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EP-3657175-B1 - DIAGNOSTIC SET OF REAGENTS FOR DETECTING CHRONIC PATHOLOGIES OF CEREBRAL ISCHEMIC ORIGIN

EP3657175B1EP 3657175 B1EP3657175 B1EP 3657175B1EP-3657175-B1

Inventors

  • DAMBINOVA, SVETLANA ALEXANDROVNA
  • IZYKENOVA, Galina Alexandrovna
  • SKOROMETS, Alexandr Anisimovich
  • GUSEV, EVGENY IVANOVICH
  • MARTYNOV, Mikhail Yurievich

Dates

Publication Date
20260506
Application Date
20171220

Claims (11)

  1. A reagent kit for aiding in diagnosis of a chronic, ischemia-linked brain pathology in a mammal, comprising: a) a hybrid peptide having at least 90% identity over its entire length with the sequence SEQ ID NO:1, wherein the hybrid peptide is immobilized on a solid carrier; b) a reagent for determining the presence of autoantibodies to said hybrid peptide in a biological fluid of the mammal, wherein the reagent has an specific binding affinity for mammalian immunoglobulins.
  2. The reagent kit according to claim 1, wherein the reagent for determining the presence of autoantibodies is a binding agent conjugated to a visualization agent, wherein the binding agent specifically binds to a constant region of mammalian antibody molecules.
  3. The reagent kit according to claim 2, wherein the visualization agent is a gold nanoparticle, an organic dye, a magnetic nanoparticle, a carbon nanotube, or a fluorescent nanocrystal.
  4. The reagent kit according to claim 1, wherein the solid carrier is a cellulose nitrate membrane.
  5. A diagnostic test strip for rapid detection of chronic, ischemia-linked brain pathology in a mammal, having at least three zones configured to be in fluid communication with one another and arranged consequently, namely, a sample application zone, a reaction zone and a detection zone, wherein the sample application zone is capable of absorbing a biological fluid of a mammal and directing it to the reaction zone and the detection zone under the action of capillary forces; the detection zone comprises a test line on which a hybrid peptide is immobilized having at least 90% identity over its entire length with the sequence of SEQ ID NO:1; the reaction zone located between the sample application zone and the detection zone comprises a reagent for determining the presence of autoantibodies to said hybrid peptide in said mammalian biological fluid, wherein the reagent has an specific binding affinity for mammalian immunoglobulins.
  6. The diagnostic test strip according to claim 5, wherein the reagent for determining the presence of autoantibodies is a binding agent conjugated to a visualization agent, wherein the binding agent specifically binds to a constant region of mammalian antibody molecules.
  7. The diagnostic test strip according to claim 6, wherein the visualization agent is a gold nanoparticle, an organic dye, a magnetic nanoparticle, a carbon nanotube, or a fluorescent nanocrystal.
  8. A method of identification of mammal patients with chronic, ischemia-linked brain pathologies, comprising: (a) applying a biological fluid sample from a mammal into the sample application zone of a diagnostic test strip according to claim 7; (b) determining the presence of a chronic, ischemia-linked brain pathology in said mammal when the visualization agent is detected on the test line in the detection zone of the diagnostic test strip.
  9. The method according to claim 8, wherein determination of the presence of the visualization agent on the test line occurs within 15 minutes or less after applying a biological fluid sample to the diagnostic test strip.
  10. The method according to claim 8, wherein the biological fluid is blood, blood plasma, serum, cerebrospinal fluid, saliva, respiratory vapors or sweat.
  11. The method according to claim 8, wherein the chronic ischemia-linked brain pathology is a disease state selected from the following list: chronic ischemia, chronic transient ischemic attacks, repeated strokes or micro-strokes, and cerebral edema.

Description

Field of the invention The invention relates to diagnostic aids, namely, a method, an apparatus and a reagent kit for rapid detection of chronic brain conditions of mammals, in particular, chronic ischemia, endotoxic and cytotoxic edema (brain edema) developing in vascular and traumatic brain lesions, as well as risk of recurrent ischemic events. The invention can be used for prophylactic medical examination or primary examination of patients with a craniocerebral trauma, a stroke or a microstroke in history, and will enable to carry out most optimal therapy measures in neurology, traumatology and sports medicine. Background of the invention Stroke, as well as other acute and chronic pathologies of the brain of ischemic genesis, represent a serious threat to the health and life of people. The importance of early and highly specific diagnostics of these conditions cannot be overestimated; the rate, severity and other parameters of patient recovery depend on the correct diagnosis. Ischemic stroke is especially important to diagnose in the first three to six hours from the onset of the disease for the possibility of performing thrombolytic therapy. Despite the successes achieved, there is still a necessity for new, objective means for diagnosing the risk of recurrence of the acute phase of ischemia against the background of chronic pathology, as well as the emergence of endotoxic and cytotoxic edema (brain edema) associated with them and subsequent small vessel diseases. Diagnostics of such conditions is generally based on methods of neuroimaging, such as computed tomography and magnetic resonance imaging (MRI), which are required to identify affected areas of the brain and the extent of their damage. According to some estimates, up to 40% of stroke patients in the UK can not be diagnosed in time by radiological methods due to contraindications, the condition instability or inaccessibility of equipment (Hand PJ et al. (2005) J Neural Neurosurg Psychiatry 76: 1525-1527). In many other countries, the problem of equipment availability is much more serious, and, consequently, the percentage of such patients is higher. A separate problem is the diagnostics and prediction of the consequences for transient ischemic attacks (TIA) or microstrokes, with symptoms lasting from one hour to 24 hours. In many cases, patients with a stroke or TIA in history, have at least one recurrent stroke recorded within a short period of time. Ignoring the TIA symptoms by the patient can result in the development of chronic brain pathologies. Despite the understanding of the role of certain factors that determine the development of recurrent or chronic strokes, such as the atherosclerosis, high blood pressure or diabetes mellitus, currently it is not possible to monitor the patient's condition with such preceding factors, especially with underlying chronic stroke, using inexpensive biochemical tests for rapid and effective risk assessment of recurrent strokes. Several immunoactive biomarkers contemplated for the diagnosis of stroke or TIA are known from the prior art in addition to the available means of neuroimaging (Bazarian JJ, et al. PLoS One 2014, 9, e94734; Wang KK, et al. J Neurotrauma 2016, 33,1270-1277; E.G. Sorokina et al., Journal of Neurology and Psychiatry 2010,110, 30-35; Guaraldi F, et al. J Clin Med 2015, 4, 1025-1035), however none of them have yet found application in clinical practice, mainly due to lack of specificity. Similarly, at present there are no effective tools on the market for predicting the development of chronic strokes or micro-strokes. Therefore, the problem of creating a specific inexpensive rapid test for the detection of chronic ischemia remains extremely urgent, especially when in association with cerebral edema (endotoxic or cytotoxic edema). This invention has a number of properties necessary to solve the task, and therefore enables to expand the arsenal of tools used to detect chronic brain damages and the risk of recurrent ischemic events. Summary of the invention It is known that the levels of circulating blood fragments of NMDA neuroreceptors can be used as a diagnostic tool for clinical evaluation of patients with stroke or TIA in history. NMDA receptors represent a subclass of ionotropic glutamate receptors that selectively bind N-methyl-D-aspartate (NMDA). The purpose of the invention is to provide a method and a device for the rapid and convenient detection of chronic brain conditions of ischemic genesis in mammals, in particular the risk of recurrent stroke or micro-stroke, delayed chronic ischemia associated with brain edema, with vascular or traumatic brain lesions leading to death of the nervous tissue cells. To solve this object, a hybrid peptide formed under pathological conditions in the form of a single fragment of two fragments of subunits of NMDA neuroreceptor subunits was obtained and tested experimentally (the possibility of the formation of such hybrids was described in the app