WO2000034336A1 - A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids - Google Patents
A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids Download PDFInfo
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- WO2000034336A1 WO2000034336A1 PCT/US1999/029023 US9929023W WO0034336A1 WO 2000034336 A1 WO2000034336 A1 WO 2000034336A1 US 9929023 W US9929023 W US 9929023W WO 0034336 A1 WO0034336 A1 WO 0034336A1
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- map
- monoclonal antibody
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Definitions
- the present invention relates to a method of determining neuronal cell body damage by detecting the presence of a neuron-specific protein in biological fluids. More specifically, the invention relates to the quantification of microtubule-associated protein 2 (MAP-2) in a biological fluid, such as blood plasma or cerebrospinal fluid (CSF), by treating the selected fluid sample with one or more MAP-2-specific monoclonal antibodies and detecting and quantifying the amount of antibody bound to MAP-2 in the sample.
- MAP-2 microtubule-associated protein 2
- CSF cerebrospinal fluid
- the method embodied in the invention can be utilized in a clinical laboratory test to aid physicians in the diagnosis of traumatic brain injuries and other central nervous system dysfunctions.
- Traumatic brain injuries most commonly take the form of injury to the cortex of the brain. Lesions resulting from such injuries involve primarily the superficial grey matter of the brain and may or may not be associated with hemorrhage. Gentry, Radiology, 191: 1-17 (1994). Currently, the predominant method for evaluating such lesions is through the use of computed tomography (CT). Gentry, ibid.
- CT computed tomography
- Computed tomography is presently preferred because of its wide-spread availability, low cost, safety and the rapid rate with which results can be obtained.
- the major disadvantage of CT is that only those contusions which are large in area or which contain regions of hemorrhage can be easily and accurately diagnosed via a CT scan. This difficulty is underscored if the scan is obtained within a short space of time after the infliction of injury. Problems associated with radiographically imaging smaller or non-hemorrhagic contusions in critical care settings are well-documented; often the edema within the contused brain is so minimal that the lesions are only faintly seen or invisible. Although undetectable by CT scans, the gravity of such injuries is comparable to that of detectable lesions.
- Neuronal degeneration is also associated with various types of central nervous system (CNS) dysfunctions, including subarachnoid hemorrhages, brain tumors, aneurysm, stroke and similar clinical conditions.
- CNS central nervous system
- the functional integrity of the neuronal compartment is compromised and the neuronal cells themselves break apart, releasing their intracellular components. Some of these cellular components are transported to the cerebrospinal fluid, bloodstream or other biological fluids.
- MAP-2 microtubule associated protein 2
- MAP-2 is a neuron- specific cytoskeletal protein localized primarily in the cell body and dendritic regions of neurons.
- the genomic and amino acid sequences of MAP-2 have been described in Kalcheva et al. Proc. Nat'l Acad. Sci. USA 92, 10894-98 (1995), the contents of which are incorporated herein by reference.
- MAP-2 is maintained in high concentrations within neuronal cells and, under normal conditions, is not found outside the neuronal cell.
- MAP-2 is released from the cells and transported to the cerebrospinal fluid and bloodstream of the head injury patient. In the absence of trauma or disruption of neuronal cells, MAP-2 is not released; thus its presence in a biological fluid of a patient is a reliable predictor of impending neuronal death.
- MAP-2 in animal models. MAP-2 is a neuronal cytoskeletal protein localized primarily in cell bodies and dendrites. Binder, et al. Ann. NY Acad. Sci. , 466:
- MAP-2 loss is seen ipsilateral to the site of cerebral injury induced
- MAP-2 released as a result of traumatic injury may possess an antigenicity different from that of MAP-2 purified from uninjured neuronal cells.
- MAPs released from neuronal cells as a result of trauma-induced degeneration undergo significant post-translational modifications. These modifications include proteolytic cleavage by calpain, and significant phosphorylation of PRO-SER-LYS motifs. As a result, portions of the secondary structure of the MAP are altered, resulting in differing epitopes, and ultimately, in recognition by a subset of antibodies unique to injury-released MAP.
- the invention described in this application achieves this objective by utilizing monoclonal antibodies which recognize only MAP-2, a protein present extracellularly only in the presence of neuronal cell degeneration, to detect the presence of this protein in a biological fluid sample and utilizing a rapid, inexpensive, highly-sensitive detection system to detect the presence of antibody-MAP-2 complexes.
- the present invention is a method of quantifying levels of MAP-2 in biological fluid to aid in the diagnosis of clinical conditions associated with neuronal degeneration including, for example, hemorrhagic and non-hemorrhagic traumatic brain injury, stroke, brain tumors, aneurysm, subarachnoid hemorrhages and other CNS dysfunctions.
- One aspect of the invention is a method of ascertaining whether an individual has suffered neuronal cell damage comprising the steps of: (1) obtaining a sample of biological fluid from a patient, and (2) evaluating said sample for the presence of the MAP-2 protein.
- this invention is directed to the purified protein product MAP-2 having a secondary structure reflective of the post- translation modifications which occur upon neuronal degeneration as a result of traumatic brain injury.
- Further aspects of the invention include the monoclonal antibodies specifically recognizing human MAP-2 and those specifically recognizing MAP-2 purified from traumatically-injured human brain.
- the invention is further directed to a kit for use in determining the presence of MAP-2 in the biological fluids of an individual.
- MAP-2 is present in CSF from CNS trauma patients but not control CSF. Western blot analysis of MAP-2 proteins in brain and CSF.
- Left Panel MAP-2 purified from post-mortem brain consists of a primary 300 kD protein corresponding to full length MAP-2 and a lower molecular weight band of proteolytically cleaved MAP-2 (lanes from two different brains, 10 ⁇ g/lane, labeled with MAP-2 specific Mab AP-14, 1 :1,000).
- Right Panel CSF from four different patients with head injury (CNS Trauma, 20 ⁇ l CSF/lane, AP-14, 1 : 1 ,000) and four different neurologic controls
- MAP-2 in CSF from head injury patients consisted of both 300 kD full length MAP-2 and lower molecular weight cleavage products while little or no MAP-2 was observed in control CSF samples. Similar results obtained with MAP-2 antibody ICN MAPs.
- the method of the present invention may be used to determine whether neuronal damage/degradation has occurred and to what extent, and thus can be used to determine the existence or likelihood of any disease state associated with neuronal damage, such as CNS injuries, including primary neuronal injuries (e.g., cortical contusion, diffuse axonal injury, subcortical gray matter injury, and primary brain stem injury), primary hemorrhages (e.g., subdural hematoma, epidural hematoma, intracerebral hematoma and diffuse hemorrhages), primary vascular injuries (e.g., arterial pseudoaneurysm, arterial dissection/occlusion), dural sinus lacertaion/ occlusion, traumatic pia-arachnoid injuries, cranial nerve injuries, and secondary traumatic lesions (e.g., infarction, hypoxic injury, diffuse brain swelling/edema, secondary hemorrhage), central nervous system tumor, neurodegenerative diseases
- CNS injuries including primary neuronal injuries (
- the present invention involves the detection of the presence of a particular intracellular protein, MAP-2, in a biological fluid sample obtained from a patient.
- the first step in the invention embodied in the method is to obtain a fluid sample from a patient.
- the sample may be obtained using any of the many different methods known in the art. For example, if the sample is to be of cerebrospinal fluid (CSF), it can be obtained via conventional methods including lumbar puncture or intraventricular catheter. Blood plasma, extracted from whole blood by any means known in the art, or urine, may also be utilized in the present invention.
- CSF cerebrospinal fluid
- Blood plasma extracted from whole blood by any means known in the art, or urine, may also be utilized in the present invention.
- the fluid must be evaluated for the presence of MAP-2 in order to determine whether the patient has suffered neuronal cell damage.
- the selected fluid sample is treated with a solution containing at least one monoclonal antibody (MAB) specific for the human MAP-2 protein.
- MAB monoclonal antibody
- the MAP-2 specific monoclonal antibodies can be raised using any acceptable method commonly practiced in the art. One such technique for obtaining specific monoclonal antibodies is described in Kohler, G. and C. Milstein, Nature 256: 495-497 (1975), the contents of which are incorporated herein by reference.
- the MAB or MABs may be raised to the post-injury modified form of human MAP-2, to unmodified human MAP-2 or to both forms.
- MABs will be raised against a highly purified preparation of MAP- 2 from adult CNS trauma brain.
- Taxol-polymerized microtubules are employed to purify microtubule binding proteins from the brain. This results in a highly purified preparation consisting of only two microtubule binding proteins, MAP-2 and MAP- tau. Vallee, J. Cell Biol. 92: 435-442 (1982). Gel excision is used to separate 300 kD MAP-2 proteins from 30 kD to 68 kD tau proteins. This procedure results in a >90% pure preparation of MAP-2.
- MAP-2 is a large protein consisting of 1,828 amino acids.
- the shared microtubule binding domain consists of the C-terminal 185 amino acids of both proteins. Therefore, about 90% of the MAP-2 sequence in not homologous with MAP-tau.
- clones may be screened by Western blot against TBI CSF that is rich in both MAP-2 and MAP-tau. Only clones binding to the higher molecular weight MAP-2 proteins (300 kD) and not binding to the lower molecular weight MAP-tau proteins (30 kD to 68 kD) are selected. Under this procedure, the selection of MAP-2 clones is restricted to those recognizing the N-terminal 90% of the MAP-2 sequence. Although this is a potential problem, a similar screening procedure has been used to develop MAP-tau specific MABs that do not cross-react with MAP-2.
- the fluid sample obtained from the patient can be treated with one monoclonal antibody, if such antibody is of high affinity as determined by ELISA dilution curves obtained employing CSF-derived MAP-2-coated plates.
- the method may also be practiced using a treatment of more than one MAB, each specific to a different epitope of the protein.
- any detection method commonly known in the art may be used.
- One such technique is gel electrophoresis.
- MAP-2-MAB complex using labeled antibodies as described in Zemlan, F.P. and
- Another detection technique which might be employed is a sandwich enzyme-linked immunosorbant assay (ELISA), as described in Zemlan, F.P. and Dean, G.E., J. Neurosci. Res. 46; 90-97 (1996), the contents of which are incorporated herein by reference.
- the antibody employed in the detection step of this method may have any form of label applied, including, but not limited to, radiolabels, fluorescent or fluorogenic labels, colored or chromogenic labels, chemically-reactive labels, such as biotin, enzyme labels, and chemiluminescent labels.
- MAP-2 purified from human CNS trauma brains, i.e., from patients who have suffered a traumatic injury to the brain or central nervous system, is used as an antigen.
- One technique that can be used to obtain the post-injury modified form of MAP-2 to use as antigen is the isolation of MAP-2 from post-mortem brain of a suitable patient employing taxol-polymerized microtubules followed by gel purification. To do this, tubulin is purified from the brain and microtubules are assembled in the presence of taxol. Endogenous MAPs are dissociated from the microtubules by suspending the microtubule pellet in an assembly buffer.
- assembly buffer can be made up of 40 ⁇ M taxol in 0.1 M PIPES, 1.0 mM EGTA, 1.0 M MgSO 4 and 1.0 mM GTP.
- Sodium chloride (NaCl) is added to a final concentration of 0.35 M.
- Sodium chloride (NaCl) is removed from the above
- microtubule pellet (600 ⁇ g) by washing in 1.3 milliliters of assembly buffer.
- Microtubules are pelleted at 30,000 x g for 25 minutes, and human heat-stable protein extract containing MAP-2 and 1.3 milliliters of assembly buffer are added. The resulting solution is vortexed, incubated for 10 minutes at 37°C, and centrifuged again at 30,000 x g for 25 minutes. Proteins not bound to microtubules are removed by two cycles of washing. Microtubule bound proteins are dissociated by the addition of 0.5 milliliters of assembly buffer with 0.35 M NaCl to the washed microtubule pellet. Following incubation for 10 minutes at 37°C, the sample is dialyzed against 50 mM Tris-HCl and the extraction procedure is repeated twice.
- MAP-2 fraction Proteins with molecular weights greater than 100 kD (MAP-2 fraction) are excised and electroeluted in a Schleicher & Schuell (Keene, NH) Elutrap device, then dialyzed against 50 mM Tris-HCl. Sample purity is assessed by Commassie stained SDS-PAGE.
- a final aspect of the invention is a kit which can be used in the laboratory or diagnostic setting to detect the presence of MAP-2 in the biological fluids of a patient.
- the contents of the kit may vary depending upon the setting or circumstances in which it is used.
- the kit includes a monoclonal antibody specific for MAP-2, and a means for measuring the amount of MAB-MAP-2 complex formed when the sample and antibody are mixed together.
- the kit can contain a monoclonal antibody specific for MAP-2, optionally a container suitable for treating the fluid sample with the monoclonal antibodies, optionally a quantity of detection antibodies, either labeled or unlabeled, and optionally a quantity of human MAP-2 protein for use as an internal standard.
- MAP-2 proteins Significant labeling of MAP-2 proteins in unconcentrated CSF samples was observed in all severe head injury patients examined while little or no labeling was observed in control samples from migraine and back pain patients (Fig. 1). Immunolabeled proteins were judged to be MAP-2 based on two criteria. First, CSF proteins were labeled with a highly MAP-2 specific monoclonal antibody, AP-14, that demonstrates no cross-reactivity with MAP-tau. Binder, et al., Ann. NY Acad. Sci. 466: 145-166 (1986). This MAP-2 specificity results from AP-14 recognizing a MAP-2 epitope that is N-terminal to the microtubule binding domain shared by MAP-2 and MAP-tau.
- AP-14 highly MAP-2 specific monoclonal antibody
- MAP-2 purified from post mortem brain (Fig. 1). This brain preparation is highly purified for microtubule-binding proteins consisting of high molecular weight MAP-2 (300 kD) and MAP-tau (30 kD to 50 kD). Vallee, J. Cell Biol. 92: 435-442 (1982). An additional AP-14 labeled band was observed at 180 kD in both brain and most CSF samples which has been shown to consist of proteolytically digested MAP-2.
Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99964148A EP1137668A1 (en) | 1998-12-09 | 1999-12-08 | A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids |
AU20450/00A AU775443B2 (en) | 1998-12-09 | 1999-12-08 | A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids |
CA002351562A CA2351562A1 (en) | 1998-12-09 | 1999-12-08 | A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids |
Applications Claiming Priority (2)
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US11156298P | 1998-12-09 | 1998-12-09 | |
US60/111,562 | 1998-12-09 |
Publications (1)
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WO2000034336A1 true WO2000034336A1 (en) | 2000-06-15 |
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PCT/US1999/029023 WO2000034336A1 (en) | 1998-12-09 | 1999-12-08 | A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids |
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EP (1) | EP1137668A1 (en) |
AU (1) | AU775443B2 (en) |
CA (1) | CA2351562A1 (en) |
WO (1) | WO2000034336A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002041691A2 (en) * | 2000-11-24 | 2002-05-30 | Commissariat A L'energie Atomique | Transgenic or recombinant non-human mammals and their uses in screening psycho-active medicines |
US6613534B2 (en) * | 2001-03-20 | 2003-09-02 | Wake Forest University Health Sciences | MAP-2 as a determinant of metastatic potential |
US8298835B2 (en) | 2004-04-15 | 2012-10-30 | University Of Florida Research Foundation, Inc. | Proteolytic markers as diagnostic biomarkers for cancer, organ injury and muscle rehabilitation/exercise overtraining |
WO2015067915A1 (en) * | 2013-11-05 | 2015-05-14 | The Secretary Of State For Defence | Biomarkers for traumatic brain injury |
CN106605146A (en) * | 2014-04-08 | 2017-04-26 | 佛罗里达大学研究基金会有限公司 | Protein biomarkers for acute, subacute and chronic traumatic injuries of the central nervous system |
Citations (1)
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WO1997027323A1 (en) * | 1996-01-23 | 1997-07-31 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Tool for detection of nb-map2 specific expression and diagnostic as well as pharmaceutical applications thereof |
-
1999
- 1999-12-08 EP EP99964148A patent/EP1137668A1/en not_active Withdrawn
- 1999-12-08 AU AU20450/00A patent/AU775443B2/en not_active Ceased
- 1999-12-08 CA CA002351562A patent/CA2351562A1/en not_active Abandoned
- 1999-12-08 WO PCT/US1999/029023 patent/WO2000034336A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997027323A1 (en) * | 1996-01-23 | 1997-07-31 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Tool for detection of nb-map2 specific expression and diagnostic as well as pharmaceutical applications thereof |
Non-Patent Citations (9)
Title |
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B. RIEDERER ET AL.: "Changes of MAP2 phosphorylation during brain development.", JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 43, no. 12, December 1995 (1995-12-01), Baltimore, MD, USA, pages 1269 - 1284, XP000891807 * |
I. FISCHER ET AL.: "Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells.", DEVELOPMENTAL BRAIN RESEARCH, vol. 25, 1986, pages 99 - 109, XP000675932 * |
J. IZANT ET AL.: "Microtubule-associated proteins: A monoclonal antibody to MAP2 binds to differentiated neurons.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 77, no. 8, August 1980 (1980-08-01), Washington, DC, USA, pages 4741 - 4745, XP000675924 * |
J. TROJANOWSKI ET AL.: "Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues.", JOURNAL OF NEUROSCIENCE METHODS, vol. 29, no. 2, August 1989 (1989-08-01), Amsterdam, NL, pages 171 - 180, XP000891806 * |
L. BINDER ET AL.: "Differential localization of MAP-2 and Tau in mammalian neurons in situ.", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 466, 1986, New York, NY, USA, pages 145 - 166, XP000891804 * |
N. KALCHEVA ET AL.: "Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 92, no. 24, 21 November 1995 (1995-11-21), Washington, DC, USA, pages 10894 - 10898, XP002135145 * |
N. KALCHEVA ET AL.: "Localization of specific epitopes on human MAP-2.", MOLECULAR BIOLOGY OF THE CELL, vol. 4, no. suppl., 1993, Bethesda, MD, USA, pages 392a, XP000891805 * |
N. KALCHEVA ET AL.: "Localization of specific epitopes on human microtubule-associated protein 2.", JOURNAL OF NEUROCHEMISTRY, vol. 63, no. 6, December 1994 (1994-12-01), New York, NY, USA, pages 2336 - 2341, XP000891813 * |
T. SCHERSON ET AL.: "Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 129, 1982, pages 295 - 302, XP000675933 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002041691A2 (en) * | 2000-11-24 | 2002-05-30 | Commissariat A L'energie Atomique | Transgenic or recombinant non-human mammals and their uses in screening psycho-active medicines |
FR2817117A1 (en) * | 2000-11-24 | 2002-05-31 | Commissariat Energie Atomique | TRANSGENIC OR RECOMBINANT NON-HUMAN MAMMALS AND THEIR APPLICATIONS IN THE SCREENING OF MEDICINES USEFUL IN PSYCHOACTIVE DISORDERS |
WO2002041691A3 (en) * | 2000-11-24 | 2003-02-13 | Commissariat Energie Atomique | Transgenic or recombinant non-human mammals and their uses in screening psycho-active medicines |
US6613534B2 (en) * | 2001-03-20 | 2003-09-02 | Wake Forest University Health Sciences | MAP-2 as a determinant of metastatic potential |
US8298835B2 (en) | 2004-04-15 | 2012-10-30 | University Of Florida Research Foundation, Inc. | Proteolytic markers as diagnostic biomarkers for cancer, organ injury and muscle rehabilitation/exercise overtraining |
WO2015067915A1 (en) * | 2013-11-05 | 2015-05-14 | The Secretary Of State For Defence | Biomarkers for traumatic brain injury |
GB2525055A (en) * | 2013-11-05 | 2015-10-14 | Secr Defence | Biomarkers for traumatic brain injury |
CN106605146A (en) * | 2014-04-08 | 2017-04-26 | 佛罗里达大学研究基金会有限公司 | Protein biomarkers for acute, subacute and chronic traumatic injuries of the central nervous system |
Also Published As
Publication number | Publication date |
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AU2045000A (en) | 2000-06-26 |
CA2351562A1 (en) | 2000-06-15 |
AU775443B2 (en) | 2004-07-29 |
EP1137668A1 (en) | 2001-10-04 |
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