RU2045759C1 - Method for screening of neuro-mental diseases - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method is carried out by immunofermentative analysis, blood serum is tested on plates being covered by absorbed protein S100, increased titers of anti-S100 antibodies indicates neuro-mental disease. EFFECT: simplifies the method, decreases its duration.

Description

 The invention relates to medicine, biotechnology, immunology and psychiatry.

 The proposed ELITEST-1 test system can be used as a diagnosticum for detecting elevated serum autoantibodies to the species-specific protein S-100, which is a universal feature of various neuropsychiatric diseases (schizophrenia, epilepsy, multiple sclerosis, manic-depressive psychosis, and cerebral infant paralysis, hepato-cerebral dystrophy). The ELITEST-1 test system can be used for the rapid and mass detection of clinically non-manifest forms of neuropsychiatric diseases in mass dispensary examinations of the population, for identifying individuals at higher risk of developing neuropsychiatric diseases (for example, for the purpose of professional selection and determining the professional suitability of individuals to perform work associated with increased responsibility), as well as to monitor (monitor) the dynamics of the clinical condition of patients and select the optimal individual drug treatment regimens.

 The ELITEST-1 test system is carried out in vitro based on instrumental laboratory analysis.

 As an analogue of the proposed method, the method of using S-100 proteins was selected for detecting delayed-type hypersensitivity reactions to these proteins in the examined individuals, carried out in vivo by the method of skin-allergic tests.

 As a prototype, a method for obtaining a set of ELITEST-24 antigens from the brain was selected. The prototype describes the technology for producing a 24-component set of brain antigens and using it for the detection and differential diagnosis of human neuropsychiatric diseases in vitro based on an enzyme-linked immunosorbent assay.

 The diagnostic method described in the prototype eliminates two main drawbacks of the analogue: firstly, to refuse to test in vivo (in the analogue, the antigen is administered subcutaneously to the test subjects, which potentially creates the possibility of a wide range of complications, up to the development of allergic shock), secondly, it allows you to objectify the results of the analysis and give a real quantitative assessment of the data obtained, which is almost impossible when using the method of skin-allergic tests.

 Enzyme-linked immunosorbent assays of samples of test persons based on the ELITEST-24 test system described in the prototype provide detailed data on the nature and characteristics of the "anti-brain" autoimmune processes of the subjects and, after appropriate computer processing of the results, confirm or reject the diagnosis of a particular patient with a high degree of certainty. mental illness. At the same time, a number of features of the prototype sharply reduces the possibility and expediency of using it for primary mass screening of large contingents of people.

The prototype method has the following disadvantages:
firstly, obtaining the components of the ELITEST-24 kit requires the consumption of significant amounts of expensive reagents (ampholines), and the preparative isoelectric focusing (the main point in obtaining the ELITEST-24 kit) of large volumes of complex mixtures (total animal brain extracts) requires high the level of professionalism of a specialist biochemist (using the ELITEST-1 method allows to achieve qualitative simplification);
secondly, carrying out diagnostic testing and processing the obtained results take at least 2 days. Moreover, one operator is capable of analyzing up to 10 test samples per day (up to 100 samples subject to the automation of the method), i.e. using the ELITEST-1 method allows to achieve acceleration in obtaining results;
thirdly, the information contained in the results of an enzyme-linked immunosorbent assay based on the ELITEST-24 kit cannot be evaluated (decoded) without the use of a sufficiently sophisticated mathematical apparatus (multivariate analysis based on the least squares method) and computer processing of data to compare them with information banks, characterizing the "spectra" of the antigenic orientation of thousands of serum samples from patients with various forms of neuropsychiatric diseases. Thus, the use of the ELITEST-1 method allows to achieve a qualitative simplification of obtaining results in comparison with the prototype;
fourthly, the analysis based on ELITEST-24 requires a relatively large consumption of standard disposable plates for enzyme-linked immunosorbent reactions and a large consumption of developing immunoreagents (on a single 96-well plate, testing of serum samples of 2 patients is performed).

 All of the noted drawbacks determine the unprofitability (essentially impossibility) of using the prototype method for primary mass screening examinations of the population, but it is completely resolved by the claimed method based on the ELITEST-1 test system.

 The technical result of the invention is a qualitative simplification of the detection of immunopathological changes in humoral immunity, characteristic of many forms of neuropsychiatric diseases, due to the identification of high levels of autoantibodies to species-specific brain-specific proteins S-100 in the tested blood serum samples. The latter is a characteristic feature of more than 75% of blood serum samples of patients with schizophrenia, epilepsy, manic-depressive psychosis, multiple sclerosis, cerebral palsy, hepatocerebral dystrophy and brain tumors, regardless of the specific nosological form of the disease.

 An additional result is the development of a simple and cheap way to objectify the diagnosis of neuropsychiatric diseases, suitable for mass screening.

The technical result is achieved by the implementation of the essential features of the proposed method, as a result of sorption on standard 96-well plates for ELISA of brain-specific proteins of the S-100 group, the test samples of sera taken in dilutions 1: 100, 1: 200 and 1: 400 and made on them combined reference sera obtained from 100 healthy individuals and taken in the same dilutions, detecting antigen-antibody complexes (S100 antibodies) formed on the solid phase using conjugates of secondary (antispecies) immunoglobulins labeled with peroxide the basics of horseradish, the manifestation of the reaction in the wells of o-phenylenediamine / -H ₂ O ₂ and comparing the level of reaction intensity in the wells containing dilutions of the test sera with reference wells.

 The positive effect of the invention is reduced to obtaining a single-component ELITEST-1 diagnosticum with the help of which, in an average of 3 hours, one can identify among individuals from several tens or hundreds of examined individuals those individuals whose sera contain elevated levels of autoantibodies to brain-specific proteins S100, which is a universal feature the presence (or onset of development) of various forms of neuropsychiatric diseases. The data obtained as a result of using the ELITEST-1 test system make it possible to quickly (in 3 hours) and objectively, based on instrumental enzyme-linked immunosorbent assay, to identify patients with certain diseases, including their latent, preclinical and latent forms, or related to group at increased risk of developing neuropsychic diseases. It should be noted that in contrast to the precision diagnostic test system of antigens ELITEST-24 (prototype), the inventive method does not allow differential diagnosis, it is intended to determine the presence or absence (or risk) of neuropsychiatric diseases in the subjects without simultaneous clarification specific diagnosis. However, based on the cost of use (ELITEST-1 is 120 times lower than ELITEST-24), the speed of obtaining results (3 hours versus 48 hours in the prototype) and the absence of the need for complex calculations (whereas the data obtained using the ELITEST set -24, it is impossible to evaluate without the use of a specialized computer analysis program) at the stages of the initial screening of sera of large populations, the most appropriate is the use of the ELITEST-1 test system. Then, those 3-5% of sera in which a positive reaction will be detected (the presumed presence of a neuropsychiatric disease or the risk of its development), it is advisable to undergo a comprehensive precision immunochemical assessment using the ELITEST-24 antigen kit to establish a specific diagnosis of the disease.

 It should be noted that the analysis is performed in vitro without any harm to the subject. Analyzes can be performed both in manual mode (throughput of one operator up to 150 analyzes per day), and in automated mode (throughput of the operator in this case is 1000-2000 analyzes per day). Moreover, the accuracy of detection of cases of neuropsychiatric pathology (on average 75%) has the same order as in the prototype (on average 80%).

 The method used to obtain S100 is well known. Enzyme-linked immunosorbent assay procedures were performed according to generally accepted ELISA sandwich variants.

The method is as follows. Enzyme-linked immunosorbent assay procedures were carried out according to generally accepted methods. In ampoules (vials) containing 0.1 mg of lyophilized S100, 1 ml of 0.1 M carbonate buffer pH 9.5 was added, dissolved, the protein concentration was adjusted to 2 μg / ml with the same buffer and added to the wells of standard 96-well enzyme immunoassay tablets of 100 μl. After 18 h incubation at ⁴ ° C nesorbirovannogo excess antigen was removed, the wells were washed 4 times with otmyvnym buffer (0.01 M phosphate buffer pH 7.4 with 0.15 M NaCl and 0.05% Tween-20). Then, 100 μl of the tested sera were introduced into the wells in 1: 100, 1: 200, 1: 400 dilutions (in 90 wells), and the same dilutions of reference serums in two replicates in the 6 remaining wells of the plate. After incubation for 1 hour at ³⁷ ° C the wells were washed 4 times with otmyvnym buffer and applied to them by 100 .mu.l of conjugate secondary antibody (directed to human IgG) labeled with horseradish peroxidase. After additional incubation for 1 hour at ³⁷ ° C the wells were washed 4-times and applied to them by 100 l of the chromogen solution (0.01% solution of o-phenylenediamine with 0.001% H ₂ O ₂₎ was incubated for 20 minutes in the dark at room temperature and quenched the reaction by adding 50 μl of 2 M H ₂ SO ₄ , after which the reaction intensity in each of the wells was measured photometrically at a wavelength of 490 nm.

где ОП₁ оптическая плотность лунок с разведением сыворотки 1:100;
ОП₂ то же с разведением 1:200;
ОП₃ то же с разведением 1:400.A quantitative assessment of the immunoreactivity of antibodies to S100 in the tested serum samples was expressed as an indicator of average immunoreactivity (SIR), taking into account the integral values of the immunoreactivity of a particular sample in different dilutions:
SIR

where OP _{1 the} optical density of the wells with a dilution of serum 1: 100;
OP _{2 is} the same with a dilution of 1: 200;
OP _{3 is} the same with a dilution of 1: 400.

The value of the obtained SIR of each of the tested sera was compared with the SIR of the reference sera taken as 100%
The results obtained in the analysis of 50 or more serum samples of patients of each of the nosological forms of neuropsychiatric diseases (clinically reliably verified forms of diseases. Altogether more than 400 samples) indicate that a significantly increased immunoreactivity of serum samples with S100 proteins is characteristic for:
85% of patients with schizophrenia (n 120);
65% of patients with epilepsy (n 76);
75% of patients with hepato-cerebral dystrophy (n 50);
70% of patients with cerebral palsy (n 142);
50% of patients with manic-depressive psychosis (n 64).

 The value of SRI was considered to exceed the reaction rate if its value was higher than or equal to 120% of the values of SRI of reference serums.

 The value of 120% represents a double error of the average immunoreactivity with the S100 protein of blood serum of mentally, neurologically and somatically healthy individuals, determined empirically at n 50.

It is important to note that the ELITEST-1 test system (enzyme-linked immunosorbent assay plates with purified S100 proteins adsorbed in wells) can be prepared immediately before use as described above, or can be prepared in advance. In the latter case, the plates were washed from excess nesorbirovannogo antigen, dried and stored at ^-20 ° C for 2-4 months. before use. Such "delayed" use of the ELITEST-1 test system did not affect the quality of the results. Obviously, this option (“delayed") using the ELITEST-1 test system can be convenient when preparing for periodic large-scale population surveys (dispensary examinations of several thousand people), moreover, the test system itself can be prepared centrally, and S100-coated tablets will be distributed to places of use (clinics, dispensaries, field expeditionary teams). And tests can be carried out both on fresh serum samples and on samples stored for up to 1 year at ^-70 ° C (delayed analyzes if necessary).

PRI me R 1. History of the primary patient with a clinical diagnosis: borderline state (depressive syndrome?)
Patient A. 54 years. The heredity of mental illness is not burdened. Born in the village, the preschool period is remembered very vaguely. I went to school from the age of 7. She studied without much interest. The character was calm, uncommunicative, in her free time she loved to do household chores. At 20, she moved to Moscow, got a job as a typist. Soon got married. From cancer has a son and daughter. By nature, she remained calm, uncommunicative, loved to do household work, take care of her husband and children. This year, after the death of her brother, whom she loved very much, the patient's mood sharply decreased, she could not do anything, sleep was disturbed, she had a feeling of anxiety, a feeling of heaviness behind her breastbone, her appetite decreased, she lost 5 kg within a month. Daily diurnal fluctuations of mood were noted, I felt especially bad in the morning. Two months later, she first visited a psychiatrist and was hospitalized in a psychiatric hospital, where she spent 45 days. Received amitriptyline, tazepam, elenium. By the time of discharge, the mood was completely normal, sleep improved, anxiety, a feeling of heaviness behind the sternum disappeared. Mental state during hospitalization: oriented in space, time, sad expression, movements, speech slowed down. Complains of hopeless anguish, a feeling of heaviness and contraction in the sternum. She believes that she is guilty of the death of her brother, as she could have warned this, but did not. Eats with duress.

 Carrying out the diagnostic test "ELITEST-1".

 A response titer with a test system corresponding to 115% was established. This value does not exceed 120% of the level accepted for the borderline level of serum immunoreactivity of normal individuals.

 Conclusion: According to the result obtained on "ELITEST-1", the presence of neuropsychiatric pathology has not been confirmed.

 PRI me R 2. History of a patient with a diagnosis of malignant "nuclear" schizophrenia.

 Patient C., 20 years old. The heredity of mental illness is not burdened. Pregnancy and childbirth in his mother took place without complications. Early development without features, started walking and talking on time. By nature he was calm, uncommunicative, he preferred to play alone, but he did not shy away from his peers, he came into contact with them quite easily. Of the diseases transferred in preschool age were: colds, measles, scarlet fever. The course of the disease was uncomplicated, for two years (from 5 to 7 years) there was a fear of darkness, fell asleep only in the light and in the presence of one of the parents. Why the darkness scared, definitely could not say. With admission to Grade 1, the fear of darkness disappeared. I went to school from the age of seven. He studied medium, without much interest. He did not take good homework; he wrote off parts from classmates. By nature he remained quiet, calm. Once he told his parents that he hears some voices inside his head. He was hospitalized in a psychiatric hospital. In the patient’s hospital, stereotypical movements, grimacing, and increased muscle tone of the cervical and chewing groups were noted. Sometimes for long hours he froze in one pose, sometimes he became sharply excited, aggressive, tore his clothes on, attacked the sick, staff. Outwardly, he was always messy, did not wash, his hair was disheveled. It was distinguished by increased appetite, often took or took food from other patients. In 1981 he was transferred to the hospital. P.P. Kashchenko. From that moment, the state remains almost unchanged: it is inaccessible to the contact, spends most of the time in bed, while singing and muttering. He makes fanciful movements with his hands, there is an increased appetite, and he can eat anything: he eats sweets with wrappers, several times he tried to swallow a plastic bag in which food is stored. All this time the patient received massive doses of antipsychotics, but his condition did not improve.

 Carrying out the diagnostic test "ELITEST-1".

 A response titer with a test system corresponding to 155% was established. This value exceeds 120% of the level accepted for the borderline level of serum immunoreactivity of normal individuals.

 Conclusion: According to the result on "ELITEST-1" confirmed the presence of neuropsychic pathology.

 PRI me R 3. History of the patient with a diagnosis of epilepsy with severe personality changes.

 Patient M., 53 years old, disabled person of the second group. Repeatedly hospitalized in a psychiatric clinic. S.S. Korsakova. He complained of recurrent seizures, weakness, lethargy, and headaches. The heredity of mental illness is not burdened. The patient was born from the first pregnancy on time. The birth was protracted, with the imposition of obstetric forceps. Early childhood was without features. A portion was observed vomiting. Diseases in childhood: childhood infections. Until the age of 13, he suffered from nocturnal enuresis. Restless in a dream, often woke up. Subsequently, these phenomena disappeared. He was released from military service in connection with the appearance of convulsive seizures. At the age of 18, a large convulsive seizure first occurred, when, according to colleagues, it suddenly fell along the road, convulsions began in the whole body, accompanied by urination and defecation. Subsequently, seizures were repeated 1-2 times in six months. At the age of 22, seizures became more frequent and became monthly. Paroxysms have become rare (1-2 times a year). At 38, the patient developed headaches. He became conflicted, slow. At the age of 50, due to a marked decrease in prof. level and significant personality changes posed the second group of disability. Currently not working. About once a week there are paroxysms of the type of outpatient automatism.

 Mental state: the patient is oriented in the place of his own personality, however, it is difficult to give an exact date, although he correctly names the month and year. Contact. He answers notes questions in detail, in detail, not always essentially, independently switches to a description of his disorders. Hallucinatory and delusional disorders are absent. All the patient's attention is focused on their own disease. The memory has been significantly changed. The mood is subject to fluctuations. Amid complacency and benevolence, bouts of irritability often occur. Conflicts with staff and patients about compliance. Vindictive. There is no critical attitude to his condition. The main goal of life is taking care of one's own health. An electroencephalographic study revealed signs of focal bilateral paroxysmal activity, the phenomenon of areactivity and desynchronization.

 Carrying out the diagnostic test "ELITEST-1".

 A response titer with a test system corresponding to 256% was established. This value exceeds 120% of the level accepted for the borderline level of serum immunoreactivity of normal individuals.

 Conclusion: According to the result on "ELITEST-1" confirmed the presence of neuropsychic pathology.

 PRI me R 4. History of a patient with a diagnosis of multiple sclerosis (pseudotumor form).

 Patient Zh. 28, an engineer, was admitted to the hospital with complaints of headaches, numbness and weakness in his right hand. Considers himself ill for about 3-4 weeks, when he first drew attention to numbness in the fingers of his right hand, sometimes his right cheek was numb. There was a heaviness in the head.

 On examination: the patient is conscious, oriented, blood pressure 120/80 mm RT. Art. pulse 68 beats per minute, rhythmic. Nystagmus when looking to the left. Slight paresis of the right hand is predominantly in the distal regions, tendon reflexes are higher on the right. Over the course of putting on, a gradual deepening of the paresis in the right hand was noted, weakness appeared in the leg and a violation of superficial and deep sensitivity in the region of the trunk and limb on the right. Muscle tone in the right extremities is increased. Skull images: bone-destructive changes are not determined. Over the next 3 years, the patient had a relapse of the disease twice, weakness in the right extremities, headache, dysarthria, choking on swallowing and impaired coordination increased again. Under the influence of treatment in the patient's condition, a noticeable improvement occurred each time.

 Carrying out the diagnostic test "ELITEST-1". A response titer with a test system corresponding to 170% was established. This value exceeds 120% of the level accepted for the borderline level of serum immunoreactivity of normal individuals.

 Conclusion: According to the result on "ELITEST-1" confirmed the presence of neuropsychic pathology.

 PRI me R 5. Collection of medical history of a patient with a diagnosis of cerebral palsy (early stage).

 Vasya P. 4 months born in difficult birth, in asphyxiation. In the first days of life, he found weakness, and then the absence of a sucking, palmar-mouth, search reflex, support reaction, crawl reflex, did not raise his head from a prone position, did not turn it to the side, etc. The tone of extensors in the muscles of the lower extremities prevailed and the tone of the flexors in the upper limbs was slightly increased. By the end of the first month, it became possible to turn and raise the head while lying on the stomach, there was a reaction of support, a crawl reflex. However, a clear activity of the tonic labyrinth and cervical reflexes was revealed. There was a fixation of the gaze, the first elements of tracking objects. He began to gain weight. Toward the end of the 2nd beginning of the 3rd month, it was possible to note a slowly but steadily increasing pathological activity of labyrinth tonic reflexes. Sharp muscle tension was accompanied by severe pain, the child almost always made hoarse cries and groans. After a week, during which ACTH was administered, the child began to periodically suck out 50-70 g of milk, and attacks of torsion dystonia became much less common. By the end of the 10th day, torsion attacks became more frequent, the tone of the extensors of the neck, back, and limbs increased sharply in any position of the child's body. After appropriate therapy, the child's condition returned to normal. For the first time there were movements of facial muscles of the face, resembling a smile.

 Carrying out the diagnostic test "ELITEST-1".

 A response titer was established with a test system corresponding to 280% of the level accepted as a borderline level of serum immunoreactivity in normal individuals.

 Conclusion: According to the result on "ELITEST-1" confirmed the presence of neuropsychic pathology.

 In total, using the ELITEST-1 test, 662 serum samples were analyzed (of which 220 samples obtained from somatically and psycho-neurologically healthy individuals and 442 samples obtained from patients with neuropsychiatric diseases: 120 patients with schizophrenia; 76 patients with epilepsy; 76 patients with hepatitis cerebral dystrophy 50; patients with cerebral palsy 142; patients with manic-depressive psychosis 64). Moreover, false-positive results (reactivity level above 120% in clinically healthy individuals) were detected in 4% of cases and false-negative results (reactivity below 120% in individuals diagnosed with a mental illness) in 25% of cases.

 The use of the ELITEST-1 diagnosticum for the detection of neuropsychiatric diseases based on an enzyme-linked immunosorbent assay allows for mass express diagnostics to obtain a result within 3 hours. The study is performed in vitro and requires 10 μl of blood of the test person.

 A quantitative assessment of the content of autoantibodies to S100 proteins by the average immunoreactivity index allows using the ELITEST-1 diagnosticum to track the dynamics and monitor the condition of patients over long periods of time, assess the effectiveness of the therapy and select the optimal drugs and treatment regimens for the disease.

 Obtaining reagents does not require complex and expensive equipment and reagents, and the analysis procedures are carried out by the laboratory operator.

 Diagnostic ELITEST-1 is characterized by significantly lower cost (120 times lower) and higher throughput (20 times higher) compared to the precision diagnostic test system ELITEST-24. The possibility of widespread implementation of the method into practice on the basis of a negotiated price is proposed.

 The feasibility, social and medical significance of new objective methods for diagnosing neuropsychiatric abnormalities is obvious, since the incidence of schizophrenia, for example, is from 1 to 4% of the world's population (according to WHO), and there are no simple, reliable and objective methods for their diagnosis.

 There are experimental indications that changes in humoral immunity detected by the described method can be detected before the development of a clinically pronounced process. In this regard, it can be assumed that the widespread use of a simple and cheap diagnostic kit ELITEST-1 will contribute to the objective identification of groups of "increased risk" of the disease, which is important for preventive measures and to assess professional suitability in the selection of individuals for certain positions and professions, for example, related with increased responsibility and / or risk to the life and health of the operator and others.

Claims (1)

 METHOD FOR NEROPSYCHIC DISEASES SCREENING, including blood sampling, serum preparation, obtaining a test system and conducting an enzyme-linked immunosorbent assay for the detection of antigen-antibody complexes using 1gG secondary conjugates labeled with horseradish peroxidase, followed by calculation of the integral immunoassay serum is added to the wells of the tablet containing the adsorbed protein S 100 and upon receipt of data on the excess of immunoreactivity of the tested serum the level of normal phon s values of no less than 20% determined by the presence of neuro-psychiatric illness.