Patent Application
20250172573
The present disclosure relates to an in vitro method for diagnosing traumatic brain injury (TBI), in particular to methods wherein the diagnosis is based on determining level of certain glycan-based biomarkers such as glycoproteins and cleavage products thereof in body fluid of a subject suspected to suffer from TBI. The disclosure also relates to use of N-glycans as biomarkers of TBI.
Traumatic brain injury (TBI) is a multifaceted condition caused by an external force to the head that occurs in, e.g., accidents in traffic, at home, at work, in sports, and on the battlefield. It is broadly defined as an alteration in brain function or other evidence of brain pathology.
Early recognition of brain injury is vital in the process of controlling TBI and starting proper care in order to secure positive prognosis for the patient. Currently, the first diagnostic measure is neurological examination based on patient's verbal response, eye movement, and motion response (Glasgow Coma Scale, GCS). However, this is not very accurate, as the symptoms of TBI are subjective, or because similar symptoms of different origin such as intoxication may overwhelm the signs of TBI. More advanced diagnostic measures in wide use are head scanning by computed tomography (CT) or by magnetic resonance imaging (MRI). However, in many cases mild TBI (concussion) does not result in detectable alterations in the CT or MRI. Moreover, scanners are expensive big devices, and they are typically available in well-equipped hospitals only. Thus, concussion diagnosis relies significantly on GCS and patient's history.
Molecular methods such as biomarker-based diagnostic tools offer a complementary or a surrogate method to detect TBI. They can offer a robust standardized tool for objective classification of TBI. They even have potential to predict the outcome (prognosis) or monitor the state of recovery. Additionally, biomarker-based approaches bear potential for reducing the time and costs of diagnosis.
Several TBI specific blood and cerebrospinal fluid (CSF) biomarker proteins have received considerable research. Examples of these include Ubiquitin C-terminal hydrolase-L1 (UCH-L1), Neuron specific enolase (NSE), Glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100β), Spectrin breakdown product (SBDP), and hyperphosphorylated neurofilaments (p-NF). The practical use that a few biomarkers have seen until today is a reduction in CT requirement among patients who are entering the emergency room with suspected mild brain injury (negative predictive value). To date, they have not been implemented for distinguishing between cases with TBI and without TBI.
Accordingly, there is need for more specific biomarkers for traumatic brain injury.
The present invention is based on the observation that increase of concentration of certain glycan-based biomarkers in urine and/or saliva can be regarded as an indication of traumatic brain injury.
Accordingly, it is an object of the present invention to provide a new in vitro method of diagnosing traumatic brain injury (TBI) in a subject, the method comprising the steps of:
It is also an object of the present invention to provide a new in vitro method of diagnosing traumatic brain injury (TBI) in a subject, the method comprising the steps of:
It is also an object of the present invention to provide a new use of at least one glycan-based biomarker comprising a N-glycan selected from a group consisting of H4N2F1, H3N2, H9N2, H4N3F1S1, and H4N3F3S1, wherein
and
as an indicator of traumatic brain injury in urine.
It is also an object of the present invention to provide a new use of at least one glycan-based biomarker comprising a N-glycan selected from a group consisting of H5N4F2P1, H5N4F3P1, H5N5F1S1, and H3N2F1, wherein
and
and
and
Manα1-6Manα1-6Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc, preferably
as an indicator of traumatic brain injury in saliva.
Further objects of the present invention are described in the accompanying dependent claims.
Exemplifying and non-limiting embodiments of the invention, both as to constructions and to methods of operation, together with additional objects and advantages thereof, are best understood from the following description of specific exemplifying embodiments when read in connection with the accompanying drawings.
The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of un-recited features. The features recited in the accompanied depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of “a” or “an”, i.e., a singular form, throughout this document does not exclude a plurality.
The present invention concerns an in vitro method of diagnosing traumatic brain injury (TBI) in a subject. The method comprises the following steps
As defined herein, the term “biomarker” refers to a molecule that is detectable in a biological sample obtained from a subject and that is indicative of a brain damage in the subject. Markers of particular interest include glycan-based biomarkers showing differences in glycosylation between a sample from an individual with a brain damage, a sample from an individual with an orthopaedic damage, and a healthy control.
As defined herein, the term “glycan-based biomarker” refers to a polysaccharide, i.e. a polymer comprising two or more monosaccharide residues, as well as to a carbohydrate portion of a glycoconjugate, such as a glycoprotein, a glycolipid, a peptidoglycan, or a proteoglycan, or a fragment thereof. Glycan-based biomarkers may comprise either homo-polymeric or hetero-polymeric monosaccharide residues, and they may be either linear or branched. As used herein, the terms “glycan”, “polysaccharide” and “carbohydrate” are interchangeable, unless otherwise indicated. The glycan-based biomarkers of suitable for the method bind lectins.
As defined herein “glycan” is an oligosaccharide or carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. The origin of the glycan can be cell content that is spilled and undergo metabolic degradation, enzymatic cleavage, or chemical breakdown. Such molecules can originate from the immune system like globulins or antibodies, blood proteins like albumin, or macromolecules originating from intracellular fluid.
As a biomarker, the glycan can occur as a mere glycan, or it can be a part of a glycoconjugate.
As defined herein, the term “diagnosis” refers to the determination of whether or not a subject has a TBI. The term is also meant to include instances where the presence of a brain damage is not finally determined but that further diagnostic testing is warranted. In such embodiments, the method is not by itself determinative of the presence or absence of a brain damage in the subject but can indicate that further diagnostic testing is needed or would be beneficial. The methods, therefore, can be combined with one or more other diagnostic methods for the final determination of the presence or absence of a brain damage in the subject. Examples of such other diagnostic methods include, but are not limited to, CT and MRI, and are well known to a person skilled in the art. As used herein, a “final determination” or “final diagnosis” refers to ascertaining the presence or absence of a brain damage in a subject. The final determination or final diagnosis can be the result of any of the methods of the invention which, in some embodiments, can include more than one diagnostic test.
As defined herein, the term “comparing” refers to assessing how the proportion, level, or cellular localization of one or more biomarkers in a sample from a subject relates to the proportion, level, or localization of the corresponding one or more biomarkers in a standard or control sample. For example, “comparing” may refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a sample from a subject is the same as, more, or less than, or different from the proportion, level, or localization of the corresponding one or more biomarkers in standard or control sample. More specifically, the term may refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a sample from a subject is the same as, more or less than, different from or otherwise corresponds (or not) to the proportion, level, or cellular localization of predefined biomarker levels that correspond to, for example, a subject having subclinical brain injury (SCI), not having SCI, is responding to treatment for SCI, is not responding to treatment for SCI, is/is not likely to respond to a particular SCI treatment, or having/not having another disease or condition. In a specific embodiment, the term “comparing” refers to assessing whether the level of one or more glycan-based biomarkers of the present invention in a sample from a subject is the same as, more, or less than, different from other otherwise correspond (or not) to levels of the same biomarkers in a control sample (e.g., predefined levels that correlate to individuals not suffering from TBI).
According to one embodiment the subject is a child. Children are the most common group suffering TBI. In addition, paediatric TBI is also the most challenging to diagnose. Youngest children, in particular, cannot reliably communicate their symptoms. The radiation exposure by CT scan is harmful for the vulnerable, developing young brain. TBI in children is fundamentally different; in children it is a chronic disease process rather than a one-time event. As defined herein a child is a subject of 0-17 years age.
According to another embodiment the subject is an old person. Elderly are at an increased risk of suffering TBI. They are prone to falls and accidents at home or in an elderly home. They are frail, thus small impacts and collisions can cause difficult injuries. A point-of-care test for TBI detection would be a great tool for assessing whether an injured person needs further medical intervention and transportation to the hospital, or not.
According to some embodiments the subject can be an animal, more specifically, a mammal.
The incidence rate for a TBI-related ED visit, hospitalization, or death is more than 1 in 200 among Americans at ages 65-74 years, and more than 1 in 50 at ages ≥75 years. Pre-existing conditions common in elderly, including past history of TBI, are further risk factors for suffering a TBI. Chronic diseases are also associated with worse outcome. Old people with pre-existing dementia may have an abnormal baseline GCS which further complicates the assessment of brain injury after a person has hurt his/her head.
The body fluid to be tested is selected from a group consisting of blood, cerebrospinal fluid, urine, saliva (spit, phlegm, sputum, nasal discharge), plasma, serum, lymph fluid, lymphatic fluid, interstitial fluid, tear, exudate, sweat and extracellular fluid, preferably urine, saliva, and plasma, more preferably urine and saliva.
The glycan-based biomarker suitable for the method is present and detectable in body fluid following a traumatic brain injury. The glycan-based biomarker can be in its intact, native conformation, in which case altered concentration of the glycan-based biomarker in body fluid, in comparison to healthy population, indicates injury. Alternatively, it can be an abnormal biodegradation product, in which case mere emergence of the abnormal product in the body fluid may indicate injury. As a consequence of cell damage, specific proteins are released that start to digest the surrounding proteins inexorably. This leads to the formation or elevation of certain glycan structures in bodily fluids that can indicate cell damage.
In certain embodiments, the glycan-based biomarker refer to a glycoprotein, i.e., a protein that has an oligosaccharide (glycan) group attached in post-translational modifications to an amino acid side chain. The glycan is attached via a glycosidic bond which forms between hemiacetal or hemiketal group of the saccharide and either hydroxyl, nitrogen, phosphorus, carbon, or sulphur group of the amino acid which are connoted O-, N-, P-, C- or S-glycosylation, respectively. Common glycosylated amino acids are asparagine, threonine, serine, tyrosine, tryptophan, and cysteine. The glycan-based biomarker can also be in the form a glycosylated fatty acid or glycolipid. For example, glycosphingolipids are present on cell surface membranes and are particularly abundant in the brain. Glycosphingolipids are a subtype of glycolipids containing an amino alcohol sphingosine. The glycan-based biomarker can also be a free-floating carbohydrate.
A screening of a panel of glycan-based biomarkers can give comprehensive information on the status of various brain tissues. It will help doctors and paramedics in targeting their preferred examination following diagnosis of a brain injury. For example, if the patient is unconscious, drunk, fainted, or unresponsive, the brain damage biomarker can serve as a means to easily obtain information on the condition of brain.
A glycan-based biomarker can also be used to monitor the effectiveness of an intervention or treatment, or healing of a specific brain tissue. These comprise monitoring the effect of a drug or medication, medical intervention like stem cells transplantation as well as perceiving spontaneously happening improvement or stabilization.
The origins of the glycan-based biomarker can originate from various brain cells (neurons, non-neuronal, glia, microglia, astrocytes, oligodendrocytes astrocytes), connective tissues, or blood vessels in the brain.
The glycan-based biomarkers can be found in any of the following body fluids:
Especially, the glycan-based biomarker serves as a signal of brain injury, including
The glycan-based biomarker suitable for TBI analysis can have several sources. One possible origin is a glycoprotein or its cleavage product which is produced in the brain due to the release and uncontrolled function of various enzymes like proteases and glycosidases. For example, excess glutamate, an excitatory amino acid that elevates in the brain following TBI, leads to high levels of calcium ions which can influx into cells and can activate a number of enzymes, including phospholipases, endonucleases, and proteases such as calpain. These enzymes go on to damage cell structures such as components of the cytoskeleton, membrane, and organelles. Alternatively, the glycan-based biomarker can be related to abnormal glycosylation by glycosyltransferases that are released from the damaged brain cells (neuronal, non-neuronal, glia). The glycan-based biomarker can be released to the CSF and the blood flow through a breakage in the BBB and ultimately to saliva and urine.
Alternatively, the glycan-based biomarker can be related to an increase in the expression of proteins related to the physiological changes induced by TBI. It is known to induce cytokine and chemokine expression, and an increase in pro-inflammatory factors.
Upon brain injury the cellular contents are released into the surrounding liquid which is cerebrospinal fluid. From there, the molecule traverse through compromised blood-brain barrier (BBB) to the blood circulation and onward to urine and saliva. Thus, a glycan that is detected in urine or saliva is obviously present in the CSF and blood as well.
The structure can be with or without addition of one or more of the following: sialic acid, fucose, phosphate, sulphate, galactose. Their natural presence or absence is dependent on the conditions (whether acidic or basic environment). Additionally, the structures can rotate freely (for example, the two or more antennas can rotate with respect to the central core mannose).
According to a particular embodiment the glycan-based biomarkers are detected and/or quantified with the use of lectins. Lectins are a well-known family of carbohydrate-binding proteins, i.e., macromolecules that are highly specific for given glycans on the basis of their sugar moiety structures and sequences. Lectins can be classified into distinct groups according to their carbohydrate specificity including, but not limited to, fucose-specific, mannose specific, N-acetylglucosamine-specific, and galactose/N-acetylglucosamine-specific lectins. Accordingly, lectins capable of identifying subjects with brain injury may be used in either individually or in any combination thereof.
According to one embodiment the method comprises steps of
According to one embodiment for urine sample the lectin array comprises one or more lectins selected from a group consisting of GAL1, ORYSATA, PALa, LENTIL, F17AG, LCH (LCA) and DISCOIDIN I.
According to a particular embodiment the method is performed using a lectin array comprising, for urine samples, one of more lectins selected from a group consisting of UDA, GRFT, CALSEPA, BANLEC, NPA (NPL, DL), HHA (HHL, AL), Con A, GAL1, BC2L-A, SAMB, ORYSATA, PALa, PTL-I (PTL, WBA-I), GS-I (GSL-I, BSL-I), PSA (PEA), LENTIL, LEA (LEL, TL), F17AG, LcH (LCA) and DISCOIDIN I, and for saliva samples one or more lectins selected from a group consisting of PTL-1 (PTL, WBA-I), CNL, GAL7-S and CSA.
The advantage of the embodiments shown above is that the structure of the glycan-based biomarker does not need to be known. The only requirement is that lectin is specific enough for glycans relevant to TBI, i.e. the diagnosis is not distorted by other pathological conditions.
According to another embodiment the method includes analysis of the glycan-based biomarkers indicative to TBI. The analysis may include treatment of the samples with N-glycosidase and purification through solid-phase extraction. MALDI TOF mass spectrometry is a particular method since it is not prone to fragment the N-glycans.
Relative abundances of neutral-N-glycans and acidic N-glycans in urine samples are shown in
It was found that suitability of the N-glycans as biomarkers of TBI cannot be judged on basis of the highest apparent increase level in body fluid of a subject suffering from TBI vs. N-glycan level in healthy controls merely by comparing the bars in the graphs. This is because not only TBI but also tissue damages such as orthopaedic injuries liberate glycans to body fluids.
Pauci-mannose N-glycans, high-mannose N-glycans, and monoantennary sialylated N-glycans represent intracellular glycosylation while being less common on cell surface. Accordingly, their elevated level implies disruption on cells upon injury and release of the “contents” to the surroundings. In other words, glycoproteins or glycopeptides carrying those glycans would be present in elevated amounts after TBI.
The pauci-mannose N-glycan structures share common N-glycan core structure and terminal α-mannose residue. Such structures are rare on the cell surface and are associated with intracellular glycosylation, for example lysosomal glycoproteins.
High-mannose N-glycan structures share in common terminal α1,2-linked mannose residues. Also these structures are rare on the cell surface are associated with intracellular glycosylation, for example endoplasmic reticulum (ER) glycoproteins.
Monoantennary sialylated N-glycan structures share common monoantennary N-glycan core structure and sialylation of the antenna. Also these structures are associated with intracellular glycosylation, for example lysosomal glycoproteins.
Pauci-mannose N-glycans present in elevated amounts in urine after TBI and suitable for the method are H4N2F1 and H3N2 as determined by MALDI-TOF mass spectrometry. Exemplary structure of H4N2F1 (1) and H3N2 (2) are shown below.
A high-mannose N-glycan present in elevated amount in urine after TBI and suitable for the method is H9N2 as determined by MALDI-TOF mass spectrometry. An exemplary structure of H9N2 (3) is shown below.
Monoantennary sialylated N-glycans present in elevated amount in urine after TBI are H4N3F1S1 and H4N3F3S1 as determined by MALDI-TOF mass spectrometry. Exemplary structure of H4N3F1S1 (4) and H4N3F3S1 (5) is shown below.
When the body fluid is saliva, the at least one glycan-based biomarker present in elevated amounts after TBI and suitable for the method comprises a glycan-based biomarker comprising a N-glycan selected from a group consisting of H5N4F2P1, H5N4F3P1, H5N5F1S1, and H3N2F1, wherein P is sulphate or phosphate ester, preferably sulphate.
Regarding H5N4F2P1, P refers to one sulfate or phosphate ester, most likely sulfate; F2 refers to two fucose residues, i.e. a core fucose, and an antenna fucose; and H5N4 is a biantennary-size complex-type N-glycan. Accordingly, the N-glycans of formula H5N4F2P1 are biantennary N-glycans with Lewis x or Lewis y on one antenna, and 3′-sulfo-LacNAc (6) or 6-sulfo-LacNAc (7) on the other antenna.
Regarding H5N4F3P1, the formula can be represented by structures (8-11).
Regarding H5N5F1S1, the N-glycan can be presented as a biantennary-size complex-type N-glycan with proposed bisecting GlcNAc structure (12; GlcNAc β1,4-linked to the branching β1,4Man residue).
Further N-glycans (13-16) also comprising “H5N5” motif and bisecting GlcNAc structure are shown below.
Regarding H3N2F1, the N-glycan is of paucimannose type, and it has four possible isoforms, namely
Its exemplary structure (17) structure is shown below.
Thus, according to another embodiment the in vitro method of diagnosing traumatic brain injury (TBI) of the present invention comprises the steps of:
The N-glycans can be determined by using one or more of mass spectrometry, lectin binding, antibody binding, aptamer binding.
The level of N-glycan in the sample can be determined e.g. by one or more of: intensity of the MS signal of the N-glycan, level of lectin binding lectin, level of antibody binding, and level of aptamer binding.
When the sample is urine, and the lectin binding is preferably determined using an array comprising one or more lectins selected from a group consisting of UDA, GRFT, CALSEPA, BANLEC, NPA (NPL, DL), HHA (HHL, AL), Con A, GAL1, BC2L-A, SAMB, ORYSATA, PALa, PTL-I (PTL, WBA-I), GS-I (GSL-I, BSL-I), PSA (PEA), LENTIL, LEA (LEL, TL), F17AG, LCH (LCA) and DISCOIDIN I, preferably GAL1, ORYSATA, PALa, LENTIL, F17AG, LCH (LCA) and DISCOIDIN I.
When the sample is urine, the lectin binding is preferably determined using an array comprising one or more lectins selected from a group consisting of PTL-1 (PTL, WBA-I), CNL, GAL7-S and CSA.
It was also surprisingly found that the N-glycan profile in saliva and urine differ significantly from each other. This phenomenon can be considered when selecting the most suitable glycans for TBI diagnosis.
Preferable glycan structure groups found to be particularly relevant biomarkers of TBI in urine and saliva are collected in Table 1.
It is also possible to provide two body fluid samples from the subject, i.e. a saliva sample and a urine sample, determine the level of the at least one glycan-based biomarker in the saliva sample and in the urine sample, and provide the diagnosis based on the determined level of the at least one glycan-based biomarker in the saliva sample and in the urine sample, wherein increased levels of the at least one glycan-based biomarker in the sample and the further sample compared to normal control levels is indicative of TBI. The use of two body fluid samples allows the use of the best TBI biomarkers in saliva and urine for diagnosis. According to an exemplary embodiment H4N3F3S1 is used as a TBI biomarker in urine sample and H5N4F2P1 is used as a biomarker in saliva samples.
The determining of the glycan-based biomarkers comprising the N-glycans from body fluid can be done by any method known in the art. Exemplary non-limiting method are mass spectrometry, chromatography and separation methods, affinity binding like lectin binding, antibody binding and aptamer binding, or gel electrophoresis.
According to an exemplary embodiment the determining comprises the following steps
According to an exemplary embodiment the separating comprises treating the sample with an alcohol such as ethanol to produce a precipitate comprising the glycan-based biomarker. Treatment of the precipitate with N-glycosidase such as N-glycosidase F enzyme liberates the N-glycan of the glycan-based biomarker and other oligosaccharides present in the sample. The oligosaccharides are preferably purified prior to mass spectrometric analysis. The strength of the MS signal of the N-glycan is compared to a control level. A level higher than the control level, such as at least 1.5 times higher, is an indication of TBI in the subject.
According to another embodiment the determining performed by using specific glycan binders such as lectins, or antibodies raised against the glycan structure or the protein structure of the glycan-based biomarker. Alternatively, a binder element can be a molecularly printed polymer (MIP) to the glycan or protein epitope of the glycan-based biomarker. The binder can also be an aptamer which is an oligonucleotide or peptide that specifically binds the target molecule.
Standard techniques of protein microarray technology can be applied to analyze the glycan-based biomarkers. In such microarrays, lectins are immobilized on a solid support, such as a slide, in a high spatial density. Each lectin may be arrayed at several concentrations and in replicates on each slide. The concentration ranges may be tailored for each of the lectins and calibrated to provide a linear response within the same range, regardless of the affinity of the lectin. A sample of intact glycan-based biomarkers is applied to the array, and its binding pattern is detected by a label, such as a fluorescent label, a radioactive label, or a chemiluminescent label, which is placed either on the biomarker itself or on the lectin directed toward the carbohydrate moieties of the biomarker. Streptavidin may be used for detecting biotinylated samples. Also, sandwich-based methods which utilize antibody detection may be employed, as is apparent to those with ordinary skill in the art.
Suitable microarray substrates include, but are not limited to, glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, gold, various clays, nitrocellulose, or nylon. In some embodiments a glass substrate is preferred. In other embodiments, the substrate may be coated with a compound to enhance binding of the lectin to the substrate. In some further embodiments, lectins have been arrayed on a nitrocellulose membrane-coated glass slide or plastic backing plate. In some still further embodiments, one or more control lectins are also attached to the substrate.
In some embodiments, a commercially available lectin array, which encompasses one standard glass slide, which is spotted with 8 wells of identical lectin arrays, may be employed. Each lectin, together with the positive controls is arrayed in duplicate. The slide comes with an 8-well removable gasket which allows for the process of 8 samples using one slide. Four-slide slides can be nested into a tray, which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously. Unlike other conventional methods, e.g., liquid chromatography and mass spectrometry, lectin microarrays enable rapid and high-sensitivity profiling of complex glycan features without the need for liberation of glycans. Target samples include an extensive range of glycoconjugates involved in cells, tissues, body fluids, as well as synthetic glycans and their mimics. Various procedures for rapid differential glycan profiling have been developed for glycan-related biomarkers and are commercially available.
When the sample is urine, the array comprises preferably one or more lectins selected from a group consisting of UDA, GRFT, CALSEPA, BANLEC, NPA (NPL, DL), HHA (HHL, AL), Con A, GAL1, BC2L-A, SAMB, ORYSATA, PALa, PTL-I (PTL, WBA-I), GS-I (GSL-I, BSL-I), PSA (PEA), LENTIL, LEA (LEL, TL), F17AG, LcH (LCA) and DISCOIDIN I. According to another embodiment the lectins are selected from a group consisting of GAL1, ORYSATA, PALa, LENTIL, F17AG, LCH (LCA) and DISCOIDIN I.
When the sample is saliva, and the array comprises one or more lectins selected from a group consisting of PTL-1 (PTL, WBA-I), CNL, GAL7-S and CSA.
According to one embodiment the method comprises performing at least one neuroimaging procedure selected from a group consisting of x-ray, computerized tomography, and magnetic resonance imaging on the subject. The neuroimaging procedure is preferably performed only if the level of the at least one glycan-based biomarker in body fluid subject is indicative of TBI.
The present invention also concerns use of at least one glycan-based biomarker comprising N-glycan selected from H4N2F1, H3N2, H9N2, H4N3F1S1, H4N3F3S1, wherein H is hexose, N is N-acetyl hexosamine, F is fucose, S is sialic acid, as an indicator of traumatic brain injury in urine. Preferable structures of the N-glycans are disclosed in table 1.
The present invention also concerns use of at least one glycan-based biomarker comprising N-glycan comprising N-glycan selected from a group consisting of H5N4F2P1, H5N4F3P1, H5N5F1S1, and H3N2F1, wherein H is hexose, N is N-acetyl hexosamine, F is fucose, S is sialic acid, and P is sulphate or phosphate ester, preferably sulphate, as a biomarker of traumatic brain injury in saliva. Preferable structures of the N-glycans are disclosed in table 1.
It is also an object of the present invention to provide a new kit or a device for use in the method. The kit or the device comprises at least one lectin, antibody, or combination thereof that selectively binds to a glycan-based biomarker comprising a N-glycan, and a control for comparing to a measured value of binding wherein the N-glycan-is selected from a group consisting of H4N2F1, H3N2, H9N2, H4N3F1S1, and H4N3F3S1 H5N4F2P1, H5N4F3P1, H5N5F1S1, and H3N2F1 wherein H is hexose, N is N-acetyl hexosamine, F is fucose, and S is sialic acid and P is sulphate or phosphate ester. The at least one lectin of the kit and the device is preferably selected from a group consisting of UDA, GRFT, CALSEPA, BANLEC, NPA (NPL, DL), HHA (HHL, AL), Con A, GAL1, BC2L-A, SAMB, ORYSATA, PALa, PTL-I (PTL, WBA-I), GS-I (GSL-I, BSL-I), PSA (PEA), LENTIL, LEA (LEL, TL), F17AG, LcH (LCA), DISCOIDIN I, PTL-1, CNL, GAL7-S and CSA
The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the capture reagents and the washing solution allows capture of the biomarkers on the solid support or column for subsequent detection by, e.g., antibodies or mass spectrometry. In a further embodiment, a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a healthcare professional or consumer about how to collect the sample, how to wash the probe or the particular biomarkers to be detected, etc. In yet another embodiment, the kit can comprise one or more containers with biomarker samples, to be used as standard(s) for calibration.
As is apparent to a skilled person, the present lectin array kit can be used with either a label-based method or as a sandwich-based method. In one embodiment, the label-based method is used for biotinylated samples containing proteoglycans and glycoproteins for direct detection on the array via a Cy3 equivalent dye-conjugated Biotin-Streptavidin complex. In another embodiment, a sandwich-based method is used for antibody detection of glycocalyx elements (glycolipids, glycoproteins, etc.) captured on the array. Labelled reporter antibodies specific for the glycocalyx elements of interest may be provided in the kit or supplied by the user of the kit. An example protocol for this procedure with a general “Antibody Cocktail” may be included in a user manual. In some non-limiting embodiments, specific antibody concentrations and conditions may need to be determined by the end user.
In one embodiment of the biomarker detection kit, HRP protein or other enzymes and fluorescent light or change in absorbance may be employed in order to detect the biomarker in a body fluid and to indicate the quantity of the biomarker in percentage. In one embodiment of the biomarker detection kit, colloidal gold, gold particles, latex particles or similar colloids or particles may be employed in order to detect the quantity of the biomarker. These enable visual detection without instrumentation. Any of the above may be incorporated into a portable application that indicates the severity of brain damage on a scale comprising, but not limited to, none, mild, moderate, and severe. In another embodiment, an analogous yes/no reply is received. These examples do not exclude other possible embodiments.
In some embodiments, the present invention provides use of at least one antibody in a kit or in a device to detect brain damage, where the antibody may be a polyclonal or a monoclonal antibody of any species, or a fragment thereof, either enzymatically cleaved or recombinantly produced, or a humanized antibody, and where the antibody recognizes and binds glycan, glycoprotein, peptidoglycan, proteoglycan, glycolipid, protein, small molecule, lectin, or antibody of another species (generally ‘antigens’). Said antibody may be used, for instance, as
The kit may also comprise a combination of antibodies for different purposes.
All embodiments, details, advantages, and the like of the present kit also apply to a device for use in different aspects and embodiments of the present invention. Also, all embodiments, details, advantages, and the like of the present methods apply to the present kit, and vice versa. In particular, one or more compounds, compositions, or reagents disclosed as suitable for carrying out the present methods may be comprised in the present kit. Likewise, anything disclosed with reference to the kit, apply to the present methods as well.
A clinical study collecting body flid samples from injured patients and uninjured healthy control subjects was carried out. Urine and saliva were collected from 24 patients diagnosed with mild traumatic brain injury (concussion), from 16 patients with an orthopaedic injury (fracture in leg or arm/hand, but no head injury) and from 29 uninjured healthy control subjects who did not have TBI nor fractures during the past year. Upon collection, samples were frozen and stored at −70° C. until analysed.
Urine and saliva samples were analysed by using either chromatographic purification followed by analysis by Mass Spectrometry (MALDI-TOF), or lectin array analysis.
The structural data was acquired by mass spectrometry (MS, MALDI-TOF). The principles for sample preparation, data collection and data analysis were same for all body fluids, and it's briefly summarized as follows.
The samples were pre-treated with ethanol precipitation and the ethanol precipitate was subjected to N-glycan detachment from sample glycoproteins with N-glycosidase F enzyme. Liberated oligosaccharides were purified by solid-phase extraction (SPE) in 96-well plate format: passing through C18 silica in water and then purification by Hypercarb graphitized carbon. The neutral and acidic N-glycan fractions were eluted separately. The two N-glycan fractions were then separately analyzed by MALDI-TOF mass spectrometric profiling using a Bruker Ultraflex III TOF/TOF instrument.
The spectra were compared to databases/libraries of glycan structures. The glycan structures represented by the peaks (m/z ratio) of the spectra were deduced based on this data analysis and knowledge of human glycan structures and synthesis pathways. Relative abundances of the glycans in a sample were calculated based on the area or height of the peak.
Lectins spotted on a glass slide captured the glycan biomarkers from the sample. The immobilized biomarkers were detected by fluorescence visualization of the array.
The lectin-binding results showed a statistically significant increased binding of specific glycans, relative to uninjured healthy samples, both in saliva and urine following a head injury.
In saliva, four lectins showed an increase of ≥70% with statistical significance level of p≤0.05 in TBI samples compared to healthy samples, and furthermore, with none or only minimal increase in orthopaedic samples, rendering TBI vs. Orthopaedic ratio of ≥60% at statistical significance level of p≤0.1 (
In urine, 20 lectins showed an increase of ≥20% with statistical significance level of p≤0.05 in TBI samples compared to healthy samples, and furthermore, with none or only minimal increase in orthopaedic samples, rendering TBI vs. Orthopaedic ratio of ≥20% at statistical significance level of p≤0.05 (
Griffithia sp. Lectin
E. coli lectin
| Number | Date | Country | Kind |
|---|---|---|---|
| 20225157 | Feb 2022 | FI | national |
This application is the U.S. national phase of International Application No. PCT/FI2023/050076 filed Feb. 8, 2023 which designated the U.S. and claims priority to FI 20225157 filed Feb. 22, 2022, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2023/050076 | 2/8/2023 | WO |
