ASSAY FOR DETECTING DAMAGE TO THE CENTRAL NERVOUS SYSTEM

Abstract: A method is disclosed for the rapid diagnosis of disorders characterized by an ischemic event, such as stroke, transient ischemic attack, head trauma, myocardial infarction, or other insults resulting in interrupted cranial blood flow. The method involves detection of the presence of the gamma isoform of protein kinase C (PKCg) in peripheral blood, which signals damage to central nervous system tissue and at least transient breakdown of the blood brain barrier. The assay may be performed, e.g., by emergency medical personnel, in a time frame that allows treatment of the patient before permanent damage to the central nervous system occurs.

Description: ASSAY FOR DETECTING DAMAGE TO THE CENTRAL NERVOUS SYSTEM FIELD OF THE INVENTION The present invention discloses a method for the rapid and accurate detection of damage to the central nervous system (CNS) caused by an ischemic event such as stroke or head trauma The method focuses on detecting the release of the gamma isoform of protein kinase C (PKCg) into the bloodstream, e g., as a result of the breakdown of the blood-bram barrier. The current invention provides a diagnostic method and a diagnostic kit that is useful for the early diagnosis and treatment of an ischemic event. Such methods and kits may be advantageously used, e.g., by emergency medical personnel, to obtain an early indication of an ischemic event within a fame penod following the event where permanent CNS damage may be avoided. The methods and kits may also be used to monitor patient progress and recovery following an ischemic event. BACKGROUND OF THE INVENTION Brain ischemia resultmg from stroke, head trauma or other events that interfere with blood flow to the brain is a leading cause of death and disability in industrialized nations. Stroke, for example, affects 0.1-0.2% of the North American and European population. Approximately 700,000 people in the United States have a new or recurrent stroke each year, with a significant number resulting in death. An estimated 3,000,000 people in the United States have survived a stroke, however many of these survivors are considered to be at πsk for recurrent episodes There are no specific neuroprotective drugs on the market to treat ischemic stroke, and consequently this condition represents a major clinical problem with 25-35% fatality for acute strokes within the first three weeks. Of the survivors, 25-50% will be totally dependent on family or institutional care for the rest of their lives. One major impediment to the establishment of effective therapies for acute CNS injury has been the lack of definitive diagnostic procedures to permit proper and rigorous clinical trial design Present diagnostic procedures are usually based on a sudden onset of neurologic signs such as hemiparesis, aphasia, hemianopia, altered consciousness, or gait disturbances. The initial evaluation of a suspected stroke patient is time-consuming and usually occurs in the hospital after permanent neuronal damage has already occurred. Although the mechanisms involved m stroke are not fully understood, it has been proposed that the pathology arises from an initial mfarct, wherein perfusion pressure and blood flow to CNS tissues are reduced by mtracellular and microvascular edema, followed by progressive compression and reduced perfusion in areas adjacent to the mfarct. As this process evolves, a series of biochemical events takes place as cell damage progresses. These biochemical events, indicative of cell damage and cell death, may include oxidation of membrane components, free radical formation, altered fatty acid metabolism, activation of the gamma isoform of protein kinase C (PKCg), calcium entry into the cell, and disruption of the blood brain barrier. If permanent brain damage from ischemic injury is to be avoided, appropπate treatment must be administered withm two hours of the suspected msult. However, at the present time, definitive diagnostic procedures for these conditions are inaccurate, expensive, and not readily available to physicians or emergency medical personnel such that accurate diagnosis of stroke or other ischemic injury can be determined or measured withm this important two-hour window This absence of effective diagnostic procedures has also contributed to the lack of new therapies for the treatment or prevention of neuronal damage from stroke or other ischemic events. Assay techniques have been suggested for a number of potential marker proteins associated with stroke, including neuron-specific enolase (NSE), myelm basic protein, glial fibπllary acidic protein, and S-100 protein (Missler et al., 1997). In most cases these substances are measured in cerebrospmal fluid (CSF), which is obtained by invasive and difficult procedures. Given the short diagnostic window for avoiding permanent injury, it is imperative that diagnostic methods be developed that can be performed using more easily obtainable samples, ideally peripheral blood samples Even though NSE and S-100 are measurable in blood, peak levels are not found until approximately two days following infarction, which makes them impractical indicators of stroke, even though NSE and S-100 protein do correlate with mfarct volume (Missler et al., 1997). Also, none of these markers are specific indicators of general brain damage, and S-100 has been shown to be a normal component of plasma (Shashoua et al , 1984). As mentioned previously, the initial evaluation of a suspected stroke patient is time- consuming and usually occurs after permanent brain damage has occurred. Therefore, it would be advantageous to develop an assay to rapidly detect or quantitate a protein that is uniquely expressed at the early onset of stroke, and that appears in easily obtainable and rapidly assayed biological samples such as peπpheral blood. SUMMARY OF THE INVENTION The present invention is based on the discovery that a particular isoform of protein kinase C, namely the gamma isoform (abbreviated PKCγ or PKCg herein), appears in the peπpheral blood very quickly after an ischemic event m the central nervous system (CNS). The present invention descπbes a procedure for the rapid and accurate diagnosis of CNS and especially brain ischemia resulting from stroke, transient ischemia attacks (TIAs), head trauma, myocardial infarction, or other events resulting in interrupted spmal or cranial blood flow. The procedure can be easily performed by ambulance or emergency room personnel and can be performed with a venous (peπpheral) blood sample and an assay kit as descπbed herein. The present invention provides an assay for the rapid detection of PKCg, a protein expressed only as a result of the type of cell damage that is characteristic of stroke, TIAs, head trauma, myocardial infarction, or other events or "insults" resulting m interrupted blood flow to structures of the CNS Other events that might lead to such interrupted blood flow to the CNS include medical interventions, such as surgical procedures, surgical or physical errors, anesthesia, and therapeutic or pharmaceutical interventions. According to the present invention, it can be determined whether a mammalian subject has suffered an ischemic event by detecting, in the subject or in a biological sample obtained from said the subject, the presence of PKCg outside (peπpheral to) the CNS. According to the preferred embodiments of the invention, a peπpheral blood sample is drawn from the affected individual then analyzed for the presence of PKCg. Preferably, a venous blood sample is contacted with a PKCg binding partner, such as an anti-PKCg antibody specific for a PKCg epitope under conditions suitable for the formation of an anti-PKCg antibody/PKCg binding complex Detection of the presence of PKCg m the blood sample, e.g., by detection of the antibody/PKCg complex, indicates a CNS ischemic injury In addition to determining whether or not damage to the CNS has occurred, i.e., by the presence or absence of PKCg in a blood sample, the present invention also provides for quantitation of the PKCg to determine the severity of the ischemic event The quantity of PKCg in the original peripheral blood sample is directly correlated to the amount or seventy of central nervous system damage Where lmmunosorbant methods are used, the anti-PKCg antibody most preferably includes a fluorescent tag or label for rapid detection using methods well known in the art In a preferred antibody-based method, after the blood sample is contacted with the anti-PKCg antibody, the solution is passed over a column such as, for instance, a DEAE/Sepharose column m order to separate the binding complexes from cells and larger proteins in the sample. According to this preferred method, the unbound (free) labeled anti-PKCg antibody will bind to the column, and the anti-PKCg antibody/PKCg complexes will flow through. Elimination of unbound labeled antibody from the sample improves the quantitative aspects of the method, making it possible to gauge progress or degree of ischemic msult to CNS tissues. The column may be washed one or more times to remove any of the larger complexes that may have bound. After collection of the flowthrough solution, the presence of any anti-PKCg antibody/PKCg complexes may be detected using methods well known in the art for detection of such complexes. In another embodiment, the present invention provides a method for determining whether or not damage has occurred to the CNS as a result of a stroke or other event such as a transient ischemia attack, head trauma, myocardial infarction, or other event resulting in disruption of cranial blood flow through the use of a sandwich antibody assay. According to the present invention, a first anti-PKCg antibody (also refeπed to as the capture antibody), raised against a specific epitope on the PKCg protein, is immobilized on a solid substrate. A sample of venous blood, drawn from an individual suspected of having a stroke or other ischemic event involving damage to the CNS, is contacted with the first antibody immobilized on the solid substrate to form an antibody/PKCg binding complex A subsequent wash step may be employed to remove any unbound PKCg protein from the rest of the sample mixture. A second anti-PKCg antibody specific for a different PKCg epitope than the first antibody and being detectably labeled, e.g., with a fluorescemated tag, biolummescent tag, coloπmetπc tag, or colored bead, is then contacted with the PKCg protein bound to the first annbody immobilized on the solid substrate under conditions suitable to form a binding complex with PKCg. A washing step may be included subsequent to this second binding step to remove any unbound labeled anti-PKCg antibody. Detection and or quantitation of PKCg m the original sample may be conducted by methods well known in the art such as, for instance, fluoπmetry, epifluorescence microscopy, confocal scanning laser microscopy, luminometer, or coloπmetπc assay In another embodiment of the sandwich antibody assay, the solid substrate on which the first anti-PKCg antibody is immobilized preferably is a magnetic bead, the surface of a well of a microtiter plate, or a piece or stπp of solid substrate mateπal that can be used as an assay strip. Assay stπps of the invention are preferably flat rectangular strips of a plastic, nylon, or other solid mateπal which contain an immobilized anti-PKCg antibody (capture antibody), which may be dipped into a sample and assay solutions, either manually or robotically, or to which a sample and assay solutions may be applied. In still another embodiment of the sandwich antibody assay of the invention, one or both of the anti-PKCg antibodies used in the assays of the invention may be a full-length major class or subclass of lmmunoglobulm, a functional fragment thereof (such as Fab, F(ab') 2 , Fv), a hybrid antibody (such as a chimeπc or humanized antibody), or a relatively small recombinant antibody molecule (such as a single chain antibody (scFv) or a diabody. In another embodiment, a PKCg binding partner is a polypeptide other than an antibody and is used in place of an anti-PKCg antibody molecule in the assays, compositions, and kits of the invention. In yet another embodiment, a kit is provided for detecting or measuπng PKCg in a blood sample or other solution. A preferred kit of the mvention compπses a first (capture) PKCg binding partner immobilized on an assay stπp, the wells of a microtiter plate, or on beads or particles; a second PKCg binding partner that also contains a detectable label or component of a system to generate a detectable signal; and instructions that indicate how to use the kit to detect or measure PKCg in a blood sample or other solution. DEFINITIONS As used herein, the term "ischemic event" refers to any potentially harmful episode resulting from temporary or permanent decrease or elimination of blood flow to tissues, particularly, with respect to the present invention, any event or physiological occurrence that interrupts blood flow to the CNS, especially cranial blood flow, and leads, without treatment, to damage m the CNS. Specific types of ischemic events include stroke, transient ischemic attacks, head trauma, myocardial infarction or other events resulting in interrupted blood flow to the CNS. Events resulting in such interrupted blood flow may be naturally occurπng or unexpected (i.e., as in the case of stroke or accidental trauma) or may be the result of mterventionary procedures, such as surgical procedures, surgical or physical mishaps, anesthesia, and therapeutic and pharmaceutical interventions, causing (undesired) interruption of blood flow, as a side effect. As used herein, a "PKCg binding partner" is any molecule that binds PKCg, including any polypeptide or lmmunoglobulm molecule, or fragment thereof, that specifically binds PKCg The term "specificity" refers to a binding partner or moiety having a higher binding affinity for one target over another. The term "PKCg specificity" and the phrase "specifically binds PKCg" refer to a PKCg binding partner having a higher affinity for PKCg as compared with another target, such as a serum protem (e.g., bovine serum albumin (BSA), human serum albumin (HSA)) or gelatin An "anti-PKCg antibody", or simply "PKCg antibody" or "PKCg lmmunoglobuhn" is a PKCg binding partner that contains at least one antibody binding site for an epitope of PKCg or that contains one or more complementanty-determmmg regions (CDRs) which bind to PKCg PKCg antibodies include any of the five full-length mammalian lmmunoglobulm classes (l e., IgG, IgM, IgA, IgE, IgD) and subclasses thereof that bind PKCg. A PKCg antibody may also be a functional fragment of a full-length lmmunoglobulm class that binds PKCg, such as a Fab, F(ab') 2 , and Fv fragment, as well as PKCg binding molecules that may be produced by protein engineering or recombinant DNA, such as a chimeπc lmmunoglobulm molecule, which compπses a binding domain or CDRs of one lmmunoglobulm fused to a portion of another lmmunoglobulm or other polypeptide; a humanized lmmunoglobulm molecule, which compπses the CDRs from a non- human antibody molecule inserted into the framework of a human antibody molecule; a single chain antibody (scFv); or a diabody (see, e.g., Holhger et al., Proc. Natl. Acad. Sci. USA, 90: 6444- 6448 (1993)). In addition, PKCg antibodies useful in the compositions and methods of the invention may be polyclonal antibodies (e.g., serum containing anti-PKCg antibodies from a mammal inoculated with PKCg antigen) or monoclonal antibodies produced by hybπdoma technology (see, e g., Milstein, Scientific American, 243: 66-74 (1980). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows PKCg dose-dependent percent fluorescence increase, from a sandwich-type immunosorbent assay measuπng PKCg captured on anti-PKCg beads, detected with anti-PKCg- FITC. Figure 2 shows PKCg dose-dependent fluorescence in mean fluorescence units, from a sandwich- type immunosorbent assay measuring PKCg captured on anti-PKCg coated beads, detected with Figure 3 shows a PKCg standard curve generated from the data depicted in Figure 2 Figure 4 shows differences in detected level of PKCg untreated (#1 - #3), sham treated (#4 - #6), and ischemic (#7 - #11) subjects from tested peπpheral blood samples. Levels of PKCg in brain extract are also shown (#12, #13) Figure 5 shows differences in PKCg levels among untreated, sham operated, and ischemic subjects detected in peπpheral blood, and m brain extract, where PKCg amounts are read from the standard curve (see Figure 3) Figure 6 shows PKCg levels (ng/ml) in blood samples collected from normal (no operation) rats, sham operated rats, and rats subjected to middle cerebral artery occlusion at 30 minutes, 90 minutes, and 3 hours after occlusion. Levels of PKCg are the mean value for three rats determined using a sandwich assay in microtiter plates. Figure 7 shows PKCg levels (ng/ml) in blood collected from rats subjected to middle cerebral artery occlusion at 0 minutes, 15 minutes, 30 minutes, 120 minutes, 180 minutes, 6 hours, and 24 hours after occlusion Blood collected approximately 30 minutes pπor to occlusion served as a reference control Levels of PKCg are the mean value for three rats determined using a sandwich assay in microtiter plates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention describes a rapid and accurate method for detecting damage to the CNS caused by an ischemic event, which may result from stroke, TIAs, head trauma, myocardial infarction or other events resulting m interrupted cranial blood flow. Traumatic events, such as stroke, cause a seπes of biochemical events as cell damage progresses. One of these biochemical events is the production of the gamma isozyme of protein kinase C (PKCγ or PKCg), which is expressed in the CNS as a result of an ischemic event. PKCg is a specific marker for CNS tissue that is activated by certain fatty acid moieties such as arachidonic acid (AA). In addition, AA is the fully competent precursor of the oxygenase pathways. In the CNS, noteworthy pathways are cyclooxygenase, which produces prostaglandms, thromboxanes, and prostacyclms, and hpoxygenases (including 5-HETE, 12-HETE, LTC 4 , LxA), which are also activated duπng ischemia and CNS injury. PKCg has been shown to be activated during and immediately following ischemia and traumatic CNS injury. PKCg is specific for the CNS and is not normally found m peπpheral blood. The discovery of the present invention follows the observation that the blood bram barπer is often compromised as a result of an ischemic event, leading to appearance of PKCg in peπpheral blood. It has further been discovered that the presence of PKCg in peπpheral blood occurs and can be detected in peπpheral blood almost immediately following an ischemic event, and, most importantly, withm the critical time window in which diagnosis and treatment of ischemic injury can prevent permanent damage to CNS tissue. Accordingly, the detection of PKCg m a peripheral blood sample is an early diagnostic indicator of an ischemic event such as stroke, TIA, head trauma, or myocardial infarction, making early and effective treatment possible Moreover, the amount of PKCg detected in a sample is proportional to the degree of the damage or msult to normal tissues, and therefore quantitative assay of the PKCg m a sample also is indicative of the extent of the trauma to the CNS. Insult to brain tissue from stroke, TIAs, head trauma, myocardial infarction, or other events resulting in the disruption of cranial blood flow share a common pathway of cell-mediated damage which originates with the activation and eventual release of PKCg from neural tissues. PKCg is normally found only in the CNS and is not known to be localized to any other tissues. In the event of ischemic damage, there is an accompanying breakdown of the blood bram barπer, which results m the release of PKCg from its normal location in the bram into venous blood. The methods of the present invention are especially contemplated to benefit human subjects, but they are of course suitable for any mammalian subject in which the presence of a PKCg isoform signals CNS damage Any means of detection for the PKCg marker is suitable, and any known means of detecting a specific protein in a sample may be employed. Preferably, PKCg is detected m a sample of blood from a mammalian subject, by contacting the sample with a binding partner for PKCg, that is, a peptide, lmmunoglobulm, small molecule, or other moiety capable of forming an association complex with PKCg. Most preferably, the PKCg m a sample is detected using antibodies specific for PKCg. Numerous PKCg antibodies are commercially available that can be used in the methods, compositions, and kits of the invention to detect or measure PKCg from humans and other mammals. For example, monoclonal antibodies that bind to epitopes of human PKCg are produced by a number of hybπdomas on deposit with the Ameπcan Type Culture Collection (ATCC), including ATCC accession no. 1021866 (commercial source: Transduction Laboratories, Lexington, KY), ATCC accession no. 1022645 (commercial source: Fitzgerald Industπes International, Inc., Concord, MA), ATCC accession no. 1022853 (commercial sources: Calbiochem Novabiochem International, La Jolla, CA; Oncogene Research Products, Cambridge, MA) The anti-PKCg monoclonal antibody produced by ATCC accession no. 1025435 (commercial source: Sigma Chemical Co., St. Louis, MO) reacts with the carboxy terminal region of PKCg and may be used m the methods, compositions, and kits of the invention to detect PKCg in samples from vaπous species. Other anti-PKCg antibodies, which react with PKCg from one or more sources, are also commercially available, for example, the polyclonal rabbit antiserum (commercial source: Calbiochem) and the monoclonal antibody (commercial source; Transduction Laboratories) used the Examples (infra). In addition, PKCg and fragments thereof may be isolated from tissues from humans and other species, produced by recombinant DNA techniques (e.g., commercial source: A Ullrich, Milwaukee, WI), or chemically synthesized by standard methods known in the art Such PKCg or fragment thereof may then be used as an immunogen in any of a variety of methods known and available in the art for producing polyclonal, monoclonal, or recombinant forms of antibodies which bind to PKCg and which may also be used in the various methods (e g , sandwich assays, infra), compositions, and kits descπbed herein. For detection or measurement of PKCg levels in a sample, fluorescently labeled antibodies are most preferred. Many other means of detecting PKCg directly or detecting a complex of PKCg with another moiety are known, including gas chromatography mass spectroscopy, thin layer chromatography, hydroxyl apatite chromatography, high pressure liquid chromatography, colloidal gold lmmunolabelmg read by electron microscopy, enzyme-linked immunosorbent assays (ELISAs), radioactively labeled tags or antibodies specific for PKCg read using a scintillation counter, biolummescently labeled antibodies read on a coloπmeter, etc.; however, most of these methods require several hours or even days for sample preparation and/or measurement of the signal, making them inferior to sensitive fluorescence-based assays such as descπbed in the examples (infra) Also, the apparatus necessary for detection in some cases (e g , mass spectrophotometer, electron microscope) would not fit mside an ambulance, making performance of the assays by emergency medical personnel impossible before the patient suspected of suffering an ischemic event is brought to a hospital. PKCg binding partners used to detect or measure (quantttate) PKCg in a blood sample according to the invention may be used in solution or immobilized on the surface of any of a vaπety of solid substrates Solid substrates to which a PKCg binding partner may be immobilized for use in the methods and compositions of the invention include, but are not limited to, magnetic or chromatographic matπx particles, the surface of a well of an assay plate (such as a microtiter assay plate), and pieces of a solid substrate material, such as pieces or stπps of plastic, nylon, wood, paper, or other solid mateπal, which may be dipped into or otherwise placed in contact with a sample or assay solution The PKCg binding partner may be immobilized to the surface of a solid substrate by adsorption (non-covalent adherence) or by covalently linking the binding partner directly to the solid surface or to a linker molecule that permits the binding partner to be tethered to the solid substrate Adsorption of a PKCg binding partner to the surface of the wells of a microtiter plate permits the detection or assay for PKCg to be carried out using the vaπous semi-automatic or automatic microtiter plate readers used m standard ELISA procedures. Such devices are particularly useful for assaying large numbers of samples for PKCg. Preferably, PKCg m a blood sample from an individual is detected using a sandwich assay in which a first PKCg binding partner (or capture molecule) serves to capture (bind) PKCg in a sample, and a second binding partner detects the complex formed by the PKCg bound to the first capture binding partner Preferably, such a sandwich assay of the invention may be earned out in solution, in wells of a microtiter plate as used in standard ELISA protocols, or on assay stπps as described herein In the ELISA-type sandwich assay protocol, a first PKCg binding partner is adsorbed to the wells of a microtiter plate Unbound PKCg binding partner may be removed from the well by inverting the plate and tapping out the solution and/or by washing the well with a buffer solution To prevent non-specific binding by potentially interfering substances, any of the vaπous blocking proteins or agents used in ELISA protocols, such as bovine serum albumin, may also be applied to the well to block any areas on the surface of the well that are not occupied by the first PKCg binding partner After the PKCg binding partner is immobilized on the surface of the well and unbound matenal removed, a sample known or suspected of containing PKCg is added to the well Unbound mateπal may then be removed or washed away A second PKCg binding partner that binds PKCg at a different site than the immobilized first PKCg binding partner is then added to the well to detect the presence of PKCg bound in the well This second (detection and/or quantitation) PKCg binding partner preferably carnes a detectable label that provides or is capable of producing a detectable signal Such detectable labels include any of a vaπety of labels known in the art that may be used to detect specific binding, for instance an antibody binding to an antigen Frequently used detectable labels include but are not limited to fluorescent labels, radioactive labels, biotin, streptavidm, biolummescent or chemiluminescent labels, and enzymes that are capable of reacting with colongemc substrates. The signal generated by such systems may be readily detected or measured by an appropriate instrument that reads microtiter plates. Colongemc labels may also be detected visually where detection of PKCg, alone, is sufficient as in an emergency situation to simply detect that an ischemic event has occurred. In another method of the invention, PKCg is detected using an assay stnp to which a PKCg binding partner is adsorbed or covalently linked. Such assay stπps provide a convenient means to detect or measure PKCg in a sample. As with the microtiter plates descnbed above, a first PKCg binding partner is immobilized on the strip and unbound material removed, e.g., by nnsing The stπps containing immobilized PKCg binding partner may also be nnsed to remove unbound mateπal and/or incubated with a blocking agent, such as bovine serum albumin, to reduce nonspecific binding by potentially mterfeπng molecules. According to the invention, an assay strip containing the immobilized PKCg binding partner is contacted with a sample suspected of containing PKCg, for example, by manually or robotically dipping the stnp into the sample. The assay strip then may optionally be nnsed to remove unbound material, and then dipped or otherwise contacted with a second PKCg binding partner that carries any of the types of detectable labels described above. If necessary, the assay stnp is further dipped or contacted with any reagents that are necessary to develop or generate a measurable signal on the strip, such as a colongemc substrate The assay strip is then observed visually or read by an appropriate signal detection instrument, as descπbed above, for evidence of PKCg in the sample. In an alternative procedure for the sandwich assays described above, a sample known or suspected of containing PKCg may be first mixed with the quantitation PKCg binding partner to form a complex between PKCg in the sample and the quantitation antibody, and the mixture then added to a well or other solid substrate containing the capture antibody for detection (see, Example 5, infra). The assays for PKCg according to the invention may be earned out on large and small scales depending on the volume of the sample or number of samples to be tested for the presence of PKCg Preferably, a single assay of the invention requires no more than about one-half of a milhliter (ml) of peπpheral or venous blood, more preferably about 50 to about 400 microhters (μl), and even more preferably less than about 50 microhters (μl) of peπpheral blood to detect or measure PKCg in a blood sample. Most preferably, an assay of the invention requires only one to several drops of blood, for example as obtained from a pm puncture of a digit or an ear, to detect or measure PKCg in the blood sample of mammalian subject. It is understood that the assays descπbed herein may be used to detect or measure PKCg in any liquid sample containing PKCg, including, but not limited to, blood samples such as samples of whole blood or blood fractions such as blood plasma, as well as aqueous solutions containing PKCg. To accurately measure (quantitate) the amount of PKCg in a blood sample, a standard curve may be generated using an assay of the invention. For example, an assay descπbed herein may be earned out on a blood sample and on a seπes of solutions containing known concentrations of PKCg. The signal obtained for each solution of known concentration of PKCg is then used to construct a standard curve that correlates the magnitude or intensity of a signal with the amount or concentration of PKCg (such as in Example 1, infra). The signal from a sample of unknown PKCg content may then be read on the standard curve to determine the amount of PKCg present in the sample The materials necessary for detection of PKCg in a sample of venous blood are conveniently assembled into a kit, so that personnel treating or transporting a trauma victim can determine quickly whether an ischemic event has been suffered by their patient and then begin treatment immediately to prevent further damage to the bram and/or central nervous system One kit useful for such diagnoses is based on PKCg binding and is capable of providing multiple levels of detection and quantitation. The level of detection provides quantitative assessment of ischemic damage based on calibration of fluorescently tagged antibodies to PKCg detected in venous blood A preferred kit of the invention compπses a first (capture) PKCg binding partner immobilized on a solid substrate mateπal, such as an anti-PKCg antibody immobilized on an assay stnp, the wells of a microtiter plate, or on beads or particles, a second PKCg binding partner containing a detectable label or component to produce a detectable signal, and instructions that indicate how to use the kit to carry out the PKCg assay Beads, assay stπps, or microtiter plates with immobilized PKCg binding partner in kits of the invention may be packaged m a vaπety conditions, including a dry, unhydrated state, a freeze-dπed or dehydrated state; or a hydrated state m a physiological buffer solution Other solutions for washing or signal generation may also be included in the kits of the invention In another preferred embodiment, the kits of the invention compnse a capture PKCg binding partner immobilized on a solid substrate, such as a bead, an assay stnp or a microtiter plate, which has also been pre-treated to prevent mterfenng signals by non-specific binding to the substrate In addition to providing rapid diagnosis of an ischemic event by emergency and medical personnel, the methods and kits descnbed herein also may be used to monitor PKCg levels as part of a routine checkup procedure or to monitor recovery from ischemic injury to CNS tissues. The nature of the methods and kits descnbed herein makes it possible to perform diagnosis and monitoring of ischemic events m many environments, for instance ambulances or other mobile medical facilities, laboratories, hospitals, emergency rooms, or even homes, samtona or other private facilities. Examples illustrating the detection of PKCg as a means of detecting an ischemic event in accordance with this invention will be set forth below. The specific materials and parameters included in the following examples are intended to illustrate the practice of the invention, and they are not presented to m any way limit the scope of the invention. EXAMPLES Example 1 : Measurement of PKCg m Ischemic Rats The animal model of middle cerebral artery occlusion (MCA-O) used m this example relies on an lntralummal filament technique in the rat (see, Zhao et al., 1994a,b). Rats were anesthetized with halothane m a nitrous oxide/oxygen mixture and the carotid artery exposed. A chamfered monofilament suture (3/0) was introduced into the ligated carotid artery, past the bifurcations of the external and common carotid, and the internal carotid and the pterygopalatine artery, into the mtracranial circulation, where it lodged in the narrow proximal anteπor carotid occluding the middle cerebral artery. The wound was then sutured and the animal allowed to recover from anesthesia. Sham operations were also performed on some animals (sham operated controls) in which the animals underwent all of the surgical procedure except for the step used to occlude the artery. After 90 minutes of occlusion, neurologic function was assessed. The animal was then decapitated and trunk blood collected into a container containing 1 5 mg/ml EDTA which was frozen until analysis Blood samples were collected from three groups of animals: (1) untreated controls (n=3); (2) sham operated controls (n=3); and (3) ischemic animals (n=5). Neurologic function was assessed and graded from normal to severe impairment The assessment scale is set forth m Table 1 The neurological impairment scores of the control groups were 0 in both cases; the average impairment score of the ischemic group was 1.9. These results demonstrate that the middle cerebral artery occlusion (MCA-O) procedure resulted in severe neurological damage, whereas neither the untreated nor sham operated control groups showed any signs of neurological damage. It was then demonstrated that the neurological impairment scores positively coπelated with the amount of PKCg measured from penpheral blood samples of ischemic animals but not with sham operated or untreated control animals. The assay of PKCg in samples of venous blood used an immobilized "capture" antibody specific for one epitope on the PKCg protein and a second "quantifying" antibody, which included a fluorescent marker attached thereto and which recognized a different epitope of PKCg than the capture antibody. In the present example, the capture antibody was a polyclonal rabbit antiserum (Calbiochem; Catalog No. 539529, used at 1 :500 dilution) recognizing the ammo acid sequence extending from ammo acids 306-318 of the PKCg protein The quantifying antibody was a monoclonal antibody (Transduction Laboratoπes; Catalog No. P20420, used at 1 :200 dilution) reactive with amino acids 499-697 at the carboxyl end of the PKCg protein. The quantifying antibody was fluorescemated using a FluoroTag kit (Sigma Chemical). A bram extract sample was used as a bram-denved PKCg positive control as supplied by the antibody manufacturer (Transduction Laboratoπes; Catalog No. B30900). The capture antibody was immobilized according to the manufacturer's instructions on tosyl-activated magnetic Dynabeads (Dynal Catalog No. 142.03,1402.4). These mateπals allowed ease of imaging and quantification of the fluorescence using a confocal scanning laser microscope (Nikon PCM200 equipped with Fluorescem filters) Paramagnetic beads coated with capture antibody and contained m a small test tube were placed m a holder containing a magnet, which attracted and pelleted the beads. Binding of the capture antibody to the magnetic beads was tested, after πnsmg the beads in buffer, by resuspendmg m goat anti-rabbit IgG-fluorescem antibody solution which binds selectively to rabbit antibodies Bright fluorescence observed by laser microscope confirmed binding of the capture antibody to the magnetic beads. Once the capture antibody binding to the magnetic beads was confirmed, a standard curve was generated using known concentrations of PKCg (obtained from Calbiochem; Catalog No. 539627): 0 pg/ml, 1 pg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, and bram extract The vaπous concentrations of PKCg were incubated with the beads and complexed with the capture antibody. Then the quantifying antibody with the fluorescemated label was added, and complexed fluorescence measured using mtensity-measunng software on the confocal microscope. Fluorescent intensity measurements from 3 separate bead preparations are shown in Figure 1 Measurement bias was minimized or eliminated by wnting a macro for the measurement software, whereby a fixed-diameter circle was used to delineate an area for intensity measurement in at least 200 beads per PKCg concentration (i.e., n=200). Only beads that were in focus withm the image plane were measured for fluorescence intensity. As seen m Figure 1, the fluorescence intensity, taken as a mean of > 100 beads per dose of PKCg, increases in a dose-dependent fashion. The data in Figure 1 are expressed as percent (%) increase in specific fluorescence, which was denved from subtracting the baseline fluorescence from the increase m fluorescence due to anti-PKCg-FITC antibody binding to the PKCg. In order to establish a standard curve from the dose response curves denved from increasing concentrations of PKCg, the fluorescence intensity was again measured from beads using the confocal microscope, however the fluorescence intensity measurements were left as fluorescence units, as seen in Figure 2 A simple curve fit was used to establish the standard curve of fluorescence intensity due to increasing concentrations of PKCg. The standard curve is shown in Figure 3 The standard curve of PKCg-mduced fluorescence was used to establish the levels of PKCg found in trunk blood of surgically treated rats. The PKCg concentrations were read directly from the standard curve Results from the blood samples demonstrate that the sandwich technique is sensitive enough to detect significant increases in the PKCg in penpheral blood samples following the MCA- O procedure. As seen in Figure 4, blood from the untreated and sham operated control animals showed significantly less fluorescence in every case when compared to the ischemic animals. In addition, brain extract controls also showed significantly higher fluorescence levels than the untreated and sham controls When fluorescence levels are converted to picograms (pg) of PKCg per ml using the standard curve shown in Figure 3, the difference in surgical treatments on PKCg levels becomes even more pronounced, as illustrated in Figure 5. Figure 5 shows a dramatic increase in the detectable levels of PKCg following the ischemic event. Levels of PKCg detected in blood collected outside of the bram following MCA-0 are more than 50% higher than levels of PKCg derived from brain extract. PKCg is activated and mobilized at the time of the ischemic event. PKCg is released from damaged cells where it initiates several damage cascades including the arachidonic acid and hpoxygenase cycles. PKCg also initiates glutamate-mduced excitoxicity cascades. One of the events which rapidly follows the ischemic episode is at least a transient breakdown of the blood- brain barπer. At 90 minutes following the MCA-0 procedure, the trunk blood samples contained levels of PKCg that are approaching 6 ng/ml as determined from the antibody sandwich technique using the magnetic beads. This ischemic level is at least ten times the limit of PKCg detection (600-1000 pg/ml) that has been shown using this method. This is an acceptable ratio of detection to actual measurements, which confirms the utility of the sandwich assay format to monitor ischemic events measured from a peπpheral blood sample. The PKCg detection can also be performed visually from confocal images of beads bound with a fluorescent quantifying antibody. Example 2: Detection and Quantitation of PKCg in a Venous Blood Sample Using Multi-well Microtiter Plate Format Expenence with the sandwich-type assay described above has led to a general protocol for the assay of this invention using a 96-well microtiter plate format. 1. Dilute capture antibody to 1:500 using a 0.1M Na-phosphate buffer (pH 7 4) containing 1% BSA (Fraction V, Sigma Chemical, St. Louis, MO) and 0.1 % Tween-20 detergent (Sigma Chemical) To make Na-Phosphate buffer use 2.62g NaH 2 P0 4 (MW= 137.99) and 14.42 grams Na 2 HP04-2H 2 0) (MW=177.99) in 1 liter distilled water and adjusted pH to 7.4. Add capture antibody to plastic 96- well microtiter plate (or other vessel) and incubate at 37°C for at least 2 hr. 2. Rinse plate with the PBS/Tween/BSA 2-3 times to remove unbound antibody from plate wells Minimal changes to this procedure will not affect antibody binding. 3. To prevent non-specific binding, block wells with 200 μl of 10% fetal calf serum m PBS for at least 2 hr. It is possible to substitute 10% horse serum or 3% BSA (Fraction V, Sigma Chemical) Rinse 2-3 times with the PBS/Tween/BSA solution mentioned above. 4. Add standards or samples to the well(s), dilute in PBS/10%serum (50-100 μl sample size) and let stand for 30 mm -1 hr. at 37°C Wash 4 times using PBS/Tween/BSA. 5 Add quantifying antibody and incubate for 30 mm. Wash 4 times using PBS/Tween/BSA. 6. Read fluorescence on a fluoπmeter, such as a CytoFluor II from Perseptives Instruments. Fluorescem optics were used with an excitation of 450 nm, and emission at 515 nm. An alternate and more preferred protocol for the sandwich-type assay using a multi-well plate format is also provided in Example 5, which was particularly useful for assaying blood samples from the middle cerebral artery occlusion (MCA-O) studies in rats descπbed m Examples 3 and 4. Example 3 Time Course of Detection of PKCg m Rats Beginning 30 Minutes After Ischemic Event Plasma PKCg levels were determined at vaπous times between 30 minutes and three hours after blocking middle cerebral artery blood flow in rats. The middle cerebral artery of rats weighing 300-325 grams each were occluded by insertion of a catheter with a blunt end into the left carotid artery and threading it up the point of bifurcation so that it blocked (occluded) blood flow to the portion of the bram supplied by the middle cerebral artery. Sham operations were performed on some of the animals in which the animals underwent all of the surgical procedure except that the blocking catheter was not placed in the artery (i.e , surgery with no arterial occlusion). Blood (2 ml) was collected before occlusion and at 30, 90, and 180 minutes after occlusion was begun, with three rats per time point. The blood samples were centnfuged at 2000 rpm for 15 minutes in a refrigerated centrifuge to obtain plasma, which was then frozen and stored at -70°C. Plasma was also prepared for analysis from three sham-operated rats and three untreated rats, which were included as controls. Plasma PKCg levels were measured by following the lmmunochemical sandwich method using the multi-well microtiter plate format as described in Example 5 (infra). Figure 6 shows that middle cerebral artery occlusion causes an increase in plasma PKCg levels at each of the collection times PKCg levels m the untreated and sham operated samples were on the order of 1-3 ng/ml, whereas PKCg levels increased to 40-50 ng/ml for each of the time points for samples from animals subjected to the artery occlusion (Figure 6 data presented as mean and standard error for an n of 3). Example 4: Time Course of Detection of PKCg in Rats Beginning 15 Minutes After Ischemic Event Plasma PKCg levels were determined at various times between 15 minutes and 24 hours after permanently blocking middle cerebral artery blood flow in rats. This experiment was conducted as described for Example 3 (see above), except that the maximum post occlusion blood collection time was increased from 3 to 24 hours. Blood collected approximately 30 minutes before occlusion (baseline) served as a reference control. Plasma PKCg levels were measured in samples by following the immunochemical sandwich method using the multi-well microtiter plate format as described in Example 5 (infra). Figure 7 shows that middle cerebral artery occlusion causes a sharp increase in plasma PKCg levels and that PKCg levels remained elevated over baseline at each of the collection times. The PKCg level in the baseline sample was on the order of lng/ml, increasing to a maximum of 67 ng/ml at 30 minutes post-occlusion. The PKCg level at 15 minutes was only one-fifth (12 ng/ml) the 30 minute level. The levels decreased gradually to 15 ng/ml by 24 hours. (Figure 7 data presented as mean and standard error for an n of 3). Example 5. An Alternate. Preferred Protocol for Detection and Quantitation of PKCg in Venous Blood Samples Using Multi-well Plate Format This Example demonstrates an alternate protocol to that in Example 2 for using a sandwich-type assay in a multi-well plate format for the detection and quantitation of PKCg in a venous blood sample. This protocol was particularly useful for assaying PKCg in blood samples from the middle cerebral artery occlusion studies in rats described in Examples 3 and 4. 1. Preparation of Multi -well Plate A 0.1 M Borate buffer (pH 9.5) was prepared by diluting 6.183 g H 3 BO 3 in 1 liter of distilled H 2 0 and adjusting the pH with 5 M NaOH. A high protein binding plate (Nunc Maxisorb, Nalge Nunc, Denmark) was rinsed 3 times (5 minutes per rinse) with the 0.1 M Borate buffer (pH 9.5) using a fluid handling robot (Quadra 96 fluid handling robot, supplied from Tomtec, Inc., Hamden, CT). 2. Immobilization of Capture Antibody on Multi-well Plate A polyclonal anti-PKCg capture antibody (Calbiochem, Catalog No. 539529) was diluted 1 : 1000 using 0.1 M Borate buffer (pH 9.5). This polyclonal anti-PKCg reacts with (binds) the region of amino acids 306-318 of PKCg. Cross reactivity of this antibody with other PKCg isozymes was determined to be less than 0.1% by ELISA. Plates were incubated at 37°C for 2-4 hours to insure stable attachment of the capture antibody to the plastic wells. This method provided immobilized capture antibody that was stable enough after incubation to withstand rinses of 10 mM Phosphate Buffered Salme (PBS, pH 7 4) using the fluid handling robot (200 μl into the well, 200 μl removed from the well). 3 Fluorescemate Labeling (Quantitation) Antibody A monoclonal anti-PKCg antibody, which binds PKCg in the region of ammo acids 676- 689 of the carboxy terminal substrate binding region of PKCg was used as the labeling (quantitation) antibody (Transduction Laboratoπes, Louisville, KY; P82820). This anti-PKCg was fluoresce -labeled using a Fluoro Tag FITC conjugation kit (Sigma Chemical, St. Louis, MO; Catalog No. FITC-1). An excess of fluorescem isothiocyanate (FITC) was dissolved in 0.1 M Na carbonate (pH 9.0) buffer according to manufacturer's specifications. The antibody was conjugated to the fluorescem in a reaction volume of 0.25 ml for 2 hours at room temperature with constant mixing. The resulting FITC-anti-PKCg antibody conjugate and unbound FITC mixture was applied to a Sephadex G30 chromatographic column. The column was eluted with 2.5-3.0 ml of PBS/TWEEN/BSA (pH 7.4) buffer, which is PBS (pH 7.4) containing 0.1 % polyoxyethylenesorbitan monostearate (TWEEN 60, Sigma Chemical) and 0.1% bovme serum albumin Fraction V (BSA, Sigma). Fractions of 300 μl were collected from the G30 column into a 96 well plate and then read on a CytoFluor II fluoπmeter (Perseptives Instruments, Framingham, MA) using an excitation wavelength of 480 nm and an emission wavelength of 515 nm. The FITC- anti-PKCg antibody eluted m the void volume and constituted a first fluorescence peak. The unbound FITC was retained and eluted as a later second peak of fluorescence. The FITC-anti- PKCg antibody was diluted to a volume that represented approximately 1500 fluorescence units in 300 μl using the PBS/TWEEN/BSA buffer (pH 7.4), descnbed above. The final dilution for this FITC-anti-PKCg preparation was to 30 ml in the PBS/TWEEN/BSA buffer (pH 7.4). 4. PKCg Standard Curve A recombinant human PKCg (Calbiochem, Catalog No. 539637) was used to construct a standard curve of PKCg concentrations extending from 0 to 600 ng PKCg/ml. PKCg was diluted in PBS/TWEEN/BSA buffer (pH 7.4). The conditions for maximal sensitivity of the assay were determined expenmentally The standard sample protocol combined 100 μl of a PKCg standard sample, 250 μl of FITC-anti-PKCg antibody, and 650 μl of PBS/TWEEN/BSA buffer (pH 7.4). Each mixture was incubated for 30-60 minutes at 37°C with constant mixing. Following incubation of the standards with the FITC-anti-PKCg, a volume of 125 μl of each mixture was placed in 8 wells of a multi-well plate containing capture antibody and incubated for 30 minutes at 37°C Fluorescence readings from the 8 sample repeats were then read in the fluonmeter using filters for FITC, excitation wavelength of 450 nm, and emission wavelength of 515 nm. Standard curves were constructed from the readings for each standard PKCg sample. 5. Venous Blood Samples from MCA-O-Treated Rats Samples of plasma from the peripheral blood of rats subjected to the middle cerebral artery occlusion (MCA-O) procedure (see, Examples 3 and 4 above) were prepared using the same protocol described above for preparing the PKCg standard samples: 100 μl of each plasma sample was added to 250 μl of FITC-anti-PKCg antibody and 650 μl of PBS/TWEEN/BSA (pH 7.4). Each sample mixture was then incubated for 30-60 minutes at 37°C as above. A volume of 125 μl of each sample mixture was placed in 8 wells of a multi-well plate containing immobilized capture antibody as described above and incubated for 30 minutes at 37°C. Fluorescence readings of each sample were collected in the fluorimeter using filters for FITC, excitation wavelength of 450 nm, and emission wavelength of 515 nm. The levels of PKCg in each sample were established from reading levels of PKCg from standard curves as described above. Mean PKCg levels (n=8 wells) and the standard errors were derived for each treatment sample. As mentioned in Example 4, samples from MCA-O-treated animals showed significantly higher levels of PKCg when compared to sham-operated and untreated control samples. Levels reached significance at times as early as 15 minutes following the ischemic event and remained at a significant level for at least 24 hours after the event (see, e.g., Figure 7). Although a number of embodiments have been described above, it will be understood by those skilled in the art that modifications and variations of the described compositions and methods may be made without departing from either the spirit of the invention or the scope of the appended claims. The publications cited herein are incorporated by reference. REFERENCES The following publications pertain to the state of the art and/or illustrate techniques that may be utilized in practicing the present invention. Adams et al., Guidelines for the Management of Patients with Acute Ischemic Stroke, Stroke, 25(9): 1901 - 1914 (September 1994). Bazan, Effect of Ischemia and Electroconvulsive Shock on the Free Fatty Acid Pool in the Brain., Biochim. Biophys. Ada, 218: 1 - 10 (1970). Buttner et al., S-100 Protein: Serum Marker of Focal Brain Damage After Ischemic Territorial MCA Infarction, Stroke, 28: 1961 - 1965 (1997). Conn's Current Therapy. R. E. Rakel, ed., W. B. Saunders Co. (1993), pp. 840 - 851 Dippel et al., We Need Stronger Predictors of Major Vascular Events in Patients With a Recent Transient Ischemic Attack or Nondisabhng Stroke, Stroke, 28: 774 - 776 (1997). Femberg et al., Guidelines for the Management of Transient Ischemic Attacks, Stroke, 25(6). 1320 - 1335 (June 1994). Madden et al., Glutamate, Arachidonic Acid, and Calcium Regulation in Cultured Hippocampal Astiocytes: Involvement in Ischemia?, m Cellular and Molecular Mechanisms of Ischemic Bram Damage Advances in Neurology. Siesjo and Wieloch, eds., Lippmcott-Raven (Philadelphia 1996), 71 : 53 - 60. Missler et al., S-100 Protein and Neuron- Specific Enolase Concentrations m Blood as Indicators of Infarction Volume and Prognosis in Acute Ischemic Stroke, Stroke, 28: 1956 - 1960 (1997). Shashoua et al., Proteins of the Brain Extracellular Fluid: Evidence for Release of S-100 Protein, J Neurochem , 42(6): 1536 - 1541 (1984). Wieloch et al., Intracellular Signal Transduction in the Postischemic Bram. m Cellular and Molecular Mechanisms in Ischemic Bram Damage. Advances m Neurology.. Siesjo and Wieloch, eds., Lippincott-Raven (Philadelphia 1996), 71: 371 - 388. Wolfe, Eicosanoids: Prostaglandins, Thromboxanes, Leukotnenes and Other Deπvatives of Carbon-20 Unsaturated Fatty Acids, J. Neurochem., 38: 1 - 14 (1982). Zhao et al., Hyperthermia complicates middle cerebral artery occlusion induced by an mtraluminal filament, Brain Res., 649: 253-259 (1994a). Zhao et al., Delayed treatment with the spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) reduces mfarct size following transient middle cerebral artery occlusion in rats, Ada Physiol Scand., 152- 349-350 (1994b). The publications mentioned herein are incorporated by reference in their entireties

Claims: CLAIMS 1. A method for determining whether a mammal has suffered an ischemic event, comprising detecting the presence of the gamma isoform of protein kinase C (PKCg) in the mammal peripheral to the central nervous system. 2. A method for detecting an ischemic event in a mammalian subject comprising: (a) contacting a peripheral blood sample obtained from said subject with a detectably labeled binding partner capable of forming a binding complex with PKCg; (b) detecting the presence of a PKCg/binding partner complex formed in step (a). 3. The method according to Claim 2, wherein said binding partner is in the form of a finely divided solid substrate dispersible in a fluid sample, which substrate exhibits a surface moiety capable of complexing PKCg. 4. The method according to Claim 2, wherein said binding partner is in the form of an anti- PKCg antibody. 5. The method according to Claim 4, wherein said anti-PKCg antibody is selected from the group consisting of a major immunoglobulin class, an immunoglobulin subclass, a Fab fragment, a F(ab') 2 fragment, an Fv fragment, a single chain antibody (scFv), a chimeric antibody, a humanized antibody, a polyclonal antibody, a monoclonal antibody, and a diabody. 6. The method according to Claim 4, wherein said anti-PKCg antibody is fluorescently labeled. 7. The method according to Claim 1 or 2, wherein the ischemic event is the result of stroke, transient ischemic attack, head trauma, myocardial infarction, or trauma resulting in interrupted cranial blood flow. 8. A method for detecting a PKCg in the blood of a mammalian subject comprising the steps of: (a) contacting a peripheral blood sample obtained from said subject with a solid substrate having a first anti-PKCg antibody immobilized thereon under conditions suitable for forming a binding complex with PKCg in said sample; (b) contacting the substrate with a second antibody comprising a detectably labeled anti- PKCg antibody under conditions suitable for the second antibody to react with PKCg, wherein said second antibody recognizes a different PKCg epitope than said first antibody; and (c) measunng the presence of the detectable label. 9. A method for detecting an ischemic event m a mammalian subject compnsmg the steps of. (a) contacting a penpheral blood sample obtained from said subject with a solid substrate having a first anti-PKCg antibody immobilized thereon, under conditions suitable for forming a binding complex with PKCg m said sample; (b) contacting the substrate with a second antibody compnsmg a detectably labeled anti- PKCg antibody under conditions suitable for the second antibody to react with PKCg, wherein said second antibody recognizes a different PKCg epitope than said first anti- PKCg antibody; and (c) measunng the presence of the detectable label. 10 The method according to Claim 9, which further compnses the step, after step (a): (a- 1) separating the solid substrate from the rest of said sample. 11 The method according to Claim 10, which further compπses the step, after step (b): (b- 1 ) removing any unbound second antibody. 12. The method according to Claim 11, which further compπses the step of quantitating the level of PKCg m the blood sample based on the amount or intensity of the detectable label measured m step (c). 13. The method according to Claim 9, wherein the substrate is a well of a microtiter plate. 14. The method according to Claim 9, wherein the substrate is a magnetic bead. 15. The method according to Claim 9, wherein the substrate is a piece of plastic, nylon, wood or paper that is manually or robotically placed m contact with the blood sample. 16. The method according to Claim 9, wherein the second antibody is fluorescently labeled 17. The method according to Claim 9, wherein the ischemic event is the result of stroke, transient ischemic attack, head trauma, myocardial infarction or trauma resulting in interrupted cranial blood flow 18 The method according to Claim 8 or Claim 9, wherein the first and second anti-PKCg antibodies are independently selected from the group consisting of a major immunoglobulin class, an immunoglobulin subclass, a Fab fragment, a F(ab') 2 fragment, an Fv fragment, a single cham antibody (scFv), a chimenc antibody, a humanized antibody, a monoclonal antibody, a polyclonal antibody, and a diabody. 19 The method according to Claim 8 or 9, wherein the peπpheral blood sample is mixed with the second antibody compnsmg a detectably labeled anti-PKCg antibody, and the mixture is then contacted with the solid substrate having the first anti-PKCg antibody immobilized thereon. 20. A method for diagnosing stroke, transient ischemic attack, head trauma, myocardial infarction, or trauma resulting in interrupted cranial blood flow m a mammalian subject comprising- detecting the presence of PKCg in the subject's penpheral blood. 21. The method according to Claim 20, wherein PKCg is detected in a sample of venous blood taken from the subject. 22 The method according to Claim 20, wherein PKCg is detected using a detectably labeled anti- PKCg antibody 23. A kit for rapid diagnosis of injury resulting from an ischemic event m a mammalian subject, compnsmg: (a) a solid substrate having immobilized thereon a first PKCg binding partner, (b) a second PKCg binding partner reactive with a different epitope of PKCg than is recognized by said first PKCg binding partner, which second PKCg binding partner is detectably labeled, and (c) instructions and reagents for performing a sandwich-type assay for the presence of PKCg in a sample of penpheral blood obtained from said subject, wherein detection of PKCg is indicative of an injury from an ischemic event. 24 The kit according to Claim 23, wherein said first PKCg binding partner, said second PKCg binding partner, or both of said PKCg binding partners are anti-PKCg antibodies. 25 A kit according to Claim 23 or 24, wherein said first binding partner is immobilized on magnetic beads. 26 A kit according to Claim 23 or 24, wherein said first bindmg partner is immobilized in wells of a multi-well microtiter plate. 27. A kit accordmg to Claim 23 or 24, wherein said first bindmg partner is immobilized on a piece of plastic, nylon, paper or wood. 28. A kit according to Claim 23 or 24, wherein said second PKCg bmdmg partner is fluorescently labeled.

Inventors: CORNELL-BELL ANN, MADDEN KATHLEEN S, RIBLET LESLIE A, CORNELL-BELL, ANN, MADDEN, KATHLEEN, S., RIBLET, LESLIE, A.
Applicants: CORNELL BELL ANN, MADDEN KATHLEEN S, RIBLET LESLIE A, VIATECH IMAGING LLC, CORNELL-BELL, ANN, MADDEN, KATHLEEN, S., RIBLET, LESLIE, A., VIATECH IMAGING, LLC
Application year: 2000
Main classification: G01N33
IPC codes: G01N33