RU2259567C2 - Method for determining nervous system pathology risk group by means of screening and health state monitoring of people suffering from neuropsychical diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves taking blood samples, producing serum, carrying out immunoenzyme analysis for determining content of anti-idiotypic antibodies to nerve tissue antigens and screening patients according to the level of determined antibodies in which idiotypic antibodies reactivity to nervous system neuromediators like GABA, glutamine, dopamine, serotonin and nerve tissue antigens like proteins S100, GFAP, MBP65 AND NGF and neuromediator receptors is additionally determined. Nervous system development disorder risk level is determined from relation between idiotypic and anti-idiotypic antibodies deviation from norm.

EFFECT: high accuracy of disorder prognosis.

Description

The invention relates to medicine and can be used for screening of neuropsychiatric diseases.

It is known that failures in the mechanisms of immunoregulation caused by various external or internal causes, including congenital ones, can be accompanied by impaired formation and functional maturation of the child’s nervous system or by development of disorders in the activity of the adult’s nervous system (V.V. Abramov, Interaction of the nervous and immune systems Novosibirsk, Nauka, 1988; A.B. Poletaev et al., Neuroimmunology, 2003, 1,1, 11-20).

The immune system of a healthy person constantly produces natural regulatory autoantibodies (e-aAT) to any antigenic components of its own body, including e-aAT to the components of nerve cells or neurotropic e-aAT (Poletaev A.B. et al., Regulatory metasystem, M .: Medicine, 2002). The synthesis of e-aAT 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 those affecting the formation and / or activity of the nervous system.

A known method for determining the risk of developing neuropsychiatric diseases by determining by means of an enzyme immunoassay an immunoreactivity of serum antibodies to a P (ab) ₂ fragment of anti-idiotypic antibodies that specifically bind antibodies to antigens of nervous tissue and when obtaining data on the deviation of immunoreactivity of the tested sera from the level of normal values and the degree of severity of deviations determine the degree of risk of developing neuropsychiatric diseases, based on the data of the "Forecast Table", obtained according to the results of clinical observations (RF patent No. 2147128, registered 03/27/2000 "Method for screening of neuropsychiatric diseases" authors: Poletaev AB, Morozov SG, Klyushnik TP, Budykina TS, Vybishchevich N.K., Gnedenko B.B.). As antigens of the nervous tissue, use the P (ab) ₂ fragment of anti-idiotypic antibodies that specifically bind antibodies with an orientation to the antigens of the nervous tissue S100, GFAP, MP65, NGF. The disadvantage of this method is its relatively low accuracy. Firstly, it allows one to evaluate the content of e-aAT to only four protein components of nerve cells (proteins S100, GFAP, MP-65 and NGF). Corresponding to changes in frequencies, but not always, are a characteristic sign of neuropsychiatric diseases.

Secondly, it allows you to register pathological changes in the serum content of only first-order e-aAT (idiotypic). This is not enough to judge the prescription of the pathological process and the stage of its development.

The objective of the invention is to improve the accuracy of the method.

The problem is solved in that they additionally determine the immunoreactivity of serum antibodies to F (ab) fragments of the neurotransmitters of the nervous system GABA, glutamate, dopamine, serotonin and their antigen-binding fragments or molecules of idiotypic antibodies to these neurotransmitters and when the immunoreactivity deviates from the levels of the tested values to the severity of deviations determine the level of risk of impaired development of the nervous system in children, the risk of pathological changes in of the nervous system in adults, and judging by the ratios of paired idiotypic / anti-idiotypic antibodies, the prescription and dynamics of the development of the pathological process are judged.

The method according to the invention allows:

1. To register pathological changes in the serum content of e-aAT interacting with both nerve cell proteins (S100, GFAP, MBP proteins) and with the main protein of the myelin sheath of nerve fibers (NGF protein), as well as e-aAT, interacting with the main neurotransmitters of the nervous system (GABA, glutamine, dopamine, serotonin), and e-aAT, interacting with membrane receptors of the listed neurotransmitters.

2. To register pathological changes in the serum content of the above-listed first-order e-aATs (idiotypes), as well as second-order e-aATs (specific anti-antibodies; anti-idiotypic antibodies).

All this significantly expands the spectrum of detectable forms of pathology of the nervous system and allows you to reliably judge the dynamics and stage of development of the pathological process in the nervous tissue and the duration of the disease.

In practice, the method is as follows.

As a result of sorption on standard 96-well polystyrene ELISA plates a) antigen-binding fragments or whole molecules of anti-idiotypic antibody-immunochemical analogues of the epitopes of S100, GFAP, MBP and NGF proteins; b) antigen-binding fragments or whole molecules of idiotypic antibodies; c) conjugates of molecules of neurotransmitters - GABA, glutamate, serotonin, dopamine, used to evaluate the content of the corresponding first order anti-mediator antibodies; d) antigen-binding fragments or whole molecules of idiotypic antibodies to these neurotransmitters used to evaluate the content of the corresponding second-order antibodies, which are simultaneously antibodies to the receptors of the corresponding neurotransmitters.

I. Application of tested serum samples and reference reference serum obtained from healthy individuals.

II. Detection of the resulting antigen-antibody complexes using conjugates of secondary (antispecies) immunoglobulins labeled with horseradish peroxidase or another enzyme.

III. IV. Manifestations of the reaction in the wells of the tablet using a chromogen substrate.

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

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

OBTAINING THE MAIN COMPONENTS USED IN THE ELI-N-TEST TEST SYSTEM

1. Obtaining antigen-binding fragments or whole molecules of idiotypic antibodies to proteins S100, GFAP, MBP and NGF. To obtain these components of the test system, rabbits were immunized with S100, GFAP, MBP, and NGF proteins; Using immune affinity chromatography on columns with immobilized S100, GFAP, MBP, and NGF proteins, the corresponding idiotypic antibodies (first-order antibodies) were isolated from the immune serum and their F (ab) - or P (ab) ₂ fragments were obtained as a result of proteolytic treatment. The resulting material (in the form of antigen-binding fragments or whole molecules of idiotypic antibodies (AT1)) was used in the work.

2. Obtaining antigen-binding fragments or whole molecules of anti-idiotypic antibodies (AT2) - immunochemical analogues of the epitopes of proteins S100, GFAP, MBP and NGF. To obtain these components of the test system, rabbits were immunized with F (ab) ₂ fragments of polyclonal antibodies to the corresponding proteins; Using immune affinity chromatography on columns with immobilized polyclonal antibodies to S100, GFAP, MBP, and NGF proteins, the corresponding anti-idiotypic antibodies were isolated from the immune serum and their F (ab) or F (ab) ₂ fragments were obtained by proteolytic treatment. The resulting material (in the form of antigen-binding fragments or whole molecules of anti-idiotypic antibodies) was used in the work.

3. Obtaining conjugates of molecules of neurotransmitters - GABA, glutamate, serotonin, dopamine, used to evaluate the content of the corresponding first order anti-mediator antibodies. To obtain these components of the test system, covalent crosslinking of the corresponding neurotransmitters with gelatin, albumin, or another macromolecular carrier was performed. The resulting material was used in the work.

4. Obtaining antigen-binding fragments or whole molecules of idiotypic antibodies to the neurotransmitters GABA, glutamate, serotonin and dopamine. To obtain these components of the test system, rabbits were immunized with conjugates of neurotransmitter molecules - GABA, glutamate, serotonin, dopamine; Using immune affinity chromatography on columns with immobilized neurotransmitters, the corresponding idiotypic antibodies (first-order antibodies) were isolated from the immune serum and their F (ab) - or F (ab) ₂ fragments were obtained as a result of proteolytic treatment. The resulting material (in the form of antigen-binding fragments or whole molecules of idiotypic antibodies) was used in the work.

DIRECT INFORMATION TECHNIQUE

1. For sorption on standard 96-well flat-bottom plates for enzyme immunoassay, solutions of each component of the test system are prepared on a pH 9-10 carbonate buffer. The prepared solutions are added to individual wells of the tablet. Tablets incubate overnight at +2 ... + 4 ° C.

2. Remove (shake) component solutions from the wells and (without additional washing) fill in a blocking buffer (for example, 0.5% gelatin solution in 0.15 M NaCl).

3. After incubation, the plates are washed with washing buffer (for example, 0.15 M NaCl with 0.05% tween-20).

4. Application of test sera:

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

b) make diluted serum in the wells.

c) the plates are incubated overnight at + 4 ° C.

5. Wash the plates with wash buffer.

6. Manifestation of the reaction:

a) the initial solution of horseradish peroxidase conjugate (or another enzyme) with antibodies to human IgG immunoglobulins is diluted to the required number of times (depending on its activity) with washing buffer; b) the conjugate solution is added to all wells of the plate; c) the plates are incubated for 60-90 min at 37 ° C, or 12-16 hours at +2 ... + 4 ° C. The conjugate is removed and the plates are washed with washing buffer; d) immediately after this, a substrate-chromogen solution is introduced into the wells (for example, 0.01-0.05% o-phenylenediamine with 0.001-0.01% hydrogen peroxide in 0.01-0.05 M citrate-phosphate buffer in the case of peroxidase conjugates). Incubate the tablets in the dark for 15-20 minutes at room temperature.

7. 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.

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

9. Processing of 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 test system OR-H-Test 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

Designations:

R (ar1) is the optical density of the analyzed blood serum in the wells with antigen - 1;

R (ar2) is the optical density of the analyzed blood serum in the wells with antigen-2; ...

R (arn) 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.

10. Interpretation of the received data:

a) if the intensity of the reaction of the test serum of adults with any of the components of the test system does not go beyond -25% to + 15% with respect to the reaction of the reference serum, this serum is assigned to the 1st classification group or normal group (the likelihood of psycho-neuropathology is minimal). For newborns and children before puberty, the norm does not go beyond -40% to 0;

b) if the intensity of the reaction of the test serum with any of the components is outside the limits of group 1, but does not exceed deviations from -40% to + 40% with respect to the reaction of the standard serum, this serum is assigned to the 2nd classification group or group of moderate deviations (low / moderate risk of developing neuropsychiatrics). For newborns and children before puberty, the norm does not go beyond 60% to +10;

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 a group of pronounced deviations (signs of the presence of a mental illness or a high risk of it development);

d) the probability of a favorable / unfavorable prognosis of the development (normal formation) of the nervous system in newborns and young children, and in terms of the normal activity of the nervous system, directly depends on the levels of normal / deviations from the norm, determined by e-aAT in the blood serum of the subjects, and the frequency abnormalities in the formation and functioning of the nervous system directly correlate with the level of deviations. The more pronounced the deviations, the higher the risk of anomalies in the functional-morphological development of the nervous system;

d) the risk of developing pathological changes in the nervous system in adults directly correlates with the levels of deviations from the norm determined by e-aAT in the blood serum of the subjects. The more pronounced the deviations, the higher the risk of developing diseases of the central or peripheral nervous system;

3 + -e) normal ratios between idiotypic and anti-idiotypic antibodies (AT1 / AT2 indices) in any of the pairs are close to 1.0 (should fall within the range of 0.8 ... 1.2). A predominantly elevated level of first-order antibodies (idiotypes) indicates a short duration (not more than 1 month) of the pathological process. A predominantly elevated level of second-order antibodies (anti-idiotypes) indicates the prescription (chronicity) of the pathological process (more than 6 months). By the dynamics of changes in the ratios of paired idiotypic and anti-idiotypic e-AAs, one can judge both the growth of negative trends and the normalization of the functions of the nervous system, for example, against the background of effective treatment.

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

Claims (1)

A method for identifying persons at risk of developing pathology of the nervous system, including obtaining serum, conducting an enzyme-linked immunosorbent assay to identify the content of anti-idiotypic antibodies (second-order antibodies) to antigens of the nervous tissue, followed by calculation of immunoreactivity indicators and screening patients according to the level of immunoreactivity, characterized in that they determine immunoreactivity as anti-idiotypic (AT2) and idiotypic antibodies (AT1) to antigens of the nervous tissue: proteins: S100, GFAP, MBP and NGF, as well as to the neurotransmitter m: GABA, glutamate, serotonin, dopamine and neurotransmitter receptors and the degree of deviation of immunoreactivity from the norm in the ratios of idiotypic and anti-idiotypic antibodies are used to judge the risk of development of the nervous system pathology.