RU2425371C1 - Method of laboratory detection of consequences of perinatal central nervous system affections and severity determination in children - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: blood serum samples are analysed for leukocyte elastase (LE) and 1-proteinase inhibitor (1-PI) activity, levels of nerve tissue antigen autoantibodies (aAT1) and related anti-idiotypic autoantibodies (aAT2). A deviation degree of the derived values of congenital and/or acquired immunity from the similar values of serum samples taken from healthy persons of respective age characterises a severity level and a compensatory potential in children with the consequences of perinatal nervous system affections. ^ EFFECT: more precise detection and mass prophylactic medical examinations for detection of the persons showing characteristic changes of the observed immunological values and referred to high-risk groups of psychological diseases. ^ 2 tbl, 4 ex, 2 cl

Description

Field of technology: medicine, biotechnology, immunology, pediatric neurology, psychiatry.

State of the art

The problem of timely detection of perinatal lesions of the nervous system (PPS) and their consequences is extremely urgent due to their widespread prevalence, high risk of adverse effects (cerebral palsy, mental retardation, persistent sensory defects, vegetovisceral disturbances, etc.) and decompensation in subsequent stages life, including in children with minimal cerebral dysfunctions. This means that the PPNS-induced destructive process can continue in the following months / years.

The use of the clinical (neuro-psychological) method for the diagnosis of PPNS and / or their consequences is often associated with certain difficulties, due to the fact that in the first year of life these disorders appear only in the form of nonspecific symptom complexes (increased neuro-reflex irritability, muscle tone disorders , violation of the pace of psychomotor development). At the same time, early diagnosis of these conditions is extremely important for the timely start of rehabilitation measures at the stages of maximum plasticity of the nervous system.

Modern ideas about the existence of close relationships between the nervous and immune systems suggest the involvement of immune mechanisms in the formation of the pathology of the nervous system, including PPNS and their consequences, which is confirmed by numerous studies.

It is known that one of the main etiopathogenetic factors of the vast majority of PPNS is ischemia, which triggers a number of neuroinflammatory reactions, including activation of microglia, increased synthesis of pro-inflammatory cytokines, specific adhesion molecules and chemotactic factors, as well as an accumulation of the blood-brain barrier (BBB) from the active poppy on the endothelium (BBB) channels, vasoactive substances. The consequence of this is a violation of the BBB permeability and the release of neuroantigens into the bloodstream. The neuroantigens that enter the bloodstream bind to the corresponding autoantibodies (aAT) from the pool of physiological regulatory autoantibodies and, in some cases, activate the corresponding clones of B-lymphocytes producing specific aAT. Changes in the level of aAT to neuroantigens is one of the markers of impaired development and functioning of the nervous system. Determining the level of aAT to neuroantigens was previously proposed to determine the degree of risk of developing neuropsychiatric diseases (RF patent No. 2218569 on "A method for identifying risk groups for the development of neuropsychiatric diseases"; authors Morozov SG, Gnedenko BB, Poleshchuk V. V., Klyushnik T.P. et al.).

However, it must be borne in mind that increased synthesis of aAT to antigens of nervous tissue is a specific reaction of the immune system to the destructive process in brain tissue. This reaction is delayed in time, and the half-life of immunoglobulin molecules in the blood is 24-32 days, therefore, the determination of aAT in the blood cannot be used to monitor the state of the child’s nervous system in short time intervals (2-3 weeks). In addition, numerous clinical and immunological studies suggest that the autoimmune component is characteristic only of the most severe lesions of the nervous system.

The earliest and nonspecific reaction to any tissue damage is the activation of innate nonspecific immunity. Phylogenetically more ancient innate immunity acts as the first line of defense on the path of a foreign agent. At the heart of its action is inflammation and phagocytosis.

The cellular link of innate immunity is represented by monocytes / macrophages, granulocytes, dendritic cells and natural killer cells. The humoral link includes the kallikrein-kinin system, acute phase proteins, lysozyme, mannan-binding lectin. Activated neutrophils secrete a proteolytic enzyme in the blood - leukocyte elastase (LE). Once in the extracellular space, leukocyte elastase breaks down the main substance, elastin and collagen fibers of the vascular basement membranes, and in some cases acts as a powerful destructive factor that contributes to the maintenance of BBB damage and impaired brain function. Clinical and biological studies have shown that impaired development and functioning of the brain are accompanied by activation of innate immunity, and a number of indicators, such as LE activity, correlate with the severity of these disorders.

All of the above was the basis for the development of a laboratory method for identifying the consequences of PPNS and determining their severity in children of the first years of life by comprehensively determining a number of parameters of innate and acquired immunity: enzyme activity - LE and α1-proteinase inhibitor (α1-PI), idiotypic level ( aAT1) and anti-idiotypic (aAT2) autoantibodies to the spectrum of various neuroantigens.

As the closest analogue of the invention, the method described in RF patent No. 2231074 on “A method for determining the nature of the pathophysiological disorders of the psychomotor development of children of the first year of life”; authors Klyushnik T.P., Scherbakova I.V., Khachatryan L.G. At the same time, this method has a number of significant disadvantages.

First, it allows you to register a change in the level of aAT to only one antigen of nerve tissue - the nerve growth factor (NGF). At the same time, in case of disturbances in the autoimmunity system with a focus on neural tissue antigens, the levels of aAT to various neuroantigens do not always change in the same direction. Therefore, a more accurate result is always obtained when determining aAT levels not to one, but to several antigens of nervous tissue.

Secondly, the enzyme-linked immunosorbent assay used in the analogue method allows only the level of idiotypic aAT to NGF to be recorded. At the same time, a more holistic view of changes in the system of autoimmunity gives a comprehensive study of the content of aAT1 together with their “functional balances” - aAT2. This is due to the fact that the ratio aAT1 / aAT2 is of great importance, which gives an idea of how aAT1 is “shielded” by the corresponding anti-idiotypes, which can limit the adverse effects of elevated levels of aAT1.

Thirdly, the analogue method allows registering only one parameter of innate immunity, namely, the activity of LE - a serine protease contained in azurophilic granules of neutrophils and secreted into the extracellular space upon activation of neutrophils by various factors, including endogenous tissue destruction products. However, to assess the possible destructive potential of LE, it is also necessary to evaluate the activity of the enzyme that regulates the activity of LE, namely α1-PI. α1-PI suppresses the activity of LE with a high association constant (> 10 ⁷ M ^-1 · s ^-1 ). In blood serum, LE is predominantly in combination with α1-PI and lacks proteolytic activity. α1-PI is a glycoprotein synthesized by hepatocytes. By controlling the proteolytic activity of LE, this inhibitor creates the conditions for limiting the focus of inflammation or destruction, therefore, the level of α1-PI in blood serum determines the course of many inflammatory and destructive processes.

In contrast to the above method, the analogue of the proposal

The proposed new method allows

- register a change in the level of aAT1 and aAT2 to a number of antigens of the nervous tissue, as well as determine the ratio of AT1 / AT2;

- register the activity of not only LE, but also α1-PI, which characterizes the severity of the compensatory potential.

A comprehensive definition of the above parameters of innate and acquired immunity associated with the processes of development and functioning of the brain allows a more accurate assessment of the state of the child’s nervous system, to reveal the destructive process in the brain tissue, to clarify its severity, as well as the severity of the compensatory potential.

The aim of the proposed method is to increase the reliability of the prognosis of normal or abnormal development of the central and peripheral nervous system in children, to identify individuals with the current destructive process in the brain induced by perinatal damage to the nervous system and to clarify its severity by determining in blood serum such parameters of innate and acquired immunity as activity of LE, α1-PI, aAT1 and aAT2 levels to antigens of nervous tissue. As the latter, brain protein S100, gliofibrillar acid protein (GFAP), basic myelin protein (MBP), NGF can be used.

The implementation of the essential features of the invention is as follows. In blood serum samples, the activity of LE and α1-PI is determined by spectrophotometric enzymatic method and the level of aAT1 and aAT2 to neuroantigens is determined by enzyme-linked immunosorbent assay. During testing, individuals are identified that contain altered (increased or decreased compared to serum samples of healthy individuals of a similar age group) activity / level of one or more determined immunological parameters, which is a characteristic immunological sign of impaired development / functioning of the nervous system. The degree of deviation of the determined indices of innate and / or acquired immunity from similar indices of serum samples of healthy individuals of the corresponding age indicates the presence and characterizes the severity of PPNS.

All determinations are carried out in blood samples taken from a finger, heel or from a vein into dry tubes. After the formation of a blood clot, a blood sample is centrifuged at 2500 g, and serum is taken using an automatic pipette, which is used for analysis. Serum can also be frozen and stored until analysis for a month at temperatures from -18 ° C to -24 ° C.

It does not follow from the prior art that, according to a comprehensive assessment of the activity of LE and α1-PI, as well as the levels of aAT1 and aAT2 to neuroantigens, it is possible to identify the effects of PPNS and to characterize their severity.

SUMMARY OF THE INVENTION

Phased determination of immunological parameters.

I. Determination of LE activity. To determine the activity of LE located in the blood serum in complex with α1-PI, the spectrophotometric method is used using N-tert-butoxy-carbonyl-alanine-n-nitrophenyl ether (BOC-Ala-ONp) in the presence of acetonitrile as a chromogenic substrate. In the presence of an organic solvent and with a certain composition of the buffer solution, the complex loosens, leading to the displacement of the inhibitor and the penetration of BOC-Ala-ONp to the active center of the enzyme.

The progress of the analysis.

1. A bottle with a buffer solution is placed in a thermostat at a temperature of + 25 ° C for 15 minutes.

2. Immediately prior to use, 500 μl of acetonitrile is added to a BOC-Ala-ONp tube and mixed thoroughly.

3. In a cuvette of a spectrophotometer preheated to + 25 ° C, add 480 μl of a buffer solution heated to + 25 ° C, add 20 μl of a BOC-Ala-ONp substrate solution, 5 μl of the analyzed serum and mix thoroughly.

4. In parallel, prepare a control sample: 480 μl of a buffer solution with the addition of 20 μl of acetonitrile.

5. The optical density of the experimental sample is measured against the control 4 times with a time interval of 1 min (exactly!) At a wavelength of 347.5 nm.

6. Calculate the change in optical density of the analyzed sample for 1 min (DE / min) according to the formula:

ΔE / min = ΔE _n / t,

where ΔE _n is the change in optical density of the analyzed samples, units opt. tight.,

t is the time interval between determinations, min.

The activity of LE in the analyzed serum sample is determined by the formula:

A = ΔE / min × 0.108 / 0.005,

where 0.108 is the conversion factor taking into account the sample volume (0.5 ml) and the molar extinction coefficient of the substrate (4.6 ml × mol ^-1 × cm ^-1 );

0.005 is the volume of serum added to the cuvette (in ml).

The activity of LEs of each serum sample is measured 3 times, after which the arithmetic average of the activity of LEs is calculated.

II. Determination of α1-PI activity. The spectrophotometric method for determining the activity of α1-PI in blood serum is based on the interaction of this inhibitor with trypsin when N-α-benzoyl-L-arginine ethyl ether (BAEE) is used as a substrate: blood serum α1-PI forms a trypsin complex that does not break down BAEE. Therefore, the activity of α1-PI is determined by the degree of inhibition of trypsin cleavage of a low molecular weight substrate.

The progress of the analysis.

1. Open the bottle with buffer solution, its contents are transferred to a graduated cylinder and distilled water is added to the level of 100 ml.

2. Dissolve the contents of the tube with BAEE in 60 ml of distilled water and mix thoroughly. Withstand both solutions in a thermostat at + 25 ° C for at least 15 minutes.

3. Transfer the trypsin dilution solution to a trypsin tube and mix gently with swaying.

4. The studied serum samples are diluted with saline in a ratio of 1:50.

5. 2.0 ml of a buffer solution and 0.01 ml of trypsin solution are added to a spectrophotometer cuvette, previously heated to + 25 ° С, mixed and incubated in a cuvette for 5 min at + 25 ° С.

6. Add 1.0 ml of the substrate solution to the cuvette, mix and measure the optical density 4 times with an interval of 1 min (exactly!) At a wavelength of 253 nm (control sample).

7. Calculate the change in optical density of the control sample for 1 min (ΔE / min _control ) according to the formula:

ΔЕ / min _control = ΔЕ _к / t,

where ΔE _to - the change in optical density of the control sample, ed. tight., t - time interval between determinations, min.

8. Then, 1.9 ml of a buffer solution, 0.01 ml of trypsin solution and 0.1 ml of a pre-diluted 1:50 sample of the analyzed blood serum are added to a cuvette preheated to + 25 ° C, mixed and incubated in a cuvette for 5 min at + 25 ° С.

9. Add 1.0 ml of the substrate solution to the cuvette, mix and measure the optical density 4 times with an interval of 1 min (exactly!) At a wavelength of 253 nm (test sample).

10. Calculate the change in optical density of the sample for 1 min by the formula: ΔE / min _sample = ΔE _n / t,

where ΔE _n is the change in optical density of the investigated blood serum samples, unit opt. tight .; t is the time interval between determinations, min.

11. Calculate the difference in the optical density of the control and test samples (ΔE) by the formula:

ΔE = ΔE / min _control -ΔE / min _sample ;

the activity of α1-PI in the test sample is determined by the formula:

A = ΔE × F,

where A is the activity of α1-PI, IE / ml; ΔЕ is the difference in the optical density of the control and test samples, unit opt. tight .; F is the conversion factor.

III. Determination of the content of aAT to neuroantigens.

As antigens of the nervous tissue, MBM, S100, GFAP and NGF can be used. It is possible to use several of these proteins in any combination or other neuroantigens. They can be taken as antigens for determining aAT1 themselves, as well as their immunochemical analogues (for example, F (ab) ₂ fragments of the corresponding anti-idiotypic antibodies). To determine aAT2, F (ab) ₂ fragments of anti-idiotypic antibodies with the orientation to those antigens that were used to determine aAT1 are used as antigens.

During the analysis, it is possible not to determine aAT2 to antigens of the nervous tissue, however, this may somewhat reduce the accuracy of the results.

Analysis progress

1. Solutions of the antigens used in the work, dialyzed against a carbonate buffer, pH 8.5-9.8, are added to individual wells of standard 96-well flat-bottom plates for enzyme immunoassay. Then the plates are incubated for 12-18 hours at + 2 ... + 4 ° C.

2. Remove (by shaking) the solutions with unbound components from the wells and (without additional washing) fill in a blocking buffer (for example, 0.5% gelatin solution with 0.15 M NaCl). Incubated for 1 h at 37 ° C.

3. Wash the plates with wash buffer (with 0.05% tween-20).

4. Make samples of the test and reference serums at a dilution of 1: 200 in washing buffer on the plates. Incubated for 12-18 hours at + 4 ° C.

5. Wash the plates with wash buffer.

6. The manifestation of the reaction is as follows. A solution of horseradish peroxidase conjugate (or other enzyme) with anti-species antibodies to human IgG (or IgG and IgM) is added to all wells of the plate. Then the plates are incubated for 60-90 minutes at 37 ° C or 12-16 hours at + 2 ... + 4 ° C, after which the conjugate is removed, and the tablets are washed with washing buffer. Immediately after this, a substrate solution is added to the wells (for example, 0.01-0.05% o-phenylenediamine with 0.001-0.005% hydrogen peroxide in 0.01-0.05 M citrate-phosphate buffer in the case of peroxidase conjugates). Incubate the tablets in the dark for 10-20 minutes at room temperature.

7. Upon reaching the optimal level of staining, the reaction is stopped by the addition of 0.2-0.4 M sulfuric acid.

8. The staining intensity is evaluated using a reader for enzyme-linked immunosorbent assay of any model.

9. Process the received data.

a) From three duplicate values of the reaction results of the reference and test serum samples, choose the average.

b) Calculate the relative intensity of the reaction with the components of the test system of the studied sera in relation to the reaction of the reference serum (in percent) according to the obtained values of the optical density of the holes. The coefficient I, which is the ratio aAT1 / aAT2, is calculated. Coefficient I is calculated for each pair of aAT1-aAT2.

As a reference serum, the pulsed serum of healthy donors aged 18 to 40 years is used. Empirically, the following standards were established for various age groups:

Age Normal levels of aAT1 and aAT2 (percent relative to the reaction of the reference serum) up to 2 months from 50 to 90 from 2 months to 1 year from 60 to 100 from 1 year to 3 years from 65 to 115 from 3 to 16 years from 70 to 125 from 16 to 25 years old from 75 to 130 The values of the coefficient I for all age groups are normally 0.7-1.3.

IV. Interpretation of the received data.

The activity of LE and α1-PI of blood serum reflect the degree of activation of innate immunity, as well as the state of antiproteolytic (compensatory potential). In children with the current destructive process in the brain induced by PPNS, there is an increase in LE activity compared to the control, which correlates with the severity of clinical symptoms. A parallel increase, in comparison with the control, of α1-PI activity, aimed at limiting destructive reactions, characterizes the safety of the antiproteolytic potential. The decreased α1-PI activity compared with the control is an unfavorable prognostic factor in terms of the further development of the destructive process.

Lowering the level of AAT to antigens of the nervous tissue corresponds to brain lesions of mild and / or moderate severity. In such pathological conditions, the binding and elimination of neuroantigens from the brain to the blood occurs due to a pool of natural (regulatory) aATs. An increase in the level of aAT compared with the control indicates the launch of specific autoimmune reactions that accompany the most severe lesions of the nervous system.

Thus, a comprehensive determination of the above indicators of immunity allows us to identify the presence of a pathological destructive process in the brain tissue and to clarify its severity, as well as the severity of the compensatory potential.

Based on the totality of immunological parameters, the following main groups of serum samples can be distinguished:

PPNS severity rating table ("Severity table") The activity of LE nmol / (min · ml) The activity of α1-PI IE / ml Levels of aAT1 and aAT2 to neural tissue antigens Conclusion 105-200 25-35 Fine There are no signs of the effects of PPNS. Similar indicators can be in children with a formed defect in the nervous system, in which there is no current destructive process in the brain. 201-250 Above 35 Normal or below normal The current destructive process in brain tissue of mild severity with pronounced compensatory potential

251-300 Above 35 Normal or below normal The current destructive process in brain tissue of moderate severity with pronounced compensatory potential 251-300 Below 25 Normal or below normal The current destructive process in brain tissue of moderate severity with depleted compensatory potential, which is an unfavorable prognostic factor in terms of further weighting the process Above 300 35-45 Above norm The current destructive process in brain tissue of a severe degree with insufficiently expressed compensatory potential Above 300 Below 35 Above norm The current destructive process in severe tissue of the brain with depleted compensatory potential, which is an unfavorable prognostic factor in terms of the relative compensation of developmental disorders of the child Note. An increase in coefficient I in cases where there is an increase in the levels of aAT to neuroantigens indicates a more severe destructive process in brain tissue.

The study should be carried out no earlier than 2-3 weeks after suffering an acute infectious disease to exclude false-positive results.

Techno-economic and social significance

Using the proposed method allows to identify the induced PPPS destructive process in the early, often preclinical stages, which can contribute to the timely appointment of adequate methods of therapy and reduce the incidence of severe forms of pathology of the nervous system in children.

Using the proposed method, the severity of the destructive process in the brain of the child can be determined, as well as the severity of the compensatory potential, which helps to optimize therapeutic measures, including drug therapy.

The proposed method allows monitoring the state of the nervous system of the child, objectively evaluate the effectiveness of therapy.

In the presence of a destructive process in brain tissue, the use of the proposed method allows to optimize the timing of vaccination of sick children, which is advisable to carry out at a time when the consequences of PPNS by this method are not detected or the detected changes are insignificant.

Examples of the use of the invention

Example 1

Patient K., age 5 months. At the first visit to a neurologist at the age of 1 month, regurgitation, trembling were observed. Clinical examination: the head does not hold firmly, the gaze does not fix. Unconditioned reflexes are fuzzy, proprioceptive reflexes R = S. Diagnosis: syndrome of muscular dystonia of ischemic origin. Laboratory examination "Neurotest": the activity of LE 229 nmol / (min · ml), the activity of α1-PI 36 IE / ml, the levels of aAT1 and aAT2 to antigens of nervous tissue 40-45% relative to the reference serum, the corresponding coefficients of aAT1 / aAT2 0.8 -1.1. The results of the analysis correspond to the current destructive process in the brain tissue of mild severity with a pronounced compensatory potential.

Upon repeated examination after 4 months, a positive dynamics of the revealed neurological disorders was revealed: the relative normalization of muscle tone, firm head retention, the child reaches for the toys and holds them. An increased reaction to sound stimuli remains. Repeated laboratory examination “Neurotest”: activity of LE 200 nmol / (min · ml), activity of α1-PI 27 IU / ml, levels of aAT1 and aAT2 to nerve tissue antigens - within normal values, corresponding coefficients of aAT1 / aAT2 0.8- 1.1. The results of the analysis also indicate the attenuation of the destructive process in the brain and correspond to the control indicators.

Example 2

Patient E., 1 year. At the first visit to a neurologist at 3 months, there were signs of a delay in psychomotor development: muscle hypertonicity, increased reaction to sound stimuli, delayed reduction in search, proboscis, sucking, grasping reflexes of Moro and Babkin, there was no insecure head retention, capture and retention of the toy, a revitalization complex was formed not enough. Diagnosis: delayed psychomotor development. Laboratory examination: LE activity of 280 nmol / (min · ml), α1-PI activity of 48 IE / ml, aAT1 and aAT2 levels to nervous tissue antigens of 40-50% relative to the reference serum, the corresponding coefficients of aAT1 / aAT2 are 0.8-1, one. The results of the analysis correspond to the current destructive process in the brain tissue of moderate severity with pronounced compensatory potential.

A repeated examination after 9 months revealed a generally positive dynamics of the noted neurological disorders: the relative normalization of muscle tone, the child sits alone, crawls, but does not walk, insufficient development of fine motor skills, general motor awkwardness, insufficient development of emotional resonance. There remains an increased reaction to sound stimuli, a violation of the circadian rhythm of mood. Repeated laboratory examination “Neurotest”: activity of LE 220 nmol / (min · ml), activity of α1-PI 36 IU / ml, levels of aAT1 and aAT2 to antigens of the nervous tissue - within normal values, the corresponding coefficients of aAT1 / aAT2 are 0.8- 1.1. The results of the analysis indicate a relative improvement in the determined parameters compared with the first examination, however, the destructive process in the brain has not completely stopped and qualifies as a mild process.

Example 3

Patient D., 4 months. At the first visit to a neurologist at the age of 2 months, severe neurological disorders: delayed reduction of unconditioned reflexes, impaired sucking reflex, muscle hypertonicity, hyperergic reaction to sounds, Moro spontaneous reflex, absence of an animation complex, and visual-motor coordination. Diagnosis: delayed psychomotor development, organic brain damage. Laboratory examination: LE activity of 320 nmol / (min · ml), α1-PI activity of 35 IE / ml, levels of aAT1 and aAT2 to neural tissue antigens 110-120% relative to the reference serum, the corresponding coefficients of aAT1 / aAT2 are 1.3-1, 5. The results of the analysis correspond to the current destructive process in the brain tissue of a severe degree of severity with insufficiently expressed compensatory potential.

At a repeated clinical examination after 2 months, there is a slight dynamics of the revealed violations: there is a violation of muscle tone and a hyperergic reaction to sounds, the child does not hold his head, the revitalization complex is not expressed enough, does not respond to toys. Repeated laboratory examination: LE activity of 300 nmol / (min · ml), α1-PI activity of 25 IE / ml, levels of aAT1 and aAT2 to neural tissue antigens 100-125% relative to the reference serum, the corresponding coefficients of aAT1 / aAT2 are 1.3-1 ,5. The results of the analysis correspond to the current destructive process in the brain tissue of severe severity with exhausted compensatory potential.

Example 4

Patient B., 8 months. Vegetative dysfunction syndrome: prolonged falling asleep, superficial sleep with frequent waking up, dyspeptic disorders, weak sucking. Delay in motor development: he began to hold his head for 4 months, turn over in 7 months. Underdevelopment of fine motor skills. Mimic disorders: hypomania, delay in the formation of a smile. Laboratory examination: LE activity 260 nmol / (min · ml), α1-PI activity 45 IU / ml, aAT1 levels to nervous tissue antigens of 40-50% relative to the reference serum. The results of the analysis correspond to the current destructive process in the brain tissue of moderate severity with pronounced compensatory potential.

Claims (2)

1. A method for laboratory identification of the consequences of perinatal lesions of the central nervous system and their severity in children, including blood sampling, obtaining serum, determining the level of serum autoantibodies to antigens of the nervous tissue aAT1 and aAT2 and their ratio (coefficient I), determining the activity of leukocyte elastase (LE) ), the activity of the α1-proteinase inhibitor (α1-PI) and
- when the activity of LE 201-250 nmol / (min · ml) is detected and when the level of aAT1 and aAT2 is below normal, a destructive process in the brain tissue of mild severity is diagnosed;
- when LE activity of 251-300 nmol / (min · ml) is detected, α1-PI activity is higher than 35 IE / ml, and at aAT1 and aAT2 level below normal, a destructive process in brain tissue of moderate severity with a pronounced compensatory potential is diagnosed;
- upon detection of LE activity of 251-300 nmol / (min · ml), α1-PI activity below 25 IE / ml and at aAT1 and aAT2 level below normal, a destructive process in brain tissue of moderate severity with depleted compensatory potential is diagnosed;
- when detecting LE activity above 300 nmol / (min · ml), α1-PI activity from 35 to 45 IE / ml and at aAT1 and aAT2 levels above normal, a destructive process in the brain tissue of a severe degree with insufficiently expressed compensatory potential is diagnosed;
- when detecting LE activity above 300 nmol / (min · ml), α1-PI activity below 35 IE / ml and at aAT1 and aAT2 levels above normal, a destructive process in the brain tissue of a severe degree with exhausted compensatory potential is diagnosed;
- with an increase in coefficient I above 1.3, a more severe destructive process in the brain tissue is judged. 2. The method according to claim 1, characterized in that the main myelin protein (MBP), S100, GFAP and nerve growth factor (NGF) are used as antigens to the nervous tissue.