WO 92/0193:~ 2 0 8 8 o ~ ~ PC~/US91/1)491~) .T. NE~ROSIS DEll~lqON q~E~I ASSAYS EOR SPECLRIN AND ~REAI
~ackaround 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 cytoskeleton breakdown products to determine cell necrosis.
This invention was made with government support under AFOSR Contract No. ~6-0099 (P.I.: Lynch), NIH Grants Nos.
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 o~ a protein either identical to or closely related to brain spectrin (also known as fodrin). Spectrin binds F-actin, and together they are generally associated with the inner face of the cell membrane, wh~re they form a `~ ~ilamentous meshwork.
Brain and many other tissues have been known for some time to express calcium-stimulated proteolytic activity.
Studies of degradation in peripheral nerves have indicated that a calcium activated neutral protease, calpain, is critically involved in the degradation of neurofilament proteins following denervation or injury. Two forms of this protease have been identified in brain and other tissues. The two forms are differentiated by their threshold for activation by calcium: calpain I requires micromolar calcium while calpain II is activated by concentrations of calcium between O.l and O.S mM. The two forms are differentially distributed in the brain. While calpain II is mainly localized in the cytoplasmic ~raction ~ -' ~
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~ -2-of brain cells, the highest activity of calpain I is found in small processes. While the two forms of calpain differ in these and other ways, the term "Calpain" shall be used herein to refer to calcium activated neutral proteases generally, including both forms of calpain.
A variety of cellular insults (e.g., toxins, anoxia, etc.) and disease states (e.g., Alzheimer's, Parkinson's, HIV-induced neuropathy, 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 reliable methods to detect degenerative events as soon as possible, preferably before the onset of pathological symptoms. Preferably such methods also have high sensitivity, wide ranging applicability and ease of administration.
Summary of the Invention Briefly, the present invention provides a method of detecting cellular death or degradation in a subject, such as a mammal, comprising analyzing a biological sample from the sub~ect for the presence of spectrin breakdown products and comparing the quantity of spectrin brŰakdown products to the quantity of spectrin breakdown products in a normal subject, wherein an increased level of spectrin breakdown products indicates cellular death or degradation in the sub;ect. In many cases, the quantity of spectrin breakdown products in the normal subject is substantially undetectable. The biological sample can be any sample from an organism, especially cerebrospinal fluid or a component of blood. The cellular death or degradation detected can, for example, be due to a non-pathological cellular insult, such as a trauma, ischemia, lesions, or exposure to toxins;
or may be due to a pathology, including those of the nervous system, such as Alzheimer's disease, Parkinson's disease and muscular dystrophy. Biological samples for the detection of cellular death or degradation in the nervous system can include neural tissue or cerebrospinal fluid.
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WO 92/0193: PCI`/US91/04910 3 20880~
The step of analyzing the biological sample for the presence of spectrin breakdown products can comprise, for example, contacting a spectrin breakdown product in the sample with a detectably labeled antibody, and can also include the steps of exposing the sample to an electrical gradient so as to separate the components in such a way that spectrin breakdown products are separated from spectrin, contacting the separated components with a detectably labeled antibody that binds to a spectrin breakdown product, and determining the presence of antibody binding, wherein the presence of the antibody binding indicates the presence of spectrin breakdown products. The step of analyzing the biological sample can also comprise the step of staining a separated product with a stain which visualizes the product, and determining the presence of stain binding, wherein the presence of the stain indicates the presence of spectrin breakdown products.
In another embodiment of the invention, a method of detecting cellular death or pathology in a sample from a subject, such as a mammal, is provided comprising obtaining a biological sample from the subject, analyzing the biological sample for the presence of spectrin breakdown products, determining a basal level o~ spectrin breakdown products, and comparing the determined level to the basal -level, wherein a higher level than the basal level indicates cellular death or degradation. The basal level of this method is usually the basal level of spectrin breakdown products in a normal subject, and in many instances can be assumed to be zero. The sample can be any biological sample from the subject, including cerebrospinal fluid, a tissue sample, or blood or any component of blood.
In still another embodiment, there is provided a method of detecting cellular death or degradation in a subject, comprising obtaining a biological sample from the subject; determining the total amount of spectrin, including intact spectrin and spectrin breakdown products, in the sample; determining a basal quantity of the total , :
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amount of spectrin; and comparing the determined quantity of the total amount of spectrin to the basal quPntity of the total amount of spectrin; wherein a quantity of the total amount of spectrin in the sample greater than the basal quantity indicates cellular death or degradation.
The total amount of spectrin can be measured as total -~
spectrin immunoreactivity through such means as an ELISA
assay or a Western Blot assay. The sample can be any biological sample from the subject, including cerebrospinal ~luid, a tissue sample, or blood or any component of blood.
Another aspect of the present invention provides a method of enriching a biological sample for spectrin breakdown products relative to intact spectrin in a biological sample, comprising precipitating intact spectrin -in said sample, leaving spectrin breakdown products in solution in said sample by altering conditions which di$ferentially affect the solubility of the intact spec~rin molecule and the spectrin breakdown products; and collecting the precipitated or soluble spectrin breakdown products. The step of precipitating intact spectrin or spectr~n breakdown products can comprise altering the pH or ionic strength of the biological sample solution.
Further ob~ects, ~eatures and other advantages of the present invention will become apparent from the ensuing detailed description, considered together with the appended figures.
Brief DescriDtion of the Figures 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 ths alpha and beta spectrin subunits with apparent Mrs of about 240 and 230 kilodaltons ("kDn), respectively, and two additional immunoreactive peptides (BDR1 and BDP2) with apparent Mrs of about 155 and 150 kD, respectively.
B. Purified brain spectrin incubated under the following conditions: Lane 1: no additions; Lane 2:
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W092/0193~ PCT/US91/04910 ~ 0 lmM CaCl2, 1.8 ~g/ml calpain I, 10 minutes: Lane 3: lmM
CaCl2, 3 ~g/ml ca}pain I, 30 minutes; Lane 4: lO~g of dentate gyrus protein homogenate two days post-lesion; Lane 5: lmM CaCl2, 13 ~g/ml calpain, 1.25 ~g/ml calmodulin, 3 minutes; Lane 6: lmM CaC12, 13 ~g/ml calpain, 1.25 ~g/m calmodulin, 30 minutes.
Figure 2. BDP1 (filled circles) and BDP2 (open circles) levels are expressed as a percentage of the total spectrin immunoreactivity, as determined by scanning reflective densitometry.
Figure 3. Levels o~ BDP's in regions of the brain of control and Brindled mice, showing the effect of treatment with copper.
Figure 4. Levels of BDP's in the dent~te gyrus and the CA1 region of the hippocampus of rats receiving trimethyltin.
Figure 5. Levels of BDP's in the dentate gyrus and the CAl region of the hippocampus from a gerbil following ischemia.
Figure 6. Standard curve showing absor~ance at 405 nm for various levels of spectrin in an ELISA assay.
Figure 7. LeSt (rostral to caudal, 1-4) and right (5-8) hippocampi of a control animal and the left (9-12) and right (13-16) hippocampi from' a kainate in~ected animal.
Top panel, western blot assay: bottom panel, ELISA assay.
Figure 8. Spectrin immunoreactivity of CSF samples, as measured by ELISA, from two patients of group 1 (unruptured aneurisms) and four patients of group 2 (ruptured aneurisms).
Figure 9. Spectrin immunoreactivity of CSF samples, as measured by ELISA for: ANEU (unruptured aneurism patients (n = 2)); SAH (subarachnoid hemorrhage patients (n = 12));
- AD (Alzheimer's Disease patients (n = 3)); STRORE (stroke patient (n = l)); IVH (intravascular hemorrhage patients (n 3S = 3)); and PICK'S tPick's Disease Patient (n = l)).
Detailed Description of the Preferred Em~odiment The present invention relates to sensitive and : . ' - ~ .. : . '- ; - - . . . . . -. - . , . . :
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. , . ~ : .. ~ . , -WO92/0193~ PCT/US91/0~910 ~ 6-efficient methods for the early detection of cellular death and degradation. The methods of the invention detect cellular death and/or degradation through an assay for spectrin or the breakdown products of spectrin.
The activation of Calpain leads to the proteolysis of many proteins including spectrin. Accordingly, Calpain is believed to cause production of spectrin breakdown products ~ -("BDP's") from spectrin in dead and degenerating cells.
Thus, the detection of BDP's is believed to advantageously serve as an indicator of the activation of Calpain.
Spectrin ~DP's are unusually stable polypeptides.
BDP's can be detected in vivo for up to as long as two months or more after release from the cytoskeleton. Thus, ;
BDP's can advantageously remain to serve as indicators of cellular death or degradation during this period of stability.
There is evidence that the activation of Calpain is an early event in cell death. This is in contrast to other known proteases which are believed to be activated only in the late stages of cell death. The activation of Calpain is believed to often occur before the onset o~ pathological symptoms associated with cell death. Thus, the detection of BDP's is believed to advantageously be useful as a method for the early detection of cell death, potentially prior to the onset o~ pathological-symptoms.
The present invention advantageously provides a method for the detection of cellular pathology by means of an immunoassay to determine the presence of BDP's of spectrin.
Two major BDP's are known, BDPl and BDP2. 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's and spectrin are separated. The separated components can then ~e visualized, as by staining with a stain such as Coomassie Blue. Alternatively, antibodies reactive with 8DP's can then be contacted with the separated sample, and antibody binding to the portion of the sample containing BDP's - - . ., ~ . . .
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determined. The determined amount of BDP's can then be compared with a basal level of BDP's in similar samples from normal patients. An increase in the level of BDP's is indicative of cellular death or degradation. In many cases, the basal level will be a level which is below the detection threshold of the methods herein described. Thus, in these cases, the detection of any immunoreactivity is an indication of cellular death or degradation.
The invention provides an additional method for the detection of cellular pathology to determine the presence of intact spectrin itself or spectrin immunoreactivity. In one embodiment of this aspect of the invention, an enzyme-linked immunosorbent assay (ELISA) for spectrin immuno-reactivity in biological samples, such as tissue extracts, cerebro-spinal fluid (CSF), or ~lood serum is provided.
One particular application of the method is the detection of spectrin or spectrin immunoreactivity in the CSF as an indication of neurodegenerative conditions such as subarachnoid hemorrhage, Alzheimer's Disease, HIV-induced neuropathy and/or stroke.
Iden~ šation Or Spectrin and BDP's BDP's were identified from a sample of purified spectrin, by exposing the sample to Calpain for varying lengths o~ time and exposing the treated samples to SDS-PAGE. Brain spectrin was purified to greater than 90%
purity by the method of Davis and Bennett, J. Biol. Che~.
258:7757-7766 (1983), which is incorporated hersin by reference. Calpain was purified to a similar level of purity from rat erythrocyte cytosol according to the method of Seubert, et al., Syna~se 1:20-24 (1987), which is incorporated herein by reference.
Spectrin at a concentration of 75 ~g/ml was incubated at 30-C with 100 ~M CaCl2, 3 ~gJml calpain I, 20mM Tris-Cl, 5mM ~-mercaptoethanol and lS0 mM NaCl at pH 7.5. Aliquots were withdrawn at 10 minutes and at 30 minutes. The aliquots were added to one-third volume of 3X SDS-PAGE
- : , . . ~ : , W O 9Z/01935 ~ PC-r/US91/04910 buffer (150mM Tris-Po4, 6%, SDS, 30% glycerol, 3.75mM EDTA, 3% ~-mercaptoethanol, pH 6.8). The samples were heated in a go 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 destained with 7% acetic acid. The foregoing method is described in Seubert, et al., SYna~se 1:20-24 (1987~, which is incorporated herein by reference.
The amount of peptide in two peptide bands of approximately lS0 kilodaltons (kD~ and 155 XD respectively (referred to ~ointly as the "150 kD bands") were found to increase with exposure time to Calpain. Correspondingly, the amount of peptide in the two peptide ~ands representing the and subunits of spectrin at 240 kD and 230 XD respectively, decreased with exposure time to Calpain. The peptides of the two 150 kD bands were termed BDPl and 8DP2.
Thus, Example I shows that spectrin produces BDP's in the presence of Calpain I. Example I also shows that staining after SDS-PAGE can be used to detect spectrin or BDP' 8 in samples having the relatively high levels necQssary for polypeptide b~nds to be visible to the naked eye upon exposure to stains such as Coomassie Blue. The method of Example I is also well suited to relatively pure samples where the bands corresponding to the BDP's and intact spectrin can be easily identified. Samples having suitably high levels and purity of spectrin and/or BDP's are, for example, obtained from homogenized neural tissues after purification. See Davis and Bennett, supra.
A more sensitive method of detecting spectrin or BDP's, even in complex mixtures of polypeptides can advantageously be obtained by exposing the separated sample to antibodies reactive with spectrin or BDP's. One assay which is suitable for this purpose has come to be known as a Western blot assay.
BDP's exhibit apparent stability towards further degradation, suggesting that antibodies directed against spectrin can recognize the BDP's in biological samples, such as tissues, fluids, etc. Both BDP's and intazt '' ' s ~, , . , . -WO92/0193~ 2 0 8 8 ~ ~ PCT/US91/04910 _9_ spectrin can be recognized by antibodies directed against spectrin. Accordingly, anti-spectrin antibodies will detect both intact spectrin and BDP's when used in a Western blot assay. The following example shows such a Western blot assay, using the gel obtained from Example I
and anti-spectrin antibodies to detect the presence of both intact spectrin and BDP's.
Western Blot Assay fo~ Smectrin and B~P's Antibodies to brain spectrin were raised in rabbits using well known procedures (see, for example, Hurn, ~.A.L.
and Chantler, S.M., Meth. Enz. 10:104-135 (1988), which is incorporated herein by reference). The anti-brain spectrin antibodies were purified from serum by brain spectrin-sepharose affinity chromatography. Briefly, antibodies to brain spectrin were isolated from the serum by adsorption to brain spectrin coupled to ~-amino hexanoic acid activated sepharose 4B (Sigma Chemical Co., St. Louis, MO).
The specifically bound antibodies were then eluted in 0.2 M
glycine, p8 2.8. These affinity purified antibodies were then equilibrated to pH 7.4 and frozen until use. The antibodies were found to be reactive against BDPl and BDP2 as well as to intact spectrin. Thus, the 150 XD bands which appeared upon exposure of spectrin to Calpain comprised polypeptides which were cross reactive with spectrin.
Purified brain spectrin was incubated as described in Example I. After SDS-PAGE, the proteins were electrophoretically transferred to a nitro-cellulose membrane using a Trans-Blotter (Bio-Rad, Richmond, CA) according to the manufacturer's recommendations for the transfer of high molecular weight proteins. The nitro-cellulose sheets were incubated with anti-spectrin antibodies and the bound antibodies detected using an Immuno-~lot assay kit (also available from Bio-Rad) according to the manufacturer's directions. Briefly, anti-rabbit IgG (Bio-Rad, Richmond, CA) conjugated to alkaline ... . . . ..
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: , W09~/~193~ PCT/US91/~4910 phosphatase was used in a 5-bromo-4-chloro-3-indolyl -phosphate/nitro blue tetrazolium substrate system of ' detection according to the manufacturer's recommendations. --Affinity purified anti-spectrin antibody was diluted l/750 (in a volume of 50 ml) and incubated overnight with the blot during the primary antibody step. The immuno-reactivity of the bands is shown in Figure lB, }anes 1-3.
Lane l shows spectrin without exposure to Calpain; Lane 2 shows spectrin after treatment with lmM CaCl2 and l.8 ~g/ml calpain I for lO minutes; and Lane 3 shows spectrin after treatment with lmM CaCl2 and 3 ~g/ml calpain I for 30 minutes. It can be seen that in the presence of Calpain, the degradation of spectrin produces BDP's, primarily BDPl and BDP2.
The findings of Example II and other similar experiments led us to the discovery that the increased Calpain activity following denervation or injury results in significant levels of BDP's being generated in injured tissues.
In accordance with the foregoing discoverv, in one aspect of the present invention, there is provided a method for detecting cellular pathology comprising the steps of extracting a sample from a subject mammal and analyzing the sample for the presence of spectrin BDP's. In order to obtain greater sensitivity, the analyzing step can involve an immunoassay using antibodies which recognize spectrin or stable breakdown products of spectrin.
In this aspect of the invention, the amount of BDP's is determined and this amount is compared to a basal level of BDP: any increase of BDP's being indicative of cellular death or degradation. Normally, the basal level is the level of BDP's from healthy cells. The basal level can be taken from a corresponding sample in a healthy subject -mammal. Alternatively, the basal level can be obtained ~5 from a sample from the same subject at a point in time prior to the insult. Thus, a series of samples can be taken from a single subject over time and analyzed for the - . . . : : . .
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, WO92/0193~ 2 0 8 ~ PCT/US91/04910 presence of BDP's, thereby advantageously providing an indication of the course of cellular death or degradation in the subject. In many samples taken from healthy subjects, the level of BDP's is below the detection threshold of the assays described herein. Accordingly, the basal level against which the datected amount of BDP's are compared is often zero. Therefore, in many samples, the detection of any BDP's is indicative of cellular death or degradation.
The following example illustrates the establishment of a basal level for human CSF.
Establishment of a Basal LeYel of BDP's in Human CSF
CSF sa~ples are obtained from a healthy human subject.
All CSF samples are concentrated by ultrafiltration. Two marker samples are also obtained to identify bands corresponding to intact spectrin and BDP's in completed gels. The first marker sample is a sample of purified spectrin without exposure to Calpain as in Lane l of Example I to show the position of intact spectrin. The other marker sample is of purified spectrin after exposure to Calpain as in Lane 3 of Example I to show the position of BDP's. Protein concentration of the samples and marker samples was determined by the method of Bradford, Anal.
Biochem. 72:248-254 (1976), the disclosure of which is hereby incorporated by reference. Ten yg, of each sample and marker sample is subjected to SDS polyacrylamide gel electrophoresis on a 3-10% gradient gel until a bromophenol marker dye reaches the front of the gel. The proteins are then transferred to nitrocellulose membrane using a transblot apparatus (Bio-Rad, Richmond, CA) according to the manufacturer's instructions for high molecular weight proteins. Antibodies are produced as in Example II and used to detect spectrin immunoreactivity on the nitrocellulose membrane. Spectrin immunoreactivity is found for each of the CSF samples at positions ; corresponding to the position of the intact spectrin as - .. - -: ~. - - . : - . ~ . . : . , , - - - -, ~ - - -~ : - ,., . : : ~ ... . -. .
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WO92/0193~ ~ PCT/US~1/04910 determined by the marker sample. However, no detectable spectrin immunoreactivity is found in the CSF samples at a position corresponding to the position of the BDP's determined by the marker sample. Thus, a basal level of BDP's for CSF of this human subject is determined to be zero.
The methods described herein can be used to measure BDP's in a variety of tissues and fluids because spectrin is found in a variety of tissues. For example, 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 79:4002-4005 (1982)). The following tissue taken from rats have been examined by the present inventors and others using the methods described herein 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 been determined by the present inventors and others to contain spectrin and BDP's, suggesting that spectrin and BDP's are common to all mammals.
Injury in the mammalian Central Nervous System (CNS) results in both the degeneration o~ 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.
Thus, to show the ability of a preferred method of the present invention to detect the well-defined dendritic zone in the dentate gyrus after lesioning the entorhinal cortex of rats, Examples IV through VIII are provided, showing the detection of cellular death or degradation in the expected tissues.
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Preparation of Dentate Gyrus SamDle 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, 0.4, l, 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, lOm~
Tris, 2mM EDTA, lmM ethylene glycol bis (~-amino-ethylester) N,N,N',N'-tetraacetic acid (EGTA), lO0 ~M
leupeptin, l ~g/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 ma~ority of CAl. The remaining tissue (10-20 mg) served as the dentate gyrus sample which was used as a tissue sample as in Example V. Contralateral and ipsilateral samples of the dentate gyrus were obtained.
Pre~ara~.ion o~ sue ~eles ~Q~I~Lectrophoresis The contralateral dentate gyrus tissue sample and the ipsilateral dentate gyrus tissue sample were each homogenized in 500 ~l of dissection buffer. An aliquot of each was added to one-third volume of 3X SD5-PAGE sample 30 buffer (consisting of 150mM Tris-PO4, 6% SDS, 30% glycerol, 3.75mM EDTA, 3% ~-mercaptoethanol, pH 6.8) and placed in a -90-C water bath for three minutes. The protein concentration of each homogenate was determined by the method of Bradford! supra. The concentration of proteins in each homogenate sample was then ad~usted to 0.33 mg/ml with a~t~El c~pl~ h~r. EXAMPLŠ VI
SeDaration o~ SamDle Proteins and Transfer to Hembranes .. .-, ,. ~ ~ , . . , . . ~ ............................. . . . .
W092/01935 ~S ~ -14- PCT/U591/049~0 Ten ~g of protein from each of the samples from Example V, were subjected to SDS polyacrylamide gel electrophoresis on a 3-10% gradient gel until a bromophenol marker dye reached the front of the gel. The proteins were then transferred to nitrocellulose membrane using a transblot apparatus (Bio-Rad, Richmond, CA) according to the manufacturer's instructions for high molecular weight proteins.
Pre~aration of Anti-Spectrin Antibodies Antibodies were produczd by the following method: For each rabbit, approximately 200 ~g of purified brain spectrin was excised from SDS-polyacrylamide gels (after electrophoretic separation) and emulsified with Freund's complete adjuvant. Multiple subdermal injections were made and the procedure repeated again after two to four weeks, using Freund's incomplete adjuvant. After an additional " two weeks, subcutaneous injections of an emulsion containing approximately lOOyg of spectrin were made. This procedure was repeated approximately one month later. Ten days following this series of in~ections, approximately 20 ml o~ blood was drawn from each rabbit and the serum collected after allowing the blood to clot overnight at 4-C.
Antibodies to brain spectrin were then affinity purified by adsorption to brain spectrin coupled to ~-amino hexanoic acid activated sepharose 4B, as described in Example II. The affinity purified antibodies were then equilibrated to pH 7.4 and frozen until use.
Determination of BDP's Resulting ~rom Brain Lesions To determine the amount of spectrin immunoreactivity on the membrane of Example VI, the membrane was exposed to the antibody of Example VII as part of a Western Blot Assay. Procedures ~or blocking, primary and secondary antibody incubations and color development were as described in Example II. Quantitation of the WO92/0193~ PCT/US91/0491n 2o88~5~o immunoreactive species was made using a soft laser scanning densitometer (Model #SLR504-XL, BioMed Instruments, Fullerton, CA). An integrator (Model 4270, Varian, Sunnyvale, CA) summed the amount of reaction product in each band and expressed them as a percentage of the total in that sample.
The anti-spectrin reactive species present in the contralateral (lane 1) and ipsilateral dentate gyrus (lane 2) two days after a unilateral entorhinal lesion are shown in Figure lA. The homogenates of the ipsilateral dentate gyrus exhibited a marked increase in the amount of two peptides, termed ~DPl and BDP2, with apparent Mrs of about 155,000 and 150,000 Daltons, respectively. ~ -The procedures of Examples IV through VIII were 15 repeated, allowing various lengths of time to elapse -between the lesion and sacrifice of the animals of Example IV. The time course of the changes in BDP1 and BDP2 in the dentate gyrus following a unilateral entorhinal cortex lesion i5 shown in Figure 2. BDP's are usually below the 20 limit o~ detection in samples from unoperated animals. A -8~ gni~icant 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 ~DP's represent 25% O~ the total immunoreactivity. Two and even ~our 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% of total spectrin immunoreactivity. 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.
The 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. It is known that aberrations in cytoplasmic calcium levels occur in the dendritic zone of the dentate :' ' :
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-3~ PCT/US91/04910 gyrus after lesioning. See, for example, Baudry, et al., J.
Neurosci., 3:252-259 (1983), the disclosure of which is hereby incorporated by reference. Thus, we believe that the elevated levels of BDP's in these tissues is the result of s the activation of Calpain in these tissues by these aberrant levels of calcium. The results of Example VIII, therefore, confirm the ability of the pres2nt invention to detect the well-defined dendritic zone in the dentate gyrus after lesioning of the entorhinal cortex in rats.
lOExamples IX through XI are provided in order to show that the methods utilized in Examples V through VIII have widespread utility in detecting cellular death or degradation. These examples show the detection of cellular death or degradation from a varisty of causes and in a variety of cellular tissues through ~ethods of the present invention. As such, these examples are intended to illustrate, not to limit the invention. While the procedures described herein, such as those of Examples V
through VIII, are typical of those that might be used, other alternative procedures known to those skilled in the art can be alternatively employed.
~ssay for BDP in the Brindled Mouse.
A Heredita ~ De~eneratiVe šo-nditiQn 25Samples from the hippocampus, cortex and striatum of 12 day mouse pups were processed as described for the dentate gyrus samples in Examples V-VIII. 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 ~, spectrin BDP's are elevated in the pathological condition and this elevation is blocked by the copper supplement.
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Assav for BDP after ExDosure to ~he Industrial Toxin Trimethyltin rTMT) Three rats were injected intraparitoneally (i.p.) with lO mg TMT/kg body weight. A fourth rat was injected i.p.
with saline alone to serve as control. At 3, 7 and 14 days post-treatment, the dentate gyrus and CAl regions of the hippocampus of the three test rats were removed and analyzed for BDP's, as described in Examples V-VII.
Massive increases in BDP's were noted in both the dentate gyrus and CAl regions, a~ depicted in Figure 4. These regions of the brain have been identified as the regions most at risk to TMT toxin (see Balabin, et al., Neurosa 26:337-361 (1988), which is incorporated herein by reference).
Assay for BDP ~ollowing ~schemia Carotid arteries were clamped for lO minutes to ;interrupt the principal blood flow to the cortex in each oS
two groups of eight Mongolian gerbils. Two control groups of gerbils were also analyzed. Samples of the CAl hippocampal region and the cerebellum were taken at 4 hours after ischemia from one group of control gerbils and one group o~ test gerbils. Samples were also taken at 24 hours after ische~ia ~rom the second control and test groups of gerbils. The test gerbils showed elevated BDP's in the CAl region compared to control animals, as shown in Figure 5.
The blood supply to the cerebellum was not interrupted and this structure showed no such increase. Analysis of BDP
levels was as described in Example V - VIII.
Thus, the foregoing examples show that the methods of the present invention can advantageously be used to detect cellular death or degradation from a variety of causes in a -variety of samples.
35The present invention advantageously provides an ` additional method for the detection of cellular pathology without the necessity of separating the sample into BDP and : ' . .
WO92/0193~ PCT/~1S91/04910 intact spectrin. This additional method is by means of an immunoassay to determine the presence of intact spectrin itself or spectrin immunoreactivity regardless of source.
Therefore, in this embodiment of the invention, total spectrin immunoreactivity, including immunoreactivity to spectrin and to BDP's, can be measured. In one embodiment of this aspect of the invention, an enzyme-linked immunosorbent assay (ELISA) for spectrin immunoreactivity in biological samples, such as tissue extracts, cerebro-spinal fluid (CSF), or blood serum is provided.
In preparation for the competltive E~ISA assay of a preferred embodiment, a spectrin sample is immobilized to polystyrene microliter plates. We have found that spectrin desorbs from conventional activated polystyrene plates after immobilization, resulting in an unexpected bell-shaped relationship between the amount of antibody bound and the amount of spectrin in the sample which is measured.
While not wishing to be bound by any particular explanation for this unexpected result, it is believed that the desorbed spectrin forms polymers with still immobilized spectrin in the presence of accessory proteins present in the sample. The spectrin polymers are believed to be more accessible to binding of anti-spectrin antibody. In addition, spectrin in the sample is believed to bind to the plate through further polymerization of the spectrin.
In order to prevent the unexpected relationship between the amount of antibody bound and the spectrin in the sample, polystyrene plates can be treated with glutaraldehyde prior to the immobilization of spectrin to the plates. Glutaraldehyde forms covalent bonds to both the polystyrene of the plates and to the spectrin molecules. The use of buffers with conditions, including salinity and pH, unfavorable to the polymerization of spectrin has also been found to prevent the unexpected ~5 re~ult~. ~igh ion concentration has been found to inhibtt formation of spectrin polymers, however, such concentrations also interfere with immunoreactivity.
W092/0193~ PCT/US91/04910 2 018 8 0 5 D . .
Addition of various other agents has also been found to prevent the unexpected results, including EGTA, sucrose and detergents. Thus, in a preferred method of the present invention, buffers with a pH slightly greater than 7.0 in physiological saline with EGTA, sucrose and detergent is used. Chaotropic salts, such as NaBr or KI, can also be used to inhibit formation of polymers.
When an unknown sample is tested, a limiting amount of anti-spectrin antibody is added to each well along with the ;~ ;
sample. Spsctrin in the sample competes for antibody with the spectrin immobilized to the plate. ~hus, the more spectrin in the sample, the less antibody will bind to the spectrin immobilized to the plate. Accordingly, the amount of antibody binding to the spectrin on the plate provides an indication of the amount of spectrin in the sample. The amount of antibody can be detected by a colorimetric reaction as in a standard ELISA procedure, or ca~ be detected in any known manner, such as through a radio-immune assay.
EXAMPL~ XII -ELISA Assay ~or Spectrin A spectrin preparation was prepared from rat brains by the method of Davis and Bennett (J. Biol. Chem.
258:7757-7766, 1983). Antibodies to spectrin were prepared by subjecting the spectrin preparation to SDS-PAGE (see Seubert, et al., Synapse 1:20-34, 1987), excising the region of the gel containing the spectrin, homogenizing the gel and immunizing rabbits with the homogenized gel according to established procedures (see, e.g., Hurn and ~-~
30 Chantler, Methods Enzymol. 70:104-135, 1980).
Microtiter plates having immobilized spectrin were -~
prepared by first treating microtiter plates (unmodified -polystyrene, such as those sold under the trade mark "Corning Easy-Wash") with glutaraldehyde 0.2% in 0.lM
sodium phosphate pH=5.0 for 4 hours at room temperature.
j After removal of glutaraldehyde, 100 ~l of a solution of spectrin (10 ~g/ml) in O.lM sodium phosphate pH=8.0 was ; .. ~ . . . ., .. , , . . ... . ,. . .. .. -. ... - .. . . . .,, . -WO92/0193~ ~ PCT/US91/04910 ~ -20-added to each well and the plates incubated an additional 4 hours at room temperature. The plates were rinsed with O.lM lysine in O.lM sodium phosphate pH=8.0, and lO0 ~l of this lysine solution was added to each well. The plates S were then incubated for 4 hours at room ~emperature.
Lysine serves to react with unreacted glutaraldehyde binding sites to prevent the further binding of spectrin to the plates.
For the measurement of spectrin immunoreactivity of an unknown, the lysine solution in each well was discarded and a sample of the unknown was placed in each well. The volume was then adjusted to 50 ~l with 20mM Tris, 0.8%
NaCl, 0.02~ RCl, 0.5% bovine serum albumin, 0.05% Tween 20, 2mM EGTA, 0.2% sodium azide pH=7.2 ("assay buffer"). To this was added 50 ~l of a 1:50,000 dilution of anti-spectrin antiserum in assay buffer. The plates were mixed and incubated overnight at 4-C. The plates were then rinsed 4 times in lOmM Tris, 0.9% NaCl pH=7.2 ("rinse buffer") and lO0 ~l of biotinylated goat anti-rabbit antiserum (available from Vector Laboratorie~), diluted in assay buffer at the concentration recommended by the manufacturer was added and the plates incubated on a rocking platform at room temperature for 4 hr. The plates were rinsed 4 times with rinse buffer and lO0 ~l of ABC
~alkaline phosphatase) reagent (also available from Vector Laboratories) prepared according to the manufacturer's instructions in assay buffer was added. The plates were incubated for 2 hrs on a rocking platform at room temperature and rinsed 6 times with rinse buffer. Color was developed by adding lO0 ~l of alkaline phosphatase substrate solution (available from Bio-Rad) made according to the manufacturer's directions and incubating for 30 minutes to 4 hours at room temperature.
In parallel with the measurements of the unknown samples, measurements of spectrin immunoreactivity of wells initially containing known concentrations of spectrin were also performed.
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W092/0193~ 2 0 8 ~ ~ S ~ PCT/US91/04910 The absorbance at 405 nm of the wells containing these standard concentrations was read using a plate reader and the standard curve shown in Figure 6 was produced from this data.
5The absorbance at 405 nm of the wells containing unknown samples was also read and the concentration of spectrin determined by comparing the absorbance of the unknown wells to the standard curve. The concentration of spectrin immunoreactivity correlates well with measurements of the same samples which are subjected to the Western Blot assay of Example VIII. The following example demonstrates the correlation between the Western blot and the ELISA
assay in neurodegenerating rats.
15Comparison of Western Assav and ~LISA Assav Adult rats were given intracerebral ventricle injections of 75 ng of kainate, a compound known to cause neurodegeneration within the hippocampus. A second set of rats were given equal volume injections of saline. The rats were allowed to recover for four days. The hippocampi were then removed and divided into four sections, rostral to caudal. Each section was analyzed using both the Western blot assay as in Example II, and using the ELISA
assay of Example XII. Results are shown in Figure 7.
25Figure 7 shoWs the left (rostral to caudal, 1-4) and right (5-8) hippocampi of a control animal and the left (9-12) and right (13-16) hippocampi from a kainate injected animal. The top panel shows the Western Assay and the bottom panel shows the ELISA assay. It can be seen that the increase in the total amount of immunoreactivity measured by the ELISA correlates well with the increase in BDP's measured by the Western Blot assay.
One particular application of the ELISA of the present invention is the detection of spectrin or spectrin immuno-reactivity in the cerebrospinal fluid (CSF) as anindication of neurodegenerative conditions, including subarachnoid hemorrhage, stroke, multiple infarction W092/0193~ 2- PCT/US91/0491~
dementia, HIV-induced neuropathy and Alzheimer's Disease.
Although it is possible to detect small quantities of spectrin in normal CSF concentrated by ultrafiltration: in normal unconcentrated CSF, virtually no spectrin immuno-reactivity is detected using the ELISA assay of the presentinvention. Therefore, the detection of either spectrin or spectrin BDP's in unconcentrated CSF would be indicative of cellular death or degradation within the nervous system Although spectrin is present in isodermal cells lining the ventriculus and in particular in speciSic cells standing between the CSF and the blood, the number of such cells is very small compared to neural cells. The death or - degradation of glial cells, the support cells for neurons, would also be capable of contributing spectrin or BDP's to the CSF. However, after death or degradation of glial cells, the death or degradation of the neural cells such cells support would follow shortly thereafter. Therefore, r the vast majority of spectrin immunoreactivity found in the CSF would be indicative of breakdown of neural cells.
The cerebrospinal fluid of humans can be assayed either directly or after concentration using lyophilization or centrifugal ultrafiltration (using materials such as those sold under the trademarks "Centricon-lO" or "Amicon"). The following example illustrates one typical method of the pre~ent invention for assaying human CSF for the presence of spectrin immunoreactivity.
Assay of SDectrin Immunoreactivity in Human CSF
Cerebrospinal fluid samples were obtained from 2 patients diagnosed as having unruptured aneurysms (group l) and from 4 patients in which the aneurysm has burst producing subarachnoid hemorrhage (group 2). Two ml of each sample was lyophilized, resuspended in lO0 ~l water, and lO
~l of the resulting solution was analyzed for spectrin ~5 immunoreactivity using the ELISA assay of Example XII.
Results are shown in Figure 8. The CSF samples from the subarachnoid hemorrhage group all showed spectrin immuno-.
- . .: . . - - . : ~, : . "
:, . . : : :: -WO92/01935 ~?n8 ~g .~ Pcr/l)s91/0491o reactivity while the unruptured aneurism group had no detectable spectrin immunoreactivity.
Thus, it can be seen from Example XIV that the presence of detectable quantities of spectrin immuno-5 reactivity in the CSF is indicative of cellular death ordegeneration in neural tissue.
In summary, the foregoing examples clearly show that neurodegeneration in vivo dramatically elevates total spectrin immunoreactivity, as measured by the ELISA. To lO demonstrate the widespread applicability of the ELISA assay in detecting neurodegeneration, the following example was perf ormed .
evels of Spectrin Immunoreactivitv in CSF
Total spectrin immunoreactivity was measured, using -the ELISA method of Example XII, in CSF taken from a number of different patients suffering from a variety of ~ -conditions known to be associated with neurodegeneration.
Results ars shown in Figure 9 . The f irst column of Figure 2 0 9, labeled "ANEU" shows the results of two patients who had brain aneurisms detected and surgically corrected before the aneurisms burst, as in Group l of Example XIV. Thus, signif icant neurodegeneration would not be expected in these patients. As can be seen in Figure 9, no spectrin immunoreactivlty was found in CSF taken from these patients . The data conf irms that no spectrin immuno-` - reactivity is detected in CSF in non-neurodegenerating mammals using the ELISA assay of the present invention.
The next column of Figure 9, labeled "SAH", shows spectrin immunoreactivity measurements in CSF of 12 -:
patients with subarachnoid hemorrhage who had had CSF
drains installed. The CSF from all 12 patients shows - spectrin immunoreactivity, indicating that -~
neurodegeneration has occurred.
The third column of Figure 9, labeled "AD" shows spectrin immunoreactivity measurements in CSF from 3 Alzheimer's disease sufferers. All three patients show - .
- -~ .
- .. , . . - , . ..
WO 92/0193~ 24-- PCr/US91/04910 spectrin immunoreactivity in their CSF, indicating neurodegeneration has occurred.
The fourth column of Figure 9 shows spectrin immuno-reactivity in the CSF from one stroke victim. It can be 5 seen that spectrin immunoreactivity is s~uite high in this patient, indicating significant neurodegeneration.
The fifth column of Figure 9, labeled "IVH" shows spectrin immunoreactivity from three premature infants suffering from intraventricular hemorrhage. Results show lO that two out of three of these patients show spectrin immunoreactivity in their CSF, indicating neurodegenerat ion .
The last column of Figure 9 shows spectrin immuno-reactivity in one victim of Pick' s disease . The results 15 show high levels of spectrin immunoreactivity in this patient, indicative of the neurodegeneration which accompanies this disease. Pick's disease is clinically very difficult to distinguish from Alzheimer's disease.
Presently, Pick's can only be readily distinguished from 20 Alzhe~mer's upon autopsy. It can be seen from the present data, that the Pick's sufferer had significantly higher levels of spectrin immunoreactivity than any of the Alzhe~mer' s sufferers. Thus, it is believed that the pre5ent method will provide a diagnostic tool in 25 distinguishing between these two diseases by the generally higher levels of spectrin immunoreactivity found in the CSF
of Pick' s patients.
Thus, it can be seen from the foregoing example that measurements of spectrin immunoreactivity in CSF are useful 3 0 indicators of neurodegeneration from a wide spectrum of clinical causes.
The spectrin immunoreactivity detected in all of the foregoing examples is, of course, due to a large number of different antigenic epitopes. It is believed that after 35 the proteolysis of spectrin to BDP's, additional or occult epitopes are exposed which are not present in intact spectrin. Thus, when performing the ELISA assay using ., . ., .. , ;. ..: . : : . :. . .:
' ' :
WO92/0193~ 208~D~ ~ PCT/US91/n4910 polyclonal antibodies raised against BDP's, BDP's can give a stronger signal than the intact spectrin. In such assays, treating the spectrin in such a way to expose the occult epitopes, can also give a stronger signal than intact spectrin.
The anti-spectrin antibodies used in the Western Blot assays of examples II - XI, were affinity purified using the affinity purification method described in Example II. :
This affinity purification step with intact spectrin, resulted in the purification of antibodies to epitopes of spectrin present and exposed in intact spectrin. However, the raw serum contained at least two other classes of antibodies which react against spectrum. One class of antibodies are to epitopes of spectrin not exposed in the intact tetrameres, but exposed in cleaved spectrin.
Another class of antibodies would be antibodies specific to spectrin-SDS complexes. This class of antibodies is expected because the spectrin used to immunize the rabbits producing the antibodies in Example II was purified from SDS-PAGE, resulting in the formation of these SDS-spectrin complqxes. In order to demonstrate that raw, not affinity puri~ied, anti-~pectrin antibodies raised against denatured rat spectrin react more efficiently with denatured spectrin than with intact spectrin, and that, therefore, occult epitopes exist and that antibodies against the occult epitopes can be u~ed to distinguish native from denatured -spectrin, the following example was performed.
Immunoreactivity of Denatured and Native Smectrin Spectrin was immobilized onto polystyrene plates as in Example XII. Each well was incubated with one of four denaturing treatments for one hour at room temperature and then washed six times in wash buffer (SOmM tris, 150 mM
NaCl pH=7.5)- The four treatments were: control (wash buffer), 1% SDS, l M acetic acid, and l M ~I. Four wells for each treatment were analyzed. The amount of spectrin immunoreactiYity on the plate was dPtermined by incubating . .' ~ ` , .
' : ' . ' ~. ' .~ :. .
' ." '`; , ' , "
~C~ 26-lates with raw serum from immunized rabbits at l:lO,000 dilution in assay buffer (100~1/well) overnight at 4'C, rinsing the plates four times with wash buffer, and detecting bound antibody using the Vector ABC-AP kit as in Example XII. Results are shown in Table 1.
TREATMENT ABSORBANC~ 1405nm) + S.D.
Control 0.997 + 0.115 10 1% SDS 2.540 + 0.281 1 M Acetic Acid 1.110 + 0.087 1 M KI 1.835 ~ 0.117 --- :
It can be seen from the results of Table 1, that raw serum from rabbits immunized with SDS-treated spectrin recognizes denatured spectrin more effectively than the native, control-treated spectrin. Not unexpectedly, the SDS-treated spectrin reacts the most strongly with this serum. Thus, it is clear from these results that denaturing spectrin exposes occult epitopes not present in intact spectrin molecules. It is expected that oth~r denaturing treatments, such as TCA, organic solvents, ethanol and guanidine, will produce similar increas~s in immunoreactivity.
It is also believed that the cleavage of intact spectrin into BDP's expos~s hidden epitopes. In order to demonstrate that cleavage of spectrin in solution exposes hidden epitopes and that antibodies directed against these epitopes can be used to distinguish intact spectrin from cleaved spectrin, the following example was performed.
EXa~PL~ ~; ..
Cleavage of Spectrin to Increase Immunoreactivity Rat brain was homogenized in 10 mM HEPES, lmM EGTA, 1 35 mM DTT pH = 7.2. The homogenate was centrifuged at 12,000 x g for ten minutes and the supernatant split into two fractions. CaCl2 was added to Fraction 2 in order to activate Calpain. The final concentration of Ca++ in Fraction 2 was 50 mM. No CaC12 was added to Fraction 1.
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WO92/0193~ PCTtUS91/04910 -27- Q~
Both fractions were incubated at 37-C for 30 minutes. The immunoreactivity of the samples was taken both before and after this incubation period using the ELISA assay of Example XII. Extensive proteolysis of the second sample, leading to formation of BDP's, was confirmed by Western Blot analysis as in Example II. No proteolysis was detected in any of the other samples. No precipitate was observed in any samples. Results of the ELISA
determination are shown in Table 2.
TABL~ 2 ABSORBANCE AT 405nm F~ACTI0~ t = O min. t = 30 min.
1 (- Ca++) 0.316 + 0.014 0.381 + 0.023 15 2 (+ Ca++) 0.334 + 0.033 0.451 + 0.022 ' :
It can be seen from Table 2 that in Fraction 2 where Calpain is activated that total immunoreactivity increased 20 from an average of 0.381 to an average af 0.4S1, an increase of 18%. In no instance did the immunoreactivity of Fraction 1 exceed the immunoreactivity of Fraction 2.
Thus, the foregoing example shows that cleavage of spectrin into BDP's in vitro increases the immunoreactivity towards raw serum of rabbits immunized with SDS-spectrin complexes.
Referring back to Figure 7, where the Western Blot assay is compared to the ELISA assay, it can be seen that the amount of spectrin immunoreactivity is most dramatically increased in those sections of rat hippocampus in which increased BDP's are found. Thus, the data of Figure 7 confirms that immunoreactivity is enhanced by cleavage into BDP's in vivo, as well as in yitro.
The availability of hidden epitopes in intact spectrin suggests an ELISA assay or other immunoassay for specifically detecting BDP's as opposed to intact spectrin.
Such an assay could use antibodies directed solely to these hidden epitopes, obtained through methods known in the art, ... . ... , ,, -- . ...... . . . - .................... - - -: . -, : . ~.: ~ . .
WO92/01935 ~ C~ PCT/US91/04910 such as through affinity purification or the production of a monoclonal antibody directed to a hidden epitope. It is expected that the affinity purification of the class of antibodies directed to epitopes present and exposed in intact spectrin as in Example II results in a fraction not bound to the spectrin-sepharose containing antibodies directed to hidden epitopes.
Using a source of antibodies directed to hidden epitopes, it is expected that a determination of the amount o~ total spec~rin immunoreactiv~ty and the amount of BDP
immunoreactivity could be separately made.
Alternatively, it is believed to he possible to separate intact spectrin from BDP's, including the BDPl and BDP2 polypeptides visualized by Western blot and other spectrin fragments, by altering the conditions of the solution to affect the solubility of the intact spectrin molecule. By altering the pH, ionic strength, or other such factors, it is believed possible to solubilize the BDP's while precipitating the intact spectrin molecules.
It is believed that treatment of samples containing spectrin immunoreactivity with an agent selected from the ~roup consisting of detergents, agents which produce an acidic or basic pH (preferably a pH of greater than 8.5 or less than 5.5), chaotropic agents and organic solvents of lowered dielectric will result in the altered solubility conditions required to precipitate intact spectrin without precipitating some or all of the BDP's. 8y removing the precipitated intact spectrin molecules, a determination of the amount of BDP's present can be obtained. For commercial utility, an enrichment for BDP's of at least ten fold is preferable, more preferably on the order of one hundred fold.
Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made - . : , ' :' : ' ' :
WO92/0193~ PCT/US91/04910 29 2Q3~$o without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
- - . . . . . . ..