Method for carrying out monitoring of diabetes mellitus patients state and neurological and vascular complications development

FIELD: medicine.

SUBSTANCE: method involves determining immune reactivity of blood serum with respect to insulin, to anti-insulin antibodies or their antigen-binding fragments, to anti-anti-insulin antibodies binding the antibodies to insulin and antigens to growth factor or their antigen-binding fragments and ANCA antigen. Blood serum immunoreactivity increase with respect to parameters under measurement relative to norm is used for determining diabetes mellitus neurological and vascular complications development.

EFFECT: high accuracy of monitoring.

 

The invention relates to medicine, in particular to therapy and endocrinology, and may find application in assessing the effectiveness of therapy of diabetes mellitus type 1 and 2.

It is known that the immune system of a healthy person constantly produces natural regulatory autoantibodies (auto-AB) to any of the antigenic components of its own body, including auto-antibodies to components of the cells of the pancreas and insulin receptors (Poletaev A.B. and other Regulatory metasystem, M.: Medicine, 2002). Synthesis of auto-antibodies in a healthy body is maintained within certain limits, required to run past certain regulatory functions, and their excessive as well as insufficient production can lead to the development of pathological conditions, including insulin-dependent diabetes mellitus (DM type 1) and non-insulin dependent diabetes mellitus (DM type 2), as well as vascular and neurological complications of the underlying disease, often leading to permanent disability.

This has been the basis for the development of the proposed method suitable for diagnostic and prognostic assessment of levels of a number of auto-antibodies, the content of which reflects the level of autoimmunogenic to insulinsecreting cells of the pancreas in DM-1, peripheral insulin receptors in DM-2, and ASU is estolate control over the development of the main complications of diabetes (cardiovascular and neurological).

The generally accepted method for the diagnosis of diabetes and monitoring of their condition is the determination of the level of sugar in the blood (Dev, Ussales. "Clinical laboratory diagnostics" Hippocrates" SPb, 1997, str).

The disadvantage of this known method is its relatively low reliability and the impossibility of monitoring the development of major vascular and neurological complications that accompany diabetes.

The objective of the invention is a method of monitoring the condition of patients with diabetes mellitus and the development of neurological and vascular complications, allowing more objectively judge the progression of diabetes and the development of neurological and vascular complications.

The problem is solved by the method lies in the fact that determine the immunoreactivity of serum in relation to insulin, to antiinsulin antibody (the binding of antibodies to insulin receptors) or their antigennegative fragments, intentionality antibodies that bind antibodies to insulin and antibodies to nerve growth factor (antiidiotype antiinsulin antibodies) or antigennegative fragments and the antigen of ANCA and with the increase in the immunoreactivity for 6-15% in relation to insulin and/or antiinsulin antibodies or their antigennegative fragments conclude about the mind is adopted progression of diabetes, when used simultaneously in the same degree of increase of immunoreactivity against intentionalism antibodies that bind antibodies to insulin and antibodies to nerve growth factor or antigennegative fragments conclude about moderate progression of diabetic neuropathy, and with a simultaneous increase in the same degree of immunoreactivity against ANCA conclude diabetic angiopathy, and at a higher immunoreactivity in relation to the listed reagents conclude a high degree of progression of diabetes and its neurological and vascular complications.

The practical implementation of the method includes the following stages:

I. Sorption on a standard 96-well polystyrene plates to ELISA (a) insulin b) molecules idiotypical antiinsulin antibodies (or fragments) - immunochemical analogues insulin receptors; b) antiidiotypic antibodies (intential), specifically interacts with antibodies to insulin; C) anionic proteins in the cytoplasm of neutrophils (ANCA);

II. The application of the tested serum samples of patients and reference reference sera obtained from healthy individuals;

III. Identifying formed antigenetically complexes using conjugates secondary (antivitamin) immunoglobulins labeled with horseradish peroxidase or ins is an enzyme;

IV. The manifestation of the reaction in the wells with substrate-Chromogen;

V. comparison of the intensity of the reaction in the wells containing test serum to the wells containing the reference serum.

For analysis ELI-DIA-Test takes about 0,03 ml of the blood serum of the subject (e.g., obtained from the finger pad).

THE BASIC COMPONENTS USED FOR THE PROPOSED METHOD (TEST SYSTEM ELI-DIA-TEST).

1. The paper uses the commercial preparation of insulin.

2. Getting antiinsulin antibodies or their antigenspecific fragments was performed using standard procedures immunoaffinity chromatography as described previously (Poletaev A.B. and others J. Neuroimmunology, 2003, 1, 1, 11-17).

3. Getting intentionality antibodies or their antigenspecific fragments was performed using standard procedures immunoaffinity chromatography as described previously (Poletaev A.B. and others J. Neuroimmunology, 2003, 1, 1, 11-17).

4. Getting ANCA antigen was carried out as follows: from whole human blood were isolated fraction of neutrophils, were extracted using a neutral buffer solution (for example, Tris-chloride), the extract was passed through the anion-exchange column (for example, DEAE-cellulose), who contacted the material was suirable salt solution (for example, 1 M chloride intothree is) and used in the work.

METHODOLOGY FOR INDIRECT ELISA.

1. For sorption on standard 96-well flat-bottomed plates to assay prepared solutions of each component of the test system in carbonate buffer pH 9-10. The prepared solutions were made in separate wells and incubated for 1-24 hours

2. After incubation, washed tablets wash buffer (for example, 0.15 M NaCl with 0.05% tween-20) and tested sera; to do this:

(a) in separate vials or trays for ELISA were prepared by dilution of sera (1:200) in wash buffer,

b) made of diluted serum in wells,

C) the plates were incubated overnight at +4°or 1-3 h at +37...+39°C

g) washed tablets and showed the reaction using standard techniques, using a conjugate of horseradish peroxidase (or other enzyme) with antibodies to immunoglobulins IgG man and a solution of substrate-Chromogen (e.g., 0.01-0.05% o-phenylenediamine with 0.001-0.01% hydrogen peroxide in 0.01-0.05 M citrate-phosphate buffer in case of application of peroxidase conjugates).

3. Incubated plates in the dark for 10-30 min at room temperature.

4. Upon reaching the optimal level of staining (usually 10-20 min incubation in the dark) reaction stopped by the addition of 0.2-0.4 M tartaric acid.

5. Check the results: intensives the e staining assessed using enzyme immunoassay analyzer (ELISA-reader) of any model.

6. Data processing:

on the obtained values of optical density of holes calculate the relative intensity of the reaction was tested sera with each of the components of the test system ELI-DIA-Test relative to the reaction control sera with the same antigen minus 100 units and expressed in arbitrary units. To calculate using the formula:

where

R(a) is the optical density of the analyzed blood serum in the wells with antigen-1;

R(AGW) is the optical density of the analyzed blood serum in the wells with antigen-n;

R(K1...KP) is the optical density of the control serum in the wells with antigen 1...n.

7. The interpretation of these data:

a) if the intensity of the response of the test serum with any of the components of the test system did not go beyond +5% relative to the reaction serum reference, this serum is referred to as the 1st classification group (those without progression of the underlying disease and no signs of complications),

b) if the intensity of the response of the test serum with any of the components outside the group-1 but does not exceed +15% relative to the reaction serum reference, this serum is dated to the 2nd classification group or a group of moderate rejected the th (persons with progression of diabetes and/or complications of moderate severity),

C) if the intensity of the response of the test serum with any of the components beyond the 1st and 2nd groups, this serum is referred to as the 3rd classification group or a group expressed deviations (persons with a high risk of rapid progression of the underlying disease and/or its complications),

g) the likelihood of progression of DM-1 proportional to the degree of abnormalities in the content of auto-antibodies to insulin,

d) the probability of progression of DM-2 proportional to the degree of abnormalities in the content of auto-antibodies to insulin receptors, detected by the reaction in the wells with adsorbed antiinsulin antibodies (idiotypical) or their fragments,

(e) the likelihood of progression of diabetic neuropathy proportional to the degree of abnormalities in the content of antibodies cross-reacting with insulin and nerve growth factor detected by the reaction in the wells with the adsorbed anti-antiinsulin antibodies (antiadiotipiceskih) or their fragments,

W) the probability of progression of diabetic angiopathy proportional to the degree of abnormalities in the content of antibodies reactive with the antigen of ANCA,

C) dynamic changes in the content of the corresponding auto-antibodies can be judged positive or negative trends in the state of the patient, for example, on the background of conduct the treatment.

It should be noted that the analysis is performed in vitro without any harm to the subject.

EXAMPLES

EXAMPLE 1

Surveyed 1

The results of the analyses ELI-DIA-Test (immunoreactivity in % of the reaction control sera):

AT the insulinAT to insulin receptorsAT reacting with insulin and NGFAT to ANCA
31542

Diagnosis: newly diagnosed diabetes mellitus type-2; disease duration of approximately 6 months. Clinical signs of complications were found.

EXAMPLE 2

Surveyed 2

The results of the analyses ELI-DIA-Test (immunoreactivity in % of the reaction control sera):

Antibodies to insulinAT to insulin receptorsAT reacting with insulin and NGFAT to ANCA
3515442

Diagnosis: diabetes mellitus type-1 with signs of insulin resistance; disease duration of about 5 years. Clinical signs of diabetic neuropathy.

EXAMPLE 3

Surveyed 3

The results of the analyses ELI-DIA-Test (immunoreactivity in % of the reaction control savoro the key):

AT the insulinAT to insulin receptorsAT reacting with insulin and NGFAT to ANCA
20451728

Diagnosis: diabetes mellitus type-2 with signs of insulin resistance (combined type 1+2); disease duration of about 8 years. Clinical signs of diabetic angiopathy with changes in the fundus, the signs of neuropathy.

EXAMPLE 4

Surveyed 4

The results of the analyses ELI-DIA-Test (immunoreactivity in % of the reaction control sera):

AT the insulinAT to insulin receptorsAT reacting with insulin and NGFAT to ANCA
1351158762

Diagnosis: diabetes mellitus type-1, state of decompensation; signs of insulin resistance; disease duration of about 3 years. Clinical signs of diabetic neuropathy. Exacerbation of chronic infectious process.

EXAMPLE 5

Surveyed 5

The results of the analyses ELI-DIA-Test (immunoreactivity in % of the reaction control sera):

Antibodies to insulinAntibodies and sulinova receptors AT reacting with insulin and NGFAT to ANCA
-5-111762

Diagnosis: diabetes mellitus type-2, compensated, with no signs of autoimmune pathogenetic component; disease duration of about 10 years. Clinical signs of diabetic angiopathy.

In contrast to the above of the prototype method the new method allows to:

1) to register pathological changes in serum levels of auto-antibodies that interact with insulin (their changes is a characteristic feature of SD-1),

2) to register pathological changes in serum levels of auto-antibodies that interact with membrane insulin receptors (their changes is a characteristic feature of the SD-2),

3) to register pathological changes in serum levels of auto-antibodies, changes which precede and accompany the development of major vascular and neurological complications of diabetes like type 1 and type 2 diabetic angiopathy, diabetic neuropathy),

4) to register pathological changes, dangerous for those suffering from diabetes, but to give a forecast of the development of the underlying disease in all persons with diabetes.

Method of monitoring patients with diabetes mellitus, zakluchalos what is to determine the immunoreactivity of serum in relation to insulin, to antiinsulin antibodies or their antigennegative fragments, to intentionality antibodies that bind insulin antibodies and antibodies to the growth factor, or antigennegative fragments and the antigen of ANCA and with the increase in the immunoreactivity for 6-15% of the norm in relation to insulin and/or antiinsulin antibodies or their antigennegative fragments conclude about moderate progression of diabetes, when used simultaneously in the same degree of increase of immunoreactivity against intentionalism antibody or antigennegative fragments conclude about moderate progression of diabetic neuropathy, and with a simultaneous increase in such the same degree of immunoreactivity against ANCA conclude diabetic angiopathy, and at a higher immunoreactivity in relation to the listed reagents conclude a high degree of progression of diabetes and its neurological and vascular complications.



 

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