MXPA01007149A - Removal of prions from blood, plasma and other liquids - Google Patents

Abstract

Devices such as flow through columns, substrates such as spherical polymer beads, and methods of using such to remove prions from any liquid sample are disclosed. A surface of a substrate is coated with a prion complexing agent, such as a metal salt (e.g. sodium) of phosphotungstic acid. Blood or plasma passing through a column containing beads coated with prion complexing agent are rendered prion free. The beads may be comprised of a ferromagnetic core to facilitate removal of the beads from the sample.

Description

ELIMINATION OF PRESSES FROM BLOOD, PLASMA AND OTHER LIQUIDS FIELD OF THE INVENTION The invention relates generally to methods for purifying samples and particularly to methods for removing prions from blood and blood products.

BACKGROUND OF THE INVENTION Prions are infectious pathogens that cause spongiform encephalopathies of the central nervous system in humans and animals. Prions are different from bacteria, viruses and viroids. The prevailing hypothesis at present is that no nucleic acid component is necessary for the infectivity of the prion protein. In addition, a prion that infects an animal species (eg, a human) will not infect another (eg, a mouse) A major stage in the study of prions and the diseases they cause was the discovery and purification of a protein designated protein Prion ("PrP") [Bolton et al., Science 278: 1309-1 1 (1982); Prusiner et al., Biochemistry 27: 6942-50 (1982); McKinley et al., Cell 35: 57-62 (1983)]. The genes encoding the complete prion protein have already been cloned, sequenced and expressed in transgenic animals. PrPc is encoded by a single-copy host gene [Basler et al., Cell 46: 417-28 (1986)] and is normally found on the outer surface of neurons. A guiding hypothesis is that Prion diseases result from the conversion "of PrPc into a modified form called PrPSc. It seems that the isoform of demyelinating encephalomyelitis of the prion protein (PrpSc) is necessary both for the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See, Prusiner, S.B., "Molecular Biology of Prion Desire," Science 252: 1515-1522 (1991). The most common prion diseases of animals are demyelinating encephalomyelitis of sheep and goats and bovine spongiform encephalopathy (BSE) and cattle [Wilesmith, J and Wells .. Microbiol. Immunol. 772: 21 -38 (1991)]. Four human prion diseases have been identified: (1) kuru, (2) Creutzfeldt-Jakob disease (CDJ), (3) Gerstmann-Strassler-Scheinker disease (GSS), and (4) fatal familial insomnia (FFI) ) [Gajdusek, DC Science 797: 943-960 (1997); Medori ef al., N. Enal. J. Med. 326: 444-449 (1992)]. The presentation of human prion diseases as an infectious, genetic or sporadic disease initially has an issue that has been explained by the cellular genetic origin of PrP. Most cases of CJD are sporadic, but approximately 10-15% are inherited as dominant disorders of an autosome that are caused by mutations in the human PrP gene [Hsiao et al., Neurology 40: 1820-1827 (1990); Goldfarb et al., Science 258: 806-808 (1992); Kitamoto eí al., Proc. R. Soc. Lond. 343: 391-398. Yatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as from hard matter grafts [Brown et al., Lancet 340: 24-27 (1992)]. Despite the numerous Attempts to link CJD to an infectious source such as the consumption of scratch infected sheep meat, none have been identified to date [Harries-Jones et al., J. Neurol. Neurosurg. Psvchiatry 57: 1 1 13-1 1 19 (1,988)] except in cases of iatrogenically induced disease. On the other hand, Kuru, who for many decades devastated the neighboring and Fore tribes of the mountainous regions of New Guinea, is believed to have spread by infection during ritual cannibalism [Alpers, M.P. , Slow Transmissible Diseases of the Nervous System, Vol., 1, S. B. Prusiner and W.J. Hadlow, eds. (New York;: Academic Press), pp. 66-90 (1979)]. The initial transmission of CJD to experimental primates has a rich history that begins with William Hadlow's recognition of the similarity between kuru and demyelinating encephalomyelitis. In 1959, Hadlow suggested that prepared extracts of patients dying from kuru be inoculated into non-human primates and that animals be observed for the disease that was predicted to occur after a prolonged incubation period [Hadlow, W.J. Lancet 2: 289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpers demonstrated the transmission of kuru to chimpanzees after incubation periods ranging from 18 to 21 months [Gajdusek et al., Nature 209: 794-796 (1996)]. The similarity of the neuropathology of kuru with that of CJD [Klatzo et al., Lab Invest 8: 799-847 (1959)] indicated similar experiments with chimpanzees and transmissions of the disease were reported in 1968 [Gibbs, Jr et al. , Science 767: 388-389 (1968)]. For the past 25 years, approximately 300 cases of CJD, GSU kuru have been transmitted to a variety of chimpanzees and monkeys.

The cost, scarcity and often perceived inhumanity of such experiments have restricted this work and in this way limited the accumulation of knowledge. Although it has been said that the most reliable transmission data emanate from studies using non-human primates, some cases of human prion disease have been transmitted to rodents but apparently less regularly [Gibbs, Jr. et al., Slow Transmissible Diseases of the Nervous System, Vol. 2, SB Prusiner and W.J. Hadlow, eds. (New York, Academic Press), pp. 87-1 10 (1979); Tateishi et al., Prion Diseases of Humans and Animáis, Prusiner et al., Eds. (London, Ellis Horwood), pp. 129-1 34 (1992)]. Infrequent transmission of prion disease from human to rodent has been cited as an example of the "species barrier" first described by Pattison in his studies of passage of the demyelinating encephalomyelitis agent between sheep and rodents [Pattison, LHNINDB Monoqraph 2. DC Gajdusek, C.J. Gibbs Jr. and M.P. Alpers. Eds. (Washington, D.C: Impression of the U.S. Government), pp. 249-257 (1965)]. In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only little development of the disease in animals. The subsequent passage in the same species is characterized by all the animals that become sick after greatly shortening the incubation times. The molecular basis for the species barrier between the Sirian hamster (SHa) and mouse was shown to reside in the PrP gene sequence using transgenic mice (Tg) [Scott et al., Cell 59: 847-857 (1989)] . SHaPrP differs from MoPrP in 16 positions out of 254 residues amino acids [Basler et al., Cell 46: 417-428 (1986); Locht eí al., Proc. Nati Acad. Sci. USA. 86: 6372-6376 (1986)]. Tg (SHaPrP) mice expressing SHaPrP have abbreviated incubation times with SHa prions. When similar studies are performed with mice expressing the sheep or human PrP transgenes, the spice barrier is not abrogated, ie the percentage of animals that become infected was unacceptably lower and the incubation times were unacceptably long. Thus, it has not been possible, for example in the case of human prions, to use transgenic animals (such as mice containing a PrP gene from another species) to reliably test a sample to determine if that sample is infected with prions. . The seriousness of the health risk that results from the lack of such a test is exemplified below. More than 45 young adults previously treated with HGH derived from human pituitaries have developed CJD [Koch et al., N. Engl. J. Med. 373: 731-733 (1985); Brown et al., Lancet 340: 24-27 (1992); Fradkin et al., JAMA 265: 880-884 (1991); Buchanan et al., Br. Med. J. 302: 824-828 (1991)]. Fortunately, recombinant HGH is now used, although the apparently remote possibility that increased expression of wtPrPc stimulated by elevated HGH may induce prion disease has been raised [Lasmezas et al., Biochem. Biophvs. Res. Common. 796: 1 163-1 169 (1993)]. That HGH prepared from pituitary contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with an expected lot of HGH [Gibbs, Jr. et al., N. Enol. J. Med. 328: 358-359 (1993)]. The Long incubation times associated with prion diseases will not reveal the full degree of iatrogenic CJD for decades in thousands of people treated with HGH worldwide. Yatrogenic CJD also appears to have developed in four infertile women treated with gonadotropin hormone derived from the contaminated human pituitary [Healy et al., Br. J. Med. 307: 517-518 (1993); Cochius ef al., Aust. N.Z. J. Med. 20: 592-593 (1990); Cochius ef al., J. Neurol. Neurosurq. Psvchiatrv 55: 1094-1095 (1992)] as well as at least 1 1 patients receiving hard matter grafts [Nisber et al., J. Am. Med. Assoc. 267: 1, 18 (1989); Thadani ef al., J. Neurosurq 69: 766-769 (1988); Willison ef al., J. Neurosurg. Psychiatric 54: 940 (1991); Brown ef al., Lancet 340: 24-27 (1992)]. These cases of iatrogenic CJD underscore the need to select pharmacists who may possibly be contaminated with prions. Recently, two doctors in France were charged with involuntary manslaughter of a child who had been treated with growth hormones extracted from corpses. The child developed Creutzfeldt-Jakob disease (See, New Scientist, July 31, 1993, page 4). According to the Pasteur Institute, since 1989, 24 cases of CJD have been reported in young people who were treated with human growth hormone between 1983 and mid-1985. Fifteen of these children died. Now it appears as if hundreds of children in France have been treated with the growth hormone extracted from the dead bodies at risk of developing (see New Scientist, November 20, 1993, page 10). In view of such, clarity is a must for a cost effective means, convenient to eliminate prions that cause CJD of blood and blood products. The present invention provides such a method.

BRIEF DESCRIPTION OF THE INVENTION Prions are removed from biological materials such as blood and plasma by contacting these materials with a complexing agent, such as a biological agent (e.g., an antibody) or a chemical agent (e.g., a substratum comprised of sodium phosphotungstate) that binds prions. The substrate can be used in any configuration that allows prion removal of biological materials, such as spherical polymer beads that are coated with a complexing agent and housed in a column through which blood, plasma and blood are allowed to flow. other material suspected of containing prions. One embodiment of the invention is a method for removing prions from a sample such as human blood by contacting the sample with a substrate comprised of a prion complexing agent such as a sodium salt of phosphotungstic acid and allowing the prions in the blood join the complexing agent for his retirement. - Another embodiment of the invention is a device that removes prions from a liquid sample, such device is comprised of a substrate such as ferromagnetic beads coated with a polymer with a complexing agent thereof, wherein the substrate is preferably placed in a housing tubular through which the liquid sample. An object of the invention is to provide a simple and economical means for removing prions from a material. An advantage of the invention is that blood products that can contain prions can be certified as prion free when processed through the invention. A feature of the invention is that the prions selectively bind to chemical agents such as heteropoly acids or metal salts of heteropoly acids. A preferred chemical agent for use in the methods of the invention is sodium phosphotungstate. Another feature of the invention is that the prions selectively bind to biological agents such as peptides, small molecules and selective PrPSc binding antibodies. One aspect of the invention is a substrate that can be in the form of spherical polymer beads having a complexing agent coated on its surface. In a particular embodiment, the spherical beads have a core of ferromagnetic metal that allows separation of the beads using magnetic forces and has an inert polymer that coats the metal core with the polymer coated with a metal salt of phosphotungstate acid. Another aspect of the invention is blood and blood products such as grass that have been treated with the method of the invention. Yet another aspect of the invention is a filter membrane used to remove prions from biological materials using methods such as hemofiltration. - > Yet another aspect of the invention is a device comprised of a (preferably cylindrical) housing having surfaces thereon coated with a PrPSc-binding composition, for example spherical guide surfaces coated with sodium phosphotungate. These and other objects, aspects, advantages and features of the invention will be apparent to those skilled in the art after reading the details of the binders, devices and methods as described more fully below.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Before the present methods and devices are described or disclosed, it should be understood that this invention is not limited to particular complexing agents, proteins, labels, analysis or method as such, it can, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limited, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary experience in the subject matter to which this invention pertains. Although any method and materials similar to or equivalent to those described in the present may be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and expose the methods and / or materials in connection with which the publications are cited. The publications described herein are provided for description only before the filing date of the present application. Nothing herein is construed as an admission that the present invention is not entitled to advance such publication by virtue of the foregoing invention. In addition, the publication dates provided are subject to change if it is found that the current application date is different from that provided herein.

DEFINITIONS The term "complexing agent" is used herein to refer to any material that selectively binds or complexes with either the constrictive conformation of a protein (e.g., PrPSc) and / or with the relaxed conformation of a protein (e.g. example, PrPc). This complexing agent can be a biological molecule such as a peptide or antibodies, for example, an antibody selective for PrPSc, or a chemical agent, for example, phosphotungstic acid (PTA), which can be added in the form of a salt, for example , sodium phosphotungstate. The complexing agents can be used singly or in combination. For example, a biological complexing agent can be used one after the other with a chemical complexing agent, such as the use of a peptide > or a chemical agent. In another example, two complexing agents for the same class can be used together, for example, a mixture of phosphotungstic acid (and salts thereof) and trichloroacetic acid. The complex formed must provide some means to separate the complex from the rest of the composition, such as immobilization of complexing agent to a surface. A preferred complexing agent that binds PrPSc more readily than binds PrPc and a particularly preferred agent that binds PrPSc with a high degree of affinity and does not bind PrPc at significant levels. Objectively, a binder binds PrPSc with double or more binding affinity than what PrPc binds and preferably five times or more binding affinity than what PrPc binds. The terms "protein" as used herein are proposed to comprise any amino acid sequence and include modified sequences such as glycoproteins. The term includes naturally occurring proteins and peptides as well as those that are synthesized recombinantly or synthetically. As used in connection with the present invention, the term "protein" is specifically proposed to cover naturally occurring proteins in at least two different conformations where both confirmations have the same or substantially the same amino acid sequence but have two or more different three-dimensional structures. The two conformations of the protein include at least one conformation that is not related to a disease state and at least one conformation that is related to a pathogenic disease state. A specific and preferred example of a protein as used in connection with the description is a PrP protein that includes the non-disease form referred to as the PrPc form and the related form of disease referred to as PrPSc. Although a prion protein or the PrPSc form of a PrP protein is infectious and pathogenic, the disease conformation of other proteins is not infectious but is pathogenic. As used herein, the term pathogenic is associated with the disease and is therefore present when the disease is present. Thus, a pathogenic protein as used in connection with this description is not necessarily a protein that is the specific causative agent of a disease. The terms "PrP protein", "PrP" and the like are used interchangeably herein and both should mean the infectious PrPSc particle form known as the causative agent of diseases (spongiform encephalopathies) in humans and animals and the non-infectious form of PrPc which, under Appropriate conditions become the form of infectious PrPSc. The terms "prion", "prion protein" and "PrPSc protein" and the like are used interchangeably herein to refer to the PrPSc infectious form of PrP, and is a contraction of the words "protein" and "infection". " The particles are greatly, if not exclusively, comprised of PrPSc molecules encoded by a PrP gene. Prions are generally PrPSc dimers. Prions are different from bacteria, viruses and viroids. The known prions infect animlaes to cause demyelinating encephalomyelitis, a degenerative, transmissible disease of the central nervous system of sheep and goats, as well as bovine spongiform encephalopathy (BSE), or "mad cow disease", cat feline spongiform encephalopathy. Four prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Strassier-Scheinker disease (GSS), and (4) fatal familial insomnia ( FFI). As used herein, "prion" includes all forms of prions that cause all or any of the diseases or others in many animals used, and in particular in humans and domesticated farm animals. The term "PrP gene" is used herein to describe genetic material that expresses proteins that include known polymorphisms and pathogenic mutations. The term "PrP gene" generally refers to any gene of any species that encodes any form of a PrP protein. Some commonly known PrP sequences are described in Gabriel ef al., Proc. Nati Acad. Sci USA §9: 9097-9101 (1992), and US Patents. 5.565, 186; 5,763,740; 5,792,901; and WO97 / 04814, incorporated herein by reference for describing and exposing such sequences. The PrP gene can be from any animal, including the "test" and "host" animals described herein and any and all polymorphisms and mutations thereof, if it is recognized that the terms include other PrP genes that are yet to be discovered . The protein expressed by such a gene may already assume the form of PrPc (not disease) or PrPSc (disease). The term "antibody" means a protein of immunoglobulin that is capable of binding an antigen. Antibody as used herein means including the entire body as well as any antibody fragment (eg, F (ab) \ Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest. Preferred antibodies for the assays of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to a protein of interest, for example, a PrP protein and specifically a PrPSc protein or PrPSc dimer. Antibodies that are immunoreactive and immunospecific for both the native disease form and the disease form (e.g., for either native PrPc or native PrPSc) can be used. The antibodies to PrP are preferably immunospecific, for example, not substantially cross-reactive with related materials. Some specific antibodies that can be used in connection with the invention are described in the published PCT application WO 97/10505 which is incorporated herein by reference for describing and exposing the antibodies. This published PCT application corresponds to the U.S. Patent. 5,846.6533 issued December 8, 1998, also incorporated herein by reference. The term "antibody" includes all types of antibodies, for example, polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies that have a relatively high degree of affinity for both native PrPc and PrPSc, and those with higher binding affinity for PrPSc are preferred. An antibody of the invention is a "complexing agent" as defined in the present. An antibody for binding to PrPc is the monoclonal antibody 263K 3F4 produced by the hybridoma cell line ATCC HB9222 deposited on October 8, 1986 in the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852 and described and discussed in the U.S. Patent 4,806,627 issued February 21, 1989, incorporated for reference to describe antibodies that selectively bind PrPc but not PrPSc. "Antigenic fragment" of a protein (e.g., a PrP protein) means a portion of such a protein that is capable of binding an antibody. By "specifically binding" is meant high affinity binding and / or high strength of an antibody to a polypeptide, for example, epitope of a protein, for example, a PrPSc. The binding of the antibody to its epitope on this specific polypeptide is preferably stronger than the binding of the same antibody to any other epitope, particularly those that may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest. , for example, binds more strongly to epitope fragments of a protein such as PrPSc so that upon adjusting the binding conditions the antibody binds almost exclusively to an epitope site or fragments of a desired protein such as an epitope fragment. of PrPSc. By "detectably labeled antibody", "detectably labeled anti-PrP" or "anti-PrP fragment" detectably "labeled" means an antibody (or antibody fragment that retains the binding specificity), having a detectable label attached The detectable label is normally adhered by chemical conjugation, but where the label is a polypeptide, it could alternatively be adhered by Genetic engineering Methods for the production of detectably labeled proteins are well known in the art Detectable labels are known in the art, but are usually radioisotopes, fluorophores, paramagnetic labels, enzymes (eg, horseradish peroxidase) or other parts or compounds that either emit a detectable signal (eg, radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate Several pairs of label / sub-substrate (eg, horseradish peroxidase) / diaminobenzidine, avidin / streptavidin, luciferase / luciferin), methods for et labeling antibodies, and methods for using the labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY)). Europium is a particularly preferred label. Abbreviations used herein include: CNS for central nervous system; BSE for bovine spongiform encephalopathy CJD Creutzfeldt-Jacob FFI disease for fatal familial insomnia; GdnHCl for guanidine hydrochloride; GSS for Gerstamnn-Strassler-Scheinker Disease; Hu for human; HuPrP for human prion protein; Mo for mouse, MoPrP for mouse prion protein; SHa for a Sirian hamster; SHaPrP for a Syrian hamster prion protein; PrPSc for the isoform of demyelinating encephalomyelitis of the prion protein; PrPc for the normal, common cellular protein content of the prion protein; PrPCJ D for the CJD isoform of a PrP protein; FVB for the standard innate strain of mice used frequently in the production of transgenic mice since the eggs of FVB mice are relatively longer and tolerate the microinjection of exogenous DNA relatively well; [PrPß] -prion protein concentration in ß sheet conformation. [DRC] -concentration of a disease related to the conformation of a protein; PTA-phosphotungstic acid NaPTA-sodium phosphotungstate TCA-trichloroacetic acid AC-affinity chromatography.

GENERAL ASPECTS OF THE INVENTION The analyzes for the detection of prions are in development but not yet commercialized. In addition, the cost, convenience and accuracy (large-scale) of such analyzes have not yet been determined. According to the above, when a material such as human plasma is suspected to contain prions is destroyed, see The Wall Street Journal November 25, 1998, page 1 article entitled: "Mad Cow'Fears Leads UK to Destroy Parts of all Donated Blood ", which indicates that England destroyed its human plasma supply. This dramatic action was taken due to (1) prions that may be present in their human plasma, (2) prion diseases are fatal and can not be treated at present, (3) tests are not commercially available at present. for prions, and (4) a method for removing prions from a sample is not commercially available at present. The present invention includes a method for eliminating prions. Some proteins such as the protein expressed by the PrP gene have more than one conformational form. For example, a PrP protein can assume its cellular form, i.e., PrPc form or its demyelinating encephalomyelitis form, i.e., PrPSc form. One form of the protein is harmless (for example, PrPc) while the other form of the protein is pathogenic (for example, PrPSc). When the pathogenic, tapered form of the protein such as PrPSc is present in an animal in very small amounts, the animal shows no symptoms of disease. However, the animal will develop a disease related to the pathogenic form of the protein, for example, will develop a prion disease. In order to prevent the progression and / or possible transmission of disease, it is important to eliminate any PrPSc present in biological flute. and pariicula rrr níe bio logical fluids r i - * -:;; introduce a subject (for example, blood products). The present invention is useful with respect to (1) removing the pathogenic form of the protein from the sample so that the material becomes "prion free" and / or (2) reducing the concentration of the pathogenic form of the protein in the a material at a level such that the material becomes "non-infectious". The present invention makes it possible to eliminate prions from a biological sample by exposing the sample to a complexing agent, which binds (preferably selectively) to a PrPSc and allows the removal of PrPSc. The present process is especially, although not exclusively, useful for the treatment and / or elimination of PrPSc from whole blood. The removal can be done through complexation with an immobilized complexing agent, i.e., exposure of the sample to an affinity column, membrane, filter or beads with immobilized complexing agent. The complexing agent will efficiently remove PrPSc from the sample, while allowing the sample to remain substantially in the same form to allow proper use of the sample, i.e., maintain the appropriate pH, structural integrity of the cells and proteins, and the similar.

PROCEDURES IN GENERAL Any type of sample can be processed using the present invention in order to eliminate a pathogenic form of a protein. Although the invention could be applied to the removal of a narrowed form of any protein having a relaxed and narrowed form, the invention is specifically described with respect to the elimination of the pathogenic form of a PrP protein, i.e., concentrating and eliminating PrPSc. A biological sample to be treated should be in a liquid flowable form at room temperature (15 ° C to 30 ° C). The solution should have a pH of about 6.4 to 8.4, preferably 7.4, and should not contain excess calcium or magnesium. The next stage is the most important in the process of the invention. A sample is exposed to a complexing agent that is immobilized on a solid surface or otherwise provided in a manner that allows separation of the prion binding complexing agent from the sample. The complexing agent forms a complex with or somehow binds preferentially or exclusively to any narrowed form (generally a pathogenic form) of the protein present in the sample, thus effectively immobilizing any PrPSc present in the sample to the solid surface in the exhibition of the sample to the immobilized complexing agent. In one embodiment, a chemical agent such as a heteropoly acid (e.g., PTA9, or preferably a metal salt thereof (NaPTA) is immobilized to a solid surface such as a filter. membrane, a magnetic pearl and the like. The sample is subjected to the complexing agent for a sufficient period of time to allow substantially all of the PrPSo in the sample to be complexed with PTA. For example, the sample could be incubated at about 30 ° C to 45 ° C (preferably 37 ° C) for a period of about 1 to 16 hours. The complexing agent forms a complex with PrPSc. What is important is that the complex formed can be separated from the rest of the sample by some means, for example, filtration, use of magnetic field, sedimentation and the like. The process of the invention produces a biological sample in which PrPSc or other pathogenic protein is substantially removed from the sample, and preferably at levels in which PrPSc is not detectable by conventional means. Methods for the elimination of PrPSc will prevent the transmission of disorders mediated by PrPSc by providing biological samples that are free of infectious levels of prions, ie, "prion-free".

COMPLEXING AGENTS Compounds that are useful as complexing agents in the present invention include antibodies, enzymes, peptides, chemical species, binding molecules, etc. These complexing agents are used in a manner that allows binding and elimination of prions from a biological solution, while maintaining the essential elements of the biological material intact, for example, cell morphology retention and protein integrity. Such complexing agents can be used in whole blood, in blood components such as plasma and pastures, and in other biological fluids as will be apparent to one skilled in the art. Chemical Agents In one embodiment of the invention, the compounds for the removal of prions from a biological material is a chemical agent that precipitates PrPSc. A preferred class of chemical agents to be used as complexing agents in the present invention are the heteropoly acids and salts thereof. The heteropoly acids are fully or partially protonated forms of oxyanions having at least one core element and at least one coordination element. The heteropoly acids may have Keggin and Dawson structures. A particular class of heteropoly acids is the protonated form of heteropolybdata. These anions contain from 2 to 18 hexavalent molybdenum atoms around one or more central atoms. Approximately 36 different elements have been identified as central atoms of these heteropololyblasts. These anions are highly oxygenated. Examples of heteropololyndata include [PMo12O0], [As2Mo18O62] 6 and [TeMo6O2] 6, where the central atoms are P5 +, As5 + and Te5 +, respectively. A more detailed description of the heteropololyblasts is provided in the Kirk-Othmer Encyclopedia of Chemical Technology 3rd ed, 15, 688-689 (1981). Another class of heteropoly acids, which is analogous to the protonated form of heteropololyblasts, is the protonated form of heteropolitungstates. In heteropolitungstates, the element of coordination is tungsten instead of molybdenum, US Patent. No. 4,376.21 9, the full disclosure of which is expressly incorporated by reference, describes the preparation of various heteropoly acids. The core elements of these heteropoly acids can be selected from the group consisting of P, Si, B, Ge, As, Se, Ti, Zr, Mn, F, V, Ce and Th. The coordinating element of these heteropoly acids include Mo and / or W. Additional coordination elements include V, Mn, Co, Ni, Cu, Zn, and Fe. The ratio of the number of coordination elements to the number of core elements may be from 2.5 to 12, preferably 9 to 12. Particular heteropoly acids, which are exemplified in U.S. Patent No. 4,376,219, include phosphotungstic acid, silicotungstic acid, 10-tungsto-2-vanadofosphoric acid, 6-tungsto-6-molibodofosphoric acid, phosphomolybdic acid, silicomolybdic acid, germanotungstic acid, tungstofluoric acid; and 18-tungsto-2-phosphoric acid as well as salts of all or any of these acids, for example, metal salts such as Na, K, Mg and Ca salts. A particular heteropoly acid for use in the present invention is phosphotungstic acid, that is, H3PW12O 0 and its salts, particularly Na salts. Such complexing agents bind effectively to PrPSc. Such chemical agents can be used alone, in combination, or with other non-bioactive chemicals such as regulators and inert binding chemicals. The heteropoly acids of the invention (eg, PTA) are preferably, but not exclusively, used in the form of a metal salt. Metallic salt includes, but is not limited to, sodium, potassium, calcium and the like. The amount of heteropoly acid or salt thereof that is combined with the present support material should be present in an amount sufficient to significantly eliminate PrPSc at undetectable levels or at least non-infectious levels. The weight ratio of heteropoly acid to support material, it can be, for example, from about 1: 20 to about 1: 1. The heteropoly acid can be combined with the support material in any manner that provides adequate dispersion of the heteropoly acid, thus increasing the effective surface area of the heteropoly acid. A preferred technique for combining these components is by impregnating the support material with the heteropoly acid. The heteropoly acid can also be combined with the support material by an ion exchange technique. The impregnation technique can include absorbing an aqueous solution of the heteropoly acid in the porous region of the support material followed by drying to remove water and leaving behind the supported heteropoly acid. Other methods for immobilizing the heteropoly acids or salts thereof can be used to immobilize these complexing agents, as will be apparent to one skilled in the art after reading this description. Biological Agents In another embodiment, the complexing agent is a protein, peptide, and another biological part that binds selectively to PrPSc. In one embodiment, the complexing agents are peptides or other small molecules designed to bind selectively to prions.

Preferably, the peptides or small molecules are designed to preferentially bind to PrPSc. By "preferentially" is meant that the peptide is designed to be at least 20 times or more, more preferably 50 times or more, more preferably 100 times or more, and even more preferably 1000 times or more likely to bind to PrPSc than at other proteins in the biological solution. The peptides of the invention are preferably designed to bind to the native form of PrPSc, as opposed to the denatured form, since biological fluids generally contain PrPSc in a native form. The peptides can be designed to maximize binding to PrPSc by designing the peptides for PrPSc areas that are more accessible to the junction, as can be predicted by one skilled in the art. Useful antibodies that bind to PrPSc are described and expressed in U.S. Patent 5,846,533, issued December 8, 1998, incorporated herein by reference for describing and exposing the antibodies and methods for making the antibodies. Portions of these antibodies that bind to PrPSc are peptides that can bind to a support surface and be used in the present invention. Alternatively, the peptides can be designed to selectively bind PrPc and both PrPSc and PrPc. Although the PrPSc form of the prion is the infectious portion, the removal of the normal cellular prion proteins can also effectively decrease or arrest the progression of a prion-mediated disorder. The complexing agent of the invention can also be a selective antibody for prions. The antibody can be immobilized -, directly or can be joined to another component (for example, a high density metal). That antibody can be bound to PrPSc, for example, the antibody described in U.S. Patent 5,846,533. To remove PrPc present in the sample, an antibody that binds selectively or exclusively to PrPc can be used. Such an antibody is described in US Pat. No. 4,806,627, filed on February 21, 1989, which describes the monoclonal antibody 263K 3F4, produced by the ATCC cell line HB9222 deposited on October 28, 1986, which is incorporated herein by reference. The cell line that produces the antibody can be obtained from the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852. In general, the infection of demyelinating encephalomyelitis fails to produce an immune response, with host organisms being tolerant to PrPSc from the same species. Antibodies that bind to either PrPc or PrPSc are described in the U.S. Patent. 5,846,533. Any antibody that binds PrPc and not PrPSc can be used, and those skilled in the art can generate such using known methods, for example, see methods for producing phage display antibody libraries in E.U. 5,223,409. Polyclonal anti-PrP antibodies have been elevated in rabbits after immunization with large amounts of formic acid or SHaPrP denatured by SDS 27-30 [Bendheim, Barry et al., (1984) Nature 310: 418-421; Bode, Pocchiari ef al., (1985) J. Gen. Virol. 66: 2471-2478; Safar, Ceroni ef al., (1990), Neurology 40: 513-517]. Similarly, a small amount of monoclonal anti-PrP antibodies against PrP 27-30 have been produced in mice [Barry and Prusiner (1986) J. Infect. Dis. 1 54: 518-521; Kascsak, Rubenstein eí al, (1 987) J. Virol. 61: 3688-3693]. These antibodies were generated against denatured PrP by SDS or acid 27-30 and are able to recognize native PrPc and treated or denatured PrPSc from both SHa and humans equally well, but not to bind to MoPrP. Not surprisingly, the epitopes of these antibodies were mapped to regions of the sequence containing amino acid differences between SHa and MoPrP [Rogers, Yehiely et al. (1993) Proc. Nati Acad. Sci. USA 90: 3182-3186]. It is not completely clear why many antibodies of the type described in the above-cited publications will bind to PrPc and PrPSc treated or denatured but not to native PrPSc. Without joining any particular theory, it is believed that such may occur because the epitopes that are exposed when the protein is enforced in the PrPc conformation are not partially exposed or hidden in the PrPSc configuration, where the protein is relatively insoluble and more fully folded together . For purposes of the invention, an indication that no binding occurs means that the equilibrium constant or affinity Ka is 106 1 / mol or less. In addition, the bond will be recognized as existing when Ka is at 107 l / mol or more, preferably 10 l / mol or more. The binding affinity of 107l / mole or more can be due to (1) a single monoclonal antibody (ie, large numbers of a class of antibodies) or (2) a plurality of different monoclonal antibodies (eg, large numbers of each five different antibodies monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use the combinations of (1) - (3). The selected preferred antibodies will bind at least 4 times more strongly to the treated or denatured forms of PrPSc of the protein when compared to their binding to the native conformation of PrPSc. The fourfold differential in binding affinity can be carried out by using several different antibodies as per (1) - (3) above, and such some antibodies in a mixture could have less than a fourfold difference. A variety of different methods can be used with or more different antibodies. Those with ordinary experience will recognize that antibodies can be labeled with different known labels and used with currently available robotic, sandwich analysis, electronic detectors, flow cytometry, and the like. In addition antibodies can bind to more dense components directly or through other intermediates such as anti-bodies.

PURIFICATION METHODS The complexing agent of the invention can be used in a variety of purification procedures to effectively remove prions from a biological material. A number of methods for use in the present invention are summarized as follows: Affinity Chromatography Affinity chromatography (AC) depends on the interaction of the protein with an immobilized ligand. AC is predicated, in part, on the interaction of ligands attached to chromatographic supports. A hydrophobic ligand coupled to a matrix is referred to several times herein as an AC support, AC gel or AC column. It is further appreciated that the intensity of the interaction between the protein and the AC support is not only a function of the ratio of non-polar to polar surfaces of the protein but by the distribution of the non-polar surface, as well. A number of shades can be used in the preparation of AC columns. Preferably, such matrices are beads, and more preferably spherical beads, which serve as a support surface for the complexing agent of the invention. Suggested materials for the matrices include, degraded dextran, polyhydroxyl ethyl methacrylate, polyacrylamide, cellulose and derivatives or combinations thereof, preferably in the form of porous spheres. Cellulose acetate has previously been used successfully in biological fluid purification devices, for example, extracorporeal blood purification devices. Polyurethane is particularly compatible with blood. Silica and its derivatives are also especially useful as a support material for use with heteropoly acids. See US Patents Nos. 5,475, 178 and 5,366,945 which are incorporated herein by reference. The preferred material for use in the methods of the present invention is agarose, a hydrophilic polymer that occurs naturally. A pearl gel with a porosity of 90-96% is formed by varying the percentage of agarose. The molecular weight of the gel varies from 0.5 million per 10% agarose to 20 million per 4% agarose. Particle diameters ranging from 20 to 200 microns are commercially available. The mechanical intensity of agarose beads can be increased either by increasing the percentage of agarose or degrading the beads with epichlorohydrin or 2,3-dibromopropanol, using the method of J. Porta ef al., In J. Chromat 60, 167 (1971) . This allows for a corresponding increase in the maximum operating pressure (a fifty percent increase in agarose beads to a two to four times increase in maximum operating pressure.) The criteria for determining the appropriate coupling method are: minimization of spill of the complexing agent of the support, maintenance of the thermal stability of the compound, and retention of the optimum quantity of complexing agent.The technique must also cause a deterioration in the support material or the production of reactive groups in the support that could bind components In vivo, the complexing agent can also maintain its activity over time.Additional factors that must be considered to optimize the affinity chromatography coupling method are: the degree of distribution of the coupling agent within the particles and / or columns; pH; temperature; the flow velocity of the biological sample through the column; the size of the attached complexing agent; and / or the diameter and size of the pore of the particular support. Each of these conditions can be optimized for a particular procedure, biological sample, and complexing agent as it will be apparent to a skilled. - The composition AC of the present invention can be contained within a filter cartridge for easy use of the composition in a biological fluid purification process. When the column composition is contained within a single cartridge, it can easily and conveniently be rced when the purification capacity of the composition becomes exhausted. Alternatively, the cartridge can be an integral part of a purification device, in which case the entire device is rced once the filtration composition has exhausted its effectiveness. The support particles with the complexing agent are placed inside the cartridge, and the solution to be reacted with the complexing agent is then circulated through the cartridge. Commercially available units for dialysis, blood exchange or oxygenation can be adapted to be used as the purification device. Filtration methods Another method can be used to remove prions from a biological sample that includes filtration through the membrane. The membrane may have the prion complexing agent conjugated directly to the membrane, either on the side facing the biological fluid or more preferably on the side remote from the biological fluid. Alternatively, the complexing agent can be divided into compartments an area behind the membrane that is inaccessible to the larger components of the biological materials, for example, blood cells. In the last example, the complexing agent can join an insoluble matrix behind the membrane. The membrane for use in the present invention may be in planar form, in the form of one or more hollow fibers, and / or in the form of flat sheets.

See, Patent of E.U. No. 4,361, 484, which is incorporated herein by reference. Suitable materials for the membrane include regenerated cellulose, cellulose acetate, non-woven acrylic copolymer, polysulfone, polyether sulfone, polyacrylonitrile, polyamide and the like. The biologically active material is immobilized in the pores and / or on the surface of the side of the membrane that is far from the biological fluid. Therefore, components such as blood corpuscles avoid contacting active material. The pores of the membrane are usually of the magnitude of the order of 0.01 to 0.8 microns, preferably 0.15 to 0.45 microns, the polymer support can be stable under the conditions of its intended use, that is, it must not be degraded chemically or enzymatically by the blood, the support and the immobilized complexing agent must be compatible with the blood, and the support should have good flow characteristics and low compressibility under clinical flow rates in the range of 150-250 ml / min. Through the anterior construction of the microporous membrane, that is, asymmetric immobilization of the prion complexing agent, the biological fluid does not need to be exposed to any subsequent filtration to eliminate possible harmful residues. Also the separation as the elimination of substances can be carried out in one and the same stage.

The microporous semipermeable membrane may be in the form of individual fibers that are tied or encapsulated within one or the same case with an inlet and outlet for the biological fluid. The ends of the fibers are glued by means of a suitable binder to keep the individual fibers essentially parallel within the case. One end of the fibers or ties of the fibers is provided in communication with the inlet, while the opposite end is provided in communication with the outlet. The biological material is pumped into the case through the entrance and through the longitudinal hollow of the fibers and out of the case through the outlet. During the passage through the case the fluid is exposed to pressure variations, so that only a fraction of penetration is caused to flow in an alternate path through the fiber walls in each direction to contact the complexing material of prion. The means for realizing the pressure variations can be made again from an expansion chamber in communication with the space between the individual fibers and fiber strips, respectively. Any subsequent filtration of the biological material for the removal of possible harmful residues is not necessary, since filtration is achieved automatically through the passage of the fluid through the walls of the fiber. Pressure variations can vary from -200 to +200 mmHg, preferably from -100 to +100 mmHg. The longer the diffusion distance for the blood, for example if the prion complexing agent binds to an insoluble matrix behind the membrane, higher compensation pressure variations will be required. In a corresponding manner, the frequency of the pressure variations can vary from about 0.05 to about 10 Hz, preferably 0.5 to 1 Hz. After passing through the treatment unit, the biological material, for example, whole blood, it can be reinserted into the patient directly, or it can be stored for future use. The treated blood can be stored completely, or it can be stored in its various components, for example, plasma, pastures, erythrocytes, etc. Alternatively, blood can be separated into its components before prion removal. When the complexing agent is an antibody, it is often desirable to have a medium that forms a molecular spacer segment to separate the antibody from the outer porous wall wall of the hollow fiber membrane. This general installation is preferred when the molecular weight of the antigen is large, for example, 100,000 Daltons or more in molecular weight. For example, a methylene group of six or eight carbons is conveniently a separate or "handle" between the antibody and the membrane surface. When the antigen is readily absorbed by albumin or is easily reacted chemically with albumin, with the material of the filter membrane surface, the spacer molecule can be a protein such as albumin. The outer surface of a membrane can be considered a relatively porous material compared to that of the inner surface which is normally the effective filter surface of an ultrafilter membrane of the asymmetric type, sometimes called anisotropic In this way, for example, the porous, outer side of a membrane can be treated with 17% human albumin solution in saline. The albumin will coat the surfaces within the porous zone of the membrane structure (i.e., the area covering the barrier layer of the membrane) and, therefore, a protein solution for example, a PrPSc antibody can be deposited at the albumin. It is often desirable to degrade the protein in some way (as with a diluted glutaraldehyde solution or some other induction agent by mild degradation); this helps hold the material in place on the membrane surface. An approach to preparing a cartridge that is capable of eliminating the pathogenic factors of blood is an extracorporeal circulation system with fiber membranes having sufficient permeability for the pathogenic blood factor to be eliminated through the membrane and in an immobilized antibody , soluble sequestered in extrafiber space. This includes the formation of a high molecular weight polymer conjugate of the PrPSc antibody and PrPSc can not cross the filtration side of the membrane to the remainder of the biological sample, i.e., where the cells are maintained. In order to form an immobilized, soluble complexing agent, the molecular weight of the immunoreactive complexing agent can be increased to such a size that it will not diffuse, from the porous, outer portion of the fiber and into the blood to be purified. This can be done by chemically reacting the complexing agent with a high molecular weight, water soluble substance such as silica gel or dextran or polymerize the complexive agent inminoreactive. The use of such macromolecular-born antibodies is advantageous for the high rate of antigen absorption, due to the increased rate of polarization effects in mass transfer and binding kinetics. Alternatively, the membrane can be composed of two membrane halves which are generally mechanically identical to each other but which can be chemically constructed of different material. In this case, it is sufficient if only half of the membrane that is far away from the biological material is able to bind to the protein complexing agent. For example, the membrane halves can be provided in a relationship of abutment to each other, wherein the complexing agent PrP.sub.c preferably binds in the porous and both surfaces of the membrane half that is far from the biological material. The complexing agent (for example, NaPTA or anti-PrPSc antibodies) can also be immobilized on the membrane so that the surface that is far away from the biological material is free of the contact reagent. This is to avoid contact between the blood corpuscles and the reagent and therefore anaphylactic and / or pyrogenic reactions. In this way, it is a form of a symmetric immobilization, where on a surface of the membrane (as well as the pores) the prion complexing agent is immobilized. The advantage of being immobilized within the pores of the membrane is that the active microscopic surface can be manipulated (>100) compared to the macroscopic surface. Since the complexing agent is immobilized in the part of membrane that is far from the biological material, the biological material will not come into contact with the material. As a result, any subsequent separate filtration of the biological material is therefore not necessary. Alternatively, the prion complexing agent can be found in an insoluble matrix behind the membrane. The treatment process is still similar, but since the required diffusion distance is approximately 10 times longer, it may be necessary to install a more real flow through the membrane. Regarding whether the prion complexing agent is immobilized in the pores or is immobilized on a soluble matrix behind the membrane, the immobilization procedure is preferably carried out so that the prion complex and the binding agent remain bound and immobilized, i.e. It is not present in the blood after the purification technique. Generally, covalent coupling is the safest immobilization. The nature of the covalent coupling depends on the choice of membrane material and the nature of the complexing agent. Magnetic particles Prions can also be removed from biological materials using magnetic particles comprised of prion complexing agent. The main components of the magnetic particles of the present invention are a magnetic core. The core consists of iron oxide particles or other magnetic materials. The PrPSc binder of the invention can be incorporated directly in the magnetic core, or incorporated directly into the magnetic core, for example, through the use of a fibrous material and a binding agent. The fibrous material may comprise an organic polymer in the form of fibers, such as carbohydrate polymers, urea formaldehyde or polynonamethylene urea, and in particular, cellulose fibers. The binder is a material that is introduced between the magnetic core and the fiber filaments as a liquid, or in solution, and solidifies during the production process of freezing, polymerization or evaporation of a solvent. Examples of suitable binders are agar, gelatin, an epoxy resin or formaldehyde furfuryl alcohol. The microparticles useful in the present method may be a variety of shape, which may be regular or irregular, preferably the shape maximizes the surface areas of the microparticles. The magnetic microparticles should be of such a size that their separation from the solution, for example by filtration or magnetic separation, is not difficult. In addition, the magnetic microparticles should not be so long that the surface area is minimized or not suitable for microscale operations. Suitable sizes vary from about 0.1 mu, from average diameter to about 100 mu. of average diameter. A preferred size is approximately 1.0 mu. of average diameter. Suitable magnetic microparticles are commercially available from PerSeptive Diagnostics and are referred to as BioMag COOH (Catalog Number 8-4124). The magnetic particles of the present invention can produced by stirring or mixing the core particles in a suspension comprising a fibrous material, a prion complexing agent, and a binder. The fibers are attached to the core particles and the binder fills the interstices. The binder then solidifies by one of the means as discussed above, in a manner that the prion complexing agent is accessible to the outer surface. An example of such a system using iron oxide as the core particles, cellulose fibers as the fibrous material and agar as the binder is described in the U.S. Patent. No. 5,705,628 which is incorporated herein by reference. The present invention also includes within its scope a composite magnetic resin comprising magnetic particles embedded in an organic polymer matrix containing either, or has attached to it, sites that are selective for prions. The composite can thus comprise magnetic particles embedded in a polymeric resin containing active or chemical sites proposed to selectively absorb the prions. For example, the polymer resin has small particles of selective absorbers attached thereto. The selective absorbers can be, for example, a metal salt of a phosphorus phosphate. The composite magnetic particles of the present invention can be used in a method for removing prions from any flowable biological sample. The elimination of prions of the human product is when contacting the solution to be treated with particles of a composite magnetic resin with complexing agent and separating by magnetic filtration the magnetic resin particles composed of the solution. These magnetic particles can be used once by subjecting the composite magnetic resin particles to regeneration using an appropriate regenerating solution, separating the composite magnetic resin particles from the regeneration solution. The magnetic resin particles composed of bound prions are selectively removed from the solution by magnetic filtration using techniques that are known in the art. The composite magnetic resin particles are then recovered from the filter and the prions are removed therefrom using an appropriate regenerating solution, for example an acidic solution. The cleaned composite magnetic resin particles can then be recovered from the regenerating solution by magnetic filtration and the clean particles recycled for further use. The purification of a patient's biological material can be through an extracorporeal treatment unit, and after treatment the purified fluid can either be stored or can be reintroduced to the patient. The biological material is pumped from for example a patient in a treatment unit comprising a semi-permeable microporous membrane having pores of 0.01 -0.8 microns, preferably 0.15-0.45 microns. During the passage through the treatment unit, the biological material is exposed to pressure variations (for example from -200 to +200 mmHg, preferably from -100 to +100 mmHg), whereby a fraction of penetration of the biological material, for example, the plasma, is caused to flow in an alternating path through the membrane wall in each direction to contact the complexing agent.

EXAMPLES The following examples are set forth to provide those of ordinary skill in the art with a description and complete disclosure of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider as their invention are not proposed. to represent that the experiments below are all or only the experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some experimental errors and deviations should be considered. Unless indicated otherwise, the parts are parts by weight, molecular weight is the weight average molecular weight, temperature is in degrees Centigrade, and the pressure is or is almost atmospheric. EXAMPLE 1 Spherical beads composed of a silicate derivative are used in a cylindrical metal chromatography apparatus. The beads are prepared by affinity chromatography by impregnating the beads with PTA prior to placement within the chromatography housing apparatus. The beads of approximately 5 mm were impregnated with 25% by weight of H3PW1 O4o by the incipient moisture method. The coated beads are dried in a vacuum oven for Remove excess water. Finally, these coated beads are calcined at 350 ° C for 1 hour in nitrogen and 4 hours in air. The coated spherical beads are cooled to about 37 ° C, placed in the chromatography column apparatus, and equilibrated with 20 mM sodium phosphate to pH 7. A preparation of human plasma is added to the mixture, which is passed over the column at a sufficient rate for the binding of the prions to immobilized PTA. An aliquot of the purified plasma is then tested for the presence of prions using Western blot analysis. EXAMPLE 2 A substituted amine nylon membrane is made as follows, as described in the U.S. Patent. No. 4,361, 484, which is incorporated herein by reference. The selective prion antibody is coupled to the substituted amine membrane for use in filtration. The IgG portion of the α-PrPSc molecule is immobilized on the membrane using glutardialdehyde. The membrane is treated with 2.5% glutardialdehyde solution in 0.1 M phosphate buffer at 9H = 6.8, rinse with distilled water and dry. A solution containing α-PrPSc is dissolved in 0.1 mM phosphate buffer (pH = 6.0, 4 ° C) and the solution is incubated with the treated membrane. The antibody coupling with the membrane is allowed to take place for 15 hours at 4 degrees C. After incubation, the membrane is rinsed in distilled water, and the unbound a-PrPSc is collected with the first rinse water for use later. The membrane with bound a-PrPSc is then used in a hemofiltration apparatus to remove the PrPSc protein from the blood human The attached a-PrPSc filter is placed in a hemofiltration device, such as that described in U.S. Patent Nos. 5,858,238, 5,855,782 and 5,851, 394 each of which is incorporated herein by reference. EXAMPLE 3 A method for purifying the blood of a living animal which includes passing the blood through an extracorporeal bypass device. A bypass device is constructed for this purpose with hollow cellulosic fibers having an ID of 200 u and an inner wall of 30 micrometers in thickness. The formed cartridge has an inside surface area of 0.6 square meter tube. The hollow fibers are perforated with a solution of anti-PrPSc antibody in saline regulated with borate to maintain pH 8.5. After three hours of recirculation, the cartridge is extensively rinsed with saline. The solution is analyzed for anti-PrPSc antibody by immunodiffusion technique, known in the art, before and after the recirculation step in order to evaluate the degree of antibody uptake by the fiber wall. Subsequently, the cartridge is dried in a stream of nitrogen, placed in a plastic bag and sealed. A cartridge containing 1, 000 asymmetric hollow fibers having a cut pore size at 500,000 Daltons MW and an inside diameter of 150-200 microns is rinsed by pumping saline solution through the fiber walls from the outside in the mode of "inverse" ultrafiltration. Therefore, 1% of a solution of human serum albumin is pumped through the fiber wall in the same in order to deposit a monomolecular layer of albumin on the porous surface on the outer side of the tubular membrane. Therefore, the anti-PrPSc antibody solution is filtered through the hollow fiber walls in the "reverse" direction to allow the antibodies to bind directly on the outer side of the membrane or in close proximity to the membrane in the membrane. external region. The activity of the solution before filtration and that of the filtrate leaving the lumen is analyzed for anti-PrPSc antibody. The balance gives the amount of immunological activity deposited in the cartridge. After cleaning the lumen space of the hollow fiber with streptomycin and saline, the cartridge is placed in a plastic bag and sealed. The cartridges thus prepared are used in extracorporeal bypasses. EXAMPLE 4 The super-paramagnetic polystyrene beads containing magnetite (average diameter of 0.8 mu.m 67% magnetic content, Sigma Chemical Co.) are coated overnight at room temperature with a native PrPSc rinsed of monoclonal antibody. The resulting antibody-coated beads are placed in an apparatus comprised of a container such as a syringe body containing a support matrix surrounded by a coiled wound copper coil, which is connected to an adequate supply of alternating electrical current through of adequate interruption means. A 10 ml sample of bovine blood containing prion protein was added to the beads and incubated for 10 minutes (in the presence of the applied magnetic field). An AC / C field (50 Hz, 50 volts) was applied to the coil to generate a magnetic field, and the magnetic beads were isolated from the blood. The complexed beads tested for the presence of prions by immuno-fluorescence staining techniques using a fluorescent anti-PrP FITC conjugate (Bradsure Biochemicals Ltd). The prion complexes detectable using the procedure clearly indicate that the specific capture of the prions is achieved. Although the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, steps or stage of the process, to the purpose, spirit and scope of the present invention. All of such modifications are proposed to be within the scope of the appended claims thereto.

Claims (15)

1. CLAIMS 1. A method for separating prions from a sample, comprising the steps of: contacting a sample in a liquid flowable state with a solid substrate comprised of a prion complexing agent; and allowing the sample to remain in contact with the substrate for a time such that the prions in the sample bind to the substrate.
2. 2. The method according to claim 1, characterized in that the complexing agent is selected from the group consisting of a heteropoly acid or salt thereof, a metal salt of phosphotungstic acid, a peptide and an antibody.
3. 3. The method according to claim 1, characterized in that the complexing agent is an antibody and the antibody binds selectively to PrPSc.
4. The method according to claim 1, characterized in that the sample comprises a human body fluid,
5. 5. The method according to claim 4, characterized in that the human body fluid comprises human blood.
6. 6. The method according to claim 4, characterized in that the substrate is in the form of spherical beads comprised of a ferromagnetic metal having a polymer coated therein and wherein the complexing agent is a metal salt of phosphotungstic acid.
7. 7. The method according to claim 6, characterized because it also comprises: removing the substrate from contact with human body fluid by the application of magnetic energy.
8. 8. A device for removing prions from a sample, comprising: a water insoluble polymer; and a prion complexing agent coated on a polymer surface.
9. The device according to claim 8, characterized in that it further comprises: a ferromagnetic metal wherein the polymer is coated in the ferromagnetic metal and the complexing agent is selected from the group consisting of phosphotungstic acid or salts thereof and an antibody that binds to PrPSc.
10. 10. A prion-free liquid, characterized in that it is subjected to the method of claim 1. eleven .
11. The prion-free liquid according to claim 10, characterized in that the liquid is further characterized by being derived from a human.
12. 12. The prion-free liquid according to claim 1, characterized in that the liquid is human blood.
13. 13. The prion-free liquid according to claim 1, characterized in that the liquid is human plasma.
14. 14. A flow through the device to remove prions from a liquid sample, comprising: a housing through which a liquid sample flows; and substrate surfaces comprised of prion complexing agent.
15. 15. The device according to claim 14, characterized in that the housing is cylindrical and the substrate is polymer beads having sodium phosphotungstate coated on its surfaces.