METHOD FOR DETECTING CELLULAR PATHOLOGY

Abstract: The invention provides a method for the detection of cellular pathology by means of an immunoassay to determine the presence of stable breakdown products, termed BDP's or BDP1 and BDP2, of the cytoskeleton component spectrin. In one aspect of the invention, the components from a sample of spectrin-containing cells are physically separated, as by exposure to an electric field, in such a way that BDP and spectrin are separated. Antibodies reactive with BDP are then contacted with the separated sample, and their binding to that portion of the sample containing any BDP determined.

Description: METHOD FOR DETECTING CELLULAR PATHOLOGY BACKGROUND OF THE INVENTION This invention relates to an in vitro method for the detection of cellular pathology, and more specifically to an assay for monitoring cytos eleton breakdown products to determine cell necrosis. This invention was made with government support under AFSOR Contract No. 86-0099 (P.I.: Lynch), NIH Grant No. NS-18427 and NIA Grant No. AG00538. The government has certain rights in this invention. The structural integrity of cells is maintained in part by the cytoskeleton, a mesh-like structure composed primarily of proteins, which lies adjacent to the inner cell surface. The cytoskeleton of many cell types (a partial list includes neurons, lymphocytes, kidney, liver, cardiac and smooth muscle, and blood platelets) contain large amounts of a protein either identical to or closely related to brain spectrin (also known as fodrin) . Calpain is an enzyme which in the presence of elevated calcium levels degrades spectrin, thereby leading to the disruption of the cytoskeleton. A variety of cellular insults (e.g., toxins, anoxia, etc.) and disease states (e.g., Alzheimer's, Parkinson's, muscular dystrophy) cause the degeneration and death of cells. Often, however, it is not possible to determine that injury has occurred until degenerative effects are irreversible. There thus exists a need for a reliable method to detect degenerative events as soon as possible after their onset before pathological symptoms appear. Preferably such method should have high sensitivity, wide ranging applicability and ease of administration. The present invention satisfies this need and provides related advantages. SUMMARY OF THE INVENTION The invention provides a method for the detection of cellular pathology by means of an immunoassay to determine the presence of stable breakdown products, termed BDP's or BDPl and BDP2, of the cytoskeleton component spectrin. In one aspect of the invention, the components from a sample of spectrin-containing cells are physically separated, as by exposure to an electric field, in such a way that BDP and spectrin are separated. Antibodies reactive with BDP are then contacted with the separated sample, and their binding to that portion of the sample containing any BDP _ determined. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. A. Spectrin immunoreactivity in blotted samples of the contralateral (left lane) and ipsilateral (right lane) dentate gyrus two days after a unilateral lesion of the entorhinal cortex. Arrows indicate the alpha and beta spectrin subunits with apparent M r s of about 240 and 230 KDa respectively and two additional immunoreactive peptides (BDPl and BDP2) with apparent M r s of about 155 and 150 KDa respectively. B. Purified brain spectrin incubated under the following conditions. Lane 1: no additions; Lane 2: ImM CaCl 2 , 1.8 μg/ml calpain I, 10 minutes; Lane 3: ImM CaCl 2 , 3 μg/ml calpain I, 30 minutes; Lane 4: 10 μg of dentate gyrus protein ho ogenate two days post-lesion. Lane 5: ImM CaCl 2 , 13 μg/ml calpain, 1.25 μg/ml colmodulin, 3 minutes; Lane 6: ImM CaCl 2 , 13 μg/ml calpain, 1.25 μg/ml colmodulin, 30 minutes. Figure 2. BDPl (filled circles) and BDP2 (open circles) levels are expressed as a percentage of the total spectrin immunoreactivity, as determined by scanning reflective densitoraetry. Figure 3. Levels of BDP's in regions of the brain of control and Brindled mice, showing the effect of treatment with copper. Figure 4. Levels of BDP in the dentate gyrus and the CAl region of the hippocampus of rats receiving tri ethyltin. Figure 5. Levels of BDP in the dentate gyrus and the CAl region of the hippocampus from a gerbil following ischemia. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates to a sensitive and efficient method for the early detection of cellular death and degradation. The method involves an immunoassay using antibodies which recognize stable breakdown products of spectrin, a component of the cytoskeleton of many cell types. Spectrin binds F-actin, and together they are generally associated with the inner face of the cell membrane, where they form a filamentous meshwork. Brain and many other tissues have been known for some time to express calcium-stimulated proteolytic activity. Studies of degeneration in peripheral nerves have indicated that a calcium-dependent protease, termed calpain, is critically involved in the degradation of neurofilament proteins following denervation or injury. Two forms of this protease have been identified in the brain and other tissues which are differentiated by their threshold for activation by calcium: calpain I re-quires micromolar calcium while calpain II is activated by concentrations of calcium between 0.1 and 0.5 mM. The two forms are differentially distributed in the brain. While calpain II is mainly localized in the cytoplasmic fraction of brain cells, the highest activity of calpain I is found in small processes. Brain spectrin can be purified to greater than 90% purity by the method of Davis and Bennett, J. Biol. Chem. 258:7757-7766 (1983), which is incorporated herein by reference. Calpain can be purified to a similar level of purity from rat erythrocyte cytosol according to the method of Seubert et al.. Synapse 1:20-24 (1987), which is incorporated herein by reference. In order td identify spectrin breakdown products, 75 μg/ml of spectrin was incubated at 30 β C with 100 uM CaCl 2 , 3 μg/ml calpain I, 20 mM Tris-Cl, 5mM mercaptoethanol, and 150 mM NaCl at pH 7.5. Aliquots were withdrawn at various times and added to one-third volume of 3X's SDS-PAGE buffer (150mM Tris-Po 4 , 6%, SDS, 30% glycerol, 3.75mM EDTA, 3% mercaptoethanol, pH 6.8). The samples were heated in 90"C water bath for 3-10 minutes, and subjected to SDS-PAGE on 3 to 10% gradient gels. The gels were stained with Coomassie blue and restained with 7% acetic acid. See Fig. 1, Seubert et al. Synapse 1:20-24 (1987), which is incorporated herein by reference. Peptide bands of approximately 150,000 Daltons were found to increase as spectrin disappeared. The peptides of these bands were termed BDP's. The apparent stability of the BDP's towards further degradation suggested that antibodies directed against spectrin may recognize the BDP's in biological samples, such as tissues, fluids, etc. Antibodies to brain spectrin, which are also reactive with BDPl and BDP2, were raised in rabbits using well known procedures (See, for example, Hum, B.A.L. and Chantler, S.M. Meth. Enz. 70:104-135 (1980) which is incorporated herein by reference) . The anti-brain spectrin antibodies were purified from serum by brain spectrin-sepharose affinity chromatography using methods well-known in the art. Purified brain spectrin was incubated as described for Figure IB. After SDS-PAGE, the proteins were electrophoretically transferred to a nitro cellulose membrane using an Immuno-Blot Assay Kit (Bio-Rad, Richmond, CA) according to the manufacturer's recommendations for the transfer of high molecular weight proteins. As shown in Figure IB, in the presence of Calpain the degradation of spectrin produces BDP's, primarily BDPl and BDP2. The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other alternative procedures known to those skilled in the art may be alternatively employed. EXAMPLE I BDPl and BDP2 RESULTING FROM BRAIN LESIONS Injury in the mammalian Central Nervous System (CNS) results in both the degeneration of damaged neurons and growth responses of undamaged neuronal elements. A well- documented paradigm for investigating the mechanisms underlying these processes involves lesioning the entorhinal cortex, resulting in the production of a well- defined dendritic zone in the dentate gyrus deprived of the majority of its axonal inputs. The anatomical consequences of denervation include dendritic atrophy, glial hypertrophy and atrophy, and a growth response in undamaged axons. In addition, aberrations in cytoplasmic calcium levels occur; see, for example, Baudry, et al., J. Neurosci 3:252-259 (1983) , which is incorporated herein by reference. Stereotaxically placed unilateral electrolytic lesions of the entorhinal cortex were made in Sprague-Dawley rats. Animals were sacrificed after postoperative survival times of 0.2, 0.5, 1, 2, 4, 7, 14 and 27 days. Immediately after sacrificing the animals by decapitation, brains were rapidly dissected in ice-cold homogenization buffer consisting of 0.32 M sucrose, lOmM Tris, 2mM EDTA, ImM ethylene glycol-bis B-amino-ethylether (EGTA) N,N,N » ,N'-tetraacetic acid, 100 μM leupetin, 1 μg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), pH 7.4 Each hippocampus was dissected free and cuts were made with a scalpel blade to isolate the dentate gyrus. With the hippocampus resting on the alvear surface, one cut was made longitudinally along the hippocampal fissure to separate the subiculum and another longitudinal cut removed most of the CA3 field. A third cut was then made in CAl, parallel to the fissure, to remove the majority of CAl. The remaining tissue (10-20 mg) served as the dentate gyrus sample. The dissected dentate gyrus was homogenized in 500 μl of dissection buffer. An aliquot was added to one-third volume of 3X's SDS-PAGE sample buffer (consisting of 150mM Tris-Po 4 , 5%, SDS, 30% glycerol, 3.75mM EDTA, 3% mercaptoethanol, pH 6.8) and placed in a boiling water bath for three minutes. The protein concentration of the homogenate was determined by the method of Bradford, Anal. Biochem. 72:248-254 (1976), which is incorporated herein by reference, and the concentration of proteins in sample buffer adjusted to 0.33 mg/ l. Ten μg of each sample was subjected to SDS polyacrylamide gel electrophoresis on 3- 10% gradient gels until a bromophenol marker dye reached the front of the gel. The proteins were then transferred to nitrocellulose membrane using a transblot apparatus (BioRad, Richmond, CA) according to the manufacturer's instructions for high molecular weight proteins. Antibodies were produced by the following method: For each rabbit approximately 200 μg of purified brain spectrin was excised from SDS-polyacryla ide gels (after electrophoretic separation) and emulsified with Freund's complete adjuvant. Multiple subder al injections were made and the procedure repeated again after 2 to 4 weeks, using Freund's Incomplete Adjuvant. After an additional two weeks subcutaneous injections of an emulsion containing approximately 100 μg of spectrin were made. This procedure was repeated approximately one month later. Ten days following this series of injections, approximately 20 ml of blood was drawn from the rabbit and the serum collected after allowing the blood to clot overnight at 4°C. Antibodies to brain spectrin were isolated from the serum by adsorption to brain spectrin which has been coupled to 6-amino hexanoic acid activated sepharose 4B (Si»gma Chemical Co., St. Louis, MO). The specifically bound antibodies were then eluted in 0.2 M glycine, pH 2.8. The affinity purified antibodies were then equilibrated to pH 7.4 and frozen ' until use. The procedures for blocking, primary and secondary antibody incubations and color development were as recommended by BioRad (Bio-Rad Immuno- Blot TM Assay Ki•t: Instruction Manual) using anti•-rabbi•t IgG (Bio-Rad, Richmond, CA) conjugated to alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazoliu substrate system of detection, according to the manufacturer's recommendations. Affinity purified anti- spectrin antibody was diluted 1/750 (in a volume of 50 ml) and incubated overnight with the blot during the primary antibody step. Quantitation of the immunoreactive species was made using a soft laser scanning densito eter (Model #SLR504-XL BioMed Instruments, Fullerton, CA) . An integrator 4270 Varion, Sunnyvale, CA) summed the amount of reaction product in each band and expressed them as a % of the total in that sample. The anti-spectrin reactive species present in the dentate gyrus two days after a unilateral entorhinal lesion ar shown in Figure 1A. The homogenates of the ipsilateral dentate gyrus exhibited a marked increase in the amount of two peptides, termed BDPl and BDP2, with apparent M r S of about 155,000 and 150,000 Daltons respectively. The time course of the changes in BDPl and BDP2 in the dentate gyrus following a unilateral entorhinal cortex lesion is shown in Figure 2. BDP's are usually below the limit of detection in samples from unoperated animals. A significant elevation of BDP's in the ipsilateral sample is evident as early as four hours post-lesion. The increase is maximal two days after the lesion, where the BDP's represent 25% of the total immunoreactivity. Two and even four weeks after the lesion, the amounts of BDP's were still significantly increased; the contralateral dentate gyri at two and four weeks showed no detectable BDP's and average BDP2 levels were less than 0.1%. Small increases in the amounts of BDP's were observed in the contralateral region during the first week following the lesion as compared to non-operated animals. These results indicate that removal of the main input to the dentate gyrus is followed by a rapid and long- lasting increased degradation of the cytoskeletal protein brain spectrin. EXAMPLE II ASSAY FOR BDP IN THE BRINDLED MOUSE, AN HEREDITARY DEGENERATIVE CONDITION Samples from the hippocampus, cortex and striatum of 12 day old mouse pups were processed as described in Example I. The experimental groups were control mice. Brindled mice, and Brindled mice receiving supplemental copper, a treatment which prevents the premature death which otherwise occurs. Brindled mice are characterized in that they have a copper deficit which, untreated causes normal degradation. As can be seen in Figure 3 spectrin BDP's are elevated in the pathological condition and this elevation is blocked by the copper supplement. EXAMPLE III ASSAY FOR BDP AFTER EXPOSURE TO THE INDUSTRIAL TOXIN TRIMETHYLTIN (TMT) Rats were injected (i.p.) with 10 mg TMT/kg body weight. At various times post treatment the dentate gyrus and CAl regions of the hippocampus were removed and analyzed for BDP's as described in Example I. Massive increases in BDP's were noted in regions most at risk to this toxin (See Balabin et al., Neurosa 26:337-361 (1988), which is incorporated herein by reference, as depicted in Figure 4. EXAMPLE IV ASSAY FOR BDP FOLLOWING ISCHEMIA Carotid arteries were clamped for 10 minutes to interrupt the principle blood flow to the cortex in Mongolian gerbils. Samples of the CAl hippocampal region taken either 4 or 24 hours after ischemia showed elevated BDP's compared to control animals, as shown in Figure 5. The blood supply to the cerebellum was not interrupted and this structure shows no such increase. Analysis of BDP levels was as described in Example I. Spectrin is found in a variety of other tissues. For example, similar BDP's of spectrin have been observed in blood platelets (Fox et al.. Blood 69:537-545 (1987)) and intestinal brush border cells (Glenney et al., PNAS 7JJ.:4002-4005 (1982)). The analysis described above can be extended to other tissues and fluids. The following tissue taken from rats have been examined using the methods described above, and found to exhibit spectrin and BDP's: submandibular gland, brush border, testes, thymus, skeletal muscular, heart muscle, lung, liver, spleen, adrenal gland, kidney, brain. Additionally humans, gerbils and mice have also been determined to contain spectrin and BDP's, suggesting that both are common to animals. Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the ' invention is limited only by the following claims.

Claims: WE CLAIM: 1. A method for detecting cytoskeleton breakdown in cells comprising the steps of: a. exposing a sample of spectrin-containing cells to an electrical gradient so as to separate the components of said sample in such a way that BDP is separated from spectrin; b. contacting said separated components with an antibody that recognizes BDP; c. determining the presence of antibody binding to that portion of the separated components which would include BDP, wherein the presence of said binding indicates the presence of BDP indicative of cytoskeleton degeneration. 2. The method of claim 1 wherein said sample is cerebro-spinal fluid. 3. The method of claim 1 wherein said sample is serum. 4. The method of claim 1 wherein said cytoskeleton breakdown is of cells of the neurons system.

Inventors: LYNCH GARY S, SEUBERT PETER A, LYNCH, GARY, S., SEUBERT, PETER, A.
Applicants: UNIV CALIFORNIA, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Application year: 1989
Main classification: G01N33
IPC codes: A61B10, C07K1, C07K14, C07K16, C12Q1, G01N33