Method for anti-immunoglobulin e antibody neutralisation activity test

FIELD: medicine.

SUBSTANCE: invention refers to immunology and represents a method for anti-immunoglobulin E antibody neutralisation activity test, which consists in binding the test antibodies with human IgE in a solution, incubating the above solution with human peripheral blood, inducing cell degranulation and determining a cell portion having a high expression level of CD63 (CD63high) surface marker in a basophile population with phenotype CD123+HLA-DR-.

EFFECT: invention enables considerably reducing the length of analysis and its labour intensity.

2 dwg, 2 ex

 

The invention relates to immunology, in particular to methods of determining a neutralizing activity of antibodies to immunoglobulin E person, and can be used in biotechnology, pharmaceuticals and medicine, for example, in the diagnosis and treatment of allergic diseases, in particular bronchial asthma.

Bronchial asthma is a serious chronic disease of the respiratory tract is the most common pathology among allergic and respiratory diseases [Allergic diseases: diagnosis and treatment. Edited by R. Patterson, L. K. Grammer, P. A. Greenberg, Moscow, Medicine, 2000]. In Russia, according to various sources, bronchial asthma suffer from 2.2 to 5-7% of the population, i.e. from 3 to 9.8 million people, most of them hurt in a severe form, in which the ineffective treatment with corticosteroids.

The manifestation of bronchial asthma is an allergic response mediated by immunoglobulin E (IgE). When an antigen, such as pollen, house dust or other allergen that enters the body, in the blood appear IgE specific to this antigen that bind to receptors on the surface of basophils and mast cells. Upon further contact with the antigen, the antigen binds to IgE with the formation of the complex antigen-IgE-receptor, which leads to an immediate activation of the receptor � held in the cell signal causing the degranulation of the cells, in which chemical mediators (mainly histamine and leukotrienes) are released from the cell pellets and enter the bloodstream with subsequent development of allergic symptoms.

Conventional approaches to the treatment of allergies, particularly asthma, are systemic therapy antagonists chemical mediators, such as antihistamines and steroids. The application of these antagonists directed to the desensitization of patients and decrease inflammation, but it provides only symptomatic therapy, as it is not aimed at interfering in the interaction of IgE with the cellular receptor that triggers an allergic reaction. Furthermore, the use of steroids leads to General inhibition of the immune response, resulting in certain unwanted side effects.

The currently developed method of treatment based on the use of polypeptides that block the binding of IgE with Fc-receptors on the cell surface and is able to displace IgE complex with the receptor, if IgE is already associated with him. The most promising is the use for this purpose antibodies to IgE. The IgE molecule consists of two light and two heavy chains, stitched together by disulfide bonds. Each chain is composed of variable and constant regions, the first of which about�is in charge of binding to the antigen, and the second - for interaction with the receptor. In this case the constant region of the heavy chain of IgE is composed of four sections, denoted by Ch1, Ch2, Ch3, Ch4. High-affinity binding of IgE with Fc receptorεRI is the result of a complex interaction plot Ch3 IgE to α-subunit of the receptor. To prevent this proposed binding antibodies, in particular omalizumab [RU 2242515], TES-C21 [WO 2004070011], BSW17 [RU 2193413]. On the basis of the humanized antibody E25 (omalizumab) is manufactured drug "our Department", which is an IgG1kappaantibody that contains human framework with complementarity determining sites of murine antibodies that bind IgE [health.mail.ru/drug/xolair/].

Currently in development other humanized and human monoclonal antibodies, however, to select the optimal treatment technology, enabling fast and reliable neutralization of the biological activity of immunoglobulin E (IgE) human antibodies it is necessary to develop methods to compare their effectiveness.

At the present time to determine the biological activity of the immunoglobulin E person is offered, generally, methods based on the use of animal cell lines stably expressing the Fc receptorεRI man or his alpha subunit [Hakimi J, Seals, ondas JA, Pettine L, Danho W, Kochan J. The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding. J Biol Chem. 1990; 265(36): 22079-81]. The binding of IgE to the cells of such lines with the subsequent interaction associated with IgE antibodies to IgE with anavilhanas activity, leads to degranulation of the cells, which may be registered, in particular, mobilization of cellular calcium [WO 2008099188]. The disadvantage of the above methods is their complexity and duration.

Also, the known methods based on the use of human basophils. The isolated human basophils or heparinized blood was incubated with IgE, and then induce the degranulation of the cells by treatment with antibodies to IgE with anavilhanas activity. Next, the cells are precipitated, the supernatant and cell lysates, obtained by repeated freezing and thawing acetylate histamine, the number of which is determined by using enzyme-linked immunosorbent assay (RU 2242515). Neutralization of the biological activity of immunoglobulin E antibodies is determined by the concentration of the antibodies that causes 50% inhibition of the biological activity of IgE in a standard experimental conditions.

Thus, in the patent WO 2008123999 cells RBL-2H3(FcεRI) was incubated with human IgE antibodies and studied within 48 hours, then washed free from IGE-anti-IgE complexes, leaving on the surface of the glue�OK only IgE, associated with the receptor FcεRI. These bind IgE polyclonal antibodies, causing degranulation of the cells, the level of which is determined by the release of histamine, as determined by ELISA. The disadvantage of this method is its length and complexity

The closest to the claimed technical essence is a method of determining a neutralizing activity of antibodies using cells basophilic cell line of rat RBL-2H3, transfected with DNA expressing the Fc receptorεRI(WO 2008099188). After isolation of a clone stably expressing FcεRI, the cells are cultured in the presence of an antibiotic that support the expression of the receptor. To determine neutralizing activity of antibodies 5×104cells seeded in cell digitizer for cell cultivation and pokasivaut at 37°C in medium containing 5% CO2with the addition of 400 μg/ml of the antibiotic G418 for 18-24 hours, after which the cells are repeatedly washed and added to them human IgE together with dilutions of antibodies. The degree of degranulation judged by mobilization of cellular calcium, the kinetics of which (80 measurements at intervals of 1 h and 40 measurements with an interval of 8 h) register high-speed photometer).

The disadvantage of this method is its length and complexity.

The problem solved by the authors, yavl�moose create a more rapid and simple method for determining the neutralizing activity of antibodies to IgE.

The technical problem consisted in the exclusion from the process the most time-consuming stages through the use of surface marker CD63, which comprises a group of proteins that are components of lysosomal membranes and transported to the cell surface upon activation of basophils and other cells.

The proposed solution to the problem is based on the binding with the receptor FcεRI basophils free (neitralizovannom) IgE. After washing cells from IgE, investigational antibody complexes and antibody-IgE binding of excess known antibodies to IgE (polyclonal antibodies goat to human IgE) with IgE-FcεRI causes the degranulation of basophils and allows to evaluate the neutralizing capacity of antibodies.

The technical result is achieved due to the fact that containing basophils drug - heparinized blood donor were incubated for not less than three mixtures studied with IgE antibodies of a person, containing different ratios of antibody at 37±1°C, washed with centrifugirovania, then add polyclonal antibodies goat to the IgE of a human and again incubated at 37±1°C for 30-40 min. the Reaction is stopped by the introduction of ethylenediaminetetraacetate (EDTA), is added, labeled with different fluorescent-labeled antibodies to CD63, CD 123 and HLA-DR, incubated at room temperature in the dark for 30-35 minutes then Lizzie�comfort erythrocytes, washed mixture by centrifugation and analyzed in a flow cytometer, followed by calculation of the percentage of activated cells with the phenotype of CD63highfrom a pool of cells with the phenotype CD123highHLA-DR-. As a positive control, use formyl-methionine-leucine-phenylalanine, and as a negative control - indicator cells incubated in the absence of immunoglobulin E and the indicator cells incubated with immunoglobulin E, previously associated with an excess of neutralizing antibodies to immunoglobulin E, and state in case of detecting a dose-dependent increase in the proportion of cells CD63higha population of cells with the phenotype CD123+HLA-DR-what investigational antibodies are referred to anavilhanas, and when it detects a dose-dependent reduction in the proportion of cells CD63high in the study population believes that the investigational antibody refers to neutralizing with the calculation of the concentration of the antibodies according to the formula:

% neutr = 100×(1-(%ABop-%ABa-IgE)/(%ABTo IgE-%ABa-IgE)),

where % neutr - the percentage of neutralization;

%ABop- the percentage of activated basophils in the presence of an appropriate concentration of the investigated antibodies;

%ABa-IgE- the percentage of activated basophils in the sample control anti-IgE;

%ABTo IgE- the percentage of activated basophils in the sample control IgE b�W of an investigational drug.

The analysis is as follows.

The investigated antibodies previously retitrement and incubated for 1 hour with a solution of human IgE with constant agitation on an orbital shaker at room temperature (50 µl of each dilution of antibody and 50 µl of a solution of human IgE with a concentration of 20 μg/ml), after which the mixture was added to 100 ál of heparinized donor blood and incubated at 37°C for 1 hour. Cells are washed twice by low-speed centrifugation, add a polyclonal antibody goat-to-human IgE (50 μg/ml) in a volume of 10 µl and again incubated for 30 minutes at 37°C. stop the Reaction by adding 10 ál of EDTA (20 mm) followed by incubation at room temperature for 15 minutes. Further to the sample add 20 ál of the mixture labeled with different fluorescent-labeled antibodies to CD63, CD 123 and HLA-DR (commercial kit BD FastImmune CD63 FITC/CD123 PE/Anti-HLA-DR PerCP). After incubation at room temperature in the dark for 30 minutes to produce the lysis of erythrocytes by introducing 2 ml of lysing solution (0/75% solution of ammonium chloride) followed a three-time washing by centrifugation. Further samples analyzed in a flow cytometer, followed by calculation of the percentage of activated cells with the phenotype of CD63highfrom a pool of cells with the phenotype CD123highHLA-DR-. As positive control, use formyl-methionine-leucine-phenylalanine (fMLP) (Sigma), as a negative control to determine the rate of cells incubated in the absence of IgE, as well as indicator cells incubated with IgE, previously associated with excess neutralizing antibodies to IgE.

The invention is illustrated by the following diagrams.

Fig.1 shows the results of neutralization of the biological activity of known immunoglobulin E antibodies against human IgE "our Department". Fig.2 presents an assessment of neutralizing activity of mouse antibodies against human IgE, data flow cytopfuorometry, analysis of CD123+HLA-DR - basophils. Numbers on histograms indicate the percentage of cells vysokoagressivnyh CD63. And control polyclonal anti-IgE antibodies; B - positive control (fMLP); - control of known neutralizing antibodies ("our Department", 5 µg/ml); D and e - 5 µg/ml and 1 μg/ml antibody clone C; f and G - 5 µg/ml and 1 μg/ml antibody clone V respectively.

Industrial applicability the invention is illustrated by the following examples.

Example 1. Determined the neutralization of the biological activity of immunoglobulin E (IgE) human recombinant humanized antibody to human IgE (commercial preparation "our Department").

Detection of activated basophils was performed using the set BD FastImmune CD63 manufacturer's instructions. As a positive control used forms�l-methionine-leucine-phenylalanine (fMLP) (Sigma) at a final concentration of 2 μg/ml. The findings were calculated using the flowing cytofluorimetry EPICS XL (Beckman Coulter) in tri-color mode. Basophils in the analysis were identified as CD123+HLA-DR-cells and then determined the percentage of basophils, vysokoagressivnyh CD63 (CD63high).

The results of the analysis are shown in Fig. 1.

From Fig. 1 shows that the antibody "our Department" dose-dependent decrease in the percentage of basophils activated as a result of incubation with human IgE and the subsequent processing of polyclonal antibodies to IgE. IC50 in the experiment amounted to 0,515 μg/ml.

Example 2. Determined the neutralizing activity of murine monoclonal antibodies clones C and V against immunoglobulin E (IgE).

Mouse monoclonal antibodies (clones C and V) against human IgE were obtained using hybrid technology. Neutralizing antibody activity was studied as described above, in the test using a final concentration of 5 µg antibody/ml and 1 μg/ml as positive control, neutralization of the drug was used "our Department" at a concentration of 5 µg/ml. the Results of running cytopfuorometry shown in Fig. 2. From Fig. 2 shows that by increasing the concentration of antibodies IS increased in the percentage CD63highactivated basophils, indicating that this antibody clone is anaphylactogenic. In the presence of antibody clone W ima�t a complete suppression of the activation of basophils, indicating that neutralizing properties of antibodies of the clone.

Comparison of proposed method with the analogues showed that when using the analysis time is reduced from 20-30 to 4 hours, thus achieves a significant reduction of labor costs.

The method for determining the neutralizing activity of antibodies to immunoglobulin E, which includes the interaction of basophils with antibodies and immunoglobulin E, human and the laundering of cells from the excess antibody, the analysis of physico-chemical parameters of the mixture and the determination of a significant parameter, wherein the pre-prepare at least three mixtures of antibodies with immunoglobulin E person, who then incubated with heparinized blood donor at 37±1°C, and after washing excess add immunoglobulin polyclonal antibodies goat to the IgE of a human and again incubated at 37±1°C for 30-40 min, then the reaction is stopped by the introduction of ethylenediaminetetraacetate, add labeled with different fluorescent-labeled antibodies to CD63, CD123 and HLA-DR, incubated at room temperature in the dark for 30-35 minutes, then lisarow red blood cells, the mixture was washed by centrifugation and analyzed in a flow cytometer, followed by calculation of the percentage of activated cells with the phenotype of CD63highfrom a pool of cells with �enation CD123 highHLA-DR, using as positive control formyl-methionine-leucine-phenylalanine, and as a negative control - indicator cells incubated in the absence of immunoglobulin E and the indicator cells incubated with immunoglobulin E, previously associated with an excess of neutralizing antibodies to immunoglobulin E, and if in the test samples when increasing doses of the investigated antibodies is not marked dose-dependent changes in the percentage of activated basophils corresponding antibodies recognized not neutralizing, in case of detection of a dose-dependent increase in the proportion of cells CD63higha population of cells with the phenotype CD123+HLA-DR-the investigated antibodies are referred to anavilhanas, and when it detects a dose-dependent reduction in the proportion of cells CD63highin the study population believes that the investigational antibody refers to neutralizing with the calculation of the concentration of the antibodies according to the formula:
% neutr = 100×(1-(%ABop-%ABa-IgE)/(%ABTo IgE-%ABa-IgE)),
where % neutr - the percentage of neutralization; %ABop- the percentage of activated basophils in the presence of an appropriate concentration of the investigated antibodies; %ABa-IgE- the percentage of activated basophils in the sample control anti-IgE; %ABTo IgE- the percentage of activated basophils in the control sample without IgE SPS�CSOs drug.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention aims at treating drug-induced dry eye syndrome (DI-DES). Treating DI-DES implies taking the past medical history, measuring tear production and eye xerosis values reduced and increased respectively in relation to the norm. Unpreserved ocular hypotensive medications are prescribed in the patient. Unpreserved artificial tears are also applied. The lachrymal fluid is analysed by a multicytokine technique. If the analysis shows increased concentrations of proinflammatory cytokines - interleukin-6, interleukin-8, interleukin-12, Th-1 - interleukin-2, interferon-gamma, and Th-2 - interleukin-4, by min 30% in relation to the patient's age norm, a chronic immune ocular inflammation is detected. That requires transpalpebral Blepharogel-1 phonophoresis and 1% hydrocortisone ointment phonophoresis on the sub-mastoidal region from both sides; the therapeutic course is 8-10 daily procedures.

EFFECT: optimal conditions for diagnosing and reasoned differentiated therapy of DI-DES that enables prescribing the pathogenetically reasoned therapy in due time and increasing the efficacy of the therapeutic exposure.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: in a premature baby the concentration of neuron-specific enolase (NSE), concentration of a brain-derived neurotrophic factor (BDNF), concentration of a vascular-endothelial growth factor (VEGF) in umbilical blood and concentration of the vascular-endothelial growth factor (VEGF) in peripheral blood are determined on the basis of the enzyme immunoassay of umbilical and peripheral blood serum on the 7-th day of life, a prognostic index (PI) is calculated by formula: PI=-0.007×X1+0.006×X2-0.05×X3+0.0004×X4-3.9, where X1 is VEGF content in umbilical blood at birth (ng/ml); X2 is VEGF content in peripheral blood on the 7-th day of life (ng/ml); X3 is NSE content in umbilical blood (mcg/l); X4 is BDNF content in umbilical blood (ng/ml); Const=-3.9. When PI is higher than 0, a conclusion about the absence of risk of occlusive posthaemorrhagic hydrocephalus formation is made, and if PI is lower than 0, a high risk of the said pathology development is predicted.

EFFECT: invention makes it possible to increase the efficiency of prediction of occlusive posthaemorrhagic hydrocephalus formation in the premature children with an extremely low body weight at birth.

2 ex

FIELD: medicine.

SUBSTANCE: group of inventions relate to medicine and deals with method of diagnosing neurodegenerative disease in individual, including the following stages (i) determination of one or several parameters, selected from group, consisting of 3ab40 or value of calculated parameter, selected from group, consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42; (ii) comparison of parameter value with standard value, corresponding to value of said parameter in standard sample; and (iii) diagnostics of neurodegenerative disease, in case if increase of parameter value in comparison with standard value is observed. Group of inventions also deals with method of detecting stage, preceding neurodegenerative disease, method of differentiating neurodegenerative disease from stage, preceding said neurodegenerative disease.

EFFECT: group of inventions provide high sensitivity and specificity of detection methods.

13 cl, 12 ex, 14 dwg, 12 tbl

FIELD: medicine.

SUBSTANCE: patient's synovial fluid is sampled, and patient's supernatant chemokines CXCL9/MIG, CXCL10/IP-10 and CXCL11/ITAC are measured. A diagnosis of rheumatoid arthritis is diagnosed, if at least one of chemokines exceeds a threshold; the chemokine thresholds make 2625.8 pg/ml for CXCL9/MIG, 3108.2 pg/ml for CXCL9/MIG and 32.4 pg/ml for CXCL11/ITAC respectively. If the measured values are below the thresholds for three chemokines at the same time, the absence of rheumatoid arthritis and potential osteoarthrosis are stated.

EFFECT: using the given method enables differentiating rheumatoid arthritis and osteoarthrosis by measuring specific markers in the synovial fluid taken from the location directly.

2 ex

FIELD: medicine.

SUBSTANCE: invention relates to method of diagnosing rheumatoid arthritis, method of determining therapeutic agent for treatment of rheumatoid arthritis and set for realisation of methods. Methods are characterised by the fact that include stage of measuring amount of talin in plasma or serum of animal subject. Said measurement is carried out, for instance, by immunologic method with application of antibody, binding with talin. If amount of talin is higher than its average value in control subject without rheumatoid arthritis, rheumatoid arthritis is diagnosed in subject. In case of reduction of talin amount after introduction of therapeutic agent in comparison with amount of talin before introduction, therapeutic effect is stated. Set in accordance with claimed invention contains solid-phase carrier, to which antibody, binding with talin, is attached.

EFFECT: increased efficiency of diagnostics.

9 cl, 4 tbl, 4 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: seromucoid concentration is measured in supernatant of a biological fluid aspirated from the nasopharynx of the newborns suffering a generalised form of the intrauterine mono-cytomegalovirus infection or mixed cytomegalovirus infection. If the seromucoid concentration is 0.110-0.140 absorbance units, the early stage of the generalised form of the intrauterine mono-cytomegalovirus infection is diagnosed. If the seromucoid concentration is 0.141-0.171 absorbance units, the early stage of the generalised form of the intrauterine mixed cytomegalovirus infection caused by a combination of the cytomegalovirus and type 1 herpes simplex virus is diagnosed.

EFFECT: using the declared method enables the effective differential diagnosis of the generalised form of the intrauterine mono or mixed cytomegalovirus infection in the newborns.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. There are presented versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a specified amino acid sequence and at least one free cysteine amino acid residue specified in A118C (according to the European Numeration) in the heavy chain and V205C (according to the Kabat numeration) in the light chain. There are disclosed: versions of a conjugate compound of the antibody and a drug preparation, wherein the antibody is bond to the drug preparation through free cysteine; an antibody-based pharmaceutical compound for treating cancer; method for detecting CD79b or cancer cells, as well as a method for inhibiting cell proliferation using the conjugate compound. What is described is a method for producing the conjugate compound.

EFFECT: invention can find further application in the therapy of CD79b-associated cancer diseases, including treating haemopoietic tumours in mammals.

70 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine.

SUBSTANCE: fine-needle aspiration of nodular thyroid growths is controlled by ultrasonic examination. A puncture needle containing an aspirate is washed twice in normal saline 1 ml, centrifuged; a supernatant is selected, and thyroglobulin is measured by enzyme immunoassay. If the thyroglobulin content is less than 272.5 ng/ml, the absence of high differentiated cancer is stated; the value falling within the range of 272.5-355.5 ng/ml shows a risk of high differentiated thyroid cancer; if the thyroid value is more than 355.5 ng/ml, high differentiated thyroid cancer is suspected.

EFFECT: invention provides the pre-operative differential diagnostics of high differentiated cancer in the patients suffering nodular forms of thyroid diseases, and also enables the further selection of an adequate method of treating.

3 ex

FIELD: medicine.

SUBSTANCE: immunological indices in umbilical blood and venous blood on 4-5 day of life are determined. Resistance coefficient (Cres) is calculated by formula Cres=4.95258-0.0143955×(PI)+0.0962295×(PN)-0.00903362×(IgG)-0.257936×(IgA)-0.324514×(IgM)+0.430186×(CD45+CD3+)-1.66224×(CD45+CD4+CD3+)+l.49266×(CD45+CD8+CD3+)+0.815254×(CD4+CD8+)+0.522212×(HLA-DR+)-23.1991×(CD3+HLA-DR+)+0.974106×(CD3+CD25+)+0.832493×(CD3+CD4+CD25+)-3.52478×(CD16+CD56+CD3-)+7.67325×(CD16+CD56-CD3-)+11.082×(CD16+CD56+CD3+)+0.305366×(CD19+)+0.0691703×(CD5+)+0.0610707×(CD3+), where PI is phagocytic index, PN is phagocytic number, IgG, M, A are immunoglobulins of respective classes, CD are lymphocyte differentiation markers. If Cres value constitutes 1.78 and higher, when calculated by values of indices, obtained from umbilical blood, and is lower than 5.72, when counted by values of indices, obtained from venous blood on 4-5 day of life, on the first day of life high degree of resistance to infectious diseases is expected during the first year of life.

EFFECT: application of claimed method makes it possible to predict frequency of development of infectious diseases in child of the first year of life and take preventive measures to reduce risk of high morbidity.

2 tbl

FIELD: medicine.

SUBSTANCE: relative pre-implantation CD4+CD25+CD127- T-cell count is determined in the endometrial tissue of the females with primary and secondary infertility of an undefined origin. If the count is less than 7.5%, the immunological infertility is diagnosed. Using the given technique enables considering the CD4+CD25+CD127- T-cell deficiency as a marker of the immunological infertility in the females with both habitual miscarriage, and primary infertility.

EFFECT: improving the diagnostic procedure.

3 cl, 3 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

Up!