AU775443B2 - A method for detecting neuronal cell damage by quantification of map-2 levels in biological fluids - Google Patents

Description

WO 00/34336 PCT/US99/29023 A METHOD FOR DETECTING NEURONAL CELL DAMAGE BY QUANTIFICATION OF MAP-2 LEVELS IN BIOLOGICAL FLUIDS FRANK P. ZEMLAN TECHNICAL FIELD This application is based on U.S. provisional patent application number 60/111,562, filed December 9, 1998.

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. 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.

BACKGROUND OF THE INVENTION In the early decades of life, traumatic injury is the most common cause of death and permanent disability. A majority of these traumatic injuries are injuries to the brain. Among people aged 15 to 25 years, the incidence of traumatic brain injury (TBI) peaks at 550 per 100,000 people, and characteristically transforms productive individuals into dependent patients requiring decades of institutionalized WO 00/34336 PCT/US99/29023 -2care. 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 Gentry, ibid. 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. Because most TBI patients are assessed in a critical care setting, frequently within hours of receiving injury, a method capable of aiding physicians in the diagnosis of both hemorrhagic and non-hemorrhagic cortical contusions, regardless of the size of lesion, and which retains the cost, safety, availability and rapid turn-around advantages of conventional CT scans, would augment a physician's arsenal of diagnostic tools and enhance his or her ability to limit TBI-related morbidity and mortality.

WO 00/34336 PCT/US99/29023 -3- A common feature of traumatic brain injuries, both hemorrhagic and nonhemorrhagic, is the degeneration of injured neurons. 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. Adams, et al. (eds),Greenfield's Neuropathology, 4 th ed; New York, Wiley, pp. 85-124 (1984). Upon trauma or other neuronal injury, 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.

At the present time, there is no laboratory test or assay for quantifying the neuronal degeneration associated with TBI or CNS brain dysfunctions. Prior attempts to develop an assay based upon a direct correlation between the presence of a neuronal cell component in biological fluids and neuronal cell damage have resulted in impracticable and unreliable assays. For example, the enzyme yy enolase is found in high concentrations in neurons, but is unsuitable for use in a head injury assay because it is also found in platelet cells. Dauberschmidt, P.J. Marangos, J.

Zinsmeyer, V. Bender, G. Klages, J. Gross., Clin. Chem. Acat., 131: 165-170 (1993).

Consequently, the origin of any concentration of yy enolase in a fluid sample is uncertain; it can be attributed to either platelet or neuronal cells. Scarna, B.

Delafosse, R. Steinberg, G. Debilly, B. Mandrand. Neurochemistry International, 4: 405-411 (1982). Similar concerns of attribution arise in measurement of glutamate levels in the CSF of head injury patients. Huber, A. Matus, J. Neurosci; 4: WO 00/34336 PCT/US99/29023 -4- 151-163 (1988). Assays for the axon-localized, microtubule-associated protein tau (MAP-tau) have been developed, but are believed to be of low sensitivity, because intracellular concentrations of MAP-tau are maintained at low levels. Vallee, J.

Cell Biol. 92: 435-442 (1982). See also U.S. Provisional Patent Application Serial No. 60/160,690, filed October 21, 1999, Method of Detecting Axonal Damage, Associated Disease States, and Related Monoclonal Antibodies and Protein Controls Therefor, Zemlan.

One of the intracellular components released from neuronal cells upon degeneration is microtubule associated protein 2 (MAP-2). MAP-2 is a neuronspecific 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'1 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. However, when neuronal cells are subjected to trauma, or are otherwise disrupted and consequently degenerate, 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.

Although elevated CSF MAP-2 has not been studied heretofore in TBI patients, recent studies document the relationship between TBI and loss of neuronal MAP-2 in animal models. MAP-2 is a neuronal cytoskeletal protein localized WO 00/34336 PCT/US99/29023 primarily in cell bodies and dendrites. Binder, et al. Ann. NY Acad. Sci., 466: 145-166 (1986). MAP-2 loss is seen ipsilateral to the site of cerebral injury induced by lateral cortical impact in animal TBI models. Posmantur, et al., J. Neurotrama, 13: 125-137 (1996). Loss of MAP-2 was documented at three hours post-injury in this TBI animal model, the earliest time point examined. Also, loss of MAP-2 immunostaining following ischemic damage or excitotoxic lesions has been reported in the hippocampus. Simon, et al., J. Neurosci. 9: 1579-1590 (1989), and Lee, et al., Proc. Natl. Acad. Sci. USA 88: 7233-7237 (1991). In these studies, loss of MAP-2 was considered a predictor of impending neuronal death.

Additionally, MAP-2 released as a result of traumatic injury may possess an antigenicity different from that of MAP-2 purified from uninjured neuronal cells.

Studies indicate that 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.

It would be highly desirable to develop a method which could provide rapid, inexpensive and unambiguous quantification of neuronal cell degeneration to aid in the diagnosis of recently-incurred or non-hemorrhaging traumatic brain injury and CNS dysfunctions. 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

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WO 00/34336 PCT/US99/29023 -6presence 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.

SUMMARY OF THE INVENTION 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: obtaining a sample of biological fluid from a patient, and evaluating said sample for the presence of the MAP-2 protein. In another aspect, this invention is directed to the purified protein product MAP-2 having a secondary structure reflective of the posttranslation 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.

DESCRIPTION OF THE DRAWING Figure 1. 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 WO 00/34336 PCT/US99/29023 -7purified 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 pg/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 1l CSF/lane, AP-14, 1:1,000) and four different neurologic controls with migraine or back pain (Controls, 20 pCl CSF/lane, AP-14, 1:1,000). 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.

DETAILED DESCRIPTION OF THE INVENTION 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 cortical contusion, diffuse axonal injury, subcortical gray matter injury, and primary brain stem injury), primary hemorrhages subdural hematoma, epidural hematoma, intracerebral hematoma and diffuse hemorrhages), primary vascular injuries arterial pseudoaneurysm, arterial dissection/occlusion), dural sinus lacertaion/ occlusion, traumatic pia-arachnoid injuries, cranial nerve injuries, and secondary traumatic lesions infarction, hypoxic injury, diffuse brain swelling/edema, secondary hemorrhage), central nervous system tumor, neurodegenerative diseases of WO 00/34336 PCT/US99/29023 -8the central nervous system including Alzheimer's disease, spinal cord injury, acute cerebral vascular accident or neuronal damage following ingestion of drugs or poison.

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.

Once the sample of biological fluid has been obtained, the fluid must be evaluated for the presence of MAP-2 in order to determine whether the patient has suffered neuronal cell damage. To do this, the selected fluid sample is treated with a solution containing at least one monoclonal antibody (MAB) specific for the human MAP-2 protein. 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.

Typically, 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 WO 00/34336 PCT/US99/29023 -9preparation consisting of only two microtubule binding proteins, MAP-2 and MAPtau. 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 pure preparation of MAP-2.

Although the antigen used for MAP-2 MAB production will be free of MAPtau, cross-reactivity with MAP-tau is still a concern as both contain a C-terminal microtubule binding domain demonstrating significant homology. Lewis, et al., Science 242: 936-939 (1988). 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. To insure that the MABs selected for MAP-2 ELISA development are MAP-2 specific, 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 WO 00/34336 PCT/US99/29023 practiced using a treatment of more than one MAB, each specific to a different epitope of the protein.

To detect the presence of MAP-2-MAB complexes, any detection method commonly known in the art may be used. One such technique is gel electrophoresis.

Another involves performing an immunoblot and detecting the presence of the MAP-2-MAB complex using labeled antibodies as described in Zemlan, F:P. and Dean, J. Neurosci. Res. 46: 90-97 (1996), the contents of which are incorporated herein by reference. Another detection technique which might be employed is a sandwich enzyme-linked immunosorbant assay (ELISA), as described in Zemlan, F.P.

and Dean, 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.

Once the amount of MAP-2 in the fluid sample of a particular patient is quantified via the above techniques, it is compared to quantities of MAP-2 found in individuals who do not suffer from TBI or CNS dysfunctions.

Further aspects of the invention concern the specific antibodies which recognize MAP-2 in its normal and post-injury modified form, as well as the postinjury form of MAP-2 itself. To obtain a monoclonal antibody that specifically recognizes MAP-2 in its post-injury-related form, MAP-2 purified from human CNS trauma brains, from patients who have suffered a traumatic injury to the brain or central nervous system, is used as an antigen.

WO 00/34336 PCT/US99/29023 11 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. The assembly buffer can be made up of 40 pM taxol in 0.1 M PIPES, 1.0 mM EGTA, M MgSO 4 and 1.0 mM GTP. Sodium chloride (NaCI) is added to a final concentration of 0.35 M. Sodium chloride (NaCI) is removed from the above microtubule pellet (600 gg) 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 0 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 milliliters of assembly buffer with 0.35 M NaCI to the washed microtubule pellet.

Following incubation for 10 minutes at 37 0 C, the sample is dialyzed against 50 mM Tris-HCl and the extraction procedure is repeated twice. Purity of thrice-cycled MAPs is assessed by Coomassie stained SDS-PAGE that reveals a primary MAP-2 band at 300 kD. 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.

WO 00/34336 PCT/US99/29023 12- 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. In general, 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. More specifically, 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.

EXAMPLE

Preliminary studies examined patients with acute neuronal degeneration secondary to severe head trauma. Head trauma was severe as judged by hospitalization for extensive intercranial hemorrhage, elevated intracranial pressure requiring intraventricular shunting and coma. The amount of CSF MAP-2 in these patients was semi-quantitatively assessed on the day of admission to the neurotrauma intensive care unit.

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 13 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. Lewis, et al., Science 242: 936-939 (1988). The MAP-2 specificity of AP-14 was confirmed in the present study as no AP-14 labeling of the abundant 30 kD to 50 kD MAP-tau present in CSF from CNS trauma patients was observed (Fig. Further, similar labeling of CSF 300 kD MAP-2 proteins was observed with antibody ICN MAPs that also recognizes MAP-2. Huber, et al., J.

Neuro Sci. 4: 151-163 (1988).

Second, AP-14 labeled CSF proteins demonstrated the same gel mobility as MAP-2 purified from post mortem brain (Fig. 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.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

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Claims (13)

1. A monoclonal antibody raised against human MAP-2 obtained from CNS trauma human brain, excluding monoclonal antibodies that have an antigenicity to MAP-2 obtained from uninjured neuronal cells.

2. A monoclonal antibody according to Claim 1 in substantially isolated form.

3. A human MAP-2 protein obtained from CNS trauma brain, in substantially isolated form.

4. A method for determining neuronal cell damage in a human individual comprising the steps of: o obtaining a sample of biological fluid from the individual; and evaluating the sample for the presence of microtubule-associated o: protein 2 (MAP-2), wherein the presence of MAP-2 is an indicator of neuronal cell degeneration. 15 5. A method according to Claim 4 wherein step is carried out by treating the sample with at least one monoclonal antibody raised against MAP-2 and detecting the presence of MAP-2 bound to said at least one monoclonal antibody.

6. The method according to Claim 5 additionally comprising the step of o comparing the amount of said MAP-2 bound to said at least one monoclonal antibody to control samples selected from the group representing a normal undamaged neuronal state and those representing a damaged neuronal state.

7. The method according to Claim 5 or 6 wherein the presence of said MAP-2 bound to said at least one monoclonal antibody is detected through gel electrophoresis.

8. A method according to any one of Claims 4 to 7 further comprising the measurement of said MAP-2 in said sample by an ELISA technique.

9. A method according to Claim 8 wherein the ELISA employs monoclonal antibodies recognizing MAP-2 present in said fluid sample. A method according to Claim 8 or 9 wherein the ELISA is a sandwich ELISA.

11. A method according to any one of Claims 4 to 10 wherein the fluid sample is collected from a patient with a neurological disease selected from the group consisting of traumatic central nervous system injury, central nervous system tumor, neurodegenerative diseases of the central nervous system, spinal chord injury, cerebral vascular accident, and neuronal damage following ingestion of drugs or poison.

12. A kit for evaluating the occurrence of neuronal cell damage in a human individual comprising, in packaged combination, a quantity of monoclonal antibody (MAB) specific for one or more forms of MAP-2, and a means for detecting the 60 presence of MAP-2-MAB complexes.

13. The kit according to Claim 12 which additionally comprises a container suitable for treatment of a fluid sample taken from the individual with said MAB.

14. A kit according to Claim 12 or 13 which additionally comprises a 65 quantity of human MAP-2 suitable for use as a standard. o* o*o o* o

16- A method according to Claim 4 substantially as hereinbefore described with reference to the example. o go o ooo