RU2289138C2 - Method for allergodiagnostics by the values of chemiluminescent phosphorescence of neutrophils - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should isolate neutrophilic leukocytes, form immune complexes, measure background luminescence of incubation medium (A), add neutrophils, measure the level of spontaneous chemiluminescence (B), introduce immune complex and detect the value of stimulated chemiluminescence (C), calculate the coefficient of stimulation (CS) and at CS≥0.3 it is possible to diagnose allergic reaction. The innovation provides higher accuracy and specificity in detecting the presence of allergic sensitization to certain antigen, the chance for screening serial assay, excludes additional body sensitization associated with introducing allergens in case of skin sample.

EFFECT: higher accuracy of allergodiagnostics.

1 cl, 5 ex, 6 tbl

Description

The invention relates to methods for detecting specific sensitization of the body to complete antigens of various nature. The method is intended to detect hypersensitivity to complete antigens, including the assessment of allergic side effects of therapeutic and prophylactic medications.

Recently, there has been a deterioration in the general allergic status in the population, an increase in the number of allergic diseases and reactions, including anaphylactic ones, which is associated with an unfavorable environmental situation, chemical pollution of the environment, widespread use of chemicals in everyday life, drug abuse, changes in the nature of nutrition, and growth number of occupational allergens. It has been established that 1/5 of the inhabitants of our planet seek medical help because of various clinical manifestations of an allergic nature (Macharidze D.Sh., Tarasova S.V. Dynamics of the prevalence of symptoms of allergic diseases according to ISAAC 1997-2000, Moscow. // Allergology and Immunology. - 2002. - Volume 3. - No. 2. - P.50-54). Currently, antigens of various nature and structure are considered as allergy inducers (Pytsky V.I., Adrianova N.V., Artomasova A.V. Allergic diseases. - M .: Medicine, 1984). Over the past 15-20 years, the incidence of bronchial asthma in the Russian Federation has increased 3-4 times. (Fedoskova T.G. et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - P.13- 19). Moreover, in 80-90% of cases of this disease it has an infectious etiology (Pytsky V.I., Adrianova N.V., Artomasova A.V. Allergic diseases. - M .: Medicine, 1984).

In principle, the identification of allergic rearrangement of the body is possible using two approaches - in vivo and in vitro.

The identification of the etiological factor of allergies to date is mainly based on the results of skin tests (Pytsky V.I., Adrianova N.V., Artomasova A.V. Allergic diseases. - M .: Triada-X, 1999. - P.102 -112). Staging of skin samples is a diagnostic method for identifying specific sensitization of the body by introducing an allergen under the skin and assessing the magnitude and nature of the edema or inflammatory reaction that has developed. The method is based on the interaction of an allergen with sensitized T-lymphocytes and antigen-presenting cells (macrophages and Langengars cells), which causes the release of allergy mediators and the development of a local reaction in sensitized individuals (Fedoskova T.G. et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - S.13-19). There are various methods of skin testing using allergens: prik-tests, scarification, application and intradermal tests (Skepyan N.A. Allergic diseases. Differential diagnosis, treatment. - Minsk: Belarus, 2000. - P.35-43).

Currently, one of the most commonly used skin allergy tests is a specific allergen pri-test. The formulation of this test is carried out by injection into the skin and the introduction of an allergen solution to a depth of not more than 1-1.5 mm. This skin test is considered less traumatic in comparison with scarification tests, it requires a smaller surface of the skin for allergy diagnostics, which allows you to explore a larger number of samples.

Scarification allergy tests are widely used to detect sensitization to food allergens, allergens of bacterial and fungal origin. However, when they are staged, false-positive results often arise, as well as when staged skin and intradermal tests, for example with tuberculin, the reaction is positive in people infected with nocardia, rhodococcus, corynebacteria. (Gushchin I.S. Allergic inflammation and its pharmacological action. M: Farmakus Priit, 1998). Currently, experts from the European Academy of Allergology and Clinical Immunology do not recommend using scarification skin tests to diagnose allergies due to their low information content (Fedoskova TG et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - S.13-19).

Intradermal tests are considered more sensitive than scarification tests, but they are less specific. (Berezhnaya N.M., Bobkova L.P., Petrovskaya I.A. et al. Allergology dictionary-reference book. Kiev: Naukova Dumka, 1986).

Thus, the general disadvantages of skin testing as a way to diagnose allergies include the following:

firstly, the method is unsafe for the subject, since the occurrence of not only (local) local, but also general reactions of the body to the introduction of the allergen is not excluded, and in the case of a high level of sensitization, the development of severe allergic complications. In addition, the formulation of allergological samples requires experienced, specially trained personnel who are fluent in the procedure for their formulation, as well as a constant supply of anti-shock drugs, which necessitates a reaction in specialized allergocenters;

secondly, insufficient expressivity - the results of the test are taken into account after 24-48 hours;

thirdly, the number of analyzed antigens is limited.

Another method of diagnosing allergies is the setting of provocative tests. Provocative tests are considered a fairly reliable method of allergy diagnosis. They are used if there is a discrepancy between the history and the results of skin testing. Depending on the method of introducing the allergen into the body, conjunctival, nasal, inhalation and sublingual provocative tests are distinguished (Fedoskova T.G. et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - C.13-19 )

The disadvantage of provocative tests is the frequent development of allergic complications, therefore, provocative tests are limited to only one study per day (Pytsky V.I., Adrianova N.V., Artomasova A.V. Allergic diseases. - M .: Triada-X, 1999 . - S.102-112). In addition, when they are staged, specially trained personnel are required, as well as a specially equipped room for an inhalation provocation test.

In connection with the foregoing, in vitro allergy diagnostic methods are of particular relevance for assessing specific sensitization, which is emphasized in the decision of the WHO international group of expert immunologists. (Fedoskova T.G. et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - C.13-19).

Known instrumental method for assessing the sensitization of the body to an allergen (antigen), based on the detection of antibodies of the IgE class in blood serum in vitro - radio allergenic sorbent test (PACT) (Berezhnaya N.M., Bobkova L.P., Petrovskaya I.A. and other Allergology dictionary-reference. Kiev: Naukova Dumka, 1986). The essence of the method is the association of the specific IgE of the test serum with an allergen placed on a paper plate. After washing the non-specific IgE, ¹²⁵ I-labeled anti-IgE antibodies are added to the sample. The resulting radioactive complex is read on a scintillation counter (Pytsky V.I., Adrianova N.V., Artomasova A.V. Allergic diseases. - M .: Triada-X, 1999. - S.102-112). PACT is an express method, highly sensitive, specific and accurate, as it is based on the use of a radioactive label (Skepyan N.A. Allergic diseases. Differential diagnosis, treatment. - Minsk: Belarus, 2000. - P.35-43).

The disadvantage of the PACT method is the need to use special expensive equipment, it has less sensitivity compared to a skin sample, it requires the organization of a regular supply of isotope-labeled components, a specially equipped workplace and compliance with radiation safety measures when working with radioactively labeled anti-IgE (Fedoskova T. G. et al. Principles for the diagnosis of allergic diseases. // Consilium medicum. - 2002. - No. 4. - C.13-19).

There is also a known instrumental method for determining the content of total and allergy-specific IgE - enzyme-linked immunosorbent assay (ELISA). The principle of the method essentially coincides with PACT. The sorbent is treated with the optimal concentration of the allergen (polystyrene in the form of microplates), the excess allergen is washed off, the test serum is added, and as a result the formation of the antigen-antibody complex occurs. After this, unbound components are removed by washing, anti-IgE (labeled with an enzyme) is added, and the obtained substrate is determined photometrically (Fradkin. VA Diagnostic and therapeutic preparations. - M .: Medicine, 1990). The method is quite sensitive, specific and express, in addition, it is now more often used to determine allergen-specific IgE (A. Asimov. On the role of immunoglobin E in pathology. // Immun. Pat. Aller., Infect. - 2002. - No. 2. - S.73-77).

The disadvantage of enzyme immunoassay is the need for special standardized drugs (test systems) for specific allergens, however, the choice of available systems is currently limited. In addition, the method places high demands on the standardization of the test and on the quality of antigenic components, that is, the reproducibility of the method substantially depends on the quality and manufacturer of the test system.

There is also a known method for diagnosing allergies - a direct and indirect test of basophil degranulation, based on the detection of activation (degranulation) of immunocompetent basophilic leukocytes of blood sensitized by IgE when exposed to such cells with a specific antigen (Sheley W / Circulating basophile as indicator of hyper-sensitivity in man. - // Arch. Dermal. - 1963. - v.88. - P.759). The method does not require expensive drugs, provides high specificity.

The disadvantage of this technique is the complexity of its reproduction, high subjectivity of the results, low expressivity, the presence of laboratory animals in the formulation of an indirect test.

A known method for assessing the presence of allergies in vitro by recording the activity of sensitized blood cells by specific damage (destruction) of neutrophils (V. A. Fradkin. Diagnosis of allergies by reactions of blood neutrophils. - M., 1985).

For this approach, the hypothesis about the interaction of a specific immune complex “allergen (antigen) - antibody” with specific neutrophil receptors is common with the claimed method.

The disadvantages of the method under consideration include subjectivity when taking into account the results of the reaction, poor reproducibility and high complexity. One of the significant sources of errors in this technique is the error in the preparation of fixed smears, the quality of their staining, and the difficulty in ensuring accurate counting of damaged cells before and after incubation of phagocytes (N.D. Beklemishev, G.S. Sukhodoeva. Allergy in the clinic and experiment. - M.: Medicine, 1979).

Closest to the claimed method is a method for detecting sensitization of the body to bacterial antigens using chemiluminescent analysis of peripheral blood neutrophils (N.K. Voznesensky, N.S. Manerkina. Chemiluminescence of neutrophils in allergy diagnostics. // Clinical laboratory diagnostics. 1999. - No. 8. . - S.18-20), including the diagnosis of allergies in persons in contact with allergens of fungal origin (C.albicans).

The method includes the following steps:

obtaining from a sample of the original blood neutrophils with a concentration of 2 × 10 ⁶ cells · ml ^-1 ;

the formation of an in vitro antigen-antibody complex;

adding a complex to the suspension;

measurement of chemiluminescence of a suspension sample.

To obtain the neutrophil fraction, 1 ml of heparinized blood is used. A suspension of neutrophils is prepared from whole heparinized blood by erythrocyte lysis. Every 300 μl of blood hemolyses 3 ml of a 0.083% solution of ammonium chloride, starting with an exposure of 40 s and decreasing it to 20, 10 and 1 s, respectively. The termination of the action of ammonium chloride is achieved by introducing 7 ml of 1.5 M phosphate buffer (pH 7.2) into the mixture, followed by washing the cells by centrifugation at 200 g for 7 min and resuspending them in the same solution with a concentration of 2 × 10 cells · ml ^-1 .

For the formation of immune complexes in vitro using different ratios of allergen and serum in the amount of 20 and 100 μl, respectively. The allergen is diluted with 5% glucose solution.

Chemiluminescent analysis is carried out using a BHL-06 device. A reaction mixture containing 400 μl of phosphate buffer (pH 7.6), 100 μl of a 5 mM glucose solution, 100 μl of a 0.37 mm magnesium sulfate solution and 200 μl of a 0.65 mm luminol solution is added to a cuvette of the device, 50 μl of a white suspension are added, measure the level of spontaneous chemiluminescence. Then, 20 μl of serum with immune complexes is added to the cuvette and the luminescence dynamics is recorded for 7-8 minutes. Autologous serum without antigen and a solution of antigen without serum are used as a control. To evaluate the results, the ratio of the maximum value of chemiluminescence (I _max ) to the level of stationary luminescence (I ₀ ) is used - the so-called stimulation index (IS). IP = I _max / I ₀ .

In common with the claimed method is the principle of induction of a chemiluminescent glow of blood neutrophils due to exposure to them by a preformed in vitro immune complex of an allergen with an antibody.

The disadvantages of this method are:

a multi-stage red blood cell lysis procedure that leads to significant damage and death of up to 60-70% of neutrophils, which negatively affects both chemiluminescent ability and significantly reduces cell concentration, i.e. level (amplitude) of the chemiluminescent signal.

In addition, to evaluate the results of studies in the prototype in the calculation formula does not use an indicator of the background chemiluminescence of the reaction mixture, which significantly distorts the measurement result. It should be noted that the concentration of neutrophils used in the prototype 2 × 10 ⁶ cells · ml ^-1 is insufficient under the measurement conditions to obtain statistically significant differences in the background chemiluminescence of the reaction medium and spontaneous chemiluminescence of neutrophils due to the low absolute values of the chemiluminescence index (units of .e.).

The objective of the invention is to develop an improved method for identifying specific sensitization of the body to antigens based on the registration of chemiluminescence of blood neutrophils in vitro.

The problem is solved due to the fact that neutrophils are isolated in the density gradient of the separating liquid, followed by washing the cells in a gentle mode; the neutrophil concentration is adjusted to (4 ... 6) · 10 ⁶ cells · ml ^-1 , the immune complexes are obtained by mixing the test blood serum and the antigen (allergen), and incubated; measures the background values of the chemiluminescence of the incubation medium, spontaneous chemiluminescence of washed neutrophils introduced into the sample, and the intensity of chemiluminescence after adding a complex of blood serum and the test allergen (antigen) to the sample; calculate the indicator of the coefficient of stimulation; the significance of differences in the indicators before and after stimulation is evaluated based on the results of at least three repeated measurements.

The problem is solved due to the fact that:

at the stage of sample preparation, the volume of blood taken from the test blood is increased to 5 ml of heparinized and 5 ml of non-heparinized blood;

at the stage of separation of the neutrophil fraction, the use of a liquid for separation of cells (ficoll-verographin, ficoll-hypak, ficoll-urographin or any commercial mixture or liquid for isolating the neutrophil fraction) is provided;

the operation of preliminary washing of neutrophils in gentle modes (1000 rpm ^-1 ), minimally damaging neutrophilic white blood cells;

the luminescence is recorded on a luminometer accessible to most laboratories;

as a liquid diluting a suspension of neutrophils, a commercial Hanks solution (pH 7.4) is used, due to which optimal conditions for maintaining cell viability are ensured;

based on the results of registration of the chemiluminescent glow, the stimulation coefficient of chemiluminescence is calculated and the significance level of its magnitude is determined.

The method includes the following steps:

1. Obtaining from a sample of the studied blood a suspension of neutrophils with a concentration of (4-6) · 10 ⁶ cells · ml ^-1 ;

2. Formation of the in vitro complex "antigen-blood serum";

3. Measurement of the background chemiluminescence level of the reaction medium, the neutrophil chemiluminescence spontaneous and after adding the complex "antigen-blood serum."

Stage 1. To obtain a suspension of neutrophils using 5 ml of heparinized blood. A blood sample is incubated in an incubator at 37 ° C for 40 minutes. The supernatant (plasma) containing the leukocyte fraction is taken and layered on 5 ml of separating liquid, centrifuged for 15 min at 1500 rpm ^-1 . As a result, the sample is divided into three layers. The lower layer consists of a separating liquid, the middle layer contains the neutrophil fraction, and the upper layer contains blood plasma. A neutrophil fraction is collected carefully with a plastic pipette. The isolated cells are resuspended and washed with a colorless Hanks solution for 10 min at 1000 rpm ^-1 . The concentration of neutrophilic leukocytes is adjusted to (4-6) · 10 ⁶ cells · ml ^-1 , diluted with Hanks solution.

Stage 2. For the formation of immune complexes in vitro, the test blood serum and antigen (allergen) are used. Serum is obtained from the same sample of non-heparinized blood by centrifugation at a speed of 1500 rpm ^-1 for 15 minutes Dilutions of antigens are prepared in physiological saline. Equal amounts of serum and antigen solution (0.1 ml each) are incubated at 37 ± 1 ° C for 40 minutes.

Step 3. Measurement of the level of chemiluminescence is carried out using a chemiluminometer.

0.7 ... 0.9 ml of Hanks solution is added to the cuvettes of the device, 0.1 ml of 0.1 ... 0.5 mM luminol solution is added and the level of background chemiluminescence is determined. Then add 0.1 ml of the prepared, as described above, suspension of neutrophils, mix, incubate for 5 minutes and measure the level of spontaneous chemiluminescence, while the values are recorded in the interval from 5 to 40 minutes from the end of the incubation period of the suspension of neutrophils. To induce a chemiluminescent neutrophil response, 0.1 ml of the antigen-serum complex formed in vitro is added to the sample, and the chemiluminescence intensity is again recorded. The results are evaluated by the rate of stimulation coefficient (CS) of chemiluminescence, which is determined by the formula:

where B is the level of CL after stimulation, mV;

B - level of spontaneous CL, mV;

A is the level of background CL, mV.

and with a value of COP ≥ 0.3 diagnosed with an allergic reaction.

To increase the reliability of the results, the chemiluminescence of samples prepared in at least three replicates (independently prepared from the initial neutrophil suspension) is recorded, and the arithmetic mean and confidence intervals are determined with a 95% probability level.

According to the research results, the significance level of the chemiluminescence stimulation coefficient is determined. The stimulation coefficient is significant provided that there are statistically significant differences between the levels of spontaneous neutrophil chemiluminescence and chemiluminescence after stimulation with the antigen-blood serum complex, i.e. confidence intervals of the average values of these indicators do not overlap. The presence of a causal relationship between the totality of the essential features of the claimed object and the achieved technical results are shown in table 1.

The possibility of implementing the proposed method is shown in the following examples.

Example 1. Evaluation of the number of neutrophils in obtaining a suspension of cells using a separating fluid and the method of lysis of red blood cells.

The stages of obtaining a suspension of neutrophils using a liquid for separating cells was carried out according to the claimed method described above, the production of neutrophils by the method of erythrocyte lysis was carried out according to the method described above by N.K. Voznesensky (prototype).

From the data presented in table 2, it follows that when the neutrophil fraction is isolated from the blood of people using a separating liquid, 4-8% of the cells are killed, while the death of neutrophils isolated by the erythrocyte lysis method was 68-82%.

Example 2. Evaluation of the chemiluminescent reaction of neutrophils (spontaneous chemiluminescence) in the preparation of a sample of cells using a separating liquid and erythrocyte lysis.

The neutrophil suspension obtained in experiment 1 was used for the study. The chemiluminescent neutrophil response was measured using an LKB-1251 luminometer. The reaction medium was prepared according to the methods described above, while the cell concentration was (4-6) · 10 ⁶ cells · ml ^-1 in the present method and (1-2) · 10 ⁶ cells · ml ^-1 in the prototype.

Analysis of the results of the registration of chemiluminescence, presented in table 3, showed that the level of spontaneous chemiluminescence of neutrophils isolated by the method of the proposed method is significantly higher than in the case of isolation of cells by the method proposed in the prototype, in addition, when registering chemiluminescence of neutrophils obtained by the method of N .K. Voznesensky, there is a lack of statistically significant differences in the intensity of the level of background and spontaneous chemiluminescence.

Example 3. Assessment of the level of sensitization of the body of guinea pigs, once immunized with brucellosis vaccine.

The specific allergization phenomenon in response to vaccines was detected in guinea pigs - animals recommended for evaluating the side effects of vaccines, using a live commercial brucellosis vaccine (pcs. 19-VA), which was administered subcutaneously at 0.5 ml with a concentration of 2 · 10 ⁹ CFU · ml ^-1 . The chemiluminescent reaction of guinea pig neutrophils was evaluated 35 days after immunization, with a final concentration of neutrophils of 2 · 10 ⁶ cells · ml ^-1 . In these experiments, the commercial drug brucellin was used as a specific antigen. To determine the specificity of the chemiluminescent reaction to brucellosis antigen, the commercial preparation of tularine was used as a non-specific antigen. At the same time, a leukolysis reaction and a skin test were performed using brucellin.

From the data presented in table 4, it is seen that in the group of animals, 11 out of 20 (55%) immunized guinea pigs showed a significant increase in the level of chemiluminescence after induction by a specific antigen-antibody complex in comparison with the values of the spontaneous chemiluminescence index (p <0.05 ) In the remaining 9 animals, the value of the chemiluminescence index after exposure to brucellin did not differ from the level of spontaneous chemiluminescence (p> 0.05), which indicates the absence of CL stimulation of neutrophils by a specific allergen of these animals. According to the results of statistical analysis, a significant level of chemiluminescence CS in these studies was ≥0.71. In the same group of animals, pronounced reactions with a skin test and a positive leukolysis reaction were observed. Statistical analysis of the results of the evaluation of the chemiluminescence stimulation coefficient, skin test and leukolysis reaction in experimental animals showed their good convergence (Spearman correlation coefficient 0.879 (p <0.001) and 0.795 (p <0.001) for skin test and leukolysis, respectively). When analyzing the results of changes in the intensity of chemiluminescence of blood neutrophils of immunized guinea pigs after the addition of a non-specific complex of tularine-serum, there is a significant absence of stimulation of chemiluminescence of leukocytes. This fact indicates the specific character of the induction of the chemiluminescent reaction of neutrophilic leukocytes by antigens of the brucellosis vaccine.

Example 4. Assessment of the level of sensitization of the human body, once and repeatedly immunized with tularemia, brucellosis and anthrax vaccines.

Chemiluminescent analysis of neutrophils of people immunized with tularemia vaccine was carried out in accordance with the method of the proposed method. In the course of chemiluminescent analysis, brucellin was used to stimulate neutrophilic leukocytes of the blood of individuals immunized with a brucellosis vaccine, tularemia vaccine was used tularine, and anthraxin was used for anthrax vaccine. In addition, to assess the specificity of the neutrophil chemiluminescent reaction, chemiluminescence of cells of immunized individuals was induced by nonspecific antigens.

In order to compare the results of different methods for assessing the sensitization of people immunized with brucellosis, tularemia and anthrax vaccines, a leukolysis reaction was performed with the corresponding antigens (brucellin, tularia or anthraxin), a skin test was performed with anthraxin (in primary vaccinated with anthrax vaccine), the level was determined serum enzyme-linked immunosorbent assay. The research results are presented in table 5.

As can be seen from the data presented in table 5, a chemiluminescent analysis of blood neutrophils from people once and repeatedly immunized against tularemia showed an increase in the level of neutrophil CL after induction of cells by tularine, a significant level of CL CL in these people was ≥0.3. At the same time, in vaccinated with tularemia vaccine with a pronounced and strong CL reaction to the antigen, positive and sharply positive reactions of leukolysis with tularin were simultaneously observed (Spearman's correlation coefficient in these studies was 0.863, p <0.01).

An analysis of the chemiluminescent reaction of blood neutrophils from people immunized with a brucellosis vaccine revealed a significant increase in the level of neutrophil CL after stimulation of the cells with the immune complex. A significant level of CL CS in these subjects was ≥0.30. A statistical analysis of the results of applying the neutrophil CL detection method and leukolysis reaction showed a high correlation (r = 0.929, p <0.001). When analyzing the results of determining the chemiluminescent reaction of neutrophils in people once and repeatedly vaccinated with an anthrax vaccine, an increased reaction to anthraxin was established. A significant level of CL CS in these studies was ≥0.32. A high degree of reliability was confirmed when the reaction of leukolysis and a skin test to anthraxin was formulated, while the Spearman correlation coefficient was 0.762 (p <0.05) and 0.875 (p <0.01), respectively.

Thus, allergic restructuring of the body occurs individually and according to CL CL can vary in wide ranges from the complete absence of CL stimulation to a significant increase in the values of this indicator. It should be noted that tularemia vaccine has the greatest sensitizing effect and anthrax ulcer less.

Example 5. Chemiluminescence of neutrophils of people with allergic reactions to allergens feather pillows.

For the study, a suspension of neutrophils of people with hypersensitivity to allergens feather pillows was used. The work to identify specific sensitization was carried out according to the method described above. As a specific stimulator of the chemiluminescent reaction of neutrophils in individuals with hypersensitivity to the pillow feather allergen, a commercial pill pill allergen preparation was used, and brucellin was used as a non-specific antigen. At the same time, a leukolysis reaction with a specific allergen was carried out.

An analysis of the data presented in table 6 shows that the CL CS values of people who are allergic to pillow feathers ranged from 1.12 to 3.12. At the same time, there is a lack of stimulation of chemiluminescence of human neutrophils when exposed to a nonspecific antigen.

Table 1.
Information about the causal relationship between the totality of the essential features of the claimed object and the achieved technical results. Types of technical result and their dimension Actual or Estimated Indicators The explanation of what (the hallmark and / or their combination) made it possible to improve the performance of the proposed object in comparison with the prototype prototype the claimed object A method of obtaining a suspension of neutrophils. Multi-stage lysis of red blood cells with ammonium chloride, followed by washing with phosphate buffer. One-stage cell isolation using a separating liquid, a single wash with Hanks solution. Single-stage isolation of the neutrophil fraction allows to reduce cell damage, to obtain a neutrophil suspension with a high content of viable highly active neutrophilic leukocytes, with fewer impurities, which provides a better analysis. The concentration of neutrophils in the final sample, cells · ml ^-1 . 2 · 10 ⁶ (4-6) · 10 ⁶ An increase in the concentration of neutrophils, along with their improved quality (viability), provides an increase in the absolute recorded chemiluminescence indices, obtaining statistically significant differences in the intensities of the background and spontaneous chemiluminescence. The indicator of background chemiluminescence in calculating the measurement results. Not taken into account Taken into account Taking into account the results of background chemiluminescence provides an increase in the reliability of the analysis. The composition of the incubation medium. Phosphate buffer, pH 7.6-0.4 ml; glucose 5 mm - 0.1 ml; MgSO ₄ 0.37 mmol - 0.1 ml; luminol 0.65 mm - 0.2 ml Hanks solution, pH 7.4 - 0.7 ml; luminol 1 mm - 0.1 ml The use of a commercial Hanks solution provides optimal conditions for maintaining the viability of neutrophils, maintaining a stable pH value. The repetition of the number of measurements of the level of chemiluminescence. Absent. In at least three replicates. Re-registration of the chemiluminescence of the sample provides an increase in the reliability of the results of the study. Assessment of the significance of differences in chemiluminescence indices before and after stimulation. Not determined. It is determined. Assessment of the significance of differences in indicators before and after stimulation of neutrophilic leukocytes allows one to determine the fact of neutrophil stimulation by a specific immune complex.

Table 2.
The number of viable neutrophils in various cell preparation methods. Sample number

The proportion of viable neutrophils in the selection of cells (%)

using a separating liquid (by the proposed method) erythrocyte lysis (prototype) one 94 ± 0.18 25 ± 0.31 2 92 ± 0.44 32 ± 0.12 3 95 ± 0.43 18 ± 0.24 four 95 ± 0.41 19 ± 0.17 5 96 ± 0.78 20 ± 0.21 Medium for sample averages 94.2 ± 1.5 22.8 ± 5.8 Table 3.
Chemiluminescence indices of blood samples under various modes of preparation of a suspension of neutrophils. Sample number The method of separation of neutrophils Chemiluminescence intensity

mV background spontaneous one Using a separating liquid (according to the proposed method). 0.698 ± 0.007 4.174 ± 0.514 2 0.762 ± 0.008 6.646 ± 0.353 3 0.696 ± 0.004 4.752 ± 0.385 four 0.681 ± 0.008 4.512 ± 0.227 5 0.762 ± 0.007 6.438 ± 0.116 one The method of lysis of red blood cells (prototype). 0.697 ± 0.067 1.007 ± 0.261 2 0.865 ± 0.082 1.114 ± 0.107 3 0.781 ± 0.154 0.757 ± 0.048 four 0.759 ± 0.203 0.821 ± 0.206 5 0.875 ± 0.203 0.862 ± 0.134

Table 5.
Comparative characteristics of diagnostic methods for allergies in people once and repeatedly immunized with tularemia, brucellosis and anthrax vaccines. Conventional number of the subject Vaccine name The coefficient of stimulation, conventional units Leukolysis coefficient, conventional units Skin test, mm Total IgE, IU · ml ^-1 one tularemia 1.80 1.80 - 410 2 4.30 4.30 - 62.6 3 0.34 0.34 - 257.6 four 0.96 0.96 - 132.9 5 1,62 1,62 - 26.0 6 3.46 3.46 - 404 7 "0" "0" - 14.5 8 "0" "0" - 44,4 9 brucellosis "0" "0" - 12.5 10 1.83 0.31 - 28,2 eleven 2.96 0.40 - 501.3 12 1.06 0.31 - 279.9 13 "0" 0.18 - 47.5 fourteen 0.30 0.14 - 64.1 fifteen 0.91 0.28 - 187.3 16 "0" "0" - 75.6 17 anthrax "0" "0" 13 40,4 eighteen "0" "0" 16 187.3 19 0.37 0.4 40 75.6 twenty "0" 0.11 fifteen 0.1 21 "0" 0.18 twenty 31.1 22 "0" 0.12 eighteen 29.8 23 0.44 0.28 40 149 24 0.35 0.12 25 26.3 Notes: 1 “0” - stimulation coefficient less than a significant level;
2 - - not determined

Table 6.
Chemiluminescence indices of human blood samples when evaluating sensitization to pillow feather allergen. Conditional number of the researched The level of background chemiluminescence (X ± I ₉₅ ), mV

мВ:Neutrophil chemiluminescence intensity

mV: Values when using tularine spontaneous After adding the complex, serum with ... The coefficient of stimulation, conventional units Leukolysis coefficient, conventional units feather pillow allergen brucellin one 0.842 ± 0.004 4.174 ± 0.356 14.570 ± 0.483 5.035 ± 0.126 3.12 0.24 2 0.811 ± 0.006 6.646 ± 0.251 13.181 ± 0.758 6.870 ± 0.543 1.12 0.28 3 0.769 ± 0.005 5.364 ± 0.139 5.257 ± 0.107 5,408 ± 0,087 "0" * 0.12 four 0.711 ± 0.006 6.943 ± 0.265 6.457 ± 0.520 6,704 ± 0,183 "0" * "0" 5 0.837 ± 0.008 4,752 ± 0,149 9.646 ± 0.543 5.251 ± 0.127 1.25 0.11 6 0.753 ± 0.007 5.815 ± 0.074 6.009 ± 0.235 5.819 ± 0.504 "0" * "0" 7 0.802 ± 0.008 5,448 ± 0,127 5.639 ± 0.154 5.085 ± 0.496 "0" * 0.12 Notes: 1 "0" * - stimulation coefficient less than a significant level;
2 "0" - negative leukolysis coefficient

Claims (2)

1. The method of allergic diagnostics, including the selection of neutrophilic leukocytes, the formation of immune complexes and the measurement of chemiluminescence levels, characterized in that neutrophilic leukocytes are isolated by layering blood plasma on a separating liquid and centrifuged for 15 min at 1500 rpm, the neutrophil fraction is isolated from the middle layer, resuspended and washed with Hanks' medium, concentration of neutrophilic leucocytes Hank's solution is adjusted to 4 × 10 ⁶ -6 10 ⁶ cells / ml, the formation of immune complexes is carried out by mixing equal the amount of test blood serum and antigen prepared in physiological saline, incubated for 40 min at 37 ° C, measure the background luminescence of the incubation medium (A), consisting of 0.7-0.9 ml of Hanks solution and 0.1 ml of 0.1- 0.5 mmoluminolum solution, add 0.1 ml of a suspension of neutrophils, incubate for 5 minutes and measure the level of spontaneous chemiluminescence (B) over a period of 5-40 minutes from the end of the incubation period of neutrophils, then add 0.1 ml of the formed immune complex and determine the value of stimulated chemiluminescence (B ), calculate the stimulation coefficient according to the formula

and with a value of COP ≥ 0.3 diagnosed with an allergic reaction. 2. The method according to claim 1, characterized in that the evaluation of the indicators is carried out according to the results of at least three repeated measurements.