Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1999-08-27
2001-07-31
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C435S007100, C435S007940, C435S975000, C436S518000, C436S523000, C436S533000, C436S538000, C436S546000, C436S063000, C530S380000
Reexamination Certificate
active
06268223
ABSTRACT:
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-brain 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 time period 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 resulting 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 500,000 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 risk 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 in stroke are not fully understood, it has been proposed that the pathology arises from an initial infarct, wherein perfusion pressure and blood flow to CNS tissues are reduced by intracellular and microvascular edema, followed by progressive compression and reduced perfusion in areas adjacent to the infarct. 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, appropriate treatment must be administered within two hours of the suspected insult. 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 within 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), myelin basic protein, glial fibrillary acidic protein, and S-100 protein (Missler et al., 1997). In most cases these substances are measured in cerebrospinal 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 infarct 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 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 peripheral 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 PKC&ggr; or PKCg herein), appears in the peripheral blood very quickly after an ischemic event in the CNS.
The present invention describes 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 spinal or cranial blood flow. The procedure can be easily performed by ambulance or emergency room personnel and can be performed with a venous blood sample and an assay kit as described 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, transient ischemic attacks (“TIAs”), head trauma, myocardial infarction, or other events or “insults” resulting in 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, a peripheral blood sample is drawn from the affected individual then analyzed for the presence of PKCg. Preferably, a venous blood sample is contacted with 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 in 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 central nervous system has occurred, i.e., by the presence or absence of PKCg in the 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 severity of central nervous system damage.
Where immunosorbant 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 preferred antibody-based methods, 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 in order to separate the binding complexes from cells and larger proteins in the sample. Most preferably, according to a preferred method, the inbound (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 insult to CNS tissues. The column may be washed one or more t
Cornell-Bell Ann H.
Madden Kathleen S.
Riblet Leslie A.
Berka Thomas R.
Chin Christopher L.
Ivory-McCaa Terri
Viatech Imagin, LLC
Yankwich Leon R.
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