RU2258934C9 - Method for predicting the flow of nervous-psychic diseases - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: one should test blood serumal samples in immunoenzymatic assay by applying a test system ELI-N-1, the components of which are being antigens of nervous tissue or their immunochemical analogs specifically binding antibodies with tendency to antigens of nervous tissue, and, also, idiotypical antibodies to them, or their variable parts. Prediction should be performed according to the level of idiotypical and anti-idiotypical autoantibodies to antigens of nervous tissue and, also, according to their ratio. Thus, a new test system has been elaborated that enables to predict the flow of available nervous-psychic diseases.

EFFECT: higher accuracy of prediction.

2 cl, 7 ex

Description

Field of technology: medicine, immunochemistry, psychiatry, neurology

BACKGROUND

Currently, there is more and more evidence showing a close functional relationship of the nervous and immune systems of the body. Various changes in the immune system are described for various pathologies of the nervous system, however, the role of these changes remains largely unclear (Kryzhanovsky GN, Magaeva SV, Makarov SV Neuroimmunopathology. - M .: printing house at the Scientific Research Institute of Labor, 1997 ; Abramov VV Interaction of the nervous and immune systems. - Novosibirsk: Nauka, 1988. Shrikant P., Benveniste EN The central nervous system as an immunocompetent organ. Role of glial cells in antigen presentation. - J. Immunol. - 1996. - Vol. 157. - ¹ 10. - P. 1819-1822).

Normally, the immune system produces antibodies to almost all antigens of the body, i.e. regulatory autoantibodies (AT1) (Jerne N.K. The generative grammar of the immune system. Nobel Lecture, December 1984. - Bioscience Reports (Printed in Great Britain). - 1985. - Vol. 5. - P. 439-451). Studies of recent years have shown that neuroantigens are no exception and there is a certain level of autoantibodies to them in the blood serum, changes of which both towards a pathological increase and an abnormal decrease can have adverse consequences for the nervous system (Poletaev AB, Morozov S. G., Klyushnik TP, Budykina TS, Vabishchevich NK, Gnedenko BB "A method for determining the degree of risk of developing neuropsychiatric diseases", patent for invention No. 2147128).

AT1 has its own “functional balances”, or anti-idiotypic antibodies (AT2), which bind them like an antigen. In our opinion, assessing the state of such an “immune network” can have enormous diagnostic potential, which is practically unclaimed at present.

We have developed a test system designated by us as ELI-N-1 (from ELISA, Neuropathology), which can be used to predict the course of neuropsychiatric diseases. Using this test system, the level of autoantibodies to a number of nervous tissue proteins (or their immunochemical analogues) (AT1), the level of the corresponding specific anti-idiotypic antibodies (AT2) and the coefficient I characterizing the ratio of AT2 / AT1 are determined.

As the closest analogue of the invention may be the work of Warren K.G. et al. (Warren KG, Catz I. Purification of primary antibodies of the myelin basic protein antibody cascade from multiple sclerosis patients. Immunoreactivity studies with homologlus and heterologous antigens. // Clin. Invest. Med. - 1992. - V. 15. - N1. - P. 13-25), which clearly demonstrated the increased titers of idiotypic autoantibodies (AT1) to the main myelin protein (MBP) in patients with multiple sclerosis in the acute stage. In patients in remission, such an increase was not observed. At the same time, this method has several disadvantages.

It does not provide for determining AT1 for MBP in order to predict the course of the disease, but determines the presence or absence of an exacerbation of the disease only at the time of the examination. In addition, the authors determined only AT1 and did not investigate the content of the corresponding AT2. Therefore, using the presented method, it was not possible to draw a conclusion about the balance in the idiotype-anti-idiotype system.

Unlike the method considered as an analogue of the invention, the proposed method allows you to:

- evaluate the serum level of antibodies (AT1) not only to antigens of the nervous tissue, but also to their immunochemical analogues;

- evaluate the serum level of anti-idiotypic antibodies (AT2) to the antigens used in the work or their immunochemical analogues;

- determine the values of the coefficient I, i.e. AT2 / AT1;

- conduct dynamic monitoring of the patient;

- predict the course of existing neuropsychiatric diseases.

The aforementioned is carried out by determining the dynamics of the level of antibodies to a number of proteins of the nervous tissue (or their immunochemical analogues) corresponding to AT2 and the coefficient of AT2 / AT1.

It is advisable to take, for example, the following proteins as antigens: a) S100, b) GFAP (gliofibrillar acid protein), c) MP65, d) NGF (nerve growth factor); e) MBP (the main myelin protein) or their immunochemical analogues, for example, F (ab) ₂ fragments of the corresponding AT2, specifically binding AT1 to these proteins. Various fractions obtained from brain tissue can also be used as antigens of nerve tissue.

As a result of such an examination, the degree of deviation of the levels of all determined AT1 and AT2 is determined both in the direction of pathological increase and pathological decrease, as well as the values of coefficient I (AT2 / AT1) from the empirically found normal range.

The implementation of the essential features of the invention is as follows.

I. Nerve tissue antigens or their immunochemical analogues, for example, F (ab) ₂ fragments of specific AT2 proteins S100, GFAP, MP65, MP-C, NGF and MBF, are adsorbed onto ELISA plates. You can use one, two, three, four or all five of these components of the test system, as well as other antigens of the nervous tissue or their immunochemical analogues.

II. Antibodies (idiotypic) are adsorbed onto ELISA plates to antigens of nerve tissue (to those that were adsorbed onto tablets, see section I.) or their variable regions, for example, F (ab) ₂ fragments of antibodies to S100, GFAP, MP65 proteins , FRN and OBM.

III. Samples of test sera and reference serum are applied to the plates.

IV. Antibodies (idiotypic or anti-idiotypic) binding from the test sera are detected using conjugates of antispecies (secondary) antibodies with an enzyme, such as horseradish peroxidase. The reaction in the wells of the tablet is exhibited by a substrate, for example, o-phenylenediamine and H ₂ O ₂ .

V. Process the received data. The intensities of the reaction in the wells containing the test sera with the wells containing the reference serum are determined. The coefficient I, which is the ratio AT2 / AT1, is determined.

It does not follow from the prior art that, according to a comprehensive assessment of the levels of autoantibodies to a number of nervous tissue proteins or fractions obtained from it (or their immunochemical analogues), levels of the corresponding AT2 and AT2 / AT1 coefficients, one can judge the course of neuropsychiatric diseases.

SUMMARY OF THE INVENTION

PRODUCTION OF NERVOUS TISSUE PROTEINS AND THEIR IMMUNOCHEMICAL ANALOGUES

We used commercial preparations of proteins S100, GFAP, OBM, NGF from Sigma (USA).

MP65 protein was obtained in the following manner. A fraction of cell membranes is isolated from fresh cattle brain by any method (for example, the brain is homogenized in 0.15 M NaCl with 0.01 M Tris-HCl pH 7.5; centrifuged for 5 min at 1000 g; the resulting precipitate is discarded. Sucrose up to 0.32 M is added to the supernatant; repeatedly centrifuged for 10 min at 3000 g; discard the precipitate: equal volume of sucrose was added to the supernatant 0.32 M sucrose, the procedure was repeated 2-3 times, then the supernatant was diluted 2 times with water and centrifuged for 60 min at 20,000 g. The cell membrane pellet was collected. The obtained membranes homogenized at 0.01 M Tris-HCl buffer pH 7.0 with 0.2 M NaCl Centrifuged, collect the precipitate, wash it with 0.01 M Tris-HCl buffer pH 7.5 Homogenize the precipitate in 0.01 M Tris-HCl buffer pH 7.5 with 0.1% X-100 Triton and 2 M urea. Centrifuged for 60 min at 20,000 g. The supernatant was applied to an anion exchanger column equilibrated with the same buffer. Stepwise protein elution was performed using NaCl gradients on the same buffer. A fraction eluted in the range of 0.1-0.3 M NaCl was selected. After dialysis against 0.01% Triton X-100 with 1 M urea in Tris-HCl pH 7.5 buffer, the proteins are applied to the anion exchanger column and eluted in a linear gradient of 0.01-0.6 M KSCN with 0.01% Triton X-100 with UV detection at 280 nm . Gather the last peak. The resulting material is used in the work.

Obtaining F (ab) ₂ fragments of AT2 molecules to nervous tissue proteins, i.e. immunochemical analogues of these proteins were carried out as follows. Chinchilla rabbits were immunized with F (ab) ₂ fragments of polyclonal antibodies to nervous tissue proteins, for example, S100, GFAP, MP65, NGF, OBM, as well as MP-C. Using the immuno-affinity chromatography on a column with immobilized polyclonal antibodies to any of these proteins, the corresponding AT2 was isolated from the resulting antiserum, and their F (ab) ₂ fragments were obtained by proteolytic treatment, which were used to activate the plate (Fab fragments can also be used and other analogues).

To obtain polyclonal antibodies (AT1) and their F (ab) ₂ fragments, the above proteins were used.

SOLID PHASE ELISA

1. Solutions of each of the components of the ELI-N-1 test system (for example, F (ab) ₂ fragments AT1 and AT2 for proteins S100, GFAP, MP65, NGF, OBM, dialyzed against a carbonate buffer pH 9.2-9, 8, they are introduced into individual wells of a standard 96-well flat-bottomed ELISA plate, and 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 (from 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 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 intensity of staining is assessed using an ELISA reader 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 tested 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 of AT2 / AT1, is calculated. The coefficient I is calculated for each pair of AT1-AT2.

10. Interpret the received data.

If the intensity of the reaction of the test serum with any of the components of the test system does not go beyond 65% to 135% compared with the reaction of the reference serum, and all the determined coefficients I are in the range of 0.7-1.3, this serum is assigned to classification group No. 1 - the norm (the prognosis of the course of neuropsychiatric disease is relatively favorable).

If the intensity of the reaction of the test serum with any of the components of the test system goes beyond group No. 1, but is at least 40% and no more than 180% with respect to the reaction of the reference serum, or any of the determined coefficients I is beyond limits 0.7-1.3, but not less than 0.5 and not more than 1.5, this serum is assigned to classification group No. 2 - moderate deviations (increased likelihood of exacerbation of the course of neuropsychiatric disease - it has occurred or should be expected )

If the intensity of the reaction of the test serum with any of the components of the test system goes beyond the groups No. 1 and No. 2 or the coefficient I goes beyond 0.5-1.5, this serum is assigned to classification group No. 3 - sharp deviations ( a high probability of an exacerbation of the course of a neuropsychic disease - it has occurred or should be expected)

The empirical data obtained by analyzing the correlations between the indicators obtained using the ELI-N-1 test system and clinical observations are presented below.

As can be seen from the table, the nature of the course of neuropsychiatric diseases correlates with the degree of deviation of the studied parameters (provided that during the study in dynamics - over several months - the indicators remain within the same group). The more pronounced these deviations, the greater the likelihood of an exacerbation of the course of the disease (if it has not already occurred). Apparently, maintaining in the blood physiological concentrations of autoantibodies to neuroantigens corresponding to AT2, as well as a certain balance between them, is necessary for the normal functioning of the nervous system.

The reference serum was taken from the pulled serum of healthy individuals of a certain age: up to 3 years, from 3 to 10 years, from 10 to 16 years, from 16 to 50 years, from 50 to 70 years, over 70 years, i.e. 5 reference sera for each age group. When examining patients and healthy individuals, the reference serum obtained from a similar age group was used for staging. This is due to the fact that each of these age groups has its own physiological level of the studied antibodies.

The more used the proposed components of the ELI-N-1 test system, the more accurately determine the risk of developing neuropsychiatric diseases and evaluate the nature of its course, which increases the cost of the analysis. We believe that the proposed five pairs of AT1-AT2 are one of the most optimal options.

The positive effect of the invention is to obtain a test system (diagnosticum) ELI-N-1, with which you can identify individuals (with various neuropsychiatric diseases), the serum of which contain abnormal levels of AT1 and AT2 to neuroantigens, abnormal relationships between them , which is a sign of an increased risk of exacerbation of the disease.

The claimed method can be used to monitor the course of a wide range of diseases of the nervous system, especially those where the destructive process takes place. So, this method was tested in patients with multiple sclerosis, Parkinson's disease, epilepsy, as well as schizophrenia, neurotic depression.

When conducting an ELI-N-1 analysis, it should be borne in mind that the presence of various infectious, endocrine, and other diseases can affect the studied parameters, which can lead to an inaccurate interpretation of the studied parameters.

EXAMPLES

Example 1

Patient K., 34 years old, suffers from 17 years of sluggish schizophrenia.

ELI-N-1 Test Results:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to
MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 91 98 1,1 124 101 0.8 76 85 1,1 one hundred 104 1,0 110 129 1,2

Group number 1.

The studied parameters after 1.5 months:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2k OBM I 78 90 1,2 110 97 0.9 77 68 0.9 96 96 1,0 105 111 1,1

Group number 1. Over the next 5 months, no exacerbation of the disease was noted.

Example 2

Patient R., 57 years old. Suffers 5 years of Parkinson's disease. Blood taken during remission. ELI-N-1 Test Results:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 130 134 1,0 one hundred 119 1,2 99 107 1,1 87 99 1,1 98 75 0.8

ELI-N-1 test results after 2 months:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 196 173 0.9 120 176 1,5 126 149 1,2 103 one hundred 1,0 154 167 1,1

Group number 3. ELI-N-1 test results 4 months after the first analysis:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 210 263 1.3 139 196 1.4 135 144 1,1 121 113 0.9 157 175 1,1

Group number 3. 1 month after taking the last analysis, the patient exacerbated the disease.

Example 3

Patient S., 17 years old. From the age of 12 he suffers from epilepsy. ELI-N-1 Test Results:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to NGF I AT1 to OBM AT2 to OBM I 89 85 1,0 90 82 0.9 61 70 1,1 80 94 1,2 90 90 1,0

Group number 1.

ELI-N-1 test results after 3 months:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to NGF I AT1 to OBM AT2 to OBM I 154 205 1.3 181 104 0.6 187 99 0.5 156 one hundred 0.6 119 126 1,1

Group number 1. 2 weeks after the last analysis, an exacerbation of the disease was observed.

Example 4

Patient P., 38 years old. Since the age of 21 suffers from multiple sclerosis. ELI-N-1 Test Results:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to NGF I AT1 to OBM AT2 to OBM I 155 132 0.9 164 155 0.9 117 130 1,1 133 80 0.6 118 119 1,0

Group number 2. ELI-N-1 Test Results:

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to NGF I AT1 to OBM AT2 to OBM I 79 79 1,0 87 99 1,1 104 124 1,2 130 111 0.9 119 131 1,1

Group number 2.

Over the next 3 months, no exacerbation of the disease was noted.

Example 5

Patient N., 27 years old. Suffers from a remitting form of multiple sclerosis. During remission, blood was taken for analysis.

ELI-N-1 Test Results:

Native protein preparations were used as antigens for the determination of AT1. For the determination of AT2, idiotypic antibody preparations were used.

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2k MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 94 one hundred 1,1 67 80 1,2 101 97 1,0 112 130 1,2 121 107 0.9

Group number 1.

A similar analysis was performed with the same blood product. Only instead of native antigens in the determination of AT1, their immunochemical analogues were used, namely, the -F (ab) ₂ fragments of the corresponding anti-idiotypic antibodies; when determining AT2, the preparations of idiotypic antibodies were not used, but their F (ab) ₂ fragments.

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 80 71 0.9 94 111 1,2 123 108 0.9 79 88 1,1 90 98 1,1

Group number 1. Over the next 2 months, exacerbation of multiple sclerosis was not observed.

Example 6

Patient K., 49 years old. Suffers from paroxysmal-progressive schizophrenia. Blood taken during remission.

ELI-N-1 Test Results:

Native protein preparations were used as antigens for the determination of AT1. For the determination of AT2, idiotypic antibody preparations were used.

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 235 116 0.5 200 145 0.7 123 178 1.4 177 164 0.9 289 112 0.4

Group number 3.

A similar analysis was performed with the same blood product. Only instead of native antigens in the determination of AT1, their immunochemical analogues were used, namely, the -F (ab) ₂ fragments of the corresponding anti-idiotypic antibodies; when determining AT2, the preparations of idiotypic antibodies were not used, but their F (ab) ₂ fragments.

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 247 124 0.5 221 167 0.8 144 160 1,1 179 157 0.9 300 124 0.4

Group number 3.

3 weeks after taking the analysis, the patient exacerbated the disease, i.e. observed an acute attack of schizophrenia.

Example 7

Patient L., 67 years old. Suffers from Parkinson's disease. ELI-N-1 Test Results:

Native protein preparations were used as antigens for the determination of AT1. For the determination of AT2, idiotypic antibody preparations were used.

AT1 to S100 AT2 to S100 one AT1 to GFAP AT2 to GFAP one AT1 to MP65 AT2 to MP65 one AT1 to NGF AT2 to FRN one AT1 to OBM AT2 to OBM one 178 114 0.6 131 128 1,0 142 123 0.9 150 147 1,0 172 120 0.7

Group number 2.

A similar analysis was performed with the same blood product. Only instead of native antigens, their immunochemical analogues were used to determine AT1, namely, Fab fragments of the corresponding anti-idiotypic antibodies; when determining AT2, the preparations of idiotypic antibodies were not used, but their Fab fragments.

AT1 to S100 AT2 to S100 I AT1 to GFAP AT2 to GFAP I AT1 to MP65 AT2 to MP65 I AT1 to NGF AT2 to FRN I AT1 to OBM AT2 to OBM I 170 108 0.6 147 131 0.9 153 129 0.8 155 156 1,0 178 125 0.7

Group number 2. The patient has a moderately progressive course of the disease.

TECHNICAL-ECONOMIC AND SOCIAL SIGNIFICANCE

The use of the diagnostic test system ELI-N-1 allows prediction of the course of neuropsychiatric diseases. This may enable clinicians to timely prevent exacerbations of various pathologies of the nervous system.

The study is conducted in the laboratory for enzyme immunoassay.

Claims (2)

1. A method for predicting the course of neuropsychiatric diseases, including blood sampling and obtaining serum, characterized in that they receive the ELI-N-1 test system — antigens of nervous tissue or their immunochemical analogues that specifically bind autoantibodies with an orientation to antigens of the nervous tissue, as well as idiotypic antibodies or their variable regions that specifically bind the corresponding antigens of the nervous tissue, conduct an enzyme-linked immunosorbent assay and patient screening the level of the determined autoantibodies determines in dynamics the level of serum antibodies to nerve tissue antigens (ATI), the level of the corresponding anti-idiotypic antibodies (AT2), coefficient I, which is the ratio of AT2 / AT1, and if the intensity of the reaction of the test serum with any of the components is test -system does not go beyond 65 to 135% compared with the reaction of the reference serum, and all the determined coefficients I are in the range of 0.7-1.3, then this serum is assigned to group No. 1 - normal - favorable prognosis; if the reaction intensity goes beyond the limits of group No. 1, but is not less than 40 and not more than 180% with respect to the reaction of the reference serum, or any of the determined coefficients I goes beyond 0.7-1.3, but is not less than 0.5 and not more than 1.5, then this serum is assigned to group No. 2 - moderate deviations - increased likelihood of exacerbation of the course of neuropsychiatric diseases; if the intensity of the reaction goes beyond the limits of groups No. 1 and No. 2 or the coefficient I goes beyond 0.5-1.5, then this serum is assigned to group No. 3 - sharp deviations - a high probability of an exacerbation of the course of neuropsychic diseases. 2. The test system for determining the immunoreactivity of serum idiotypic and anti-idiotypic autoantibodies to antigens of the nervous tissue, containing standard flat-bottomed plates for enzyme immunoassay, washing buffer, serum-standard, a solution of the conjugate of the enzyme with antispecies antibodies to human immunoglobulins, a substrate solution and a solution of 0.2 -0.4 M sulfuric acid to stop the reaction, characterized in that the tablets contain sorbed antigens of the nervous tissue or their immunochemical analogues, specifically binding autoantibodies with a focus on antigens of the nervous tissue, as well as idiotypic antibodies or their variable regions that specifically bind the corresponding antigens of the nervous tissue.