Method for allergodiagnostics by the values of chemiluminescent phosphorescence of neutrophils

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

 

The invention relates to methods of identifying specific sensitization of the organism to complete antigens of different nature. The method is designed to detect hypersensitivity to complete antigens, including assessment of allergic side effects of therapeutic and preventive medicines.

Recently there has been a General deterioration of allergic status of the population, the increase in the number of allergic diseases and reactions, including anaphylactic, due to unfavorable environmental conditions, chemical pollution, extensive use of chemicals in everyday life, abuse of drugs, the changing nature of power, the increase in the number of occupational allergens. Found that 1/5 of the population of our planet seeking medical help because of the different clinical manifestations of allergic nature (Macarize DS, Tarasova S.V. Dynamics of the prevalence of symptoms of allergic diseases according to ISAAC 1997-2000, Moscow. // Allergology and immunology. - 2002. Tom. 3. No. 2. - P.50-54). At present the antigens of different nature and structure is considered as inductors allergies (Picky VI, Adrianova, NV, Artamonova A.V. Allergic diseases. - M.: Medicine, 1984). On the territory of the Russian Federation C the last 15-20 years, the incidence of asthma has increased 3-4 times.(Fedosova YEAR and other Principles of diagnosis of allergic diseases. // Consilium medicum. - 2002. No. 4. - S-19). In 80-90% of cases of this disease has an infectious etiology (Picky VI, Adrianova, NV, Artamonova A.V. Allergic diseases. - M.: Medicine, 1984).

In principle, the detection of allergic realignment of the body is possible using two approaches : in vivo and in vitro.

Identification of the etiological factor of Allergy to the present time is based mainly on the results of skin tests (Picky VI, Adrianova, NV, Artamonova A.V. Allergic diseases. - M.: Triada-X, 1999. - Pp. 102-112). Statement of skin samples is diagnostic for the detection of specific sensitization of the organism by injection under the skin allergen and evaluating the magnitude and character developed under this edema or inflammatory reaction. The method is based on the interaction of allergen with sensitized T-lymphocytes and antigen-presenting cells (macrophages and cells of Langemark)that triggers the release of mediators of Allergy and development of local reactions in sensitised individuals (Fedosova YEAR and other Principles of diagnosis of allergic diseases. // Consilium medicum. - 2002. No. 4. - S-19). There are different methods of skin testing using allergens: prick-tests, scratch, application and intradermal about the s (Scepan N.A. Allergic diseases. Differentially diagnosis, treatment. - Minsk: Belarus, 2000. - P.35-43).

Currently one of the most commonly used skin allergodil is a prick-tests on specific allergen. Setting this test is performed by injection into the skin and the introduction of a solution of the allergen to a depth of no more than 1-1,5 mm, This skin test is considered less traumatic compared to scratch samples, it requires less skin surface for alergodiagnostiki, allowing you to explore a larger number of samples.

Scratch allergiesthe widely used for the detection of sensitization to food allergens, allergens, bacterial and fungal origin. However, their production often have false positive results, as well as in the formulation of transdermal and intradermal tests, e.g. tuberculin reaction is always positive in persons infected with nocardiae, Rhodococcus, corynebacteria. (Gushchin I.S. Allergic inflammation and its pharmacological action. M: Farmacos The Priit, 1998). Currently, experts of the European Academy of Allergology and Clinical Immunology not recommend the use of scratch skin test for the diagnosis of allergies due to their low information content (Fedosova YEAR and other Principles of diagnosis for allergic is olivani. // Consilium medicum. - 2002. No. 4. - S-19).

Intradermal tests are considered to be more sensitive than scratch, but they are less specific. (Gentle NM, Bobkova, L.P., Peter I.A. and other Allergology dictionary. Kiev: Naukova Dumka, 1986).

Thus, the common deficiencies of skin testing as a way to diagnose allergies include the following:

first, the method is unsafe for the subject, so as not eliminated the appearance of not only the (local) local and General reactions to the introduction of the allergen, and in the case of a high level of sensitization is the development of severe allergic complications. In addition to setting out samples requires an experienced, specially trained staff, impeccable technique of their performances, as well as the presence of a constant stock antishock drugs, which necessitates carrying out the reaction in the conditions of specialized allergotsentr;

secondly, the lack of expressnet - test results are taken into account in 24-48 hours;

third, the limited number of analyzed antigens.

Another method for diagnosing allergies is staging provocative tests. Provocative tests considered sufficiently reliable method of alergodiagnostiki. They are used in case of discrepancy of data of the anamnesis and R. the results of skin testing. Depending on the method of introduction of the allergen into the body distinguish the conjunctival, nasal, inhalation and sublingual provocative tests (Fedosova YEAR and other Principles of diagnosis of allergic diseases. // Consilium medicum. - 2002. No. 4. - C.13-19).

The lack of provocative tests is the frequent development of allergic complications, so provocative tests, limited to only one study per day (Picky VI, Adrianova, NV, Artamonova A.V. Allergic diseases. - M.: Triada-X, 1999. - Pp. 102-112). In addition, if the formulation requires specially trained personnel, as well as specially equipped premises for the conduct of inhalation provocation test.

In connection with the foregoing, is of particular relevance for the assessment of specific sensitization acquire methods of diagnostics of an Allergy in vitro, which is emphasized in the decision of the international group of experts-immunologists who. (Fedosova YEAR and other Principles of diagnosis of allergic diseases. // Consilium medicum. - 2002. No. 4. - C.13-19).

Famous instrumental method of assessing sensitization of the body to the allergen (antigen), based on detection of antibodies of class IgE in serum in vitro - radioallergosorbent test (PACT) (gentle NM, Bobkova, L.P., Peter I.A. and other Allergology dictionary. Kiev: Naukova Dumka, 986). The essence of the method lies in the connection specific IgE investigated civardi with allergen placed on a paper plate. After washing off the non-specific IgE are added to the sample labeled125I antibodies against IgE. The resulting radioactive complex is read on a scintillation counter (Picky VI, Adrianova, NV, Artamonova A.V. Allergic diseases. - M.: Triada-X, 1999. - Pp. 102-112). PACT is a rapid method, highly sensitive, specific and accurate as it is based on the use of radioactive labels (Scepan N.A. Allergic diseases. Differential diagnosis, treatment. - Minsk: Belarus, 2000. - P.35-43).

The disadvantage PACT is the need to use special expensive equipment, it has a lower sensitivity compared to skin breakdown, requires a regular supply of isotope-labeled components, specially equipped workplace and compliance with radiation safety measures when working with radioactively labeled anti-IgE (Fedosova YEAR and other Principles of diagnosis of allergic diseases. // Consilium medicum. - 2002. No. 4. - C.13-19).

Known also instrumental method for the determination of the content of General and allergenspecific IgE - setting enzyme-linked immunosorbent assay (ELISA). The principle of the method is essentially the same as PACT. Optima is Inoi concentration of allergen treated sorbent (polystyrene in the form of a blade), excess allergen pan, add the whey, resulting in the formation of complex antigen-antibody". Thereafter, unbound components are removed by laundering add anti-IgE (enzyme labeled), the resulting substrate is determined by a photometric method (Fradkin. VA Diagnostic and therapeutic drugs. - M.: Medicine, 1990). The method is sufficiently sensitive, specific and rapid, in addition, currently more often used to determine the allergen-specific IgE (Asimov A.G. ON the role of immunoglobin E in pathology. // Immun. Pat. Aller., infects. - 2002. No. 2. - S-77).

The disadvantage of immunoassay is the need for a special standardized preparations (test systems) to specific allergens, however, the choice of available systems is currently limited. In addition, the method demands high standard of production test and quality antigenic components, i.e. the reproducibility of the method significantly depends on the quality and manufacturer of the test system.

Also known a method of diagnosing Allergy - direct and indirect test degranulation of basophils, based on the detection of activation (degranulation) of immunocompetent basophilic leukocytes blood sensitized with IgE, when exposed to such cells specific what nigena (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 playback, high subjectivity of the results obtained, low expressnet, availability of laboratory animals in the indirect test is performed.

There is a method of assessing the presence of allergies in vitro by recording the activity of sensitized red blood cells by specific damage (destruction) of neutrophils (Vagrancy. Diagnosis of allergic reactions of neutrophils in the blood. - M., 1985).

For this approach, in common with the claimed method is a hypothesis about the interaction of specific immune complex allergen (antigen - antibody with specific receptors on neutrophils.

The disadvantages of this method include subjectivity, taking into account the results of the reaction, poor reproducibility and greater complexity. One of the major sources of error of this technique are errors in the preparation of fixed smears, as coloring, as well as the difficulty of ensuring accurate estimate of damaged cells before and after incubation of phagocytes (Ndibalema, Gschedule. Allergies in clinic and experiment. - M.: Medicine, 1979).

Closest to the claimed method is a method of detecting when debilizatsii of the body to bacterial antigens using chemiluminescent analysis of neutrophils in peripheral blood (Nchwaning, Nsemukila. Chemiluminescence of neutrophils in allergodiagnostic. // Clinically. laboratory diagnosis. 1999. No. 8. - P.18-20), including diagnosis of allergies in persons exposed to allergens fungal origin (C.albicans).

The method includes the following steps:

obtaining from a sample source blood neutrophil concentration 2×106CL·ml-1;

the formation in vitro of complex antigen-antibody;

adding the complex to the suspension;

the measurement of the chemiluminescence of the sample suspension.

To obtain the fraction of neutrophils using 1 ml of heparinized blood. A suspension of neutrophils prepared from heparinised whole blood by the method of lysis of erythrocytes. Every 300 ál blood emolicious 3 ml 0,083% solution of ammonium chloride, since the exposure 40 and reducing it to 20, 10 and 1 respectively. Termination of ammonium chloride achieve the introduction of a mixture of 7 ml of 1.5 M phosphate buffer (pH 7,2) followed by washing of the cells by centrifugation at 200 g for 7 min and resuspending in the same solution with a concentration of 2×10 CL·ml-1.

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

Chemiluminescent analysis is carried out with the COI is whether the device while BCL equals thousandth points-06. In cuvette make a reaction mixture containing 400 μl of phosphate buffer (pH 7,6), 100 µl of 5 mm glucose solution, 100 μl of 0.37 mm solution of magnesium sulfate and 200 μl of 0.65 mm solution lyuminola add 50 ál of lykousis, measure the level of spontaneous chemiluminescence. Then in the cuvette is made of 20 µl of serum immune complexes and record changes in luminescence for 7-8 minutes In the quality control of the use of autologous serum without antigen and the antigen solution without serum. Results using the ratio of the maximum chemiluminescence (Imax) to the level of the stationary glow (I0) - the so-called stimulation index (IP). IP=Imax/I0.

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

The disadvantages of this method are:

multistage procedure lysis of erythrocytes, causing significant damage and death to 60-70% of neutrophils, which negatively affects both chemiluminescent ability, and significantly reduces the concentration of cells, i.e., the level (amplitude) of the chemiluminescent signal.

In addition, results of studies in the prototype in the formula nailpolishes indicator background chemiluminescence reaction mixture, significantly distort the measurement result. It should be noted that used in the prototype, the concentration of neutrophils 2×106CL·ml-1is insufficient in terms of the measurement to obtain statistically significant differences of the values of the background chemiluminescence reaction medium and spontaneous chemiluminescence of neutrophils due to the lower absolute value of the digit indicator chemiluminescence (units c.u.).

The task of the invention to provide an improved method for the detection of specic sensitization of the body to antigens on the basis of registration chemiluminescence of blood neutrophils in vitro.

The problem is solved due to the fact that neutrophils isolated in the density gradient separating liquid, followed by washing of the cells in a sparing mode; the concentration of neutrophils leads to (4...6)·106CL·ml-1immune complexes receive, mixing the serum analyzed and the antigen (allergen), and incubated them; measure performance background values chemiluminescence incubation medium, spontaneous chemiluminescence included in the sample washed neutrophils and the intensity of chemiluminescence after addition of sample complex serum and analyzed allergen (antigen); calculate the ratio of the stimulus is tion; the evaluation of the reliability of the differences in performance before and after the stimulation is performed according to the results of not less than triplicate measurements.

The problem is solved by the fact that:

at the stage of preparation of the sample volume taken from the analyzed blood increased to 5 ml of heparinized and 5 ml najprimitivniji blood;

at the stage of allocation fractions of neutrophils is provided the use of liquids for the separation of cells (ficoll-urografin, ficoll-hipac, ficoll-urographine or any commercial mixture or liquid to allocate fractions of neutrophils);

the operation of the preliminary washing of neutrophils in a sparing mode (1000 rpm·min-1)minimally damaging neutrophilic leukocytes in the blood;

glow is logged on affordable for most laboratories the luminometer;

as a liquid, distributing a suspension of neutrophils, used commercial Hanks solution (pH 7.4)which provided optimal conditions for preservation of cell viability;

according to the results of the registration of the chemiluminescent light emission expect the coefficient of stimulation chemiluminescence and is determined by the significance level of its size.

The method includes the following steps:

1. Obtaining from a sample of blood suspensions of neutrophils with conc what Tracia (4-6)· 106CL·ml-1;

2. The formation in vitro of complex antigen-serum";

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

Step 1. To obtain suspensions of neutrophils using 5 ml of heparinized blood. The blood sample is incubated in a thermostat at 37°C for 40 minutes Away supernatant (plasma)containing fraction of leukocytes, and layered on 5 ml separating liquid, centrifuged for 15 min at 1500 rpm·min-1. The result is the division of the sample into three layers. The bottom layer consists of separating fluid in the middle layer is the fraction of neutrophils in the upper layer of the blood plasma. Carefully using a plastic pipette, collect the fraction of neutrophils. Highlighted cells resuspended colorless and washed with Hanks solution for 10 min at 1000 rpm·min-1. The concentration of polymorphonuclear leukocytes lead to (4-6)·106CL·ml-1breeding with Hanks solution.

Step 2. For the formation of immune complexes in vitro using serum analyzed and the antigen (allergen). Serum obtained from the same selected sample najprimitivniji blood by centrifugation at a speed of 1500 rpm·min -1within 15 minutes Cultivation antigens producing physiological solution. Equal amounts of serum and antigen solution (0.1 ml) incubated at 37±1°C for 40 minutes

Step 3. Measurement of chemiluminescence carried out using chemiluminometer.

In the cell of the device make 0,7...0,9 ml Hanks solution, add 0.1 ml of 0.1...0.5 mm solution lyuminola and determine the level of background chemiluminescence. Then add 0.1 ml prepared as described above, the suspension of neutrophils, mix, incubate 5 min and measure the level of spontaneous chemiluminescence, with registration value is in the range from 5 to 40 min after the end of the incubation period of suspension of neutrophils. For the induction of chemiluminescence response of neutrophils in the sample contribute 0.1 ml formed in vitro complex antigen-serum", again record the intensity of the chemiluminescence. The results were evaluated in terms of coefficient of stimulation (CS) chemiluminescence, which is determined by the formula:

where the level of CHL after stimulation, mV;

B - the level of spontaneous chemiluminescence, mV;

A - level background chemiluminescence, MB.

and when the value of KS≥0.3 to diagnose an allergic reaction.

To improve the reliability of results record the chemiluminescence samples prepara is built in at least three replicates (independently prepared from the initial suspension of neutrophils), determine the arithmetic mean and the confidence interval with 95% level of probability.

According to the results of studies to determine the significance level of the coefficient of stimulation chemiluminescence. The coefficient of stimulation meaningful subject to the availability of statistically significant differences in levels of spontaneous chemiluminescence of neutrophils and chemiluminescence after stimulation of antigen-serum", i.e. confidence intervals of the mean values of these parameters do not overlap. The causal connection between the set of essential features of the claimed object and achievable technical results are shown in table 1.

The ability of the proposed method is shown in the following examples.

Example 1. An estimate of the number of neutrophils when receiving the cell suspension using a separating fluid and method of lysis of erythrocytes.

The stages of obtaining a suspension of neutrophils through the fluid to separate the cells was performed according to the claimed method described above, the receipt of neutrophils method of lysis of erythrocytes was performed according to the above method Nchwaning (prototype).

Presented in table 2, the data suggest that the allocation of fractions of neutrophils from human blood using a separating liquid is observed the death of 4-8% glue is OK, while the death of neutrophils, isolated by the method of lysis of erythrocytes, was 68-82%.

Example 2. Evaluation of chemiluminescent reactions of neutrophils (spontaneous chemiluminescence) in the preparation of sample cells using the separating fluid and method of lysis of erythrocytes.

For studies used a suspension of neutrophils obtained in experiment 1. The chemiluminescent reaction of neutrophils was measured using a luminometer LKB-1251. The reaction medium was prepared according to the above methods, the concentration of the cells was (4-6)·106CL·ml-1in the present method and (1-2)·106CL·ml-1in the prototype.

Analysis of the results of the Desk chemiluminescence, are presented in table 3, showed that the level of spontaneous chemiluminescence of neutrophils, isolated by the method proposed method is much higher than in case of separation of cells according to the method proposed in the prototype, in addition, when you register chemiluminescence of neutrophils obtained by the method Nchwaning, we observed no statistically significant differences in the intensity level of the background and spontaneous chemiluminescence.

Example 3. Assessment of the level of sensitization of the organism Guinea pigs, once immunized with Brucella vaccine.

Identify specific phenomenon is legislatie in response to the introduction of vaccines was conducted on Guinea pigs - animals that are recommended for the assessment of side effects of vaccines, using a live commercial Brucella vaccines (units 19-VA), which was injected subcutaneously with 0.5 ml with a concentration of 2·109SOME·ml-1. The chemiluminescent reaction of neutrophils Guinea pigs was estimated at 35 days after immunization, with the final concentration of neutrophils was 2·106CL·ml-1. As a specific antigen in these experiments used a commercial preparation of brucellin. To determine the specificity of the chemiluminescent reaction to Brucella antigen as a non-specific antigen used commercial preparation of tolaria. Once served the reaction lakulisa and production of skin tests using Brucella.

From the data presented in table 4, it is seen that in the group of animals in 11 of 20 (55%) immunized Guinea pigs showed a significant increase in the level of chemiluminescence after the induction of specific complex antigen - antibody compared with the values of spontaneous chemiluminescence (p<0,05). The remaining 9 animals the value chemiluminescence after exposure of brucellin did not differ from the level of spontaneous chemiluminescence (p>0,05) indicating the absence of stimulation by chemiluminescence of neutrophils specific and what lerena of these animals. According to the results of the statistical analysis significant level KS chemiluminescence in these studies was ≥0,71. In this same group of animals was observed reactions expressed in skin test and a positive reaction lakulisa. Statistical analysis of the results of the assessment factor stimulation chemiluminescence, skin tests and reaction lakulisa in the experimental animals showed good convergence (Spearman correlation coefficient 0,879 (p<0.001) and 0,795 (p<0,001) for skin tests and lakulisa respectively). When analyzing the intensity of the chemiluminescence of neutrophils in the blood of immunized Guinea pigs after addition of the nonspecific complex cularin serum reliably observed no stimulation of leukocyte chemiluminescence. This fact indicates the specific nature of the induction of the chemiluminescent reaction of neutrophilic leukocytes antigens of Brucella vaccines.

Example 4. Assessment of the level of sensitization of humans, once and repeatedly immunoserologic tularemia, brucellosis and anthrax vaccines.

Chemiluminescent analysis of neutrophils people immunized tularemia vaccine was carried out in accordance with the methodology of the proposed method. During the chemiluminescent analysis to stimulate neutrophil l is nocito blood of persons immunized with Brucella vaccine, used brucellin, tularemia vaccine - cularin, anthrax vaccine - anthracen. In addition, to assess the specificity of the chemiluminescent reaction of neutrophils was carried out by the induction of chemiluminescence cells of immunized persons nonspecific antigens.

In order to compare results of different methods for assessing the sensitization of people immunized with Brucella, tularemia and anthrax vaccines, performed the reaction leucolepis with the corresponding antigens (brucellin, toleenum or anthracinum), the production of skin tests with anthracinum (initially vaccinated anthrax vaccine) and determined the levels of total IgE in serum enzyme-linked immunosorbent assay. The results are shown in table 5.

As can be seen from the data presented in table 5, chemiluminescent analysis of neutrophils in the blood of people, once and repeatedly immunized against tularemia, showed higher level of chemiluminescence of neutrophils after induction of the cells with toleenum, significant level KS CHL these people amounted to ≥0,3. While in vaccinated tularemia vaccine with rezkovyrazhennom and strong chemiluminescence reaction to the antigen at the same time reported a positive and strongly positive reaction leucolepis with toleenum (Spearman correlation coefficient is these studies put 0,863, p<0,01).

The analysis of the chemiluminescent reaction of neutrophils in the blood of people immunized with Brucella vaccine, it was found a significant increase in the level of chemiluminescence of neutrophils after stimulation of cells of the immune complex. Significant level KS CHL these studied amounted to ≥0,30. Statistical analysis of the results of the application of the method of registration chemiluminescence of neutrophils and reaction leucolepis showed a high correlation (r=0,929, p<0,001). When analyzing the results of determination of the chemiluminescent reaction of neutrophils people, once and repeatedly vaccinated anthrax vaccine, was established increased reaction to anthracen. Significant level KS CHL in these studies was ≥0,32. A high degree of reliability is confirmed when setting reaction lakulisa and skin tests on anthracen, while the Spearman correlation coefficient was 0,762 (p<0.05) and 0,875 (p<0,01), respectively.

Thus, allergic reorganization of the organism occurs individually and on CA PI can vary in wide ranges from no stimulation of PI to a significant increase of the values of this index. It should be noted that the greatest sensitizing action has tularemia vaccine and less - anthrax.

Example 5. Chemiluminescence of neutrophils people with al is ergicheskie reactions to allergens feather pillows.

For studies used a suspension of neutrophils people with hypersensitivity to allergens feather pillows. The identification of specific sensitization was performed according to the method described above. As a specific stimulator of the chemiluminescent reaction of neutrophils in individuals with hypersensitivity to the allergen feather pillows used commercial preparation of allergen feather pillows, as a non-specific antigen - brucellin. At the same time carried out the reaction of lakulisa with a specific allergen.

Analysis of the data presented in table 6 shows that the values of KS holdem people who are allergic to feather pillows, made from 1,12 to 3.12. At the same time there is a lack of stimulation of the chemiluminescence of neutrophils in the blood of people when exposed to a non-specific antigen.

Table 1.

Information about the causal relationships between the set of essential features of the claimed object and achievable technical results.
The technical result and their dimensionThe actual or estimatedExplanation due to which (hallmark and/or their combination) is made possible improvement is giving indicators of the proposed facility compared to the prototype
prototypedeclare object
A method of obtaining a suspension of neutrophils.Multi-lysis of red blood cells with ammonium chloride followed by washing with phosphate buffer.One-step selection of cells by using a separating liquid, single washing with Hanks solution.Single-stage allocation fractions of neutrophils reduces damage to cells, to obtain a suspension of neutrophils with high content of viable high level of neutrophilic leukocytes, with fewer impurities, which provides a more qualitative analysis.
The concentration of neutrophils in the final sample, CL·ml-1.2·106(4-6)·106The increase in the concentration of neutrophils along with superior quality (viability) provides an increase in the absolute measured characteristics chemiluminescence, obtaining statistically significant differences of intensity values of the background and spontaneous chemiluminescence.
Indicator background chemiluminescence when calculating the measurement results.IgnoredRecordedRecords of the results of the background chemiluminescence provides significantly increase the tee analysis.
The composition of the incubation medium.Phosphate buffer, pH of 7.6-0.4 ml; glucose 5 mm, 0.1 ml; MgSO40,37 mm - 0.1 ml; luminal 0.65 mm - 0,2 mlThe Hanks solution, pH 7.4, 0.7 ml; 1 mm luminal - 0.1 mlThe use of commercial Hanks solution provides optimal conditions to maintain the viability of neutrophils, to maintain a stable pH.
The repetition number of measurements of the level of chemiluminescence.Is missing.At least three replications.Re-register chemiluminescence of the sample ensures the reliability of research results.
Reliability estimation of differences in rates of chemiluminescence before and after stimulation.Not determined.Defined.Reliability estimation of differences in performance before and after stimulation of polymorphonuclear leukocytes allows you to determine whether stimulation of neutrophil specific immune complex.

Table 2.

The number of viable neutrophils with different ways of preparation of cells.
No. sampleThe proportion of viable neutrophils in the allocation of cells (%)
using the separating fluid (proposed method)method of lysis of erythrocytes (prototype)
194±0,1825±0,31
292±0,4432±0,12
395±0,4318±0,24
495±0,4119±0,17
596±0,7820±0,21
The average for the selective medium94,2±1,522,8±5,8
Table 3.

Performance chemiluminescence samples blood samples at various modes of preparing suspensions of neutrophils.
No. sampleThe method of separation of neutrophilsThe intensity of chemiluminescencemV
backgroundspontaneous
1With the help of separating liquid (the proposed method).0,698±0,0074174± 0,514
20,762±0,0086,646±0,353
30,696±0,004of 4.752±0,385
40,681±0,0084,512±0,227
50,762±0,0076,438±0,116
1Method of lysis of erythrocytes (the prototype).0,697±0,0671,007±0,261
20,865±0,0821,114±0,107
30,781±0,1540,757±0,048
40,759±0,2030,821±0,206
50,875±0,2030,862±0,134

td align="center"> -
Table 5.

Comparison of methods for the diagnosis of allergies in people, once and repeatedly immunized tularemia, brucellosis and anthrax vaccines.
Conditional number issleduemogoName of vaccineThe coefficient of stimulation, usledThe coefficient l is nalysis, usedSkin test, mmTotal IgE, IU·ml-1
1tularemia1,801,80-410
24,304.30-62,6
30,340,34-257,6
40,960,96-132,9
51,621,62-26,0
6of 3.46of 3.46-404
7"0""0"-14,5
8"0""0"-44,4
9brucellosis"0""0"-12,5
101,830,31-28,2
112,960,40-501,3
121,060,31-279,9
13"0"0,18-47,5
140,300,1464,1
150,910,28-187,3
16"0""0"-75,6
17anthrax"0""0"1340,4
18"0""0"16187,3
190,370,44075,6
20"0"0,11150,1
21"0"0,182031,1
22"0"0,121829,8
230,440,2840149
240,350,122526,3
Notes: 1 "0" - factor stimulation less than significant level;

2 not defined

Table 6.

Performance chemiluminescence samples blood samples of people, when evaluating the sensitization to the allergen feather pillows.
The conditional number of the investigated The level of background chemiluminescence (X±I95), mVThe intensity of the chemiluminescence of neutrophilsmV:Values when using tolaria
spontaneousAfter adding complex serum with...The coefficient of stimulation, usledThe ratio lakulisa, usled
allergen pen down pillowsbrucellin
10,842±0,0044,174±0,35614,570±0,4835,035±0,1263,120,24
20,811±0,0066,646±0,25113,181±0,7586,870±0,5431,120,28
30,769±0,0055,364±0,1395,257±0,1075,408±0,087"0"*0,12
40,711±0,0066,943±0,2656,457±0,5206,704±0,183"0"*"0"
50,837±0,008of 4.752±0,1499,646±0,5435,251±to 0.1271,250,11
6 0,753±0,0075,815±0,0746,009±0,2355,819±0,504"0"*"0"
70,802±0,0085,448±to 0.1275,639±0,1545,085±0,496"0"*0,12
Notes: 1 "0"* - stimulation coefficient less than significant level;

2 "0" - factor leucolepis negative

1. How alergodiagnostiki, including the allocation of neutrophilic leukocytes, formation of immune complexes and measurement of the levels of chemiluminescence, characterized in that neutrophilic leukocytes secrete layup by blood plasma for separating liquid and centrifugation for 15 min at 1500 rpm, the fraction of neutrophils isolated from the middle layer, resuspended and washed environment Hanks, the concentration of polymorphonuclear leukocytes with Hanks solution was adjusted to 4·106-6·106cells/ml, the formation of immune complexes is carried out by mixing equal amounts of the studied serum and antigen prepared in saline solution, incubated for 40 min at 37°To measure background luminescence incubation medium (A), consisting of 0.7 to 0.9 ml of Hanks solution and 0.1 ml of 0.1-0.5 mm solution lyuminola, add 0.1 ml of a suspension of neutrophils, in ubermut 5 min and measure the level of spontaneous chemiluminescence (B) in the time interval 5-40 min after the end of the period of incubation of neutrophils, then make a 0.1 ml of the formed immune complex and determine the value of the stimulated chemiluminescence (B)calculate the coefficient of stimulation by the formula

and when the value of KS≥0.3 to diagnose an allergic reaction.

2. The method according to claim 1, characterized in that the assessments are carried out on the results of at least three repeated measurements.



 

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SUBSTANCE: method involves preparing smears from clinical material taken in inspected patients and carrying out analysis for the presence chlamydia, mycoplasma and ureaplasma in smears by method of fluorescent antibodies. The total amount of cells damaged by these pathogens is determined. In 2-7 days from the treatment onset smears are taken again and analysis of clinical material is repeated by the same method. The high sensitivity to antibiotic in this microorganism is estimated in detection <5% of epithelial cells in smear containing bright-green granules of the corresponding agent, and the prescribed treatment course with the same antibiotic is continued. The low sensitivity to antibiotic is estimated in detection ≥5% of above indicated specifically luminescent granules of antigen, and in this case a medicinal preparation is replaced. Method provides carrying out determining the effectiveness of antibiotics effect for the first days after their prescription and to correct their prescription operatively. Method is simple and doesn't require the preliminary isolation of the pathogen culture.

EFFECT: improved method for express-control.

5 ex

The invention relates to medicine, in particular to obstetrics, and can be used for prediction of preeclampsia in women undergoing severe preeclampsia

The invention relates to medicine and relates to a method of diagnosis of salmonellosis
The invention relates to medicine, namely to Allergology and immunology, and can be used to diagnose allergies
The invention relates to medicine, more specifically to the spectral-fluorescent methods diagnosis of benign and malignant neoplasms

The invention relates to immunodiagnostics, in particular to methods for detecting low concentrations of the analyte in the sample by improving the signal/background

The invention relates to immunological methods of analysis and can be used for mass screening of congenital anomalies in newborns, control of the immune and hormonal status, some infectious diseases and reproductive function of women

The invention relates to medicine, in particular for Microbiology, and the receipt antitoxoplasma immunoglobulin to detect Toxoplasma in pathological material direct fluorescent antibody (IFA)

The invention relates to medicine, in particular to immunology, and can be used in clinical and laboratory practice
The invention relates to medicine, namely, Allergology and can be used to identify food allergies in adults and children

FIELD: medicine, microbiology.

SUBSTANCE: method involves preparing smears from clinical material taken in inspected patients and carrying out analysis for the presence chlamydia, mycoplasma and ureaplasma in smears by method of fluorescent antibodies. The total amount of cells damaged by these pathogens is determined. In 2-7 days from the treatment onset smears are taken again and analysis of clinical material is repeated by the same method. The high sensitivity to antibiotic in this microorganism is estimated in detection <5% of epithelial cells in smear containing bright-green granules of the corresponding agent, and the prescribed treatment course with the same antibiotic is continued. The low sensitivity to antibiotic is estimated in detection ≥5% of above indicated specifically luminescent granules of antigen, and in this case a medicinal preparation is replaced. Method provides carrying out determining the effectiveness of antibiotics effect for the first days after their prescription and to correct their prescription operatively. Method is simple and doesn't require the preliminary isolation of the pathogen culture.

EFFECT: improved method for express-control.

5 ex

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

FIELD: medicine, in particular oncology and hematology.

SUBSTANCE: claimed method includes determination of blood cell composition wherein absolute lymphocytes and CD34-lymphocytes in peripheral blood and percent ratio of CD34-cells to lymphocytes amount is calculated according to the next equation: CD34 % = CD34 abs./lymphocytes abs. x 100 %, wherein CD34 % is relative amount of CD34-lymphocytes; CD34 abs.; lymphocytes abs. is absolute lymphocyte amount. When patient treated by chemotherapy has CD34-lymphocyte content from to 0-7 %, infective complication will not be observed, and when said value is 7.1 % or more, there is danger of generalized infection.

EFFECT: earlier and more perfect determination of chemotherapy infective complications in patients.

3 ex, 1 tbl

FIELD: biology, medicine.

SUBSTANCE: invention relates to nano-size composite material for DNA/RNA adsorption and desorption in form of nano-particles comprising of 80-99.5 mass % of tin oxide (SnO2) with tetragonal crystal lattice and 20-0.5 mass % of substance selected from group containing Fe, Fe2O3, Fe3O4 or mixture thereof. Said material makes it possible to increase specific capacity of DNA/RNA adsorption and desorption at desired magnetization of composite material, in particular being necessary for magnetic deposition.

EFFECT: new nano-size composite material for DNA/RNA adsorption and desorption.

1 tbl

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to infectious diseases. It involves simultaneous analysis of blood serum for reaginic and treponema-specific antibodies to cardiolipin antigen and recombinant T pallidum protein complex with molecular weight 15, 17, 39, 41, 42, 44.5, 47 kDa, immobilised on microscope aldehyde slides to detect antibodies by a conjugate solution containing Cy5 phosphor tagged human IgG antibodies and Cy3 phosphor tagged human IgM antibodies; slide scanning in a multichannel biochip scanner; automatic data analysis in a program used to convert fluorescent signals to digital positiveness coefficients; and qualitative presentation. Reaginic and treponema-specific antibodies found in a sample indicate syphilis, while no reaginic and treponema-specific antibodies found in the sample shows the absence of syphilis in a patient.

EFFECT: method allows for differentiated detection of antibodies to the most immunogenic T Pallidum antigens produced at the different stages of disease, for the objective automated data record, for cutting examination and diagnosing time.

4 cl, 2 ex

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method involves preparing a biochip containing immobilised antibodies to various antigens, incubating the biochip with analysed cell suspension for 1-2 hours at room temperature, washing off the biochip from unconjugated cells and detecting an antigen-antibody reaction with using an UV light source. A bacterial cell suspension is taken in the concentration of min. 1x105 microbial cell/ml inactivated in any method not destroying the analysed antigen and resuspended in a buffer solution containing Tris HCl 50 mM, 0.02 % Tween 20 and NaCl 50 mM and propidium iodide in the concentration of 1.0-2.0 mcg/ml and used to coat the biochip surface in an amount sufficient to coat all its segments.

EFFECT: invention provides more simple and cheap method to detect the presence of bacterial antigens.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: for the purpose of diagnosing metabolic compensation disturbances and predicting a risk of developing complications in the patients suffering type 2 diabetes mellitus (type 2 DM), peripheral venous blood is examined for a percentage of apoptotic lymphocytes An+/Pl-. The lymphocyte An+/Pl- value below 3.5 % enables diagnosing a condition of carbohydrate metabolism compensation and predicting a favourable clinical course of the disease for the following year. The lymphocyte An/PI- content within the range 3.5 to 6% provides diagnosing a condition of subcompensation and a moderate risk of developing complications. The An+/Pl- value exceeding 6 % described a condition of decompensation and a high risk of developing chronic complications of type 2 diabetes mellitus.

EFFECT: higher accuracy of prediction and reduction of the risk of developing complications in diabetic patients.

4 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for producing a polyclonal antiserum which is immunospecifically bound to a biologically active parathyroid hormone (PTH) in a part of three or four N-terminal amino acid residues of (1-84) PTH. The first peptide antigen is introduced in a host animal other than a human. A titre of produced antibodies is monitored. Antiserum is extracted. At least one antibody is separated and recovered from antiserum by affine chromatography with using the second peptide antigen. It is followed by removing at least one antibody possessing specificity with respect to the peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH by affine chromatography with using the third peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH respectively. There is recovered polyclonal serum possessing binding affinity with respect to no more than the first three or four amino acid residues of the N-terminal part of PTH and possessing no affinity with respect to any amino acid residues in the fifth amino acid residue or after the fifth amino acid residue of the N-terminal of PTH.

EFFECT: determining the level of biologically active intact PTH in serum, plasma or cell culture medium.

6 cl, 4 dwg

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