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Title:  Detection of spectrin and spectrin proteolytic cleavage products in assessing nerve cell damage
United States Patent: 
7,291,710
Issued: 
November 6, 2007

Inventors: 
Hayes; Ronald L. (Gainesville, FL), Wang; Kevin K. W. (Gainesville, FL), Pike; Brian R. (Derwood, MD)
Assignee: 
University of Florida Research Foundation, Inc. (Gainesville, FL)
Appl. No.: 
10/660,069
Filed: 
September 11, 2003


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Methods for detecting a cell damage relate to the discovery that proteases are selectively activated in subjects suffering from nervous system damage compared to samples from healthy subjects. Breakdown products reflecting activation of proteases that degrade spectrin are produced. A cell injury is detected by providing a biological sample derived from the subject; detecting in the sample the presence of these breakdown products generated by multiple proteases, and correlating the presence of these breakdown products with the presence or type of cell damage.

SUMMARY OF THE INVENTION

The invention relates to the discovery of the accumulation of non-erythroid .alpha.II-spectrin and its calpain- and caspase-3-specific breakdown products (SBDPs) in the cerebrospinal fluid (CSF) of human subjects with traumatic brain injury as well as in rodent models of traumatic or ischemic nerve cell injury. The ability to detect and monitor calpain and caspase-3 concurrently after nervous system damage should facilitate (i) determining the presence and/or severity or nerve damage, (ii) selecting the best course of treatment for a subject suspected of having nerve damage, and (iii) analyzing the effectiveness of treatment for nerve damage.

Accordingly, the invention features a method for analyzing nerve cell damage in a subject. The method includes the steps of: (a) providing a biological sample isolated from a subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject (e.g., cerebrospinal fluid, blood, plasma, and serum); (b) detecting in the sample the presence or amount of at least one marker selected from .alpha.II spectrin and an .alpha.II SBDP generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and (c) correlating the presence or amount of the marker with the presence or type of nerve cell damage in the subject.

Typically, the subject will be a human patient suspected of having a damaged nerve cell. For example, the method might be applied to a human subject that has sustained trauma (e.g., a blow to the head) or one that presents with symptoms of acute ischemia of a nervous system tissue such as brain (e.g., a patient who appears to have suffered a cerebrovascular accident). The marker(s) being assessed can be one, two, three, four or all of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, or SBDP120.

The step (b) of detecting in the sample the presence or amount of at least one marker selected from .alpha.II spectrin and an .alpha.II SBDP generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain can include contacting the sample or a portion of the sample with an agent (e.g., an antibody) that specifically binds the marker. The agent can be one that does not specifically bind at least one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120 (i.e., one that binds only a subset of this group); or one that specifically binds only one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, or SBDP120 (i.e., a mono-specific agent). In some variations of the method of the invention, the step (b) includes immobilizing the biological sample or a portion of the sample on a substrate, and/or contacting the substrate with an agent that specifically binds the marker.

The step (c) of correlating the presence or amount of the marker with the presence or type of cell damage in the subject can include comparing the presence or amount of the marker in the sample with that in a standard sample known to not contain the marker (e.g., a negative control); and/or comparing the presence or amount of the marker in the sample with that in a standard sample known to contain a known amount of the marker (e.g., a positive control or a comparative control for quantifying the amount of the marker in the sample).

In another aspect, the invention features a mixture that includes: (a) a biological sample isolated from a human subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; and (b) an agent (e.g., an antibody) that specifically binds at least one marker selected from .alpha.II spectrin and an .alpha.II spectrin breakdown product (SBDP) generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain. The marker(s) being assessed can be one or more of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120. The agent can be one that does not specifically at least one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120; or one that specifically binds only one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120.

The mixture of the invention can be immobilized on a substrate, e.g., to facilitate detection of the marker(s) in an immunoblot or similar assay. The mixture can further include a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent (e.g., a detectably labeled secondary antibody).

The invention further includes a kit for analyzing cell damage in a subject. The kit includes: (a) a substrate for holding a biological sample isolated from a human subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; (b) an agent that specifically binds at least one marker selected from .alpha.II spectrin and an .alpha.II SBDP generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and (c) printed instructions for reacting the agent with the biological sample or a portion of the biological sample to detect the presence or amount of the at least one marker in the biological sample.

In the kit, the marker(s) being assessed can be one or more of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120. The agent can be one that does not specifically at least one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120; or one that specifically binds only one of .alpha.II spectrin, SBDP150i, SBDP1150, SBDP145, and SBDP120. The kit can also include a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods, compositions, and kits for detecting and quantifying neurochemical markers to detect nerve cell damage, determine the seriousness of the damage, determine the anatomical and cellular pathology of the damage, and help determine an appropriate treatment for the damage. The invention is based on the characterization of cellular protease (i.e., calpain and caspase-3) activation that occurs in response to nerve cell damage.

Through a complex series of signaling events, nerve injury upregulates both calpain and caspase-3-mediated proteolysis of a variety of intracellular substrates including non-erythroid .alpha.II-spectrin, a cytoskeletal protein particularly abundant in nerve cells. Referring to FIG. 1A (see Original Patent), activated calpain and caspase-3 both bind to .alpha.II-spectrin, but cleave it at different sites to yield distinct SBDPs. In particular, calpain initially cleaves .alpha.II-spectrin between Tyr.sup.1176 and Gly.sup.1177 resulting in the formation of calpain-signature SBDPs of 150 kDa (SBDP150). A second cleavage between Gly.sup.1230 and Ser.sup.1231 results in the formation of a second calpain-signature SBDP of 145 kDa (SBDP145). In the same fashion, caspase-3 cleaves .alpha.II-spectrin at Asp.sup.1185 and Ser.sup.1186 and at Asp.sup.1478 and Ser.sup.1479 to yield caspase-3-signature SBDPs of 150 (SBDP150i) and 120 (SBDP120) kDa, respectively. The residues number used are based on human alpha II spectrin (Homo sapiens; accession U83867.1, protein number AAB41498). However, as caspase-3 and calpain cleavage sites are fairly conserved among different mammalian species, similar SBDPs are generated in other species in response to nerve cell damage.

Detection and quantification of SBDPs such as SBDP150, SBDP145, SBDP150i, and SBDP120 can therefore be used to detect and characterize nerve cell damage. To illustrate, referring to FIG. 1B (see Original Patent), a Western blot can be used to concurrently detect full length .alpha.II-spectrin (280 kDa) and calpain- and caspase-3-generated SBDPs. In the absence of calpain or caspase-3 activation, only full length .alpha.II-spectrin is detected (lane 1). Activation of caspase-3 only leads to the generation of an additional 150 kDa band and a 120 kDa band (lane 2), whereas activation of calpain only leads to the generation of an additional 150 kDa band and a 145 kDa band. Activation of both caspase-3 and calpain leads to the generation of 4 bands corresponding to the intact 280 kDa .alpha.II-spectrin, the 150 kDa fragments, the 145 kDa fragment, and the 120 kDa fragment (lanes 3 and 4). As described below, the two different 150 kDa fragments (i.e., SBDP150 and SBDP150i) can be distinguished from one another, e.g., using an antibody that specifically recognizes the unique N-terminal region each different fragment.

General Biological Methods

Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.

Detecting Nerve Cell Damage

Nerve cell damage in a subject is analyzed by (a) providing a biological sample isolated from a subject suspected of having a damaged nerve cell; (b) detecting in the sample the presence or amount of at least one marker selected from .alpha.II spectrin and an .alpha.II SBDPs generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and (c) correlating the presence or amount of the marker with the presence or type of nerve cell damage in the subject.

Biological Samples

After insult, nerve cells in in vitro culture or in situ in an animal subject express higher levels of .alpha.II spectrin, SBDP150, SBDP145, SBDP150i, and/or SBDP120 than do such cells not subjected to the insult. Thus, samples that contain nerve cells, e.g., a biopsy of a central nervous system or peripheral nervous system tissue are suitable biological samples for use in the invention. In addition to nerve cells, however, other cells express .alpha.II-spectrin including, for example, cadiomyocytes, myocytes in skeletal muscles, hepatocytes, kidney cells and cells in testis A biological sample including such cells or fluid secreted from these cells might also be used in an adaptation of the above method to determine and/or characterize an injury to such non-nerve cells.

In addition to increased cell expression, .alpha.II spectrin, SBDP150, SBDP145, SBDP150i, and/or SBDP120 also appear in biological fluids in communication with injured cells. Obtaining biological fluids such as cerebrospinal fluid, blood, plasma, serum, saliva and urine, from a subject is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. Thus, samples which are biological fluids are preferred for use in the invention. CSF, in particular, is preferred for detecting nerve damage in a subject as it is in immediate contact with the nervous system and is readily obtainable.

A biological sample can be obtained from a subject by conventional techniques. For example, CSF can be obtained by lumbar puncture. Blood can be obtained by venipuncture, while plasma and serum can be obtained by fractionating whole blood according to known methods. Surgical techniques for obtaining solid tissue samples are well known in the art. For example, methods for obtaining a nervous system tissue sample are described in standard neuro-surgery texts such as Atlas of Neurosurgery: Basic Approaches to Cranial and Vascular Procedures, by F. Meyer, Churchill Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain Tumors, 1st ed., by David G. T. Thomas, WB Saunders Co., 1993; and Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999. Methods for obtaining and analyzing brain tissue are also described in Belay et al., Arch. Neurol. 58: 1673-1678 (2001); and Seijo et al., J. Clin. Microbiol. 38: 3892-3895 (2000).

Any animal that expresses .alpha.II spectrin might be used as a subject from which a biological sample is obtained. The subject can be, e.g., a mammal such as a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, rat, or mouse. Because the experiments presented herein relate to human subjects, a preferred subject for the methods of the invention is a human being. Particularly preferred are subjects suspected of having or at risk for developing traumatic or non-traumatic nervous system injuries, such as victims of brain injury caused by traumatic insults (e.g. gunshots wounds, automobile accidents, sports accidents, shaken baby syndrome), ischemic events (e.g. stroke, cerebral hemorrhage, cardiac arrest), neurodegenerative disorders (such as Alzheimer's, Huntington's, and Parkinson's diseases; Prion-related disease; other forms of dementia), epilepsy, substance abuse (e.g., from amphetamines, Ecstasy/MDMA, or ethanol), and peripheral nervous system pathologies such as diabetic neuropathy, chemotherapy-induced neuropathy and neuropathic pain.

Markers of Calpain and Caspase-3 Activation

The method of the invention features a step of detecting in a biological sample the presence or amount of at least one marker selected from .alpha.II spectrin and an .alpha.II spectrin breakdown product (SBDP) generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain. SBDPs generated from proteolytic cleavage of .alpha.II spectrin by caspase-3 include SBDP150i and SBDP120. SBDPs generated from proteolytic cleavage of .alpha.II spectrin by calpain include SBDP150 and SBDP145. Depending on the species of animal subject being analyzed, the migration patterns of SBDPs generated from digestion of .alpha.II spectrin by caspase-3 and calpain may vary somewhat. Using the methods taught herein, these can be determined empirically.

Detection of .alpha.II Spectrin and SBDPs

The invention encompasses methods for detecting the presence of the marker .alpha.II spectrin or one of its SBDPs (e.g., SBDP150, SBDP145, SBDP150i, or SBDP120) in a biological sample as well as methods for measuring the level of such marker in a biological sample. An exemplary method for detecting the presence or absence of .alpha.II spectrin or one of its SBDPs in a biological sample involves obtaining a biological sample from a subject (e.g., a human patient), contacting the biological sample with a compound or an agent capable of detecting of the marker being analyzed (e.g., an antibody or aptamer), and analyzing binding of the compound or agent to the sample after washing. Those samples having specifically bound compound or agent express of the marker being analyzed.

Methods of the invention can be used to detect .alpha.II spectrin or one of its SBDPs in a biological sample in vitro as well as in vivo. The quantity of expression of .alpha.II spectrin or one of its SBDPs in a sample may be compared with appropriate controls such as a first sample known to express detectable levels of the marker being analyzed (i.e., a positive control) and a second sample known to not express detectable levels of the marker being analyzed (i.e., a negative control). For example, in vitro techniques for detection of a marker include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Furthermore, in vivo techniques for detection of a marker include introducing a labeled agent that specifically binds the marker into a biological sample or test subject. For example, the agent can be labeled with a radioactive marker whose presence and location in a biological sample or test subject can be detected by standard imaging techniques.

Any suitable molecule that can specifically bind .alpha.II spectrin and/or one or more of its SBDPs might be used in the invention. A preferred agent for detecting .alpha.II spectrin or one of its SBDPs is an antibody capable of binding to the marker being analyzed, preferably an antibody conjugated with a detectable label. Such antibodies can be polyclonal, or monoclonal. An intact antibody, a fragment thereof (e.g., Fab or F(ab').sub.2), or an engineered variant thereof (e.g., sFv) can also be used. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

Particularly useful antibodies include those that can distinguish among .alpha.II spectrin and/or one or more of its SBDPs. Antibodies that bind only a particular marker or a subset of markers can be made according to known methods. See, Coligan et al, supra. As described below, antibodies that specifically bind one or a subset of .alpha.II spectrin SBDPs have been made against the N-terminal ends of individual SBDPs by immunizing animals with peptides corresponding to such N-terminal ends.

Antibody-based assays are preferred for analyzing a biological sample for the presence of .alpha.II spectrin and/or one or more of its SBDPs assays. Suitable Western blotting methods are described below in the Examples section. For more rapid analysis (as may be important in emergency medical situations), immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation assays may be used. See, Coligan et al., supra. As one example, the biological sample or a portion thereof is immobilized on a substrate (e.g., a membrane made of nitrocellulose or PVDF; or a rigid substrate made of polystyrene or other plastic polymer such as a microtiter plate), and the substrate is contacted with an antibody that specifically bind .alpha.II spectrin and/or one or more of its SBDPs under conditions that allow binding of antibody to the marker being analyzed. After washing, the presence of the antibody on the substrate indicates that the sample contained the marker being assessed. If the antibody is directly conjugated with a detectable label (e.g., an enzyme, fluorophore, or radioisotope), its presence can be detected by examining the substrate for the detectable label. Alternatively, a detectably labeled secondary antibody that binds the marker-specific antibody can be added to the substrate. The presence of detectable label on the substrate after washing indicates that the sample contained the marker.

Numerous permutations of these basic immunoassays may also be used in the invention. For example, the marker-specific antibody (rather than the biological sample) is immobilized on a substrate, and the substrate is contacted with a marker (e.g., one or more of .alpha.II spectrin and/or one or more of its SBDPs) conjugated with a detectable label under conditions that cause binding of antibody to the labeled-marker. The substrate is then contacted with a biological sample under conditions that allow binding of the marker being analyzed to the antibody. A reduction in the amount of detectable label on the substrate after washing indicates that the sample contained the marker.

Although antibodies are preferred for use in the invention because of their extensive characterization, any other suitable agent (e.g., a peptide, an aptamer, or a small organic molecule) that specifically binds .alpha.II spectrin and/or one or more of its SBDPs might be used in place of the antibody in the above-described immunoassays. For example, an apatamer that specifically binds .alpha.II spectrin and/or one or more of its SBDPs might be used. Apatamers are nucleic acid-based molecules that bind specific ligands. Methods for making aptamers with a particular binding specificity are known. See, e.g., U.S. Pat. Nos. 5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588; 5,683,867; 5,637,459; and 6,011,020.

Myriad detectable labels that may be used in a diagnostic assay for marker expression are known in the art. Agents used in methods for detecting .alpha.II spectrin and/or one or more of its SBDPs may be conjugated to a detectable label, e.g., an enzyme such as horseradish peroxidase. Agents labeled with horseradish peroxidase can be detected by adding an appropriate substrate that produces a color change in the presence of horseradish peroxidase. Several other detectable labels that may be used are known. Common examples of these include alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, magnetic particles, biotin, radioisotopes, and other enzymes.

Correlating Marker Expression with Nerve Cell Damage

The invention employs a step of correlating the presence or amount of .alpha.II spectrin and/or one or more of its SBDPs in a biological sample with the severity and/or type of nerve cell (or other .alpha.II spectrin-expressing cell) injury. The amount of spectrin and/or its SBDPs in the biological sample directly relates to severity of nerve tissue injury as a more severe injury damages a greater number of nerve cells which in turn causes a larger amount of .alpha.II spectrin and/or its SBDPs to accumulate in the biological sample (e.g., CSF). Whether a nerve cell injury triggers an apoptotic and/or necrotic type of cell death can also be determined by examining the SBDPs present in the biological sample. Necrotic cell death preferentially activates calpain, whereas apoptotic cell death preferentially activates caspase-3. Because calpain and caspase-3 SBDPs can be distinguished, measurement of these markers indicates the type of cell damage in the subject. For example, necrosis-induced calpain activation results in the production of SBDP150 and SBDP145; apoptosis-induced caspase-3 activation results in the production of SBDP150i and SBDP120; and activation of both pathways results in the production of all four markers. The results of such a test can help a physician determine whether the administration of calpain and/or caspase inhibitors might be of benefit to a patient. This invention is believed to be the only available approach for concurrent detection of the relative magnitude of apoptotic and necrotic cell death from the same biological sample. This application may be especially important in detecting age and gender difference in cell death mechanism.

Kits

The invention also provides a kit for analyzing cell damage in a subject. The kit includes: (a) a substrate for holding a biological sample isolated from a human subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; (b) an agent that specifically binds at least one marker selected from .alpha.II spectrin and an .alpha.II SBDP generated from proteolytic cleavage of .alpha.II spectrin by at least one protease selected from the group consisting of caspase-3 and calpain; and (c) printed instructions for reacting the agent with the biological sample or a portion of the biological sample to detect the presence or amount of the at least one marker in the biological sample.

In the kit, the biological sample can be CSF or blood, and the agent can be an antibody, aptamer, or other molecule that specifically binds at least one of .alpha.II spectrin, SBDP150i, SBDP150, SBDP145, and SBDP120. Suitable agents are described above. The kit can also include a detectable label such as one conjugated to the agent, or one conjugated to a substance that specifically binds to the agent (e.g., a secondary antibody).


Claim 1 of 29 Claims

1. A mixture comprising: a biological sample isolated from a subject suspected of having a damaged nerve cell, the biological sample being a fluid in communication with the nervous system of the subject prior to being isolated from the subject; and at least two added antibodies that specifically and independently bind to at least two markers selected from .alpha.II-spectrin and an .alpha.II-spectrin breakdown product (SBDP) selected from at least one of SBDP150i, SBDP150, SBDP145 and SBDP120 generated from proteolytic cleavage of .alpha.II-spectrin by at least one protease selected from the group consisting of caspase-3 and calpain.

 

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