Diagnostic kit of reagents "cis-test for the detection of ischemic brain mammals

 

(57) Abstract:

The invention is intended to detect the state of the brain of mammals and, in particular, for the diagnosis of neurological diseases. The set of reagents "CIS-test is intended for the quantitative immunoassay for determination of levels of autoantibodies to synthetic fragment benzylideneamino membrane protein (FMB) of the human brain in the serum of people with coronary artery disease brain. FMB is a component of the receptor complex protein membranes of neurons, the violation of which can lead to pathological changes in the CNS. Increased levels of autoantibodies to the FMB in the serum correlates with the presence and susceptibility to ischemic brain. The invention will now identify the ischemic brain. 11 C.p. f-crystals.

The invention relates to biotechnology, and in particular to a reagent kit for detecting the state of the brain of mammals, in particular for the diagnosis of neurological diseases.

The claimed diagnostic kit of reagents for detection of ischemic brain is new and analogues n is epicheskoi disease brain is carried out by the comparative determination in the patient's blood autoantibodies to benzylideneamino membrane protein.

The essence of the invention.

The principle of operation of a diagnostic kit of reagents CIS test is the detection of antibodies to synthetic fragment FMB human brain. Sorption of synthetic fragment FMB on the surface of solid media allows you to selectively remove autoantibodies from serum (20 microliters) taken from the patient from a vein or from a finger. The formed complex antigen - antibody is detected using a second antibody against human immunoglobulins conjugated to peroxidase. The level of antibodies to GMB is estimated by a colour change of the substrate mixture, registered with the vertical scanning spectrophotometer at a wavelength of 405 nm.

Synthetic peptide fragment benzylideneamino membrane protein is applied on a solid support (polystyrene microplate for immunological studies, or nitrocellulose) and incubated for 14-16 h at +4oC to obtain immunosorbent assay. During testing, the samples of blood serum from patients and healthy individuals was applied to immunosorbant. As positive control served as the rabbit anticavity to synthetic peptide fragment phencyclidine 1 to 50 ng/ml for protein. After applying samples immunosorbent incubated 1 hour at a temperature of 20-25oC and the level of binding immunogenic fragment benzylideneamino membrane protein was determined by the standard technique of enzyme-linked immunosorbent assay using rabbit antibodies against human immunoglobulins conjugated to horseradish peroxidase. The count of the results was made by using a spectrophotometer vertical scanning Dynatech 4000 (UK) - largest absorption solutions (E405) at a wavelength of 405 nm. Build a calibration curve for the positive control, putting the abscissa shows the concentration of antibodies in the y - axis the value of E405. The concentration of IgG in the samples was determined by a calibration curve based on the values of E405.

The content of autoantibodies to benzylideneamino membrane protein in the blood of healthy individuals was 0,9-1,5 ng/ml Content of autoantibodies over 1.5 ng/ml was detected in the blood of patients with a diagnosis of ischemic disease of the brain."

The quantitative content of antibodies to benzylideneamino membrane protein in the blood is closely correlated with the functional status of patients with coronary heart disease grown the work of the body in response to the metabolism of certain proteins in the brain during acute hypoxia. Reliable stable level of antibodies to benzylideneamino membrane protein indicates a predisposition to ischemic brain and change, including abrupt accumulation of these antibodies in patients, serves as a criterion of their functional status.

The invention is illustrated by the following examples.

Example 1.

CIS-test consists of the following components:

- tablets, 96-well polystyrene strip with immobilized on the inner surface of the hole FMB - 2 pieces;

- conjugated rabbit polyclonal antibody to immunoglobulin G man peroxidase labeled lyophilized - 1 FL.;

- positive control sample (polyclonal antibodies to FMB at a concentration of 50 ng/ml), lyophilized - 1 FL.;

phosphate-saline buffer - 1 FL. (10 tablets);

- citrate buffer - 1 FL. (4 tablets);

the detergent tween-20 - 1 FL. (0.6 ml);

- 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) - 1 FL. (2 tablets);

- hydroponic - 1 FL. (2 tablets).

The set of CIS-test is designed to test triplicate 43 analyzed serum samples and 5 samples positive control at a concentration of 1-50 n is reallylong window to analyze a drop of blood of the patient 30 pcs.

B. Scarifiers (sterile) - 30 PCs

The patch bactericidal 30 pcs.

Packaging cardboard 1 piece

For each set comes with instructions for use of the test. CIS-test is based on a qualitative solid phase competitive lateral flow binding antigen brain autoantibodies in the patient's blood. The nitrocellulose contains a coloured complex synthetic peptide fragment of FMB with a positive control at a concentration of 2 ng/ml and the second antibody, conjugated with horseradish peroxidase, painted substrate ABTS. A drop of blood from a finger of the patient is applied to nitrocellulose window containing colored stripes, after 15 min the blood is shaken and recorded the result of the analysis. In the presence of an ischemic process in the brain autoantibodies from the blood of the patient enter into a competitive reaction with the antigen on the nitrocellulose and replacing them, resulting dyed stripe disappears.

The claimed diagnostic kit of reagents CIS-test allows you to quickly and effectively diagnose ischemic brain in a significant number of patients simultaneously. In addition, the inventive diagnostic kit who their risk for disease.

1. The set of reagents for diagnosis of neurological diseases, characterized in that it contains antigen a synthetic peptide fragment of phencyclidine-binding membrane protein, immobilized on a solid medium and a reagent for determining the presence of autoantibodies to phencyclidine-binding membrane protein.

2. Set under item 1, characterized in that it contains the antigen immobilized on the polymer carrier.

3. Set under item 1, characterized in that it contains the antigen immobilized on polystyrene hole carrier.

4. Set under item 1, characterized in that it contains the antigen immobilized on the nitrocellulose.

5. Set under item 1, characterized in that it contains a reagent for determining the presence of autoantibodies, which represents a conjugate antibodies to human immunoglobulins labeled with horseradish peroxidase.

6. Set under item 1, characterized in that it further comprises a substrate for holding peroxidase-catalyzed reactions.

7. Set under item 1 or 6, characterized in that it contains 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and hydroponic.

8. Set under item 1 or 3, characterized in that it contains a detergent.

9 further comprises a buffer solution.

11. Set under item 1 or 10, characterized in that it contains a phosphate-saline buffer and/or citrate buffer solutions.

12. Set under item 1, characterized in that it further comprises polyclonal antibodies to synthetic fragment benzylideneamino membrane protein as a positive control.

 

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