RU2291437C2 - Method for carrying out monitoring of diabetes mellitus patients state and neurological and vascular complications development - Google Patents

Abstract

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.

Description

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

It is known that the immune system of a healthy person constantly produces natural regulatory autoantibodies (auto-AT) to any antigenic components of their own body, including auto-AT to pancreatic cell components and insulin receptors (Poletaev AB and others. Regulatory metasystem, M .: Medicine, 2002). The synthesis of auto-antibodies in a healthy body is maintained within certain limits necessary for the latter to perform certain regulatory functions, and their excessive, as well as insufficient production can lead to the development of pathological conditions, including insulin-dependent diabetes mellitus (type 1 diabetes) and non-insulin-dependent sugar diabetes (type 2 diabetes), as well as vascular and neurological complications of the underlying disease, often leading to persistent disability.

The above was the basis for the development of the proposed method, suitable for diagnostic and prognostic assessment of the levels of a number of auto-antibodies, the content of which reflects the level of autoimmuno-aggression to pancreatic insulin-secreting cells in DM-1, to peripheral insulin receptors in DM-2, and also to monitor the development the main complications of diabetes mellitus (vascular and neurological).

A common way to diagnose diabetes and monitor their condition is to determine the level of sugar in the blood (V.V. Medvedev, Yu.Z. Volchek. "Clinical laboratory diagnostics" "Hippocrates" St. Petersburg, 1997, p. 154).

The disadvantage of this known method is its relatively low reliability and the inability to monitor the development of the main vascular and neurological complications accompanying diabetes.

The objective of the invention is a method for monitoring the condition of patients with diabetes mellitus and the development of its neurological and vascular complications, which makes it possible to more objectively judge the progression of diabetes and the development of neurological and vascular complications.

The problem is solved by the method, which consists in determining the immunoreactivity of blood serum with respect to insulin, anti-insulin antibodies (binding antibodies to insulin receptors) or their antigen-binding fragments, anti-anti-insulin antibodies, binding antibodies to insulin and antibodies to anti-insulin and nerve growth factors ( antibodies) or their antigen-binding fragments and ANCA antigen and with an increase in immunoreactivity of 6-15% with respect to insulin and / or anti-insulin antibodies or their antigen-binding fragments conclude that diabetes mellitus is moderately progressive, while at the same time increasing immunoreactivity with respect to anti-anti-insulin antibodies that bind antibodies to insulin and antibodies to nerve growth factor or antigen-binding fragments conclude that there is a moderate progression of diabetic neuropathy, and an increase in the same degree of immunoreactivity in relation to ANCA conclude that diabetic angiopathy, and with a higher immunoreactivity in In relation to the listed reagents, they conclude about a high degree of progression of diabetes mellitus and its neurological and vascular complications.

Practical implementation of the method includes the following stages:

I. Sorption on standard 96-well polystyrene ELISA plates a) insulin, b) idiotypic anti-insulin antibody molecules (or fragments thereof) - immunochemical analogues of insulin receptors; b) anti-idiotypic antibodies (anti-antibodies) that specifically interact with antibodies to insulin; c) anionic neutrophil cytoplasm proteins (ANCA);

II. Application of test samples of patient sera and a reference reference serum obtained from healthy individuals to them;

III. Detection of the resulting antigenic complexes using conjugates of secondary (antispecies) immunoglobulins labeled with horseradish peroxidase or another enzyme;

IV. The manifestation of the reaction in the wells of the tablet using a chromogen substrate;

V. Comparison of the reaction intensity in the wells containing the test sera with the wells containing reference serum.

For the ELI-DIA-Test analysis, about 0.03 ml of the blood serum of the subject (for example, obtained from a fingertip) is required.

OBTAINING THE MAIN COMPONENTS USED BY THE SUGGESTED METHOD (EL-DIA-TEST TEST SYSTEM).

1. The work uses a commercial insulin preparation.

2. The preparation of anti-insulin antibodies or their antigen-binding fragments was carried out using standard immunoaffinity chromatography procedures, as described previously (Poletaev AB, et al. J. Neuroimmunology, 2003, 1, 1, 11-17).

3. The preparation of antiantiinsulin antibodies or their antigen binding fragments was carried out using standard immunoaffinity chromatography procedures as described previously (Poletaev AB and others J. Neuroimmunology, 2003, 1, 1, 11-17).

4. The ANCA antigen was prepared as follows: a neutrophil fraction was isolated from whole human blood, it was extracted with a neutral buffer solution (for example, Tris-chloride), the extract was passed through an anion exchange column (for example, DEAE-cellulose), the bound material was eluted with saline (for example, 1 M sodium chloride) and used in the work.

METHODOLOGY OF THE INDIRECT IF.

1. For sorption on standard 96-well flat-bottomed plates for enzyme immunoassay, solutions of each component of the test system were prepared on a carbonate buffer pH 9-10. The prepared solutions were introduced into individual wells of the plate and incubated for 1-24 hours.

2. After incubation, the plates were washed with washing buffer (for example, 0.15 M NaCl with 0.05% tween-20) and the sera were tested; for this:

a) in separate bottles or trays for ELISA prepared dilutions of sera (for example, 1: 200) in washing buffer,

b) made diluted serum in the wells of the tablet,

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

g) washed the plates and showed the reaction using standard methods using a horseradish peroxidase conjugate (or other enzyme) with antibodies to human IgG immunoglobulins and a substrate-chromogen solution (for example, 0.01-0.05% o-phenylenediamine with 0.001-0.01% hydrogen peroxide per 0.01-0.05 M citrate-phosphate buffer in the case of peroxidase conjugates).

3. The plates were incubated in the dark for 10-30 minutes at room temperature.

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

5. Registration of reaction results: staining intensity is evaluated using an enzyme immunoassay analyzer (ELISA reader) of any model.

6. Processing the received data:

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

Where

R (ag1) is the optical density of the analyzed blood serum in the wells with antigen-1;

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

R (k1 ... kn) is the optical density of the control blood serum in the wells with antigens 1 ... n.

7. Interpretation of the received data:

a) if the intensity of the reaction of the test serum with any of the components of the test system did not exceed + 5% with respect to the reaction of the reference serum, this serum is assigned to the 1st classification group (individuals without progression of the underlying disease and without signs of development complications)

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

c) if the intensity of the reaction of the test serum with any of the components is outside the limits of the 1st and 2nd groups, this serum is assigned to the 3rd classification group or the group of pronounced deviations (persons with a high risk of rapid progression of the underlying disease and / or its complications)

d) the probability of progression of SD-1 is proportional to the degree of deviation from the norm in the content of auto-AT to insulin,

d) the likelihood of progression of DM-2 is proportional to the degree of deviations from the norm in the content of auto-AT to insulin receptors, detected by the reaction in wells with adsorbed anti-insulin antibodies (idiotypic) or their fragments,

f) the likelihood of progression of diabetic neuropathy is proportional to the degree of deviation from the norm in the content of antibodies cross-reacting with both insulin and nerve growth factor, detected by the reaction in the wells with adsorbed anti-anti-insulin antibodies (anti-idiotypic) or fragments thereof,

g) the likelihood of progression of diabetic angiopathy is proportional to the degree of abnormality in the content of antibodies reacting with the ANCA antigen,

h) by the dynamics of changes in the content of the corresponding auto-antibodies, one can judge the positive or increase in negative trends in the condition of the subjects, for example, against the background of the treatment.

It should be noted that the tests are performed in vitro without any harm to the subject.

EXAMPLES

EXAMPLE 1

Examined 1

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

AT to insulin AT to insulin receptors AT reacting with insulin and NGF AT to ANCA 3 fifteen four 2

Diagnosis: Newly diagnosed type 2 diabetes mellitus; disease duration about 6 months. There were no clinical signs of complications.

EXAMPLE 2

Examined 2

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

AT to insulin AT to insulin receptors AT reacting with insulin and NGF AT to ANCA 35 fifteen 44 2

Diagnosis: Type 1 diabetes mellitus with signs of insulin resistance; the disease is about 5 years old. Clinical signs of diabetic neuropathy.

EXAMPLE 3

Examined 3

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

AT to insulin AT to insulin receptors AT reacting with insulin and NGF AT to ANCA twenty 45 17 28

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

EXAMPLE 4

Examined 4

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

AT to insulin AT to insulin receptors AT reacting with insulin and NGF AT to ANCA 135 115 87 62

Diagnosis: Type 1 diabetes mellitus, decompensation state; signs of insulin resistance; the disease is about 3 years old. Clinical signs of diabetic neuropathy. Exacerbation of a chronic infectious process.

EXAMPLE 5

Examined 5

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

AT to insulin AT to insulin receptors AT reacting with insulin and NGF AT to ANCA -5 -eleven 17 62

Diagnosis: Type-2 diabetes mellitus, compensated, without signs of an autoimmune pathogenetic component; the disease is about 10 years old. Clinical signs of diabetic angiopathy.

In contrast to the prototype method discussed above, the new method allows:

1) register pathological changes in the serum content of auto-antibodies interacting with insulin (their changes are a characteristic sign of type 1 diabetes),

2) to register pathological changes in the serum content of auto-ATs that interact with membrane insulin receptors (their changes are a characteristic feature of CD-2),

3) to register pathological changes in the serum content of auto-AT, the changes of which precede and accompany the development of the main vascular and neurological complications of diabetes of both type 1 and type 2 (diabetic angiopathy, diabetic neuropathy),

4) register pathological changes that are dangerous for people with diabetes, but give a forecast of the dynamics of the underlying disease in all people with diabetes.

Claims (1)

A method for monitoring the condition of patients with diabetes mellitus, which consists in determining the immunoreactivity of blood serum with respect to insulin, anti-insulin antibodies or their antigen-binding fragments, anti-anti-insulin antibodies that bind antibodies to insulin and antibodies to growth factor, or their antigen-binding fragments ANCA and with an increase in immunoreactivity of 6-15% to normal with respect to insulin and / or anti-insulin antibodies or their antigen-binding fragments conclude that moderate of diabetes mellitus, and with an equally simultaneous increase in immunoreactivity with respect to antiantiinsulin antibodies or antigen-binding fragments, a conclusion is made about a moderate progression of diabetic neuropathy, and with a simultaneous increase in the same degree of immunoreactivity with respect to ANCA, a conclusion is made about diabetic angiopathy, and higher immunoreactivity in relation to the listed reagents conclude a high degree of progression of diabetes mellitus and its neurological and vascular complications.