borde - janggu reaction

Borde Zhangu reaction (complement fixation reaction, RSK) - immunological reaction, based on the property of the "antigen-antibody" complex to fix free complement. CSC proceeds in two phases: 1) adsorption of antibodies on antigens; 2) expression of the reaction in the presence of a hemolytic system (hemolysis). To establish CSC, the studied objects (antibodies and antigens) are placed in conditions that ensure their interaction in the presence of complement; then an "indicator" - the hemolytic system - is added to the analyzed immune system. If the complement was fixed by the immune system, hemolysis does not occur (positive RSK; Fig., a); if the immune system did not fix the complement and its components do not correspond to each other, hemolysis occurs (negative CFR; Fig., b). Reaction ingredients: 1) test serum inactivated by heating for 30 minutes. at t° 56°; 2) antigen (suspensions of bacteria, extracts of organs and tissues, viruses); 3) complement (fresh guinea pig serum); 4) hemolytic system (sensitized sheep erythrocytes incubated with hemolytic serum for 30 minutes at t° 37°). To dilute the ingredients, an isotonic sodium chloride solution is used. The first stage of the RSC lasts 60 minutes. at t° 37° or 18-20 hours. at t° 4-6° (more sensitive RSK), the second stage - 60 min. at t° 37°.

In diagnostic practice and forensic medicine, various modifications of RSC are used - qualitative and quantitative. See also Wassermann reaction, Serological studies.

Borde Zhangu reaction - an immunological reaction based on the property of the antigen-antibody complex to fix free complement. CSC proceeds in 2 phases: 1) adsorption of antibodies on antigens, 2) expression of the reaction (bacteriolysis or hemolysis), which requires the presence of complement. The dependence of bacteriolytic reactions on the presence of complement makes it possible to identify the interaction of antibodies with antigens in the Bordet-Jangou reaction. An indicator of the compliance of antibodies in this reaction is complement fixation by the corresponding immune systems, detected by adding an "indicator" hemolytic system to the reaction mixture.

The Borde Zhang reaction is carried out in two stages: first, the studied antigens and antibodies are placed in conditions that ensure their interaction in the presence of complement, and after adsorption is completed, the hemolytic system (hemolysin + erythrocytes) is added to the analyzed immune system. When the antigens and antibodies match, they interact and the complement present in the reacting mixture is fixed by the immune system. In this case, when the indicator system is added, hemolysis does not occur due to the lack of complement (positive reaction). If the analyzed antibodies are not adsorbed on antigens, then the complement in the reacting mixture remains free, and when a hemolytic system is added, hemolysis occurs (negative reaction).

The principle of the method developed by Borde and Zhang serological analysis formed the basis of numerous modifications of the reaction used in diagnostic practice and research work.

Conventional RSK, in which 100% erythrocyte lysis is recorded, requires a preliminary complement titration; the latter is added to the immune system at a dose not exceeding that required for a hemolytic reaction. Reaction formulation schemes are determined by its purpose. In the experiment, various doses of the antigen can be tested at a constant content of serum and complement, various dilutions of serum at constant doses of the antigen and complement, etc. The reaction ingredients are: 1) the test serum heated for 30 minutes. at 56 ° in order to inactivate the complement present in it; 2) antigens (which are used as suspensions of bacteria, proteins extracted from them, polysaccharides, etc.), extracts from plant and animal cells, viruses, etc., 3) complement - fresh serum of guinea pigs, 4) hemolytic system ( sensitized sheep erythrocytes) - 5% suspension of erythrocytes in contact with hemolytic serum for 30 minutes. at t° 37°.

To dilute the ingredients, physiological NaCl solution. Anticomplementarity of individual ingredients is tested in control samples. The first stage of the reaction (adsorption of antibodies) is carried out at t° 37° for 60 min. or at 4-6 ° for 18-20 hours. (RSK in the cold). In the latter case, more complete fixation of the complement occurs, which increases the sensitivity of the method. At the second stage, the reaction mixtures are kept at t° 37°. The result is recorded at the time of 100% hemolysis in control samples.

In diagnostic practice, active as well as "indirect" methods of the complement fixation reaction are used, which are modifications of the Bordet Zhangu reaction. In the first of these, the change in the complement content in the test serum serves as an indicator of the reaction. The latter are based on the fact that in some diseases, sera do not contain complement-fixing antibodies, but are able to prevent reactions with sera with complement-fixing activity.

The greatest sensitivity and accuracy is inherent in quantitative RSC, in which hemolysis is recorded in the zone of partial lysis. The sensitivity of this modification of the Bordet-Jangou reaction is due to the fact that in middle zone lysis (20-80%), the ratio of the amount of complement to the number of lysed erythrocytes is linear. This reaction is based on special method complement titration, the amount of which is expressed in 50% units (CH50) and calculated on the basis of the Krogh equation, representing the course of hemolysis in the zone of partial lysis.

The degree of hemolysis is determined using a spectrophotometer or photoelectric colorimeter.

Quantitative RSC, in turn, has a number of modifications. In particular, the micromethod provides greater accuracy of determinations with a low consumption of ingredients.

With whooping cough, irritants are Bordet-Zhangu stick and its toxins, which for a long time cause constant irritation of nerve receptors on the mucous membrane respiratory tract, which leads to the creation in the central nervous system (in the respiratory and cough centers) of a congestive focus of excitation, which affects the nature of breathing in children.

According to I. A. Arshavsky, V. D. Soboleva (1948), in spasmodic period at the height of inhalation, the breath is held (inspiratory apnea). The respiratory muscles at this moment come into a state of tonic spasm, which is especially pronounced in the diaphragm. The latter at this time practically does not participate in the act of breathing.

Duration of inspiratory convulsions - 3 - 45 s, against the background of which there are short expiratory shocks of small force, carried out by the costal mechanism. They continue until the child exhales all the air taken. This is followed by a forced noisy breath, accompanied by a whistle (reprise). And all this is repeated several times during the attack. I. A. Arshavsky, V. D. Soboleva (1948) believe that the Traube phenomenon is the basis of inspiratory convulsions. Traube (1847), irritating the central segment with a faradic current vagus nerve observed cessation of breathing in the phase of inspiration.

Whooping cough with increasing irritability, respiratory arrest may occur in one or another phase (expiration or inspiration), especially in children during the first months of life.

A congestive focus of excitation in the central nervous system acquires all the signs of a dominant according to Ukhtomsky: the excitability of this focus is increased, as a result of which it attracts and summarizes all irritations and impulses entering the central nervous system. nervous system; excitation from the dominant focus radiates to neighboring centers - vascular (pressure rises), muscle (convulsions of the muscles of the face and limbs are observed), vomiting (vomiting appears at the end of the attack); the focus is persistent, lasts a long time and leaves a trace reaction.

This is well confirmed in life. Often, a child who has had whooping cough develops in 2-6 months paroxysmal cough with other diseases of the respiratory tract (flu, viral catarrhs of the upper respiratory tract, pneumonia, etc.).

"Guide airborne infections”, I.K.Musabaev

Color and transparency of the finished medium. Uranium chain reaction and sensational chain reaction

119. Pandey and Nonne - Apelt reactions

As a reagent for carrying out the Pandey reaction, a clear supernatant liquid is used, which is obtained by vigorously shaking 100 g of liquid carbolic acid with 1000 ml of distilled water. For sediment and clear liquid(reagent), this mixture is first placed in a thermostat for 3-4 hours, and then kept at room temperature for 2-3 days.

Place a watch or glass slide on dark paper and apply 2-3 drops of the reagent on it, then 1 drop cerebrospinal fluid. If the drop becomes cloudy or a filamentous turbidity appears along its periphery, the reaction is considered positive.

To carry out the Nonne-Apelt reaction, clean test tubes, a saturated solution of ammonium sulfate, distilled water, and dark paper are required. A saturated solution of ammonium sulfate is prepared in the following way: 0.5 g of chemically pure neutral ammonium sulfate is placed in a 1000 ml flask, then 100 ml of distilled water heated to 95 ° C is poured, shaken until the salt is completely dissolved and left for several days at room temperature. After 2-3 days, the solution is filtered and the pH is determined. The reaction must be neutral.

0.5-1 ml of the resulting solution is poured into the test tube and the same amount of cerebrospinal fluid is carefully added along the tube wall. After 3 minutes, evaluate the result. The appearance of a whitish ring indicates a positive reaction. Then the contents of the test tube are shaken, the degree of turbidity is determined by comparing with a test tube containing distilled water. The results of the reaction are evaluated against the background of black paper.

120. Bordet-Jangu reaction

Valuable diagnostic test in identifying chronic gonorrhea among people with chronic inflammatory diseases genitourinary system. According to the literature, with correct use This method reveals up to 80% of cases of gonorrhea not detected by bacterioscopic or bacteriological method.

The Bordet-Jangu reaction may be trace as a result of a previous illness or the use of a gonovaccine for diagnostic (immunobiological method of provocation), as well as therapeutic (treatment of chronic inflammatory processes of the genitourinary system in women according to Baksheev) purpose. Therefore, before carrying out it, it is necessary to carefully collect an anamnesis,

The reaction can also be false-positive with the introduction of milk, use in medicinal purposes pyrogenal.

Therefore, a positive Bordet-Jangu reaction does not serve as indisputable evidence of the presence gonococcal infection, as well as negative cannot be evidence of the absence of gonorrhea. However positive results it for a long time should be directed by a doctor to search for a focus of gonococcal infection in the body.

As an antigen, a killed gonococcal culture is used, containing 3-4 billion microbial bodies per 1 ml. Gonococcal antigen is preserved with a formaldehyde solution and poured into 1-5 ml ampoules. Unopened ampoules are suitable for 6 months, opened can be stored for 2-3 days in a sterile test tube in a refrigerator at a temperature of 3-5 ° C,

The Borde-Zhang complement fixation reaction is carried out similarly to the Wasserman reaction (see No. 121). Gonococcal antigen is diluted with isotonic sodium chloride solution according to the titer indicated on the label of the ampoule. The reaction is most often carried out in a volume of 2.5 ml, therefore, to 0.5 ml of a diluted 1: 5 test serum, 0.5 ml of the diluted antigen is added to each test tube. The remaining 1.5 ml is 1 ml of the hemolytic system and 0.5 ml of complement.

The reaction is considered positive in the presence of varying degrees hemolysis delays in the tested serum. In control (blood serum healthy people) complete hemolysis is observed.

121. Wasserman reaction

In the blood serum of patients with syphilis, there are reagins and antibodies. Reagins have the ability to enter into compounds with cardiolipin antigen. Specific antibodies against Treponema pallidum combine with specific antigens. The resulting antigen-antibody complexes are sorbed by the complement added to the reaction. The indication is made by introducing a hemolytic system (sheep erythrocytes + hemolytic serum).

For the reaction to take place:

a) isotonic sodium chloride solution;

b) ultrasonified treponemal (stored in the refrigerator at +4 °C) and cardiolipin (stored at room temperature) antigens;

c) complement, which is the blood serum obtained by heart puncture of 5-10 healthy guinea pigs. Can be stored in the refrigerator for up to 2 months provided
canning 4% solution boric acid and 5% sodium sulfate solution;

d) hemolysin - hemolytic rabbit blood serum immunized with sheep erythrocytes with different titers (stored in a refrigerator at a temperature of 4 ° C);

e) sheep erythrocytes obtained by puncture jugular vein. The blood is collected in a sterile jar with glass beads (for mixing), shaken for 15 minutes. Fibrin clots are separated by filtration through sterile gauze. Defibrinated blood can be stored in the refrigerator for up to 5 days.

Sometimes there is a need for a longer storage of sheep's blood, and therefore it is preserved with a special preservative (6 g of glucose, 4.5 g of boric acid, 100 ml of isotonic sodium chloride solution), which is boiled in a water bath for 20 minutes a day for 3 day For 100 ml of defibrinated sheep blood, 15 ml of a preservative is required. The defibrinated blood preserved in this way is stored in a refrigerator.

The main experiment is preceded by several stages.

In a patient from cubital vein take 5-10 ml of blood and process the serum. In children, blood can be taken from the temporal vein or an incision in the heel. The puncture is done with sterile instruments, a syringe and a needle
pre-washed with isotonic sodium chloride solution.

Blood for research is taken on an empty stomach; 3-4 days before the study, the patient is forbidden to use drugs, digitalis preparations, take alcohol.

The study should not be performed in patients with elevated temperature body, after trauma, surgery, anesthesia, recent infectious diseases, in women during menstruation, in pregnant women (in the last 10 days of pregnancy), women in labor (in the first 10 days after childbirth), as well as newborns (in the first 10 days of life) .

The obtained blood in a sterile tube is placed for 15-30 minutes in a thermostat at a temperature of 37 °C. The resulting clot is separated from the walls of the test tube with a sterile glass rod and placed in the refrigerator for a day. The separated transparent serum (above the clot) is sucked off with a Pasteur pipette using a rubber pear or carefully poured into another sterile tube and inactivated in a water bath for 30 minutes at a temperature of 56 °C. Serum prepared in this way for the experiment can be stored in the refrigerator for up to 5-6 days.

Antigens are diluted according to the method and titer indicated on the label.

Prepare hemolytic system. To do this, defibrinated blood or sheep erythrocytes in the amount necessary for the reaction is centrifuged, the plasma is carefully separated, and the precipitate is washed with 5-6 volumes of isotonic sodium chloride solution until the supernatant becomes completely colorless. A 3% suspension of erythrocytes in an isotonic solution of sodium chloride is prepared from the sediment according to a triple titer.

A solution of hemolytic serum and a suspension of sheep erythrocytes are quickly mixed and placed in a thermostat for 30 minutes.

Dry complement is diluted with isotonic sodium chloride solution in a ratio of 1:10, normal inactivated human serum - 1:5.

Complement is titrated in 30 tubes placed in a rack of 10 tubes in 3 rows. In two rows it is titrated in the presence of two antigens, in the third - with isotonic sodium chloride solution. Five test tubes are control: two for the corresponding two antigens and one each for control of complement, hemolytic serum and isotonic sodium chloride solution for hemotoxicity; fill them in like this:

Reagents, ml	Row of test tubes

3% suspension of sheep erythrocytes

hemolytic serum, diluted by triple titer
Complement 1:10
Triponemal antigen diluted by titer
Cardiolipin antigen diluted by titer
Isotonic sodium chloride solution

The tubes are placed in a thermostat for 45 minutes, after which they are checked. Hemolysis should not be in any test tube.

Complement at a dilution of 1:10 is poured into 10 test tubes of the 1st row of the rack in doses: 0.1, 0.16, 0.2,0.24, 0.3, 0.36,0.4,0.44, 0.5 and 0.55 ml. To the contents of each tube add isotonic sodium chloride solution up to 1 ml and mix thoroughly. 0.25 ml of the mixture from each tube is transferred to the corresponding test tubes of the 2nd and 3rd rows. The rack with test tubes is shaken, 0.5 ml of the hemolytic system is added to the 3rd row of test tubes, shaken again and placed for 45 minutes in a thermostat at a temperature of 37 °C. Normal human blood serum diluted with isotonic sodium chloride solution in a ratio of 1:5, 0.25 ml is poured into all 30 test tubes.

Antigen I (treponemal ultrasound), diluted in titer with isotonic sodium chloride solution, 0.25 ml is added to 10 test tubes of the 1st row; antigen II (cardiolipin in the same dilution and in the same amount) - in 10 test tubes of the 2nd row. 0.25 ml of isotonic sodium chloride solution is poured into 10 test tubes of the 3rd row, the remaining 0.25 ml is poured. After incubation in a thermostat, 0.5 ml of the hemolytic system is added to 20 test tubes (1st and 2nd row), shaken and placed again in a thermostat for 45 minutes.

After 45 minutes, the working dose of complement is determined, that is, its titer with an allowance in the range of 15-20%.

Complement titer is considered to be minimal amount, causing complete hemolysis of ram erythrocytes in the presence of antigen and normal human blood serum.

The main experience is that each tested inactivated serum, diluted in a ratio of 1: 5 with isotonic sodium chloride solution, is poured into 0.25 ml in three test tubes. Add 0.25 ml of antigen I to the first tube, 0.25 ml of antigen II to the second, and 0.25 ml of isotonic sodium chloride solution to the third (control). Add 0.25 ml of complement diluted to the working dose to all test tubes. All test tubes are placed in a thermostat for 45 minutes, then 0.5 ml of the hemolytic system is added to them, shaken and placed in a thermostat for 45-50 minutes. The result of the experiment is recorded after the onset of complete hemolysis in control tubes.

The results of the reaction are evaluated with pluses: complete delay in hemolysis (strongly positive reaction) ++++, significant (positive reaction) +++, partial (weakly positive reaction) ++, insignificant (doubtful reaction) - ±, no delay in hemolysis (negative reaction) - .

At quantitative method carrying out the Wassermann reaction, the experience is set with decreasing volumes of serum diluted with isotonic sodium chloride solution.

Scheme for conducting the main experience of the Wasserman reaction

Ingredients (in ml). Total volume 1.25 ml	No. of tubes
Ingredients (in ml). Total volume 1.25 ml
The test serum is inactivated, diluted 1:5 Antigen I (treponemal), diluted by titer Antigen II (cardiolipin), diluted by titer Isotonic sodium chloride solution Complement, diluted according to the working dose

The clinical significance of the Wasserman reaction is difficult to overestimate. It is carried out for all patients before the start of treatment, but special meaning she has at latent syphilis, defeat internal organs and nervous system.

The results of the Wasserman reaction characterize the quality of the treatment, which gives grounds to deregister treated patients within a certain period of time.

In primary syphilis, the Wasserman reaction is usually positive at the end of the 6th week from the moment of infection; with secondary fresh syphilis, it is positive in almost 100% of cases, with secondary recurrent - in 98-100%; tertiary active - in 85%; tertiary hidden - in 60% of cases.

The Wasserman reaction is carried out twice for all pregnant women, patients with somatic, nervous, mental and skin diseases, as well as the decreed contingents of the population. At the same time, positive and weakly positive results of the reaction should be treated critically, since they can be at the end of pregnancy and after childbirth, with hyperthyroidism, malaria, leprosy, decay malignant tumor, infectious diseases, collagenoses, etc. Therefore, in the presence of clinical manifestations diseases, they should be taken into account, along with the data of bacterioscopy, in the first place.

At the same time, there are factors that can distort the true nature of the results of the Wasserman reaction: poorly washed laboratory glassware (traces of acid and alkali in test tubes), long-term storage of blood taken for research, consumption of fats and alcohol by patients before the examination, menstruation period, etc.

In all cases, it is desirable to conduct a comprehensive examination of the patient, including, in addition to the Wassermann reaction, also RIBT (see 122, 123, 124), etc.

122. Reaction of Sachs - Vitebsky (cytocholic)

Used to detect syphilis. It is based on the formation of a precipitate when the patient's blood serum is mixed with an antigen.

To prepare the antigen, the muscles of several bovine hearts are cleaned of tendons, fascia, fat, minced in a meat grinder and extracted with a 5-fold volume of 96% ethanol for 15 days with daily 20-minute shaking. The extract obtained is evaporated on a water bath in a porcelain cup under vacuum. Hot 96% ethyl alcohol is added to the residue (bright yellow mass of lipoids) in the amount of 1/3 of the volume of muscles taken for extraction.

The extract is kept for 3 days at room temperature, filtered (in cold water) and add 0.3-0.6% solution of crystalline cholesterol, depending on the results of titration with positive and negative sera. Store cytocholic antigen at room temperature in sealed ampoules or sealed test tubes.

Methodology. To 2 ml of isotonic sodium chloride solution, 1 ml of cytochole antigen is quickly poured and left at room temperature for 15 minutes until flakes appear. Add 0.1 ml of antigenic emulsion to 0.2 ml of inactivated serum, shake for 3 minutes and leave alone for 30 minutes, then add 1 ml of isotonic sodium chloride solution.

1.2 ml of isotonic sodium chloride solution and 0.1 ml of antigenic emulsion are added to the control tube with antigen. There should be no flakes in the control. The results of the reaction are taken into account visually or with a magnifying glass and are indicated depending on the amount of flakes that have fallen out with pluses (++++, +++, ++). With a negative reaction, there are no flakes, but the contents of the tube may be slightly opalescent.

In 1931, P. Sachs and E. Witebsky proposed a modification of this technique, which consists in diluting the antigen in two phases: 2 parts of isotonic sodium chloride solution are added to 1 part of the antigen, kept at room temperature for 5 minutes, and another 9 parts of isotonic solution are added sodium chloride. It is also recommended to dilute the antigen with 2% sodium chloride solution, which, according to the authors, increases the sensitivity of the reaction. A quantitative modification of the reaction with serum dilution is also possible (1:4, 1:8, 1:16, 1:32, 1:64, etc.).

The reaction is quite sensitive, takes a short time (about 1 hour), and its results are read immediately.

123. Pale treponema immobilization reaction (RIBT)

With the help of a reaction in the blood serum of patients with syphilis, antibodies and complement are detected that immobilize pale treponema.

With primary syphilis, RIBT is predominantly negative, with secondary - positive in 92-96% of cases, with tertiary - in 92-100%, with syphilis of the nervous system and congenital - in 86-89% of cases.

Positive results of RIBT were noted in sarcoid, erythematosis, diabetes, malignant tumors, viral hepatitis, cirrhosis of the liver, malaria, leprosy, infectious mononucleosis, some diseases of hot countries (pint, yaws, etc.).

The antigen is a suspension of pale treponema taken from the testicles of a rabbit infected with syphilis by introducing into the testicle 0.75-1 ml of a suspension of pale treponema in isotonic sodium chloride solution (50 individuals per field of view).

Testicular material should be taken 6-8 days after infection of the animal. Before taking the antigen, the rabbit is slaughtered by exsanguination (cardiac puncture or carotid artery). The testicular tissue is crushed and filled with healthy rabbit blood serum diluted with isotonic sodium chloride solution, shaken for -30 minutes, and then centrifuged for 10 minutes (1000 rpm). The supernatant is microscopically examined. There should be at least 10-15 pale treponemas in each field of vision.

Complement is being prepared in the usual way from the blood of guinea pigs.

For RIBT, you need: 1.2 ml of 0.2% gelatin solution, 2.8 ml of 5% albumin solution, 1.6 ml of pale treponema suspension; medium pH - 7.2. To this mixture is added 0.15 ml of complement (cocktail). In parallel, two samples are put: with active (experiment) and reactive (control) complements.

The studied serum is collected into the melangeurs up to the “1” mark, and then the cocktail is collected up to the “2” mark and they are closed with a sterile rubber ring.

At the same time, a similar reaction is carried out with obviously positive and obviously negative sera.

Melangers are numbered and placed in a thermostat at a temperature of 35 ° C for 18-20 hours, after which they are removed from the thermostat in pairs (experiment - control) and the contents are poured into the appropriate test tubes. 2 drops are applied to the glass slide: on the left - experience, on the right - control, covered with a cover slip and microscoped in a dark field of view.

First, the results of the control are studied: the percentage of mobile and immobile pale treponemas is determined. Counting formula: X = (A - B) / A * 100, where A is the number of mobile pale treponemas in the control; B - the number of mobile pale treponemas in the experiment.

Example: (24 - 19) / 24 * 100 = 21%.

Estimates of the results of RIBT: below 20% - negative. 21-30% - doubtful, 31-50% - weakly positive, over 50% - positive.

Antigen, complement and auxiliary, indicator or hemolytic system - hemolytic serum and sheep erythrocytes.

If a specific antigen + antibody complex is formed in the first system, then the complement is adsorbed (combined with this complex) and hemolysis does not occur in the second system.

The reaction requires:

1. Test serum, which is obtained from blood taken by puncture of the patient's cubital vein. After blood clotting, the serum is aspirated into a separate tube and inactivated for 30 minutes at 56 °C in a water bath.

2. Antigen - a suspension of killed gonococci.

3. Sheep erythrocytes are obtained from sterile defibrinated blood. They are washed by centrifuging 3 times with new portions of saline. A 3% suspension of erythrocytes is used in the reaction.

4. Hemolytic serum is prepared in advance by immunizing rabbits with ram erythrocytes. Serum is titrated before use.

5. Complement - fresh guinea pig serum. Blood is sucked from the heart of a guinea pig with a syringe, after clotting, the serum is separated. Before the experiment, the working dose of complement is titrated. To do this, take the main dilution of complement 1:10 and pour it into test tubes from 0.1 to 0.5, after which the volume in each test tube is adjusted with saline to 1.5 ml.

At the same time, a hemolytic system is prepared - diluted in a triple titer, hemolytic serum + 3% suspension of sheep erythrocytes. Both ingredients are in different volumes and kept in a thermostat for 30 minutes (sensitization of the mixture), after which the mixture is added in 1 ml to test tubes and placed in a thermostat for 30 minutes. 2 test tubes serve as control:

1. 1 ml of hemolytic system + 1.5 ml of saline;

2. 0.5 ml of complement 1:10 + 0.5 ml of erythrocyte suspension + 1.5 ml of saline.

Complement titer is the minimum amount at which hemolysis still occurs. To set up the reaction, a working dose of complement is taken, increased against the titer by 20-25%, i.e., usually the amount of complement that is in the penultimate test tube with hemolysis. An increase in the dose of complement is necessary because in the reaction, complement activity may be somewhat suppressed by other ingredients of the reaction (antigen, serum).
43) RSK Wasserman

Used to diagnose syphilis in order to detect antibodies, as well as to determine the effectiveness specific therapy. It is based on the principle of the Borde-Gangou complement fixation reaction. A significant difference in the Wasserman reaction is the nonspecificity of the antigen: lipoid extracts from normal organs animals

To set up the Wasserman reaction, it is necessary to have the patient's serum, diagnostics, cross-reacting antigens No. 1, No. 2, complement, hemolytic serum, ram erythrocytes, saline.

Diagnosticum No. 1 - specific, treponemal.

Diagnosticum No. 2 - non-specific, cardiolipin antigen, which are highly purified bovine heart extracts that have a constant chemical composition of lipoids. Lipoids, by chemical composition are close to treponema pallidum lipoids, therefore, although they are not specific, they fix antibodies against spirochetes.

These antigens are produced centrally and used in the reaction diluted according to the titer indicated on the label.

Simultaneously with the main experiment, 2 controls are placed: with obviously negative and with obviously positive sera.

Setting up the main experience

First, inactivated and diluted 1:5 test serum is poured into 4 test tubes. Then, antigens (No. 1, No. 2) are poured into 2 test tubes, physiological saline is poured into the 3rd test tube. After that, a working dose of complement is added to all tubes. After mixing the ingredients, the rack with test tubes is placed in a thermostat at 37 °C for 30 minutes. After keeping in a thermostat, a hemolytic system is added to all test tubes. The tubes are again placed in a thermostat for 2 hours, then left at room temperature. The next day, the result is noted. The degree of intensity of the reaction is estimated, four pluses (++++), three (+++), two (++) and one (+) - depending on the intensity of the color of the liquid and the size of the erythrocyte sediment at the bottom. Complete hemolysis is indicated by (-) minus. With a sharp discrepancy between the results with different antigens, the experiment is repeated with a new portion of blood.
44) RIT-r. Immobilization of pale treponema.

This reaction is used in diagnostic purposes, and for the recognition false positive results standard serological reactions, especially with latent syphilis.

RIBT lies in the fact that in the presence of immobilizins in the serum of patients with syphilis and active complement, pale treponemas lose their mobility.

RIBT is placed in sterile boxes. The test serum, complement and antigen participate in the reaction.

In RIBT, the antigen is a suspension of pale treponema from a rabbit testicle in early dates(7-8 days after infection) syphilitic orchitis. A special suspension medium retains the viability of pale treponema for at least a day. Complement is used in RIBT guinea pigs. RIBT proceeds under anaerobic conditions. Test tubes with ingredients are placed in a microaerostat, from which atmospheric air and a gas mixture is injected (95 parts of nitrogen and 5 parts carbon dioxide). The microanaerostat with test tubes is placed in a thermostat (35°C) for 18-20 hours.

In parallel with the formulation of the reaction, control studies with positive and negative blood serum taken from previous experience.

The results of RIBT are evaluated after removing the test tubes from the thermostat (i.e., after 18-20 hours of experience). With a Pasteur pipette, a drop of the contents of the test tube is applied to a glass slide, which is covered with a cover slip and examined in the dark field of a microscope (objective 40, eyepiece 10). With immobilization of up to 20% of pale treponemas, the reaction is considered negative, from 21 to 30% - doubtful, from 31 to 50% weakly positive, from 51 to 100% - positive. The percentage of immobilization of pale treponema is determined according to a special table.

45) Opson-phagocytic reaction

Mechanism: increased phagocytosis of microbial cells under the influence of the friendly effects of antibodies, immune serum and complement.

Reaction components:

1) antigen - daily microbial culture;

3) complement - fresh guinea pig serum;

4) phagocytes - leukocyte suspension.

Opsonins are antibodies found in normal and immune sera and prepare microbes for phagocytosis.

The reaction is placed in special test tubes at t=37° minutes. Then, smears are prepared from each tube, 100 phagocytes are counted, and the number of phagocytosed microcontrols is determined.

Opsonic index = phagocytic index. serum/phagocytic index of normal serum

The higher the opsonic index (should be >1) of the serum under study, and therefore the higher it! brucellosis).

It is used to determine opsonins - antibodies that stimulate the phagocytic activity of leukocytes, i.e. serodiagnosis of infections such as brucellosis.

The enhancement of phagocytosis occurs due to the attachment of opsonins with active centers (Pav-fragment) to bacterial determinants, and then, with the help of Pc-fragments, to the Pc-receptors of phagocytes. Normal serum contains small amounts of opsonins, which act in the presence of complement. There are more opsonins in the immune serum, and their activity is less dependent on complement.

Components:

Serum under study

normal serum

Daily microbial culture (e.g. staphylococcal)

Phagocytes - suspension of neutrophils

Incubation at 37°C for 30 minutes. Smears are prepared from each tube, stained according to Romanovsky-Giemsa, and the number of microbes is counted under a microscope, in 100 or more neugrophiles, i.e. determine the phagocytic index.

Phagocytic index - the number of microbes absorbed by one neutrophil.

Opsonic index phagocytic index of immune (tested) serum / phagocytic index of normal serum.

The higher the opsonic index (should be > 1), the higher the immunity.

The opsono-phagocytic index is a digital indicator = the number of phagocytes x the phagocytosis score (depending on the number of microbes ingested). The maximum score is -75.

46) RN on mice in order to establish exotoxin

This reaction is based on the ability of a specific antitoxic serum to neutralize exotoxin.

Immune serum antibodies are able to neutralize the damaging effect of microbes or their toxins on sensitive cells and tissues, which is associated with the blockade of microbial antigens by antibodies, i.e., their neutralization.

The neutralization reaction (RN) is carried out by introducing an antigen-antibody mixture into animals or into sensitive test objects (cell culture, embryos). In the absence of the damaging effect of microorganisms or their antigens, toxins in animals and test objects, they speak of the neutralizing effect of immune serum and, therefore, the specificity of the interaction of the antigen-antibody complex.

To carry out the reaction, the test material, in which the presence of exotoxin is expected, is mixed with antitoxic serum, kept in a thermostat and administered to animals (guinea pigs, mice). Control animals are injected with the filtrate of the test material, not treated with serum. In the event that the neutralization of exotoxin with antitoxic serum occurs, the animals of the experimental group will remain alive. Control animals will die as a result of the action of the exotoxin.

The reaction of neutralization of exotoxin occurs when it interacts with antitoxic serum (antibodies-antitoxins). As a result of the formation of the antigen-antibody complex, the toxin loses its toxic properties.

The neutralization reaction is carried out in order to detect and titrate toxins, toxoids or antitoxins.

Toxins are obtained by filtering liquid growth medium or test material where toxigenic bacteria have proliferated. When processed with formalin for 30-45 days at a temperature of 37°C, the toxin turns into anatoxin, which is used to immunize animals in order to obtain antitoxic serum.

Neutralization reaction in vivo. To determine the type of toxin, it is mixed with a diagnostic antitoxic serum and this mixture is administered to white mice. When the toxin is neutralized with antitoxic serum, mice do not die.

The neutralization reaction is used to determine antitoxic immunity in children with diphtheria (Schick's test) and scarlet fever (Dick's test). To do this, a certain amount of the corresponding toxin (1/40 DLM) is intradermally injected into the forearm area. If there are antitoxins in the body, the toxin will be neutralized and the reaction will be negative. In the absence of antitoxins in the body, inflammatory response at the injection site.
47) RN (Neutralization Reaction) on mice to identify the virus tick-borne encephalitis

The TBE virus is pathogenic for a number of laboratory and wild animals. Newborns and young white mice are most sensitive. After infection in the brain, intraperitoneally, intramuscularly, these animals develop encephalitis, ending in the death of animals. Of the farm animals, goats are most susceptible to TBE virus, sheep and cows are less susceptible, and horses are weakly susceptible. The TBE virus has a cytopathogenic effect (CPE), causing cytopathic changes in primary and transplanted cultures of porcine embryonic kidney cells (SPEV and RPE) and multiplies without pronounced CPE in many others. cell cultures. In the brain of infected mice and the cultural fluid of infected cultures, the TBE virus and specific viral antigens accumulate: complement-fixing, hemagglutinating, precipitating, etc.

TBE virus long time preserved at low temperatures ( optimal mode-60 deg. C and below), tolerates lyophilization well, in the dried state it remains for many years, but is quickly inactivated at room temperature. Boiling kills it after 2 minutes, and in hot milk at 60 degrees. C dies after 20 minutes.

Formalin, phenol, alcohol and other disinfectants, ultraviolet radiation also have an inactivating effect.

The neutralization reaction is based on the ability of specific immune sera to extinguish the infectious effect of viruses. It is applied in two directions:

1) for typing isolated viruses;

2) for titration of antibodies in the sera of recovered patients.

Setting up the reaction:

1. On laboratory animals. Criteria for the presence of a virus

is the death of laboratory animals. LD50 is calculated

The maximum dilution of the viral suspension, which

caused the death of 50% of infected animals. For

identification of the tick-borne encephalitis virus is carried out

intracerebral infection of white mice.

2. In tissue cultures. Accounting for the results is carried out

By cytopathic action (CPE)

By color test method based on color change

By haemadsorption.

3. In the chicken embryo. Accounting for results is carried out according to

the appearance of pockmarks along the chorioallantoic membrane.

48) RTGA

This reaction is used in virological practice:

To determine the type of virus (on glass);

For detection in the serum of patients (deployed).

When setting up an approximate hemagglutination inhibition reaction, 1 drop of type-specific immune sera is applied to the glass, then 1 drop of the test material and 1 drop of a 5% suspension of erythrocytes are added.

Accounting for results: the type of virus is determined by the serum with which the reaction did not occur, since the immune serum specific for the isolated virus suppresses its hemagglutinating activity, and erythrocytes do not agglutinate.
49) RIF

Luminous fluorochrome dyes (fluoriscein isothiocyanate, etc.) are used as a label.

There are various modifications of the RIF. For express diagnostics of infectious diseases - to detect microbes or their antigens in the test material, RIF according to Koons is used.

There are two methods of RIF according to Koons: direct and indirect.

Direct RIF components:

1) the material under study (a bowel movement separated by the nasopharynx, etc.);

2) labeled specific immune serum containing AT-la to the desired antigen;

3) isotonic sodium chloride solution.

A smear from the test material is treated with labeled antiserum.

An AG-AT reaction occurs. Luminescent microscopic examination in the area where the AG-AT complexes are localized reveals fluorescence - luminescence.

Indirect RIF components:

1) the material under study;

2) specific antiserum;

3) antiglobulin serum (AT-la against immunoglobulin) labeled with fluorochrome;

4) Isotonic sodium chloride solution.

A smear from the test material is first treated with immune serum to the desired antigen, and then with labeled antiglobulin serum.

Luminous AG-AT complexes - labeled AT are detected using a fluorescent microscope.

The advantage of the indirect method is that there is no need to prepare a wide range of fluorescent specific sera, and only one fluorescent antiglobulin serum is used.

A 4-component variety of indirect RIF is also isolated, when complement (guinea pig serum) is additionally introduced. With a positive reaction, an AG-AT complex is formed - labeled - AT-complement.

The reaction refers to serological reactions involving labeled antigens or antibodies.

It is carried out by direct and indirect methods.

At direct method detect antigen in the material from the patient. A diagnostic fluorescent serum is used, which contains immunoglobulins isolated from immune sera and labeled with fluorochromes.

The specific antigen is found in the form of bright green luminescent conglomerates, and the background of the preparation turns orange-red.

Components of the direct immunofluorescence reaction:

1) the material under study;

2) specific luminescent serum;

3) luminescent microscope.

When diagnosing influenza, differentiation is carried out from the pathogens of parainfluenza and adenovirus infection. The nasopharyngeal swab is treated with fluorescent influenza serum or influenza immunoglobulin.

"+" result in the form of a green glow is obtained after 3 hours.

In the indirect method, specific antibodies are detected and titrated. Serum titer is its maximum dilution, at which fluorescence is noted at "+ +".

Components of the indirect immunofluorescence reaction

To search for an antigen in express diagnostics:

1) test material (antigen);

2) specific serum;

3) antiglobulin luminescent serum

4) luminescent microscope.

/ phase specific

Serum antibodies adsorb to the antigen.

// non-specific phase

Fluorochrome-labeled antiglobulin serum is used. An antigen + antibody complex (I) + antiglobulin serum (II) is formed, which glows.

116. Provocations in gonorrhea

Additional research method. It is used in the case when gonococci cannot be detected, and the clinical picture is suspicious for gonorrhea, and also as a criterion for the quality of therapy after its completion. They use certain methods of influencing the body as a whole and the genitourinary apparatus (mainly the urethra), which leads to an exacerbation of the existing inflammatory process. After the provocation, the material is taken from the patient for 3 days for research on the content of gonococci. Apply the following types provocations.

Alimentary - the appointment of a patient with spicy and salty foods, beer, which includes hops containing lopulin, excreted from the body mainly by the kidneys. Passing through the urethra, it irritates its mucous membrane.
Immunobiological - intramuscular injection of 0.5 ml (500 million microbial bodies) of gonococcal vaccine. IN Lately provocation with gonovaccine is combined with intramuscular injection 50-200 MPD of pyrogenal or prodigiosan. For women in stationary conditions, gonovaccine at a dose of 50-100 million microbial bodies is injected into the cervix.
Mechanical - the introduction of a straight bougie into the urethra, the size of which corresponds to the size of the external opening urethra. Instead of a bougie, you can use the tube of Valentine's ureteroscope, especially in cases where further research requires ureteroscopy. The sterilized and cooled bougie is lubricated with glycerin and injected for 10 minutes into the urethra, for men - into its anterior section. Then produce light massage urethra on the bougie in order to obtain the secretion of the glands of the urethra (Fig. 13).
Chemical - the introduction of chemical irritants into the urethra, more often silver nitrate (3-4 ml of a 0.5-1% solution for men, 3-4 ml of a 1-2% solution for women; 3-5% solution for lubricating the cervical canal uterus). Lugol's solution can be used to irritate the mucous membrane of the cervix. It is better to take smears after a chemical provocation after 2-3 days. In the first days in the discharge from the urethra and cervical canal, many
epithelial cells of the rejected mucosa, which often makes it difficult microscopic examination(Fig. 14).
Physiotherapeutic, or thermal, - the use of diathermy, less often - intravaginal mud tampons. Abdominal-vaginal-sacral diathermy is prescribed to women daily for 30-40 minutes for 3 days. Smears are taken 2 hours after each procedure.
Physiological - taking a smear in women from the cervical canal during menstruation and within 3 days after it.

Rice. 13. Massage of the urethra on a metal bougie

Rice. 14. Installation of the solution in the urethra
In practice, combined provocation is most often used.

We consider the most successful combinations: immunobiological and mechanical provocation - in men; immunobiological, thermal and physiological - in women.

The first provocation is usually carried out a week after the end of treatment, the next - a month later.

It should be noted that the negative results of the provocation can not always serve as an indisputable criterion of cure.

117. Lymph node puncture

Produced with suspicion of syphilis in the seronegative primary period in cases where, during the study tissue fluid the surface of the chancre does not reveal pale treponema or when, due to phimosis, its examination is impossible. This manipulation also produced in those patients who, by the time they went to the doctor, had epithelialization of the erosive chancre.

Regional scleradenitis is examined, the skin over which is treated with a 5% alcohol solution of iodine. Then, with a syringe with a capacity of 2 or 5 ml with a well-ground piston and a short thick needle, a puncture is made in the center of the bubo along its long axis to a depth of 1-1.5 cm. An energetic massage of the node is performed with a gradual removal of the needle from it and raising the piston.

If the needle was inserted correctly, then there should be lymphocytes in the punctate.

The liquid taken in this way is examined under a microscope in a dark field of view (see No. 68).

In the presence of syphilis and correct technique performing a puncture, pale treponema is found in 80-85% of cases.
118. Lumbar puncture

A lumbar puncture, or collection of cerebrospinal fluid, is a small surgical intervention and therefore must be carried out under conditions of strict observance of the rules of asepsis and antisepsis. For its implementation, it is necessary to have sterile material: napkins, cotton balls, Lugol's solution, ethyl alcohol 96%, tweezers or sticks, cleol, special puncture needles with mandrains sterilized by boiling. The diameter of the needle is 1.5-0.4 mm, the length is 8-12 cm. It is better to use a needle with a diameter of 0.5-0.6 mm, which reduces the trauma of the procedure. Lumbar puncture is done with the patient sitting or lying down. We consider the first option more convenient. The half-naked patient is seated on the edge of the table or, better, on a high bedside table. A high stool is placed under the feet so that the knees are as close to the stomach as possible. The patient is asked to fold his hands on his stomach, tilt his head forward so that the chin touches the chest, while the back arches in the form of an arc, the body should not be tilted down. In this position, the spinous processes of the vertebrae move apart as much as possible, leaving room for the introduction of a needle. The puncture site is chosen between IV and V or III and IV lumbar vertebrae. With a stick wound around its end with cotton wool soaked in Lugol's solution, draw a horizontal line connecting the upper edges of the ridges ilium(Jacobi line). The intersection of this line with a vertical line passing through the middle of the spinous processes will correspond to the puncture site (Fig. 15). When probing, a slight retraction is revealed. In this place, a sign in the form of a cross is put with a fingernail (puncture site). The doctor washes his hands thoroughly with soap, dries with sterile swabs, lubricates with Lugol's solution and wipes thoroughly. ethyl alcohol. The patient's skin is lubricated with Lugol's solution or iodine solution with alcohol 5%, then vigorously rubbed with ethyl alcohol and dried with sterile gauze swab. 0.5-1 ml of a 1% solution of novocaine is administered intradermally until a "lemon peel" is formed for the purpose of anesthesia, especially for patients with a labile nervous system. After 1-2 minutes, the puncture needle is inserted directly under the spinous process IV lumbar vertebra along the midline, slowly moving it forward and slightly up. The right hand of the doctor, which is like a guide, is helped by the left, pushing the needle forward. Having advanced the needle by 4-5 cm, the doctor feels a slight crunch (a puncture of the yellow ligament), after which he removes the mandrin, and cerebrospinal fluid begins to flow out of the needle. Normally, it is transparent, flows out in drops that can be counted. If the liquid flows out in a jet, then this indicates high blood pressure in the spinal canal. 3-4 ml of liquid is collected in two sterile test tubes and sent to the laboratory for analysis. With cotton wool moistened with ethyl alcohol, hold the skin at the base of the needle and remove it with slow helical movements. The puncture site is lubricated with a 5% alcohol solution of iodine and a sterile colloidal dressing is applied.

Rice. 15. Scheme of spinal puncture

After puncture, the patient is carefully placed in horizontal position belly down in bed and not allowed to move for 3-4 hours. Then you can turn in bed, it is advisable not to get up during the day. Patients with a labile nervous system are given sedatives(chlosepide, or elenium, trioxazine, bromine preparations, valerian). In order to prevent possible complications - irritation meninges- in the next 2-3 days, 5-10 ml of a 40% solution of hexamethylenetetramine (urotropine) is administered intravenously.

The examination of the cerebrospinal fluid is necessary condition For correct diagnosis, choice of treatment tactics and evaluation of its effectiveness in syphilis. Unfortunately, many doctors underestimate the practical importance of this method. In the cerebrospinal fluid, the protein content is determined (the norm is up to 0.4%), the amount is calculated shaped elements(norm - less than 8 cells),

carry out reactions: Nonne - Apelt and Pandy (norm ++), one of the colloidal (preferably with gold chloride), Wasserman in dilutions of 0.1; 0.25; 0.5 if there is 2.5 ml of liquid in the test tube. If possible, they put an immunofluorescence reaction (RIF) and a pale treponema immobilization reaction (RIBT). The Wasserman reaction (with cardiolesh and lipid antigens), RIF and RIT are carried out by conventional methods (see No. 119, 121, 123, 124).
119. Pandey and Nonne - Apelt reactions

As a reagent for carrying out the Pandey reaction, a clear supernatant liquid is used, which is obtained by vigorously shaking 100 g of liquid carbolic acid with 1000 ml of distilled water. To obtain a precipitate and a clear liquid (reagent), this mixture is first placed in a thermostat for 3-4 hours, and then kept at room temperature for 2-3 days.

Place a watch or glass slide on dark paper and apply 2-3 drops of reagent on it, then 1 drop of cerebrospinal fluid. If the drop becomes cloudy or a filamentous turbidity appears along its periphery, the reaction is considered positive.

To carry out the Nonne-Apelt reaction, clean test tubes, a saturated solution of ammonium sulfate, distilled water, and dark paper are required. A saturated solution of ammonium sulfate is prepared as follows: 0.5 g of chemically pure neutral ammonium sulfate is placed in a 1000 ml flask, then 100 ml of distilled water heated to 95 ° C is poured, shaken until the salt is completely dissolved and left for several days at room temperature . After 2-3 days, the solution is filtered and the pH is determined. The reaction must be neutral.

A valuable diagnostic test in the detection of chronic gonorrhea among individuals suffering from chronic inflammatory diseases of the genitourinary system. According to the literature, with the correct use of this method, up to 80% of cases of gonorrhea not detected by a bacterioscopic or bacteriological method are detected.

The reaction can also be false-positive with the introduction of milk, the use of pyrogenal for medicinal purposes.

Therefore, a positive Bordet-Jangu reaction does not serve as indisputable proof of the presence of gonococcal infection, just as a negative one cannot be evidence of the absence of gonorrhea. However, its positive results for a long time should direct the doctor to search for the focus of gonococcal infection in the body.

The reaction is considered positive if there is a hemolysis delay expressed to varying degrees in the test serum. In the control (blood serum of healthy people), complete hemolysis is observed.

121. Wasserman reaction

For the reaction to take place:

a) isotonic sodium chloride solution;

b) ultrasonified treponemal (stored in the refrigerator at +4 °C) and cardiolipin (stored at room temperature) antigens;

c) complement, which is the blood serum obtained by heart puncture of 5-10 healthy guinea pigs. Can be stored in the refrigerator for up to 2 months provided

preservation with 4% boric acid solution and 5% sodium sulfate solution;

d) hemolysin - hemolytic rabbit blood serum immunized with sheep erythrocytes with different titers (stored in a refrigerator at a temperature of 4 ° C);

e) sheep erythrocytes, obtained by puncture of the jugular vein. The blood is collected in a sterile jar with glass beads (for mixing), shaken for 15 minutes. Fibrin clots are separated by filtration through sterile gauze. Defibrinated blood can be stored in the refrigerator for up to 5 days.
Sometimes there is a need for a longer storage of sheep's blood, and therefore it is preserved with a special preservative (6 g of glucose, 4.5 g of boric acid, 100 ml of isotonic sodium chloride solution), which is boiled in a water bath for 20 minutes a day for 3 day For 100 ml of defibrinated sheep blood, 15 ml of a preservative is required. The defibrinated blood preserved in this way is stored in a refrigerator.
The main experiment is preceded by several stages.

5-10 ml of blood is taken from the patient from the cubital vein and the serum is processed. In children, blood can be taken from the temporal vein or an incision in the heel. The puncture is done with sterile instruments, a syringe and a needle
pre-washed with isotonic sodium chloride solution.

Blood for research is taken on an empty stomach; 3-4 days before the study, the patient is forbidden to use drugs, digitalis preparations, and take alcohol.

You should not conduct a study in patients with elevated body temperature, after trauma, surgery, anesthesia, recent infectious diseases, in women during menstruation, in pregnant women (in the last 10 days of pregnancy), women in labor (in the first 10 days after childbirth), and also newborns (in the first 10 days of life).

Antigens are diluted according to the method and titer indicated on the label.
Prepare hemolytic system. To do this, defibrinated blood or sheep erythrocytes in the amount necessary for the reaction is centrifuged, the plasma is carefully separated, and the precipitate is washed with 5-6 volumes of isotonic sodium chloride solution until the supernatant becomes completely colorless. A 3% suspension of erythrocytes in an isotonic solution of sodium chloride is prepared from the sediment according to a triple titer.

A solution of hemolytic serum and a suspension of sheep erythrocytes are quickly mixed and placed in a thermostat for 30 minutes.

Dry complement is diluted with isotonic sodium chloride solution in a ratio of 1:10, normal inactivated human serum - 1:5.

Reagents, ml	Row of test tubes
	1st	2nd	3rd	4th	5 and
3% suspension of sheep erythrocytes	0,25	0,25	0,25	0,25	0,25

hemolytic serum, diluted by triple titer	-	0,25	-	-	-
Complement 1:10	0,25	-	-	-	-
Triponemal antigen diluted by titer	-	-	-	-	-
Cardiolipin antigen diluted by titer	-	-	-	-	0,5
Isotonic sodium chloride solution	0,75	0,75	1,0	0,5	0,5

The tubes are placed in a thermostat for 45 minutes, after which they are checked. Hemolysis should not be in any test tube.

After 45 minutes, the working dose of complement is determined, that is, its titer with an allowance in the range of 15-20%.

Complement titer is considered to be its minimum amount, causing complete hemolysis of ram erythrocytes in the presence of antigen and normal human blood serum.

With the quantitative method of carrying out the Wasserman reaction, the experiment is performed with decreasing volumes of serum diluted with isotonic sodium chloride solution.
Scheme for conducting the main experience of the Wasserman reaction

Ingredients (in ml). Total volume 1.25 ml	No. of tubes
Ingredients (in ml). Total volume 1.25 ml	I	C	III
The test serum is inactivated, diluted 1:5 Antigen I (treponemal), diluted by titer Antigen II (cardiolipin), diluted by titer Isotonic sodium chloride solution Complement, diluted according to the working dose	0,25 0,25	0,25	0,25
	-	0,25	-
	0,25	0,25	0,25 0,25

The clinical significance of the Wasserman reaction is difficult to overestimate. It is carried out for all patients before the start of treatment, but it is of particular importance for latent syphilis, damage to internal organs and the nervous system.

The results of the Wasserman reaction characterize the quality of the treatment, which gives reason to deregister treated patients within a certain period of time.

The Wasserman reaction is carried out twice for all pregnant women, patients with somatic, nervous, mental and skin diseases, as well as for the decreed contingents of the population. At the same time, positive and weakly positive results of the reaction should be treated critically, since they can be at the end of pregnancy and after childbirth, with hyperthyroidism, malaria, leprosy, decay of a malignant tumor, infectious diseases, collagenoses, etc. Therefore, in the presence of clinical manifestations diseases, they should be taken into account, along with the data of bacterioscopy, in the first place.