Detection of cytokines

 

(57) Abstract:

The invention relates to medicine, namely to identify the expression of cytokines in the cells. The essence of the invention is the use of enzyme reactions in the processing of blood smears 1% potassium bichromate, 0.03% hydrogen peroxide, monoclonal antibodies to cytokines 0,3% Triton X-100, individuum conjugate and 0.05% aqueous solution of 3.3 diaminobenzidine tetrachloride. The detection is performed according to the presence of colored granules in mononuclear cells. The technical result is a simplification of the method of detection of cytokines, evaluation of the severity of the infection and the timely use of pathogenetic therapy. 2 Il.

The invention relates to the field of medical diagnostics, the detection of the expression of cytokines in the cells. Among the many factors that determine the development of those or other morphological changes special role of cytokines as mediators of intercellular interaction in immune response and inflammation, identifying which, with a high degree of certainty, allows in-depth study of the pathogenesis of the disease and to use that information to assign individual pathogenetic rentego analysis (LIS), proposed Hamish R. Michie, M. C., Kirk, R. Manogue, Ph. D. David R. Spriggs, M. D., E. A. 1988. (The New England Journal of Medicine, 1988, 318, N23, 1482-3).

1. Centrifuged and filtered spenomegaly liquid was fixed at 1% Bachem serum albumin in a special tablets at room temperature.

2. Was treated with 2% sheep serum in carbonate buffer with pH=9 at room temperature.

3. Washed in carbonate buffer with pH=7,5.

4. Put anti-rabbit serum against tumor necrosis factor related to horseradish peroxidase.

5. Was applied and incubated at room temperature with conjugate anti-rabbit lgG.

6. Was treated with O-phenylenediamine with hydrogen peroxide in citrate buffer with pH=5.

Records of the results of the reaction were conducted in a standard spectrophotometer at a wavelength of 490-600 nm.

This method allowed the authors to obtain good results. However, the disadvantage of this method is that it is laborious and time to get the results you need special equipment (microplates and spectrophotometer), drugs cannot long keep and take pictures. This method does not allow to determine the expression of cytokines in the cells, without the indirect immunoperoxidase method described j. Polak, S. Van Norden. Introduction to immunocytokine, M.: Medicine, 1986:

1 - rinse the slices in the buffer

2 - processing of 0.03% hydrogen peroxide solution,

3 - rinse buffer,

4 - application to smear the first specific antibodies,

5 - rinse buffer,

6 - application of a second antibody coupled to horseradish peroxidase,

7 - rinse buffer,

8 - handling 0.03% solution of diaminobenzidine (DUB),

9 - pokraska with hematoxylin,

10 - conclusion in balm. Microscopy.

But evidence from the literature and our own experience of the scheme only in General reflects the progress of the production methods, depending on the size, structure and nature of detected antigen required detailed design all stages, starting with commit and before the application of diaminobenzidine.

The aim of the invention is the simplification of the method of detection of cytokines by defining them in the cells for possible use in health care practice, assess the severity of the infection and the timely appointment of pathogenic (anticytokines) therapy.

To achieve this goal, we first proposed a modified naprave glass. This method applies to immunocytochemical and based on the specific reaction antigen-antibody. In contrast to enzyme-linked immunosorbent (ELISA) it is easy setting reaction, not giving specificity and sensitivity, allowing you to have constant drugs, because the intensity of the color does not fall. Drugs can be viewed in normal light microscope and photographed.

The proposed method is that the strokes of blood before treatment with hydrogen peroxide 0.03% and monoclonal antibodies to cytokines treated with bichromate of potash 1%, and before applying individualo conjugate - 0.3% Triton X-100.

The proposed method first proposed processing smears 1% solution of bichromate of potassium, which increases the permeability of cell membranes, to the elimination of cross-links between proteins. The bichromate of potash is indifferent substance, in contrast to the enzyme trypsin used for this purpose. After exposure of bichromate of potash antigen becomes more accessible to antibodies, which is a necessary condition for their interaction. In addition to the above bichromate of potassium inhibits the activity of endogenous peroxidase, eliminating thereby nespeca recommended because in the first case, the antigen remains inaccessible to antibodies, as in the second disturbed the fullness of his identification.

One of the difficult problems is the presence in the serum of contaminated antibodies. Purification of serum leads to the reduction of its avidity. Significant breeding specific serum also not desirable, as it lengthens the time of the reaction may decrease the concentration of detectable antigen. To address these shortcomings, we proposed to treat strokes of 0.3% Triton X-100 for 15 min at a temperature of 37oWith that precedes the first serum. Used detergent adsorbs contaminated antibodies, largely eliminating nonspecific staining. In addition to the above Triton X-100, and bichromate of potash, increases the permeability of cell membranes, facilitating the penetration of the antibodies used in the cell. First proposed a new approach to the detection of cytokines and a set of methodological techniques that are performed in the process immunoperoxidase detection of cytokines, is required. Failure to follow them does not give objective results or distort the end result of the research, it is not possible to carry out the corresponding interpret the TCA blood, obtained by centrifugation is applied on a glass slide (smear).

2. Dried at room temperature.

3. Fixation in pure acetone - 5 minutes

4. Washed in phosphate-buffered saline (pH 7,2-7,4) for 3-4 minutes.

5. Treatment in a 1% solution of potassium bichromate for 3-4 minutes.

6. Processing of 0.03% hydrogen peroxide for 20 minutes.

7. Rinse in two portions of phosphate-saline buffer (pH 7.2) for 5 minutes each.

8. Incubation in 0.3% Triton X-100 at a temperature of 37oC for 15 minutes.

9. Drawing on strokes monoclonal antibodies to cytokines (TNF, entertaini). Incubation with monoclonal antibodies at a temperature of 37oC for 45 minutes.

10. Rinsing in water for 5 minutes.

11. Rinse in two portions of phosphate-saline buffer (pH 7.2) for 5 minutes each.

12. Drawing on strokes antivitamin antibodies conjugated with peroxidase. Incubation for 45 min in a dark chamber at room temperature.

13. Rinse in two portions of phosphate-saline buffer (pH 7.2) for 5 minutes each.

14. The processing material in an aqueous solution containing 0.05% 3,3-diaminobenzidine tetracha the 16. Microscopy.

Microscopy stained preparations is carried out in a conventional microscope. The reaction product in the study of mononuclear cells containing cytokine detected in the form of light - and dark-brown granules intracellularly and on the cell membrane. When evaluating the data, microscopic examination it is important to consider the occurrence of artifactual changes that mimic the positive coloration, Often a cellular artifacts found on the edge of the smear. In this case, it is necessary to evaluate reactions to other sites.

The necessary controls.

To obtain reliable results and to exclude possible non-specific reactions is mandatory to conduct inspections on the quality of the reagents and the specificity of the antisera.

1) To control the specificity of the antisera as the first layer is applied non-immune serum or buffer (results must be negative.

2) Nespecificnomu serum resolve by the maximum dilution of highly concentrated serum and shortening the incubation time.

The described method is supported by examples of specific performance.

Was conducted and the AI in 10 of them (31,2%) was revealed the expression of tumor necrosis factor and interleukin - 6 in mononuclear cells (lymphocytes, monocytes) blood. All of these 10 children had severe clinical form of Haemophilus influenzae. The rest of the children (22 children) disease was spent in light and moderate form, and the expression of cytokines in this group were not found.

As an example, here micrograph of peripheral blood lymphocytes with clearly defined expression of tumor necrosis factor (photo 1) and interleukin - 6 (photo 2) the patient Burawoy Mary, 2 years, 10 months, N history 3316 from 13.05,99,, Haemophilus infection, acute phase, severe course of the disease.

Thus, the proposed method allows a high degree of specificity and accuracy to detect the expression of cytokines in mononuclear blood.

The method is simple enough, it may have wide application in medical practice for the rapid determination of cytokines in mononuclear cells of the blood and, therefore, assess the severity of the disease and timely appointment of pathogenic (anticytokines) therapy.

The proposed method has economic value because it does not require profane enzyme reaction, characterized in that take blood smears, treated with potassium bichromate 1%, hydrogen peroxide 0.03%, the monoclonal antibodies to cytokines 0,3% Triton X-100, individuum conjugate and 0.05% aqueous solution of 3,3-diaminobenzidine tetrachloride, for washing at some stages of the use of phosphate-saline buffer pH 7,2-7,4 and identification carried out microscopically, the presence of light - and dark-brown granules in poorly decrasing with hematoxylin mononuclear cells.

 

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