US 9,850,518 B2 · Biomarkers for rapid detection of an occurrence of a stroke event

Abstract

A diagnostic assay for detecting an occurrence of a stroke event in a mammalian subject. The assay comprises the steps of: (i) separating a plasma fraction from a blood sample collected from the mammalian subject; (ii) quantifying in the plasma fraction a L-glutamine hydroxylamine glutamyl transferase (L-GHGT) activity; (iii) quantifying in the plasma fraction a gamma glutamyl hydroxamate synthetase (GGHS) activity; (iv) adding together or alternatively calculating the combinatorial probability for the quantified L-GHGT activity and the quantified GGHS activity to produce a value for the net glutamine synthetase activity, and (v) correlating the net glutamine synthetase activity value with net glutamine synthetase activity values from healthy subjects to detect an occurrence of a stroke event in the mammalian subject. Also disclosed are kits comprising reagents and instructions for performing a diagnostic assay to detect and quantify L-GHGT activity and/or GGHS activity.

Drawings

Drawings are temporarily unavailable. Open the PDF at USPTO

Drawings

Claims5

Dependent claims

A diagnostic assay for detecting an occurrence of a stroke event in a mammalian subject, comprising:

(1) separating a plasma fraction from a blood sample collected from the mammalian subject;
(2) quantifying in the plasma fraction a L-glutamine hydroxylamine glutamyl transferase (L-GHGT) activity detected with an assay comprising three sets of steps wherein
the first set of steps comprises
(i) mixing together a pH 6.5 imidazole buffer, a NH2OH solution, a MnCl2 solution, a Na-ADP solution, and a L-glutamine solution to form a first mixture;
(ii) mixing together the first mixture with water to form a second mixture;
(iii) mixing together the second mixture with an aliquot of the plasma fraction to form a third mixture;
(iv) incubating the third mixture at about 30° C. for production therein of a L-glutamic acid γ-monohydroxamate product;
(v) mixing together the incubated third mixture with a L-methionine sulfoximine solution and a hydrochloric acid solution to form a fourth mixture;
(vi) mixing together the fourth mixture with a ferric chloride solution to form a fifth mixture;
(vii) incubating the fifth mixture at about 37° C. for production therein of a ferric hydroxamate complex;
and
(viii) measuring the optical density of the fifth mixture at an excitation wavelength of about 500 nm to produce a first optical density;
the second set of steps comprises
(ix) mixing together a pH 6.5 imidazole buffer, a NH2OH solution, a MnCl2 solution, a Na-ADP solution, and a L-glutamine solution to form a sixth mixture;
(x) mixing together the sixth mixture with a L-methionine sulfoximine solution and a hydrochloric acid solution to form a seventh mixture;
(xi) mixing together the seventh mixture with an aliquot of the plasma fraction to form an eighth mixture;
(xii) incubating the eighth mixture at about 30° C. for production therein of a control reaction;
(xiii) mixing together the incubated eighth mixture with water to form a ninth mixture;
(xiv) mixing together mixture;
(xv) incubating the tenth mixture at about 37° C. for production therein of a ferric hydroxamate complex;
and
(xvi) measuring the optical density of the tenth mixture at an excitation wavelength of about 500 nm to produce a second optical density wherein said second optical density is a control optical density;
and
the third set of steps comprises
(xvii) subtracting the second optical density from the first optical density of the fifth mixture to produce a third optical density;
and
(xviii) correlating the third optical density to a L-GHGT activity of glutamine synthetase enzyme and expressing said L-GHGT activity in Units per liter of plasma;
(3) quantifying in the plasma fraction a gamma glutamyl hydroxamate synthetase (GGHS) activity detected with an assay comprising three sets of steps wherein
the first set of steps comprises
(xix) mixing together a pH 7.2 imidazole buffer, a NH2OH solution, a Mg-ATP solution, and a L-glutamine solution to form a first mixture;
(xx) mixing together the first mixture with water to form a second mixture;
(xxi) mixing together the second mixture with an aliquot of the plasma fraction to form a third mixture;
(xxii) incubating the third mixture at about 37° C. for production therein of a L-glutamic acid γ-monohydroxamate product;
(xxiii) mixing together the incubated third mixture with a L-methionine sulfoximine solution and a hydrochloric acid solution to form a fourth mixture;
(xxiv) mixing together the fourth mixture with a ferric chloride solution to form a fifth mixture;
(xxv) incubating the fifth mixture at about 37° C. for production therein of a ferric hydroxamate complex;
and
(xxvi) measuring the optical density of the fifth mixture at an excitation wavelength of about 500 nm to produce a first optical density;
the second set of steps comprises
(xxvii) mixing together (x) a pH 7.2 imidazole buffer, a NH2OH solution, a Mg-ATP solution, and a L-glutamine solution to form a sixth mixture;
(xxviii) mixing together the sixth mixture with a L-methionine sulfoximine solution and a hydrochloric acid solution to form a seventh mixture;
(xxix) mixing together the seventh mixture with an aliquot of the plasma fraction to form an eighth mixture;
(xxx) incubating the eighth mixture at about 37° C. for production therein of a control reaction;
(xxxi) mixing together the incubated eighth mixture with water to form a ninth mixture;
(xxxii) mixing together the ninth mixture with a ferric chloride solution to form a tenth mixture;
(xxxiii) incubating the tenth mixture at about 37° C. for production therein of a ferric hydroxamate complex;
and
(xxxiv) measuring the optical density of the tenth mixture at an excitation wavelength of about 500 nm to produce a second optical density wherein said second optical density is a control optical density;
and
the third set of steps comprises
(xxxv) subtracting the second optical density from the first optical density of the fifth mixture to produce a third optical density;
and
(xxxvi) correlating the third optical density to a GGHS activity of glutamine synthetase enzyme and expressing said GGHS activity in Units per liter of plasma;
(4) producing a value for glutamine synthetase activity by adding together the quantified L-GHGT activity and the quantified GGHS activity or alternatively, by estimating combinatorial probabilities of the quantified L-GHGT activity and the quantified GGHS activity;
and
(5) correlating said glutamine synthetase activity value with a glutamine synthetase activity value from a healthy subject to detect an occurrence of a stroke event.

53 more elements

Dependencies are inferred from the claim text.

Description9,957 words

Summary757 words

#The present disclosure pertains to the fields of neurology and medicine. More particularly, the present disclosure relates to biomarkers, kits comprising the biomarkers and their use for detecting and/or diagnosing the occurrence of ischemic stroke events.

#Ischemic stroke events are clinical manifestations of rapid loss in brain functions as a result of a lack of blood supply to the brain region due to blockages in the vascular system exemplified by thrombosis and arterial embolisms. A transient episode of ischemia causing neurological dysfunctions for a short period of time, usually less than 24 hours, is called transient ischemic attack (TIA). Rapid diagnosis and treatment of ischemic strokes can ameliorate long-term debilitating neurological damage responsible for partial or full paralysis of limbs and musculature, and/or cognitive impairments.

#Common diagnostic procedures for clinical diagnosis of ischemic stroke include the use of un-infused computerized axial tomographic (CT) imaging and CT angiogram imaging to exclude intra-cerebral hemorrhage and other brain pathologies such as tumors and vascular abnormalities. The limitation with imaging studies is that only a very small percentage of cases of ischemic strokes show any change within the time window of 4.5 hours from the time of onset of stroke symptoms, a critical period during which clot-dissolving medications are useful in dissolving the clot. Therefore, in the majority of cases, a diagnosis of ischemic stroke is made by a reasonable assumption based on clinical presentations. There are numerous diseases that mimic stroke events as their presenting features. They are called stroke-mimics and include diseases exemplified by Todd's paralysis, migraine with prolonged aura, focal neurological deficits associated with hypoglycemia, and hypertensive and metabolic encephalopathies. Currently there are no quick methods available for identification of stroke-mimetic diseases for the purpose of excluding such diseases, and for making a confirmed diagnosis of ischemic stroke in an emergency presentation. Therefore, it is not clear whether all the patients that are diagnosed with ischemic stroke are actually suffering from this clinical condition. Even though magnetic resonance imaging (MRI) scans are available for clinical use, they are even more time-consuming, costly and therefore not used routinely for diagnostic purposes in emergency clinical settings.

Brief description of the drawings6 figures

#The present disclosure will be described in conjunction with reference to the following drawings in which:

#FIG. 1 is a schematic flow chart showing the steps of an exemplary method of the present disclosure for rapid detection of an ischemic stroke event;

#FIG. 2 is a chart showing a standard curve for a ferric hydroxamate complex formed from a reaction of γ-glutamyl hydroxamate with FeCl3 in an acidic medium, in the presence of reagents used for detecting L-GHGT catalytic activity;

Detailed description8,971 words

#Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In order that the disclosure herein described may be fully understood, the following terms and definitions are provided herein.

#The word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.

#The phrases “prevention of” and “preventing” include the avoidance of the onset of a disease, disorder, or a symptom thereof.

Biomarkers for rapid detection of an occurrence of a stroke event

Life of this patent

Biomarkers for rapid detection of an occurrence of a stroke event

Life of this patent

Life of this patent

Abstract

Drawings

Drawings

Claims5

Description9,957 words

Drawings

Classification9 codes

Life of this patent

Description9,957 words