A 3% suspension of washed sheep erythrocytes is prepared for the entire number of test tubes in the experiment. To prepare a hemolytic system, 30 minutes before it is introduced into the experiment, equal volumes of ram erythrocytes are mixed with an antibody to them.

3. Complement is usually diluted 1:10. It must be titrated before each experiment. Complement titer is its smallest amount, when added to the hemolytic system, complete hemolysis occurs within 1 hour at 37°C.

4. The antigen is usually received ready-made with an indication of its titer, i.e. the amount that, after dilution of the antigen, should be contained in 1 ml. For example, at a titer of 0.4, it is diluted in 0.96 ml of isotonic solution. In the experience take the amount of antigen, equal to half the titer (0.5 ml). This is his working dose. Prepare a total antigen dilution for the entire experiment by multiplying 0.5 ml by the number of tubes in the experiment.

5. Antibody - patient's serum. Fresh serum is inactivated before the experiment in order to destroy the complement present in it. To do this, it is heated for 30 minutes at 56°C in a water bath or in an inactivator with a thermostat. The latter method is preferable: it eliminates the possibility of overheating

Statement of the complement fixation reaction (RSC).

The reaction is put in a volume of 1 cu. cm (0.2 cubic cm of each component).

The test sera are examined at a dilution of 1: 5 and 1: 10 (the dose of serum is 0.04 cc and 0.02 cc with a specific antigen, at a dilution of 1: 5 with a control antigen (for specificity) and without an antigen (for anticomplementarity). ).

The test and control sera are inactivated on the day of the reaction in a diluted form at 56-65°C (depending on the type of animal) for 30 minutes. (also in RDSC).

Both stages of the reaction are carried out in a water bath at 37-38°C, the bacteriolytic system is kept for 60 minutes, the second stage of the reaction (with the hemolytic system) is 20 minutes.

Controls of the main RSK experience:

positive serum diluted 1:5 without antigen and with control antigen; in dilutions from 1: 5 to its titer with a specific antigen;

negative serum in dilutions 1: 5 and 1: 10 with a specific antigen, in a dilution of 1: 5 with control antigen and without antigen - specific and control antigens in a double dose - for anticomplementarity (complement +) and for hemotoxicity (complement);

hemolytic system for hemotoxicity (complement).

Accounting for results. There should not even be traces of hemolysis in the controls, since one of them does not have a complement, while the other does not contain hemolysin. Controls indicate the absence of hemotoxicity reactions (the ability to spontaneously lyse erythrocytes) in the components.

In the experiment, complement activity may decrease due to its nonspecific adsorption by other reaction components, therefore, for the experiment, the amount of complement is increased: the dose following the titer is taken. This is the working dose.

There are various options for staging RSC: classic. the method of setting in the form of macro- and microvariants, the reaction of long-term complement fixation (RDSC) in the cold, the method of quantities. RSC by 50% hemolysis of sensitized erythrocytes, etc. For the diagnosis of a number of diseases, a more sensitive method is used - RDSC.

Statement of the reaction of long-term complement fixation (RDSC).

The first phase of the reaction is carried out in a refrigerator at 2-6°C for 16-18 hours, the second phase in a water bath for 20 minutes at 37-38°C.

The complement is used in a working dilution of 1:25 or 1:30 (when determining its working dilution).

On the first day, sera and antigen are bottled for the main experiment. The reaction is put in a volume of 1 cu. cm (0.2 cubic cm of each component). Controls of RDSC are the same as in the setting of RSC.

Then, a complement of 0.2 cu. cm, depending on its working dilution, the test tubes are shaken and placed in the refrigerator for 16-18 hours. at 2-6°C.

The next day, the racks with the first phase are removed from the refrigerator and after keeping for 20-30 minutes. at room temperature, the hemolytic system is poured into all test tubes in a dose (in the given example, 0.6 cubic cm). The racks are shaken and placed in a water bath for 20 min. at 37-38°C.

The scheme of the main experiment RDSC is presented in Table 3.

Accounting for the results of RSC, RDSC is carried out twice: the first time - immediately after the water bath, the second - after the erythrocytes settle to the bottom of the test tube (3-4 hours after the water bath) or the next day when stored in a refrigerator from 2 ° to 6 ° C.

To objectively determine the results of the reaction, it is recommended to evaluate the percentage of hemolysis. To do this, 5 tubes with complete (100%) hemolysis are selected from the reaction and the liquid from them is poured into one tube. Dilutions with a lower percentage of hemolysis are prepared from it, the so-called "Standard" scale.

The degree of hemolysis in test tubes with test sera is determined by comparison with the degree of hemolysis in "standard" tubes and the percentage of hemolysis is expressed in crosses.

The degree of delay is inversely proportional to the percentage of erythrocyte hemolysis:

++++ (4 crosses) - no hemolysis, supernatant liquid transparent, colorless;

+++ (3 crosses) - hemolysis of 25% of erythrocytes;

++ (2 crosses) - hemolysis of 50% of erythrocytes;

+ (1 cross) - hemolysis of 75% of erythrocytes;

(minus) - complete hemolysis of erythrocytes, no sediment, the liquid is intensely stained with hemoglobin.

Evaluation of the results of RSK, RDSK.

Evaluate the test sera in dilutions of 1: 5 and 1: 10 (0.04 cc, 0.02 cc) with complete hemolysis of erythrocytes in control serum without antigen and with control antigen.

A positive reaction is assessed when hemolysis of erythrocytes is delayed by 3-4 crosses in a dilution of 1: 10.

Doubtful - with a delay in hemolysis of erythrocytes by 1 cross in a serum dilution of 1: 10 and from 2 to 4 crosses in a dilution of 1: 5.

Negative - with complete hemolysis of erythrocytes in dilutions of serum 1: 5 and 1: 10.