RU2371727C2 - Method of immunity assessment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of immunity assessment involves source material obtainment from peripheral blood and inflammation spot, obtainment of cell suspension based on medium 199, division of the suspension in two parts. Stimulation agent for Staphylococcus aureus is added to one part of cell suspension at 1:10 ratio in concentration of 2×10⁷ microbial bodies per ml. Further the stimulated and non-stimulated cell suspensions are placed in indents of flat-bottom tray for immunological analysis separately for each parametre examined. Activity of cation proteins, enzymes such as myeloperoxidase, adenosinetriphosphatase, acid phosphatase, cytochromoxydase, lactate dehydrogenase, succinate dehydrogenase is measured, and HCT test is performed. Stimulation index is defined for each parametre. Conclusion on immunity state is drawn on stimulation index. Index value under 1 indicates insufficient anti-infection protection. The more stimulation indices are under 1, the less is body resistance to infection penetration and the harder is the disease course.

EFFECT: reliable assessment of anti-infection organism protection condition.

2 cl, 5 tbl, 4 ex

Description

The invention relates to medicine, namely to immunology, allergology, dermatology, pulmonology, infectious microbiology, veterinary medicine, and can be used to assess the immune status of a healthy and sick body.

The initial state of the body’s anti-infection protection affects the development and severity of the disease, therefore, determining the state of immunity allows you to assess how much the body is able to withstand the introduction of an infectious agent. One of such indicators of condition includes cellular mechanisms of nonspecific protection, which are characterized by indicators of phagocytosis and functional activity of phagocytic cells. The identification of the degree of tension of the functional state of these cells related to the systemic and local immunity of the body, due to their rapid reaction in response to the introduction of bacteria, is of particular prognostic value. In this case, a significant increase in the activity of phagocytic cells in response to the introduction of a stimulating agent in vitro can serve as a criterion that makes it possible to accurately assess the state of immunity both in pathology and in the characterization of a relatively healthy organism. However, the currently used research methods do not always reliably detect the presence of a violation of the anti-infection protection of the body as one of the most important immunity conditions.

Known methods for assessing violations of the antimicrobial protection of organisms in infectious diseases with the determination of changes in the state of non-specific immunity in comparison with the corresponding indicators of healthy organisms:

1) In this case, the activity of the functional potential of neutrophilic granulocytes is investigated in case of purulent-septic and allergic diseases based on indicators of the intracellular content of cationic proteins in the blood of patients. The coefficient of realization for this enzyme was calculated with an additional load of neutrophils (Staphylococcus aureus was introduced as a stimulating agent to the cells). The magnitude of this indicator judges the severity of purulent-septic and allergic processes (Nesterova V.I., Svetlichnaya M.A. Importance of studying the functional potential of neutrophilic granulocytes in purulent-septic and allergic diseases in children // Pediatrics, 1989, No. 9, S.63-65).

2) A known method for determining the insufficiency of antimicrobial protection, in which the patient determines the phagocytic activity of blood leukocytes simultaneously with the control face (reference) with a predetermined level of phagocytosis in relation to its average level in the group of healthy people. As an indicator of phagocytosis, the ratio of the sum of objects absorbed by active phagocytes to the total number of inactive and inactive phagocytes is additionally determined. According to the results of the study, the average level of phagocytosis in healthy people is calculated, and the phagocytosis in a patient is expressed as a percentage of this average level. When the phagocytic index is below its minimum level, healthy people are diagnosed with insufficient antimicrobial protection. (V.N. Kaplin. Alternative immunology of infections. - Perm, Publishing House of the Perm State Medical Academy, 1996, 163 pp.).

The disadvantages of these methods is that only one indicator of the activity of phagocytes of systemic immunity (blood) is studied and the results do not reflect the state of local immunity of patients.

Closest to the claimed technical solution is a method for assessing the functional activity of phagocytic cells to detect increased sensitivity to infectious agents (PMKhaitov, B.V. Pinegin, Kh.I. Istamov. Ecological immunology. - M., 1995, p.149- 155).

In this case, a suspension of cells from peripheral blood is prepared at a concentration of 2 million / ml (parameters of systemic immunity) of a person and introduced into the wells of flat-bottomed plates for immunological analyzes separately for each enzyme and adhesion test, in triplets for each sample. For cell adhesion, the plate is placed in a thermostat for 45 minutes, then non-adherent cells are removed by washing three times with phosphate buffer (PBS, pH 7.2-7.4). Zimosan solution is added to half of the adherent cells for stimulation, then unstimulated and stimulated cells in medium 199 are placed in a thermostat for 30 minutes. Then medium 199 is removed and cells are fixed by adding 100 μl of 96% ethanol. After 30 minutes, the alcohol is removed and, to activate the enzymes (myeloperoxidase, acid phosphatase, cationic proteins and nitrosine tetrazolium), the corresponding substrates are added to the cells. After completion of the reaction of the interaction of enzymes with substrates, the cell monolayer is stained with PBS dye, then the cells are washed three times from the dye that is not included, and the intracellular dye is dissolved and dissolved with a 40 mM HCl solution in isopropanol. The optical density of the solution was measured on a Titertek Multiscan Plus microprocessor ("Flow lab.") At the appropriate wavelength for each enzyme.

The obtained indicators are calculated taking into account the number of cells adhered to the plastic, which is calculated by the formula: Y = exp ^{lnX2.09718556-9.78437829} , where Y is the number of cells adhered to the plastic, and X is the optical density in units × 10. Then, obtained by testing either another parameter of the functional activity of cells, the optical density is multiplied by one hundred thousand and divided by the number of adherent cells, determined by the test of adhesion of phagocytic cells to plastic.

The disadvantage of this method is that only indicators of systemic immunity (blood cells) are studied, parameters of the functional activity of cells are evaluated only for individual enzyme systems of cells, the complexity of calculating indicators, which require an additional test for the adhesive activity of cells, as well as the results do not reflect the state of local immunity of patients.

The objective of the proposed technical solution is to increase the accuracy and reliability of assessing the state of immunity by determining the degree of damage to cellular elements of the immunophagocytic system of the body.

The problem is solved in that in a method for assessing immunity, including collecting the starting material from peripheral blood, obtaining a cell suspension based on medium 199, placing it in the wells of a flat-bottomed tablet for immunological analyzes separately for each enzyme, adhering the cells, washing the cells, dividing them into two parts, adding a stimulating agent to one part of the adherent cells, incubating stimulated and non-stimulated cells, removing the incubation medium, fixing the cells, introducing substrates for testing determination of the enzymatic activity of stimulated and non-stimulated cells by the optical density of the solution and calculation of the index of stimulation index, according to the invention, the selection of the starting material is additionally carried out from the site of inflammation of the body, the cell suspension is divided into two equal parts, the stimulating agent is applied directly to the cell suspension before placing it in the wells flat-bottomed tablet for immunological analyzes, while Staphylococcus aureus at a concentration of 1:10 in concentration is used as a stimulating agent 2 × ¹⁰ July bw / ml. Cell fixation is carried out with formalin vapors for 10-15 minutes, the enzymatic activity of the cells is determined for 6 or more enzymes, and the stimulation index is determined by dividing the optical density of the solution of stimulated cells by the optical density of the solution of unstimulated cells, on the state of immunity judged by the value of the stimulation index less than or greater than one.

In this case, the adhesion of stimulated and non-stimulated cells is carried out by incubation for 45 min at a temperature of 37 ° C. Cells are washed three times with phosphate buffer (PBS, pH 7.2-7.4). The enzymatic activity of cells is determined for enzymes: myeloperoxidase, adenosine triphosphatase, acid phosphatase, nitro-blue tetrazolium, cytochrome oxidase, lactate dehydrogenase, succinate dehydrogenase and cationic proteins. The optical density of the solution was measured on a Titertek Multiscan Plus microprocessor ("Flow lab.") At a wavelength corresponding to the substrate solution for each enzyme.

The essence of the method is illustrated by the following sequence of operations.

As a material for the study, a cell suspension is used at a concentration of 2 million / ml, obtained simultaneously from peripheral blood and the site of inflammation of a person or animal. For this, venous peripheral blood and rinsing from the site of inflammation (sputum, rinsing from the wound) are treated in a known manner, namely, the starting material in the amount of 4 ml is centrifuged for 30 min at 1500 rpm, after which the supernatant is taken. The cell suspension is washed 2 times in 1 ml of a balanced Hanks saline solution without phenol red at 1500 rpm for 10 minutes. In the Goryaev’s chamber, the concentration of nucleated cells is calculated. Dilution (addition of sterile saline or Hanks solution without phenol red) gives the cell suspension necessary for research at a concentration of 2 million in 1 ml.

Prepared cell suspensions (from peripheral and flushing from the site of inflammation) based on medium 199 are divided into equal parts and a stimulating agent is added to each half. As a stimulating object of phagocytosis, Staphylococcus aureus is used. Suspension S.aureus is diluted to a concentration of 2 × 10 ⁷ MK / ml with physiological saline and added to a suspension of cells at the rate of 10 bacteria per phagocyte. The obtained stimulated and non-stimulated cell suspensions are placed in the wells of a flat-bottomed tablet for immunological analyzes separately for each enzyme. For cell adhesion and stimulation, the plate is placed in a thermostat with a temperature of 37 ° C for 45 minutes, then non-adherent cells are removed by washing three times with phosphate buffer (PBS, pH 7.2-7.4). After that, medium 199 is removed and the cells are fixed with formalin vapors for 10-15 minutes.

In order to activate the enzymes myeloperoxidase, adenosine triphosphatase, acid phosphatase, nitro blue tetrazolium, cytochrome oxidase, lactate dehydrogenase, succinate dehydrogenase and cationic proteins, the substrates suitable for the enzyme under study are added to the cells. After completion of the reaction of the interaction of enzymes with substrates, the optical density of the stimulated and non-stimulated solutions is measured on a Titertek Multiscan Plus microprocessor ("Flow lab.") At a wavelength corresponding to the substrate solution for each enzyme.

1) Intravital test (HCT test)

The isolated cell suspension with a concentration of 2 × 10 ⁶ cells / ml based on medium 199, including HCT (nitro blue tetrazolium 0.5 mg / ml), is divided into two parts and half of the samples are added with S.aureus at a concentration of 2 × 10 ⁷ m.k./ml. Then a suspension of cells with and without bacteria is distributed 100 μl into the wells of a flat-bottomed 96-well plate, three for each sample. After incubation at 37 ° C for 45 min, the cell monolayer is washed three times with warm Hanks solution without phenol red from the HCT solution and non-absorbed bacteria and dried. A monolayer of cells is fixed in formalin vapors for 15 minutes. Then the cells are destroyed by adding 50 μl of dimethyl sulfamide (DMSO, manufactured by ICN) to the wells preheated to 83 ° C. The optical density of the obtained solutions is measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 540 nm.

2) Tests on fixed samples

The determination of enzyme activity was carried out on fixed samples, which are prepared as follows.

The isolated cell suspension with a concentration of 2 × 10 ⁶ cells / ml is divided into two parts and half of the samples are added S.aureus at a concentration of 2 × 10 ⁷ m.k. / ml. Then a suspension of cells with and without bacteria is distributed 100 μl into the wells of a flat-bottomed 96-well plate, three for each sample. After incubation at 37 ° C for 45 min, the cell monolayer is washed three times with a warm Hanks solution without phenol red from unabsorbed bacteria and dried. Then they are fixed in formalin vapors for 15 minutes, after which the enzymatic activity of the cells is examined.

a) determination of the activity of adenosine triphosphatase (ATPase)

50 μl of ATP substrate 8 mg adenosine-5 * -triphosphate (ICN) per 1 ml of substrate solution, which is prepared on the basis of 0.05 M Tris-Hcl, is added to plastic 96-well plate and fixed unstimulated and stimulated suspension cells. buffer (pH 7.8), by dissolving in 100 ml of this buffer 870 mg NaCl, 287 mg KCI and 52 mg MgCl ₂ ). Samples are left in a thermostat at 37 ° C for 60 minutes. The reaction was stopped by adding 100 μl of a mixture of ascorbic and molybdenum acids in a ratio of 1: 1. After 20 minutes, the absorbance of the obtained substrates was measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 620 nm.

b) determination of myeloperoxidase activity

Freshly prepared substrate solution (to 10 ml of phosphate-citrate buffer (pH 5.0) add 4 mg of o-phenylenediamine (ICN) and 500 μl of 0.33% hydrogen peroxide) 100 μl is added to the wells of the tablet containing plastic-adhered and fixed cells. The plate is incubated at room temperature for 10 minutes, then the reaction is stopped by the addition of 10% sulfuric acid solution at 100 μl per well. The optical density of the solution is determined on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 492 nm.

c) determination of the activity of cationic proteins

50 μl of a solution containing 10 mg of strong green ("Fast Green", "Serva") dissolved in 10 ml of methanol Tris buffer (pH 8.0-8.2) is added to wells with cells fixed on plastic. The plate is incubated for 30 minutes at 37 ° C. Then the unincorporated dye is washed twice with Hanks solution. The dye bound to the cationic proteins of cells is dissolved by the addition of dimethyl sulfoxide (50 μl), preheated in a water bath to 80 ° C. The results are taken into account using a Titertek Multiscan Plus spectrophotometer at a wavelength of 620 nm.

g) determination of acid phosphatase activity

50 μl of a solution of paranitrophenyl phosphate (PNPF solution, ICN) is added to the wells of a flat-bottomed 96-well plate containing fixed cells. To prepare it, 0.09 g of PNPF and 0.31 g of NaCl were dissolved in 37 ml of 1M sodium citrate buffer (pH 5.5). Samples are incubated at 37 ° C for 30 minutes. Then, by adding 0.2 M NaOH solution, 100 μl per well, the reaction is stopped. The optical density of the solution is measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 405 nm.

d) determination of cytochrome oxidase activity

Freshly prepared substrate solution (to 10 ml of 0.1 M acetate buffer, pH 5.5, add 20 mg of 3.3 * -diaminobenzidine (ICN), 100 mg of MnCl ₂ and 300 μl of 0.33% hydrogen peroxide) 100 μl are added in the wells of the tablet containing fixed cells. The plate is incubated at room temperature for 10 minutes, then the reaction is stopped by the addition of 10% sulfuric acid solution at 100 μl per well. The optical density is determined on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 492 nm.

d) determination of the activity of lactate dehydrogenase

100 μl of a solution of iodonitrotetrazolium violet (iodonitrotetrazolium, JNT, manufactured by ICN) with a concentration of 2 mg / ml is added to adherent and fixed on plastic cells. For this, 20 mg of NNT is first dissolved in 0.5 ml of 70 ° alcohol, and then 9.5 ml of 0.1 M phosphate buffer (pH 7.2-7.4), including 4 ml of a 1% MnCl ₂ solution, is added. The cell monolayer together with the solution of NNT is incubated at 37 ° C for 30 min, after which the supernatant is removed, the cell monolayer is washed twice with Hanks solution and dried. The intracellular granules of diformazan are dissolved in 100 μl of isopropyl alcohol, acidified with 0.04 M HCl, for 20 minutes. The optical density of the solution is determined on a microplate raider at a wavelength of 492 nm.

e) determination of succinate dehydrogenase activity

Determination of enzyme activity is carried out in the MTT test, where 3- (4,5-dimethylthiazolyl-2) -2,5-diphenyl tetrazolium bromide (methylthiazolyltetrazolium bromide, MTT, manufactured by ICN) is used as a substrate.

100 μl MTT (2 mg / ml) is added to the wells to the fixed cells based on 0.1 M phosphate buffer (pH 7.2-7.4), including 4 ml of a 1% MnCl ₂ solution. The cell monolayer, together with the MTT solution, is incubated at 37 ° C for 30 min, after which the supernatant is removed, the cell monolayer is washed twice with Hanks solution and dried in air. For counting the granules of diformazan bromide, the cells are destroyed by adding 100 μl of isopropyl alcohol, acidified with 0.4 M HCl. The optical density is determined on a microplate raider at a wavelength of 540 nm.

Reactions are placed in triplets of wells of a flat-bottomed 96-well plate with non-stimulated and stimulated cells. The optical density of the solution was measured on a Titertek Multiscan Plus microprocessor ("Flow lab.") At the appropriate wavelength for each enzyme.

The results of spectrophotometric analysis of enzyme activity are expressed as a stimulation index, which is defined as the ratio of the optical density of a solution of stimulated cells to the optical density of a solution of non-stimulated cells.

The value of the stimulation index greater than unity indicates the body's ability to withstand pathogens, while a value of less than unity means impaired immunity according to the studied parameters. A comprehensive assessment of the state of systemic and local immunity when comparing all the values of the obtained stimulation indices allows us to judge the degree of violation of the anti-infection protection of the body. The more values of the stimulation index are less than unity in the studied parameters, the less the body is able to withstand the introduction of an infectious agent and the more severe its disease progresses.

Thus, this method of assessing the state of immunity allows you to make objective accurate conclusions about the degree of violation of the body's defense, not only in various pathological conditions (disease), but also in relatively healthy individuals.

The technical result of the claimed invention is as follows.

1. At the same time, peripheral blood (systemic immunity) and flushing from the site of inflammation (wound, induced sputum, or the site of infection — local immunity) are used as material for the study of cells. This material, as accurately as possible reflects the parameters of systemic and local immunity, when comparing such indicators, we can objectively judge the protection of the body from pathogens.

2. The stimulating agent is applied directly to the cell suspension before placing it in the wells of a flat-bottomed tablet for immunological analyzes. This allows you to more evenly distribute the stimulating agent in the cell suspension, as well as reduce the complexity of this process and avoid preliminary stimulation of the cells in the process of adhesion.

3. The bacterium S. aureus is introduced as a stimulating agent, which is physiologically closer to specific cell stimulants in the body at a rate of 1:10 at a concentration of 2 × 10 ⁷ mt / ml.

4. Cell fixation is carried out with formalin vapors for 10-15 minutes, which allows preserving the initial enzyme activity for more accurate determination of the intracellular concentration of enzymes.

5. Stimulation of enzyme activity in cells is carried out for 6 or more enzymes, which helps to increase the accuracy of determining the state of immunity of the studied organism.

6. The index of stimulation is determined by dividing the optical density of the solution of stimulated cells by the optical density of the solution of non-stimulated cells.

7. The state of immunity is judged by the value of the stimulation index, which at values less than unity indicates the lack of anti-infection protection of the body. When using this indicator, the method of determining the state of the body's immunity is simplified, its information content and reliability are increased.

Case Studies

We have studied the functional activity of cells of the systemic (peripheral blood) and local (wound, induced sputum) immunity of healthy and sick people, as well as animals. Materials for the study from patients with community-acquired pneumonia of moderate and severe degree and acute bronchitis, as well as healthy people, were obtained in the pulmonology departments of the Main Hospital of the Pacific Fleet (Vladivostok) and the Hospital TORPU FPS of the Russian Federation (Vladivostok) during the period from 2002 to 2004 The diagnosis of community-acquired pneumonia was established on the basis of generally accepted criteria (community-acquired pneumonia in adults: practical recommendations for diagnosis, treatment and prevention / A.G. Chuchalin, A.I. Sinopalnikov, S.V. Yakovlev, etc. - Smolen to: Aventis, 2003. - 54 c).. Assessment of the severity of the condition of patients at the time of admission was carried out according to the criteria proposed by L.I. Butler in 1996 (A. Okorokov. Diagnosis of respiratory diseases / A.N. Okorokov. - M .: Med. Lit., 2000. - 448 p.). Materials from healthy and sick Far Eastern scarlet fever and purulent aseptic inflammation caused by staphylococcus animals were studied in the laboratory of pathomorphology and electron microscopy of the Research Institute of Epidemiology and Microbiology of the Siberian Branch of the Russian Academy of Medical Sciences.

Example No. 1

Healthy volunteer K., age 19 years.

The patient was inhaled with 3-5% sterile hypertonic saline solution using an Omron CX-3 compression nebulizer (Japan) with 7-minute sessions. Then he rinsed his mouth and coughed up sputum in a sterile polymer disposable Petri dish with a diameter of 90 mm (GREINER BIO-ONE, Germany). After that, the induced sputum was transferred using a sterile disposable 10 ml syringe into centrifuged graduated plastic tubes with a screw cap. A 0.1% dithiothreitol solution was added in a 1: 1 ratio. The mixture was shaken for 10 minutes and filtered through nylon gauze.

The filtered induced sputum was centrifuged at 1200 rpm for 10 minutes, then the supernatant was drained, and the cell suspension was washed 3 times for 10 minutes in 1 ml of Hanks balanced saline without phenol red at 1200 rpm. In the Goryaev’s chamber, the concentration of nucleated cells was calculated. Dilution (by adding sterile physiological saline) yielded the necessary cell suspension for research at a concentration of 2 × 10 ⁶ cells / ml, S.aureus was introduced into half of the samples at a concentration of 2 × 10 ⁷ c./ml.

4.0 ml of blood was taken from the ulnar vein, centrifuged for 30 min at 1500 rpm, after which the supernatant was taken. The cell suspension was washed 2 times in 1 ml of balanced Hanks saline without phenol red at 1200 rpm for 10 minutes. In the Goryaev’s chamber, the concentration of nucleated cells was calculated. Dilution (adding sterile saline or Hanks solution without phenol red) gave a cell suspension at a concentration of 2 million in 1 ml, half the samples were added S.aureus at a concentration of 2 × 10 ⁷ MK / ml.

The obtained stimulated and non-stimulated cell suspensions were placed in the wells of a flat-bottomed tablet for immunological analyzes separately for each enzyme. For cell adhesion, the plate was placed in a thermostat with a temperature of 37 ° C for 45 min, then non-adherent cells were removed by washing three times with phosphate buffer (PBS, pH 7.2-7.4). After that, cells were fixed with formalin vapors for 15 minutes.

To determine the activity of the enzymes myeloperoxidase, adenosine triphosphatase, acid phosphatase, nitro-blue tetrazolium, cytochrome oxidase, lactate dehydrogenase, succinate dehydrogenase and cationic proteins, the substrates corresponding to these enzymes were added to the cells. After completion of the reaction of the interaction of enzymes with substrates, the enzymatic activity of phagocytes was evaluated by spectrophotometric determination of the optical density of solutions.

Reactions were set in triplet wells for each sample of a flat-bottomed 96-well plate with non-stimulated and stimulated cells.

1) Intravital test (HCT test)

The isolated cell suspension at a concentration of 2 × 10 ⁶ cells / ml based on medium 199, including HCT (nitro blue tetrazolium 0.5 mg / ml), was divided into two parts and S. aureus at a concentration of 2 × 10 ^{7 was} introduced into half of the samples m.k. / ml. Then, a cell suspension with and without bacteria was distributed 100 μl into the wells of the tablet, three for each sample. After incubation at 37 ° C for 45 min, the cell monolayer was washed three times with warm Hanks solution without phenol red from the HCT solution and unabsorbed bacteria and dried. A monolayer of cells was fixed in formalin vapors for 15 min.

Then the cells were destroyed by adding 50 μl of dimethyl sulfamide (DMSO, manufactured by ICN) to the wells preheated to 83 ° C. The optical density of the obtained substrates is measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 540 nm.

2) Tests on fixed samples

The determination of enzyme activity was carried out on fixed samples, which were prepared as follows: after incubation at 37 ° C for 45 min, the cell monolayer was washed three times with warm Hanks solution without phenol red from non-absorbed bacteria and dried. They were fixed in formalin vapors for 15 minutes.

a) determination of the activity of adenosine triphosphatase (ATPase)

50 μl of ATP substrate 8 mg adenosine-5 * -triphosphate (ICN) per 1 ml of substrate solution, which was prepared on the basis of 0.05 M Tris-Hcl-, was added to plastic 96-well plate and fixed unstimulated and stimulated cells. buffer (pH 7.8), which included 870 mg of NaCl, 287 mg of KCl and 52 mg of MgCl ₂ . Samples were left in an incubator at 37 ° C for 60 min. The reaction was stopped by adding 100 μl of a mixture of ascorbic and molybdenum acids in a ratio of 1: 1. After 20 minutes, the absorbance of the obtained substrates was measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 620 nm.

b) determination of the activity of myeloperoxidase (MPO)

Freshly prepared substrate solution (to 10 ml of phosphate-citrate buffer (pH 5.0) was added 4 mg of o-phenylenediamine (ICN) and 500 μl of 0.33% hydrogen peroxide) 100 μl were added to the wells of the tablet containing plastic-adhered and fixed cells. The plate was incubated at room temperature for 10 minutes, then the reaction was stopped by adding 10% sulfuric acid solution at 100 μl per well. The optical density was determined on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 492 nm.

c) determination of the activity of cationic proteins (KB)

50 μl of a solution containing 10 mg of strong green ("Fast Green", "Serva") dissolved in 10 ml of methanol Tris buffer (pH 8.0-8.2) was added to wells with cells fixed on plastic. The plate was incubated for 30 min at 37 ° C. Then, the unincorporated dye was washed twice with Hanks solution. The dye bound to the cationic proteins of the cells was dissolved by the addition of dimethyl sulfoxide (50 μl), preheated in a water bath to 80 ° C. The results were taken into account using a Titertek Multiscan Plus spectrophotometer at a wavelength of 620 nm.

g) determination of the activity of acid phosphatase (CF)

50 μl of a paranitrophenyl phosphate solution (PNPF solution, ICN) was added to the wells of a flat-bottomed 96-well plate containing fixed cells. To prepare it, 0.09 g of PNPF and 0.31 g of NaCl were dissolved in 37 ml of 1M sodium citrate buffer (pH 5.5). Samples were incubated at 37 ° C for 30 minutes. Then the reaction was stopped by adding 0.2 M NaOH solution at 100 μl per well. The optical density of the solution was measured on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 405 nm.

d) determination of the activity of cytochrome oxidase (TSXO)

Freshly prepared substrate solution (to 10 ml of 0.1 M acetate buffer, pH 5.5, add 20 mg of 3.3 * -diaminobenzidine (ICN), 100 mg of MnCl ₂ and 300 μl of 0.33% hydrogen peroxide), 100 μl were added in the wells of the tablet containing fixed cells. The plate was incubated at room temperature for 10 minutes, then the reaction was stopped by adding 10% sulfuric acid solution at 100 μl per well. The optical density was determined on a Multiscan Titertek Plus spectrophotometer (Flow lab.) At a wavelength of 492 nm.

d) determination of the activity of lactate dehydrogenase (LDH)

100 μl of a solution of iodonitrotetrazolium violet (iodonitrotetrazolium, JNT, manufactured by ICN) with a concentration of 2 mg / ml was added to adherent and fixed on plastic cells. For this, 20 mg of NNT were first dissolved in 0.5 ml of 70 ° alcohol, and then 9.5 ml of 0.1 M phosphate buffer (pH 7.2-7.4), including 4 ml of a 1% MnCl ₂ solution, was added. A monolayer of cells together with a solution of UNT was incubated at 37 ° C for 30 min, after which the supernatant was removed, the monolayer of cells was washed twice with Hanks solution and dried. The intracellular granules of diformazan were dissolved in 100 μl of isopropyl alcohol, acidified with 0.04 M HCl, for 20 minutes. The optical density of the solution was determined on a microplate raider at a wavelength of 492 nm.

e) determination of the activity of succinate dehydrogenase (LDH)

The enzyme activity was determined in the MTT test, where 3- (4,5-dimethylthiazolyl-2) -2,5-diphenyl tetrazolium bromide (methylthiazolyltetrazolium bromide, MTT, manufactured by ICN) was used as a substrate.

100 μl MTT (2 mg / ml) was added to the wells to the fixed cells based on 0.1 M phosphate buffer (pH 7.2-7.4), including 4 ml of a 1% MnCl ₂ solution. The cell monolayer together with the MTT solution was incubated at 37 ° C for 30 min, after which the supernatant was removed, the cell monolayer was washed twice with Hanks solution and dried in air. To count the granules of diformazan bromide, the cells were destroyed by adding 100 μl of isopropyl alcohol, acidified with 0.4 M HCl. The optical density was determined on a microplate raider at a wavelength of 540 nm.

The index of stimulation was determined by dividing the optical density of the solution of stimulated cells by the optical density of the solution of non-stimulated cells.

The results are presented in table 1.

Table 1 Induced sputum and blood cell stimulation index values from a healthy volunteer Research enzymes The value of stimulation indices for induced sputum cells The value of stimulation indices for blood cells HCT test 1,206745 1,362218 ATPase 1,0941315 0.939959 IGO 1,037406 1,068123 KB 1,063939 1,047014 CF 1,250909 1,030239 CHO 1,56321 1,03528 LDH 1,15603 1,14563 LDH 1,82411 1.65324

The value of the stimulation index for all cells of a healthy volunteer was more than unity, which indicated the body’s ability to withstand pathogens and indicated the absence of a violation of the body’s anti-infection protection.

Example No. 2

Patient V., age 19 years.

Diagnosis: Community-acquired pneumonia of moderate severity.

The state of the parameters of the systemic and local immunity of the patient was evaluated analogously to example No. 1. The data are presented in table 2.

table 2 Indices of cell stimulation of induced sputum and blood of a patient with community-acquired pneumonia of moderate severity Research enzymes The value of stimulation indices for induced sputum cells The value of stimulation indices for blood cells HCT test 1,100629 1,082969 ATPase 1,102564 1,139591 IGO 0.713415 0.765412 KB 0.951731 1,032768 CF 0.96977 1,153571 CHO 0,87456 0.78923 LDH 0.79562 1,02657 LDH 1,23567 1,009546

The values of the stimulation index for blood cells of a patient with community-acquired pneumonia of moderate severity were predominantly greater than unity, while for cells of induced sputum (local immunity factors) less than this value. A comparison of the obtained parameters of systemic and local immunity indicated an insufficient ability of the body to withstand the causative agent of the disease and indicated the presence of a partial violation of the anti-infection protection of the body.

When comparing the obtained indicators with the values of the stimulation index obtained as a result of a study of the systemic and local immunity of a patient with severe community-acquired pneumonia (Table 3), one can judge the severity of the disease. Mostly, the latter obtained stimulation index values less than unity, which indicated the absence of anti-infection protection of the body.

Patient S., age 19 years.

Diagnosis: Community-acquired pneumonia of a severe degree.

Table 3 Indices of cell stimulation of induced sputum and blood of a patient with severe community-acquired pneumonia Research enzymes The value of stimulation indices for induced sputum cells The value of stimulation indices for blood cells HCT test 0.678937 0.748447 ATPase 1,021484 1,189655 IGO 0.857043 0.515102 KB 0.643931 0.790894 CF 0,874317 0.89392 CHO 0,423645 0.954123 LDH 0.654312 0.564712 LDH 0.789965 0.678912

Example No. 3

Guinea pig with purulent-septic wound

The state of the parameters of the systemic and local immunity of the animal was evaluated analogously to example No. 1. The data are presented in table 4.

Table 4 Indicators of the stimulation index of cells of the site of inflammation (wound) and blood of the animal Research enzymes The value of stimulation indices for wound cells The value of stimulation indices for blood cells HCT test 0.929454 1,23564 ATPase 0.641791 0.862197 IGO 0.903716 0.755435 KB 0.85432 1,12472 CF 0.929661 0.938395 CHO 1,017157 0.803493 LDH 0,995556 0,51423 LDH 1,086364 0,879641

The value of the stimulation index for cells of an animal with a purulent-septic wound is predominantly lower than unity, which indicates an insufficient ability of the body to resist the causative agent of the disease and indicates the presence of a violation of the anti-infection protection of the body.

Example No. 4

Guinea pig, sick with Far Eastern scarlet fever, intraperitoneal injection of bacteria, peritoneal exudate was taken to study the cells of the site of inflammation.

The state of the parameters of the systemic and local immunity of the animal was evaluated analogously to example No. 1. The data are presented in table 5.

Table 5 Indices of stimulation of cells of the site of inflammation (peritoneal exudate) and blood of an animal suffering from Far Eastern scarlet fever Research enzymes The value of stimulation indices for peritoneal exudate cells The value of stimulation indices for blood cells HCT test 0.769817 1,091447 ATPase 1,107339 1,091712 IGO 1,030097 1,027063 KB 0.836939 0.917153 CF 0,993098 0.817337 CHO 1,023654 1,23145 LDH 0.715614 0.67851 LDH 0.975102 1,142361

The stimulation index values for blood cells of an animal suffering from Far Eastern scarlet fever-like fever were mostly greater than unity, while for peritoneal exudate cells (local immunity factors) it was less than this value. A comparison of the obtained parameters of systemic and local immunity indicated an insufficient ability of the body to withstand the causative agent of the disease and indicated the presence of a partial violation of the anti-infection protection of the body.

Thus, our proposed method for assessing the state of immunity, namely, the degree of damage to cellular elements of the immunophagocytic system of the body by at least six criteria after their additional load, allows us to reliably assess the state of anti-infection protection of the body. This state of this immunity parameter must be taken into account both when characterizing a relatively healthy organism, and when characterizing an organism with the presence of damaging infectious factors, while revealing the severity of pathological processes.

Claims (2)

1. A method for assessing the state of immunity by determining an index of cell stimulation index, including collecting the starting material from peripheral blood, obtaining a cell suspension based on medium 199, placing it in the wells of a flat-bottomed tablet for immunological analyzes separately for each enzyme, cell adhesion, cell washing, cell division into two parts, adding a stimulating agent to one part of the adherent cells, incubating stimulated and unstimulated cells, removing the incubation medium, fixing the cells, carrying substrates for determining the enzymatic activity of cells by the optical density of a solution of stimulated and unstimulated cells and calculating a stimulation index, characterized in that the selection of the starting material in addition to the peripheral blood is carried out from the site of inflammation of the body, a cell suspension is obtained, it is divided into two equal parts, the stimulating agent make directly into the cell suspension prior to placing its wells flat-bottomed tablet for immunological analyzes, while as a stimulating cient use of Staphylococcus aureus 1:10 calculating a concentration of 2 × ¹⁰ July bw / ml and the cells incubated with adhesion are carried out simultaneously at 37 ° C for 45 min, cell fixation pairs carried formalin for 10-15 min, evaluate the activity of cationic proteins, enzymes: myeloperoxidase, adenosine triphosphatase, succinate dehydrogenase, lactate dehydrogenase, cytochrome oxidase, acid phosphatase, and the HCT test (nitrosine tetrazolium reduction test), the stimulation index is determined by dividing the optical density of the solution of stimulated cells by the value of the optical density of the solution of unstimulated cells, and the state of immunity and the severity of pathological processes are judged by the value of the stimulation index, which at values less than unity indicates the lack of anti-infection protection of the body, while the more stimulation index values are less than unity in the studied parameters, the less the body is able to withstand the introduction of an infectious agent and the more severe the disease. 2. The method according to claim 1, characterized in that the optical density of the solution is measured on a Titertek Multiscan Plus microprocessor ("Flow lab.") At a wavelength in accordance with the substrate solution for each enzyme.