JP2020509337A - Predicting the course and outcome of unconscious coma and post-coma states (including vegetative state) using blood tests - Google Patents

Abstract

The present invention relates to the field of diagnostics in emergency medicine, trauma, intensive care and surgery, and can be used in medical practice. In order to objectively assess a subject's brain status in an unconscious or coma state, assess damage to nerve tissue, and predict the progress of a patient's condition, the present invention provides a method for identifying GluR6 and / or GluR7 in body fluids. It is proposed to measure the level of antibodies against fragments of the KAR receptor. KAR peptide fragments and antibodies thereto show structural damage to the brain and are specifically present only in unconscious patients. In the present invention, the magnitude and kinetics of these changes in antibody levels are used to predict both the recovery of the subject's condition in favorable outcomes and the potential for prolonged vegetative status or fatal outcome. [Selection diagram] FIG.

Description

  The present invention relates to emergency medicine, in particular emergency and emergency medicine, trauma, surgery, critical care, and neurosurgery, i.e., by using a blood test to measure brain-specific protein biomarkers, such as antibodies to kainate receptors, for trauma. Methods for assessing unconsciousness and vegetative status as a result of structural damage to the brain of sexual, ischemic or metabolic origin, and for predicting the outcome of coma.

  The unconscious state can be represented by coma and different options of post-coma disease of consciousness (vegetative state, ataxia / hyperactivity muteia) (EV Aleksandrova et al., "Recovery from a controlled unconscious state due to"). severe diffuse axonal brain damage ", Neurology, Neuropsychiatry, Psychosomatics, Issue No. 2/2013, p.51-58). Structural (traumatic) and non-structural (metabolic) injuries can cause coma. A common feature of all types of unconsciousness is the dysfunction of the cerebral cortex, hypocortex and brainstem, and impairment of ascending reticular formation that activates the cerebral cortex.

  Severe cranial brain injury (CCI) is characterized by an acute coma state, either as a direct traumatic injury or as a result of associated secondary (non-traumatic) factors such as edema, translocation, and increased intracranial pressure. Most often due to brain stem dysfunction. In about 35% of cases, recovery from coma is accompanied by a transition to another unconscious state (vegetative state, ataxia), the duration of which affects the degree of further recovery of mental function, work capacity, and deep brain. Determined by the presence and extent of structural damage (stems, basal ganglia and thalamus) (EV Aleksandrova et al., "Recovery from a controlled unconscious state due to many years of radiation, a review of a variety of physicians and stakeholders." No. 2/2013, p.51-58).

  Prediction of coma outcomes includes clinical and biochemical diagnoses. Neurodiagnosis of coma using computed tomography (CT) and nuclear magnetic resonance imaging (MRI) involves setting the nature, cause and severity of hypoconsciousness (using the Glasgow Coma Scale (GCS)). Performing brain MRI, the most informative diagnostic method, is not possible due to the instability of the patient's condition, the inability to move out of the intensive care unit, and the presence of contraindications (the presence of metallic objects in the body) Is often found. In addition, dysfunctions (blood flow, biochemical processes) in the brain are not often evaluated in the normal MRI mode, which causes a false negative diagnosis of brain stem dysfunction. The identification of the cause of the coma also includes the evaluation of the following system parameters: arterial pressure, blood gas and acid-base equilibrium (pH), biochemistry (glucose and ketones, lactic acid and creatine) and electrolyte indicators. However, the specified indicators are not specific to predicting coma outcome, because their values depend on the speed of peripheral circulation and the severity of personal injury.

  It is difficult to predict recovery from coma in the acute phase of severe CCI using only conventional methods. Except for mere medical significance, determining predictions in coma is of great economic importance due to the need to redistribute material resources and limit intensive activity in desperate cases. The rapid assessment of specific parameters of brain status in kinetics is still an unsolved problem. In this regard, identifying specific brain biomarkers that reflect disease onset and are measured in reliable amounts of peripheral blood is an important challenge to monitor and predict brain status in coma .

Russian Patent Application Publication No. 2435165 Russian Patent Application Publication No. 2112243

E. FIG. V. Aleksandrova et al. , "Recovery from a controlled unconscious state due to severe diffuse axonal brain damage", Neurology, Neuropsychology, Psychososomiation, Psychososomiation. 2/2013, p. 51-58 http: // klinrek. ru / calcs / glasgo. htm http: // refdb. ru / look / 23664618-p51. html

  The essence of the present invention is that the concentration of antibodies to a specific neuroreceptor, a fragment of the kainate receptor, which can be detected in the body fluids of patients by appropriate biochemical tests, is unconscious as a result of structural damage to the brain. And increasing in comatose patients. Based on the measurement of objective indicators in the patient's body fluids, reasonably appropriate treatment can be assigned without delay.

  Ion-channel glutamate neuroreceptors are known to be classified into three subtypes: NMDA-type receptors, AMPA-type receptors, and kainate receptors, and are involved in different structures of the central nervous system that perform high-speed signal transmission. Can be seen. NMDA type receptors are mainly located on the microvascular endothelial surface of the cerebral cortex. AMPA-type receptors are localized at synaptic terminals and are found in many subcortical brain structures. Finally, kainate receptors (KARs), particularly subtypes such as GluR6 and GluR7, are located primarily in the brainstem and spinal cord pathways and are involved in regulating cerebral venous circulation, and these receptors are responsible for basic life. Adjust the maintenance function.

  KAR is a receptor protein of the synaptic membrane and is involved in the neurotoxic cascade in brain injury with the formation of secondary vasogenic and / or cytotoxic edema in deep brain structures. The appearance of KAR peptide fragments in body fluids, including blood, refers to structural disorders, ie, pathological changes in the postsynaptic membrane of neurons, and impaired venous blood circulation in the brainstem. The present inventors have found increased levels of KAR fragments formed from GluR6 and / or GluR7 in unconscious patients. In healthy individuals, the formation of KAR fragments in the blood occurs in much smaller amounts or not at all. Excessive concentrations of KAR fragments entering the bloodstream can provoke an immune response, including specific antibody production, whose levels correlate with the degree of severity and the magnitude of structural damage to the brain. In this regard, both the KAR peptides and antibodies thereto can function as biomarkers of acute and chronic brain structural damage.

  It is an object of the present invention to objectively assess the brain status of unconscious and comatose subjects, and also to restore the subject's consciousness (favorable outcome) or to transition to a chronic vegetative or fatal outcome Predicting the development of a patient's condition that may cause (unfavorable outcome of disease onset).

  A solution for this purpose is to measure the level of antibodies to KAR in the body fluids of unconscious subjects and comatose subjects to assess nervous tissue damage, and then to recover from the unconscious state based on this. Is to predict gender. The essence of the proposed method of assessing a patient's brain status is based on the assessment of brain-specific molecular biomarkers, which immediately determines the presence and extent of both structural and functional damage to the patient's stem and subcortical brain structures. This cannot be done with common neurovisualization methods alone (changes occur within 5-7 days).

  In some embodiments of the present invention, a method of predicting a subject's recovery from an unconscious state comprises the following steps: (a) obtaining a biological sample from the unconscious subject; and (b) one of the biological samples. Or measuring the level of an antibody against a plurality of GluR6 and / or GluR7 kainate receptor fragments at least once; and (c) comparing the measured level of the antibody with the level of the relevant antibody in a sample collected from a healthy subject. (D) predicting favorable recovery from an unconscious state when the level of antibody in a sample taken from an unconscious subject does not exceed the level of control recorded in a sample taken from a healthy subject; (e) Antibody levels in samples from unconscious subjects were comparable to control levels recorded in samples taken from healthy subjects. When obtaining includes predicting the onset undesirable disease, the.

  In some embodiments of the invention, it is preferred to use a dynamic measurement of the level of KAR receptor fragments released in brain injury and circulating in the bloodstream. Assess the presence and extent of injury and monitor the restoration of deep structural function, particularly the course of traumatic brain disease, and thus predict outcome under long-term chronic symptoms. In these cases, at least two, three or more biological samples are taken from the unconscious subject at intervals of 1 hour to 90 days after the onset of the unconscious state. Thereafter, the method of predicting the recovery of the subject from the unconscious state is as follows: (a) obtaining at least two biological samples from the unconscious subject at each sampling time interval of 1 hour to 90 days; Measuring the level of antibodies against one or more GluR6 and / or GluR7 kainate receptor fragments in (c); and (c) comparing the levels of the relevant antibodies measured in different samples taken from unconscious subjects with each other. And comparing with the level of the relevant antibody in a sample taken from a healthy subject; and (d) favorable recovery from the unconscious state when the level of the relevant antibody in a sample taken from an unconscious subject decreases over time. (E) the level of the relevant antibody in a sample taken from an unconscious subject over time When pressurized, including this level if more than the level of control that is recorded in samples taken from healthy subjects, and to predict the onset undesirable disease, the. It is desirable to select three or more samples to improve the accuracy of the prediction. To assess the overall status of the glutamate system responsible for cortical and subcortical functions of the brain, determine the time interval between samplings based on kinetic changes in antibody levels to KAR receptor fragments and other glutamate receptor fragments It is reasonable (for example, according to Russian Patent Application Publication Nos. 2435165 and 2112243).

  In a preferred form of the invention, the unconscious subject is a patient, wherein the unconscious state is a coma of traumatic, ischemic and / or metabolic cause.

  In certain embodiments of the invention, whole blood, serum, plasma, cerebrospinal fluid, respiratory air, sweat and / or saliva obtained from a subject are used as a biological sample.

  In the method specified in the present invention, the antibody specific for the kainate receptor fragment is mainly used for immunization such as, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RAI) or an enzyme immunoassay (EIA). Measurement techniques; and other methods of analyzing the measured protein after interaction with a specific ligand (eg, mass spectrometry, NMR spectroscopy, liquid chromatography, etc.), using one or more analytical systems to quantify Detected and / or evaluated.

  According to another aspect, the present invention relates to the application of a kit for use in the method, or the area of application described above, wherein said kit is capable of binding to an antibody against a fragment of the kainate receptor or It includes a ligand that can be specifically recognized by the antibody, and a reporter means.

  In a preferred form of the invention, predicting a subject's recovery from unconsciousness also includes additional clinical, experimental and / or instrumental methods of testing. For example, additional methods are recommended for the appearance of cytotoxic edema, diffuse axonal injury and / or hematomas, and used special modes of nuclear magnetic resonance imaging (T1, T2, FLAIR, DWI, SWI and SWAN). Confirmed by nerve visualization methods and by measuring whole body blood indicators.

  The technical result of the present invention is that the present invention solves the problem of objective prediction of favorable or unfavorable outcomes after brain structural damage causing unconsciousness. The present invention evaluates the presence and extent of structural and functional damage to the brainstem and subcortical structures. In addition, the present invention can assess the time (hours, days, weeks, months) of a patient's recovery from unconsciousness, depending on the value of a number of biochemical indicators. The prediction improves the accuracy and objectivity of assessing brain structural status in unconscious and comatose individuals. Such an approach eliminates subjectivity and improves the accuracy of predicting recovery from unconsciousness when brain structures of different origin are damaged. This will provide optimal therapy and improve the patient's condition.

Terms and Definitions As used herein, the terms "include" and "include" are considered to mean "include among others". It is not intended that these terms be interpreted as "consisting solely of".

  The term "favorable recovery from an unconscious state" refers to a restoration of the subject's (patient's) consciousness, while "unfavorable outcome of disease onset" refers to a chronic vegetative or fatal outcome.

  "Subject" means a human or another less preferred conscious mammal.

  The kainate receptor (KAR) is one of the subtypes of ionotropic glutamate receptors found in structures of the central nervous system of humans and animals. There are five different subunits of the kainate receptor (GluR5, GluR6, GluR7, KA1 and KA2). These form homo- or hetero-tetramers to form functional receptors. In one aspect, the invention relates to a method of measuring antibody levels against a fragment of GluR6 and / or GluR7 that occurs in an unconscious subject. The GluR6 subunit may alternatively be called GluK2 or GRIK2 and is determined by the sequence of SEQ ID NO: 1 (see FIG. 1) or its options. The GluR7 subunit may alternatively be referred to as GluK3 or GRIK3, and is determined by the sequence of SEQ ID NO: 2 (see FIG. 1) or its options.

Means a test using the Glasgow Coma Scale or Pittsburgh scale, whereas "experimental inspection methods" are acid-base equilibrium (pCO 2, _pH) and biochemical parameters (ketones blood "clinical method of examination", Includes measurement of systemic blood parameters, including measurement of lactate, creatine). By "instrumental method of examination" is meant nuclear magnetic resonance imaging, including, for example, T1 and T2 enhancement, FLAIR, DWI, SWI and SWAN modes.

  As used herein, “GluR6 and / or GluR7 kainate receptor fragment” refers to both total protein molecules as determined by the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, which may include partial fragments of these molecules. Different options of the molecules, covalent modifications of fragments of these molecules (eg, phosphorylation, acetylation, modifications under the influence of oxidative stress, amidation, sulfonation, etc.), partial fragments of these molecules and other Cross-linking with other molecules (polyglutamyl, saturated acid, urea, carbamate, aldehydes, ethers, nitroanilides, aminomethylcoumarins, polyethylene glycols, etc.) and the formation of specific antibodies in the body of the subject Other variants that can be induced are meant. All protein molecules of these receptors and their fragments, as well as cross-linking of all protein molecules, partial fragments, partial fragments, and proteins or low molecular weight compounds of these molecules or fragments thereof (biotin, avidin, peroxidase, GABA, AEA, AVA) , Ahx), the various recombination options for GluR6 and / or GluR7 kainate receptor fragments can be used to detect antibodies specific for GluR6 and / or GluR7 kainate receptor fragments. it can.

  A "control" or "threshold" value of an antibody to a fragment of the kainate receptor means the upper limit level of the antibody of interest as measured in healthy subjects. The absolute value of this level may vary depending on the particular detection method of the antibody, and should be determined experimentally for each method.

Amino acid sequences of GluR6 (SEQ ID NO: 1) and GluR7 (SEQ ID NO: 2) kainate receptor Test card for semi-quantitative measurement of KAR antibody based on immunochromatographic assay using nanoparticles. 1-Window for input of body fluid sample, 2-2 duplex filter, one containing nanoparticles (3) with detection reagent, 4- Applied to nitrocellulose membrane (5) with additional control strip (6) Test strip with engineered recombinant GluR6 / 7, 7-result window, 8-filter to adsorb excess reaction solution. The test card having the holding bar (9) is assembled on the main frame of the plastic cassette (10). Dynamic results of KAR antibodies in serum of patients after CCI were taken at different coma durations (see Examples). The red line indicates the KAR antibody threshold (1.5 ng / ml) measured in healthy subject serum.

  KAR proteins are specific to the central nervous system and are involved in a neurotoxic cascade in brain injury with the formation of secondary vasogenic and / or cytotoxic edema in the subcortical brain structure. In the case of structural disorders, the formation of peptide fragments of KAR occurs in body fluids of the deep brain, including the formation of new "heterologous" autoimmune antigens, further causing specific antibody production against peptide fragments of KAR. In the case of CCI, the formation of cytotoxic edema and secondary ischemia (onset of ischemia) in the hemorrhage is associated with a marked increase in KAR antibodies, is a late onset disease of the cerebral circulation, and worsens the outcome of coma cause. By analyzing the body fluids of individuals without CCI, a threshold for the level of antibodies specific for a peptide fragment of a KAR can be determined. An ever-increasing concentration of KAR antibody (ie, above a predetermined threshold) is associated with negative values of systemic blood parameters and predicts the risk of an unfavorable outcome of disease development. Conversely, a gradual decrease in the level of the specific KAR antibody toward the threshold allows a favorable outcome (restored consciousness) to be predicted.

Different biological samples, such as blood, serum or plasma, cerebrospinal fluid, saliva, etc., taken from unconscious patients can be used to measure antibodies. Antibodies are preferably measured by an enzyme-linked immunosorbent assay (ELISA). The ELISA involves reacting the analyzed sample with an antigen that is the recombinant GluR6 / 7 of the kainate receptor immobilized on a solid support, followed by immunization formed by an antibody conjugate to human immunoglobulin G with the corresponding detection enzyme. Detecting the complex. Semi-quantitative or quantitative detection of an increase in the level of specific antibodies is determined by the difference between the value of the analyzed sample (optical density, fluorescence, luminescence or color change value) and the similarity index of the control sample (threshold measurement), Identify brain structural injury patients with low recovery potential. In addition, this method performs a dynamic measurement of systemic blood parameters (lactic acid, creatine, pCO ₂ , ketones and pH) together with the measurement of KAR antibody, and can survive in a traumatic, metabolic or ischemic coma. The method is characterized by allocating treatment without delay to patients with persistent gender.

  The principle of operation of the ELISA-KAR antibody reagent kit involves the quantitative or semi-quantitative detection of antibodies to peptide fragments of the human brain kainate receptor that are present in body fluids and do not bind to the immune KAR complex. The sorption of the specifically selected recombination option of KAR (formed from GluR6 and / or GluR7) can be performed on the surface of the solid carrier with the patient's body fluid (20-100 microliters of blood, serum, cerebrospinal fluid, respiratory fluid, (Ki, saliva) to selectively extract the KAR antibody. The formed antibody-antigen complex is detected by an enzyme-labeled secondary antibody against human immunoglobulin G. Enzyme immunoassays are performed by a vertical scanning device (450/630 nm) with a movable optical density or by a color camera of a mobile device (phone, tablet, etc.) and by the optical density change recorded by a software application (fluorescence or (Depending on luminescence). The color reaction can also be measured visually on a color scale (semi-quantitative option). Changes in KAR antibody concentration, including significant accumulation in the patient's blood, are a measure of the functional status of the patient's brain.

Additional methods of examining patients include the Glasgow Coma Scale (http://klinrek.ru/calcs/glasgo.htm) and the Pittsburgh Scale for brain structural status (http://refdb.ru/look/2236618-p51. html see) and clinical methods, including determination of the severity of the condition. The experimental method of testing involves the measurement of the following systemic blood parameters: lactic acid (reference 0.8-4.0 mmol / h * l); pCO ₂ (reference 32.5-47 mmHg) to determine the degree of gas acidosis And pH (<7.3); creatine to detect renal failure (baseline 76.3 to 114.5 μmol / l in plasma); ketones to evaluate metabolic disease (metabolic coma) (baseline <0.6 mmol) /); All of the above parameters are evaluated cumulatively. Once the patient is hospitalized, nuclear magnetic resonance imaging is also performed using a number of modalities, including T1 and T2 weighting to assess brain structural damage, FLAIR, DWI to determine cerebral edema, and SWI / SWAN to detect cerebral hemorrhage. You.

  The following examples are for the purpose of illustrating the method according to the invention and should not be regarded as means to limit the scope of the invention.

Example of a method for determining antibody content against recombinant variants of the GluR6 / 7 kainate receptor Preparation of analytical serum samples for quantitative ELISA: Analytical serum samples are diluted 1:50 in ratio before measurement; glass or plastic Transfer 20 μl of serum to a test tube, add 980 μl of phosphate buffer, and mix the contents thoroughly by rotary motion. Diluted serum samples can be stored only at + 2-8 ° C for 7 days.

Performing the quantitative ELISA The KAR antibody level in the body fluid of the patient can be analyzed by an enzyme-linked immunosorbent assay using an enzyme-linked immunosorbent assay using a DRD LLC kit (Uran-Ude, Russian Federation). Standard solutions, conjugate solutions labeled with horseradish peroxidase, working buffers and test samples are prepared according to the kit instructions. Wash all wells of the plate coated with the recombinant GluR6 / 7 of the kainate receptor once with buffer (200 μl / well) using an automatic ELISA microplate washer. Add 100 μl of the standard solution or 100 μl of the prepared test sample to the corresponding wells and incubate for 30 minutes at + 37 ° C. in a microplate incubator shaker. At the end of the incubation, wash the wells of the plate with working buffer three times with an automatic ELISA microplate washer. 100 μl of a solution containing an anti-human IgG antibody conjugate with the enzyme (horseradish peroxidase) is added to all wells and the plate is incubated at + 37 ° C. for 30 minutes with constant shaking on a shaker. At the end of the incubation, wash the plate wells three times with working buffer and finally wash the wells with distilled water as described above; with each wash of the plate wells, wash the plate on a shaker at + 37 ° C. for 2 minutes. Need to incubate. Add 100 μl of tetramethylbenzidine (TMB) solution and incubate for 10 minutes in the dark at room temperature. The reaction is stopped by adding 100 μl of stop solution and the light blue / blue color changes to yellow. Each well is carefully mixed to a uniform color and the optical density is measured with a vertical scanning photometer at a wavelength of 450/630 nm. The KAR antibody concentration in the test sample is measured at ng / ml using the antibody concentration-optical density calibration curve in the instructions.

Performing a semi-quantitative ELISA The semi-quantitative measurement of the KAR antibody is performed on a test card by an immunochromatographic assay (lateral flow test) (FIG. 2) using diluted samples just taken from the patient's body fluids. The reaction is activated by adding one drop (about 20 μl) of the sample, and the antibody reacts with the GluR6 / 7 peptide. The formed antigen-antibody complex is detected by visualizing an anti-human IgG antibody conjugate with horseradish peroxidase on a colorful banded control strip. The expression intensity and rate of the band correspond to the antibody concentration in the sample and are measured relative to the color of the band on the reference card provided with the kit. If only the control band appears, the result is considered negative.

Study on correlation between antibody levels to specific KAR (GluR6 / 7) fragments and recovery kinetics of unconscious patients Patient K (female, 19 years old) fell comatose on day 8 after falling from height. He was admitted to the Neurosurgical Laboratory from the day and night primary clinic with non-open severe CCI (sCCI) [Glasgow Coma Scale (GCS) 4 points].

  Diagnosis: Non-open CCI. Grade II brain diffuse axonal injury, diffuse cerebral edema. Judgment on neurological condition at admission: coma 2. Stem damage to mainly metabolic brain.

  Since the female patient had 11 days of coma and 8 days of vegetative state, the duration of the unconscious state was 19 days overall. Dynamic measurement of antibodies to the kainate receptor in serum for the first 7 days (FIG. 3A) showed that the levels were moderately elevated (4.7 to 1.5 ng / ml threshold). 4.4 ng / ml). The overall symptoms of the stem are not obvious and fall into metabolic dysfunction rather than structural damage. MRI performed in T1, T2, FLAIR, and SWAN modes showed no damage to brainstem and subcortical structures.

  Neurological condition 1 month after sCCI: clear consciousness (intelligence disorder syndrome). Predominantly left limb paralysis with low muscle tone. After 6 months, the Glasgow Outcome Scale (GOS) was 5 out of 5 points (good recovery).

  By the end of the first month, antibody levels against KAR (FIG. 1A) tended to drop to the threshold (1.5 ng / ml), indicating a restoration of stem function as noted in the neurological assessment.

  Thus, in patients without damage to the deep brain structures, KAR antibody levels during the coma were somewhat elevated and were immediately normalized with complete regression of stem symptoms and regained consciousness. Overall, after 6 months the patient had a good outcome from sCCI.

  Patient Sh (male, 44 years old) was hospitalized from the day and night primary clinic with severe open CCI (GCS = 4) on the second day in a coma after falling from a height. Diagnosis: acute non-open sCCI. Severe contusion of the left frontal lobe with formation of an intracerebral hematoma (removed on the day of admission). Brain edema. Judgment on neurological condition at admission: coma 2. Comprehensive symptom of the midbrain, trunk from both sides at the top and bottom of the bridge. MRI revealed significant ischemic and hemorrhagic damage to the brainstem and subcortical structures.

  Dynamic measurement of KAR antibodies in serum showed that antibody levels rose sharply from the threshold on the first day to 8.0 ng / ml by day 7 (FIG. 3B). Nine days after sCCI, the level of consciousness decreased to coma 3 (GCS = 3 points), and negative neurodynamics were evident in the form of enhanced overall symptoms in the midbrain, the upper pons, and the trunk at the lower pons. Was.

  On the 14th day, a fatal outcome was recorded against the background of further enhancement of the neurological symptoms. The GOS score was 1 point.

  Thus, a marked increase in antibody titers to KAR with worsening neurological symptoms due to stem translocation was evident in patients with significant structural damage deep in the brain. The outcome of the disease is death 14 days after trauma.

  Patient L (male, 39 years old) was hospitalized 3 days after a traffic accident (motorcycle accident) with a severe CCI coma (GCS = 3 points). Diagnosis: Acute complications of sCCI, severe brain contusion with formation of type 3 bruises in frontal and right temporal and parietal lobes, brainstem. Grade 4 diffuse axonal injury. Severe subarachnoid hemorrhage was removed on the day of admission, and plastic surgery for frontal bone defects was performed on two sides. Judgment on neurological status at admission: coma 3, general symptoms of severe trunk in midbrain position, less pronounced in upper and lower pons, medulla oblongata.

  According to brain CT, hemorrhagic lesions are evident in the frontal, medial, and subarachnoid spaces of the brain. Dynamic measurements of antibodies to the kainate receptor in serum showed that antibody levels were threshold on the first 3 days, 8 days, and 14-17 days of hospitalization (FIG. 3C). However, these values alternate with ultra-high levels of KAR antibody on days 7 and 10-12 (18-28 ng / ml), indicating worsening of the brainstem structure and instability of the patient's condition. By the end of the second month of hospitalization, the patient had recovered from coma and was in a vegetative state for more than 90 days, so the overall duration of unconsciousness was more than 120 days. The patient was discharged from the clinic for vegetative status, GOS 2 points.

  Thus, a clear increase in antibody titers to KAR was also evident in patients with severe structural damage of the deep brain, prolonged coma and vegetative status. The outcome of the disease is chronic unconsciousness.

Characteristics of reagent kit operation The test results of 143 blood samples in 25 unconscious patients (post-traumatic and metabolic coma) and 47 controls show that the concentration of KAR antibody measured by quantitative ELISA is <0.6- It was shown to vary over a wide range of 60 ng / ml. KAR antibody values were distributed as follows: 89 out of 90 serum samples, including controls and favorable outcomes, showed a KAR antibody concentration of ≦ 1.5 ng / ml. KAR antibody levels were 1.6-60 ng / ml in 51 of the 53 serum samples from unconscious and unfavorable individuals. Measurement of the main characteristics of the test to diagnose recovery from coma [disease / positive test (true positive) = 51, disease / test negative (false negative) = 2, no disease / positive test (false positive) = 1, disease None / negative test (true negative) = 89] showed an initial sensitivity of 96% and a specificity of 98%, with a positive likelihood ratio of 86.6.

  Although the present invention has been described in terms of a form of embodiment of the invention, the specific details set forth in the experiments are for illustrative purposes only and are considered to limit the scope of the invention It should be clear to those skilled in the art that this should not be the case. It is to be understood that other embodiments, including various modifications, are possible without departing from the spirit of the invention.

Claims (10)

A method for predicting a subject's recovery from an unconscious state,
a) obtaining a biological sample from said unconscious subject;
b) determining at least once the level of an antibody against one or more GluR6 and / or GluR7 kainate receptor fragments in said biological sample;
c) comparing the measured level of antibody to the level of the relevant antibody in a sample collected from a healthy subject;
d) predicting a favorable recovery from the unconscious state when the level of the antibody in the sample taken from the unconscious subject does not exceed the level of control recorded in the sample taken from a healthy subject;
e) predicting the onset of undesired disease when the level of the antibody in the sample from the unconscious subject exceeds the level of the control recorded in a sample taken from a healthy subject.   2. The method of claim 1, wherein the unconscious state is a coma of traumatic, ischemic or metabolic cause.   The method according to claim 1, wherein the biological sample is a sample of whole blood, serum, plasma, cerebrospinal fluid, respiratory air, sweat or saliva.   2. The method of claim 1, wherein said antibody level is measured by an enzyme-linked immunosorbent assay.   The method of claim 1, wherein the method comprises a clinical, experimental or instrumental additional method of testing. A method for predicting a subject's recovery from an unconscious state,
a) obtaining at least two biological samples from said unconscious subject at time intervals between each sampling of 1 hour to 90 days;
b) measuring the level of antibody to one or more GluR6 and / or GluR7 kainate receptor fragments in said biological sample;
c) comparing the levels of the relevant antibodies measured in different samples taken from the unconscious subject with each other, and comparing the levels of the relevant antibodies in the samples taken from healthy subjects;
d) predicting a favorable recovery from the unconscious state when the level of the relevant antibody in the sample taken from the unconscious subject decreases over time;
(E) if the level of the relevant antibody in the sample taken from the unconscious subject increases over time and this level exceeds the level of the control recorded in the sample taken from a healthy subject, Predicting the onset of undesirable disease;
A method that includes   7. The method of claim 6, wherein the unconscious state is a coma of traumatic, ischemic or metabolic cause.   7. The method according to claim 6, wherein the biological sample is a sample of whole blood, serum, plasma, cerebrospinal fluid, respiratory air, sweat or saliva.   7. The method of claim 6, wherein said antibody levels are measured by an enzyme-linked immunosorbent assay.   7. The method of claim 6, wherein the method comprises an additional clinical, experimental or instrumental method of testing.