MXPA05010726A - Prion protein binding materials and methods of use - Google Patents

Abstract

Prion protein binding materials and methods for using the binding materials to detect or remove a prion protein from a sample, such as a biological fluid or an environmental sample. The binding materials are capable of binding to one or more forms of prion protein including cellular prion protein (PrPc), infectious prion protein (PrPsc), recombinant prion protein (PrPr), and proteinase resistant prion protein (PrPres). Prions from various species, including humans and hamsters, are bound by the binding materials.

Description

WO 2004/090102 A2 E lili! Itlfllil 11 IIES1 Lilli lltll lliiffl I ff fll ULLL lllil lllll iilll llill llllít lili fill my ip CO. CR, CU CZ, D DK. DM, D ?. EC, lili, HG, LS, II, GH, GR, lili, Ili, IT. Ui, MC. NI., Pl .. l, RO, SE, SI, SK, GB, GD. Gli. Gil, ÜM, HR, III), ID, 11..1N, 1S. JP. KJ;, 1 R), O? PI (BE, BJ, CT, CG, CI, G ?, GN, GQ, GW, KG, KP, KR, KZ, C, IK1 R, LS, IT IV, M, MD, MI, MR, NE, SN, ID, TG) MG. MK, MN, MW. MX, MZ, N ?, NI, NO, NZ, OM, PG, PH. PL PT. RO, Rl !, SC, SD. SI :, SG, SK, SI., SY.TJ.1 M, TN.1 R, TT. TZ U ?, UG, US, UZ, VC, VN, YU, YA, ZM, Published: ZW - Hillumi mlernaiional h repon nd to be upon re ect? F that report (84) Designated States I urden otherwne mdtialed j'i / re \ erv ind irf regional p ie, non reludable):? RIPO (BW, Gl I, GM, KL LS MW, MZ, SD, SL, SZ.17, UG, ZM, ZW), hor rw o-lettet and oi er abbrevtano. refer to the lite "CuidHurasian (? M,? Z, BY. KG, KZ, MD, RU, l'J.TM.) Euro- ara e Note-, on and? bbiev iliirrti" uppearing al ¡he begtn- pean ( ? T, Bli BG, C:.. il, CY CZ, DE, DK, BUNDLE, IS, II, IR, riing ofetu h nj regulate l / ie PCI Gazetle MATERIALS OF UNION TO THE PROTEIN OF THE PRION AND METHODS OF USE FIELD OF THE INVENTION This invention relates to the field of protein binding and more particularly relates to materials that bind prion proteins and methods of using prion protein binding materials to detect or remove prions from samples biological BACKGROUND OF THE INVENTION The native or cellular prion protein "PrPc" is widely distributed in all mammals and has a particularly well-preserved amino acid sequence and protein structure. It is thought that infectious prions are composed of a modified form of the cellular normal prion protein (PrPc) and are called "PrPsc". Prions have some properties in common with other infectious pathogens, but apparently do not contain nucleic acid. Instead, it has been proposed that a post-translational conformational change is involved in the conversion of the noninfectious PrPc to the infectious PrPsc during which the a-helices are transformed to β-sheets. PrPc contains three a-helices and has a small β-sheet structure; in contrast, the PrPsc is rich in ß-sheet. It is believed that the conversion of PrPc to PrPsc leads to the development of transmissible spongiform encephalopathies (TSEs) during which PrPsc accumulates in the central nervous system and is accompanied by neuropathological changes and neurological dysfunction. PrPsc is often referred to as the "pruritus lumbar" form of the prion protein, and is considered necessary, and possibly adequate, for the transmission and pathogenesis of these transmissible neurodegenerative diseases of animals and humans. Specific examples of TSEs include lumbar pruritus, which affects sheep and goats; bovine spongiform encephalopathy (BSE); transmissible mink encephalopathy, feline spongiform encephalopathy and chronic devastating disease (CWD). In humans, TSE diseases can present themselves as kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal insomnia and variant Creutzfeldt-Jakob disease (vCJD). Recently vCJD emerged in humans as a result of epidemic BSE in Britain and is most likely caused by the consumption of food products derived from cattle infected with BSE or "mad cow disease". An unknown number of people in the UK ingested food potentially contaminated with nervous from BSE-infected cattle during the mid-1980s until the early 1990s tissue. Because the incubation period for orally contracted disease may be longer than 20 years in humans, the true incidence of vCJD may not be evident for many years. To date, it is known that more than 150 people have contracted the disease, mainly in the United Kingdom; however, cases have been reported in Canada, France, Hong Kong, Ireland, Italy, and the United States. The export of contaminated bovine food products from the United Kingdom worldwide indicates a possible global presence of BSE and therefore the likelihood of vCJD. Consistent with these observations is the detection of BSE in most European countries, Japan, Canada, the United States and Israel. Consequently, the ability to detect and remove infectious prion protein from a variety of materials including food products is of great importance. A characteristic of all TSEs is the absence of a measurable immune response by the host to the agent. Consequently, no specific antibodies for TSCs have been identified to date. In addition, the absence of a known nucleic acid sequence prevents the use of diagnostic methods based on the polymerase chain reaction. Therefore, conventional serological tests can not be used to identify infected animals. Recently, improved techniques based on immunological methods have been used to identify PrPsc in brains from sacrificed animals.

In addition to the ingestion of infected products of bovine origin, blood transfusion and organ transplantation represent another mode of transmission of vCJD among humans. The risk of transmission of vCJD in humans by blood transfusion is currently unknown, but is based on data from experimental animal models including transmission from orally infected sheep experimentally with BSE and sheep naturally infected with lumbar pruritus. , seems to be a very likely possibility and has already been considered more likely for a human-to-human transmission of vCJD. Unlike other human TSEs, PrPsc is present in the linio-reticular system of patients with vCJD, therefore increasing the probability of the infectious agent found in blood and its transmission through blood transfusion. Other factors that raise concerns about the risk of transfusion transmission include the unknown, but presumably high, numbers exposed to BSE and the absence of a preclinical diagnostic test for vCJD. In addition, the virulence of vCJD seems to improve after adaptation of the species in primates and mice suggesting that human-to-human transmission may be more efficient than from cow to human. Therefore, there is an urgent need for methods to prevent the transmission of vCJD by blood transfusion. Such measurements may include the early identification of infected donors and their deferral, removal and inactivation of TSE agents in food and health products derived from the animal that are intended for consumption or applications in animals or humans, blood products derived from human and bovine, and organ transplants. Unfortunately, the infectivity of TSE is remarkably resistant to chemical and physical inactivation methods and a method of selective inactivation is elusive. Numerous materials that bind to prion protein have been identified. Pertinent combinatorial libraries have been selected for ligands that bind to the octapeptide repeat sequence (PHGGGWGQ) (SEQ ID NO: 1) found in all known proteins of the mammalian prion and a number of ligands were discovered, as described in PCT / US01 / 11150. Other materials include ligands that interact with the amyloid plaque for example, Congo Red (Ingrosso, L., et al., Congo Red Prolongs the Incubation Period in Scrapie-infected Hamsters, J Virology 69: 506-508 (1995)); 4-iodo, 4-deoxy doxorubicin (Tagliavini, F., et al., Effectiveness of Anthracycline Against Experimental Prion Diseases in Syrian Hamsters, Science 276: 1119-1122 (1997)); amphotericin B, porphyrins and phthalocyanines (Priola, S.A., et al., Porphyrin and Phthalocyanine Antiscrapie Compounds, Science 287: 1503-1506 (2000)); metals (Stockel et al., Biochemistry, 37, 7185-7193 (1998)); peptides that interact with PrP to form complexes (see U.S. Patent 5,750,361 to Prusiner et al., and Soto, C. et al., Reversion of Prion Protein Conformational Changes in Synthetic ß-sheet Breaker Peptides, Lancet, 355: 192- 197 (2000)); heparin and other polysulphated polyanions (Caughey, B., et al., Binding of the Protease-sensitive Form of Prion Protein PrP to Sulphated Glycosaminoglycan and Congo Red, J. Virology 68: 2135-2141 (1994)); antibodies (Kascsak, R.J., et al., Immunodiagnosis of Prion Disease, Immunological Invest. 26: 259-268 (1997)); and other proteins, for example plasminogen (Fischer, M.B. et al., Binding of Disease-associated Prion Protein to Plasminogen., Nature 408: 479-483 (2000)). Ion exchange chromatography has been used to purify blood components, such as hemoglobin, from contamination by the prion (U.S. Patent No. 5,808,011 to Gawryl et al.). However, the chromatographic material taught by Gawryl et al. it binds to hemoglobin, and the purified hemoglobin is subsequently collected by gradient elution. Currently, no material has been fully characterized or found to be capable of joining the prion from a variety of media. To date, human TSE diseases are 100% fatal Unfortunately, even when numerous compounds have been reported including amphotericins, sulfated polyanions, Congo Red dye and anthracycline antibiotics as presumed therapeutic agents, all have shown only a modest potential to prevent the spread of the prion, and none have been shown to have any effect on the removal of pre-existing prions from an infected host in a controlled clinical study. Therefore, there remains an urgent need for novel therapeutic agents.

It is thought that the assembly and disassembly of normally soluble proteins into conformationally altered forms and insoluble forms is a process causing a variety of other diseases, many of which are neurological diseases. The relationship between the onset of the disease and the transmission from the normal protein to the conformationally altered protein is understood unsatisfactorily. Examples of such insoluble proteins in addition to the prion include: β-amyloid peptide in amyloid plaques of Alzheimer's disease and cerebral amyloid angiopathy; deposits of a-synuclein in the Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in temporal frontal dementia and Pick's disease; superoxide dismutase in amyotrophic lateral sclerosis; and huntingtin in Huntington's disease. Frequently these highly insoluble proteins form aggregates composed of unbranched fibrils with the common characteristic of a β-folded sheet conformation. Methodologies that can easily separate or that can distinguish between two or more different conformational forms of a protein, for example, PrPc and PrPsc, are necessary to understand the conversion process and to find structures that will interact specifically with the form associated with the disease. . Current methodologies for separating or distinguishing between protein isoforms include: differential mobility in polyacrylamide gels in the presence of a chaotrope such as urea, for example, transverse urea gradient gels (TUG); differential sensitivity to protease treatment, e.g., proteinase K (PK) and detection of PK resistant digestion product of PrPsc referred to as PrPres; differential stability of temperature; relative solubility in non-ionic detergents; and the ability of fibrillar structures to bind to certain chemicals, eg, Congo Red and isoflavin S. However, an unmet need remains to identify additional prion binding materials. There also remains a need to identify materials with high binding affinity that are specific for the conformationally altered protein and especially for forms associated with the disease. These reagents could be useful to develop possible equipment for diagnosis, separation and purification of the various forms of protein, for the removal of infectious forms of the disease from therapeutic agents, biological products, vaccines and food products, and for therapy .

BRIEF DESCRIPTION OF THE INVENTION The materials that bind to the prion proteins and the methods for using the prion protein binding materials (hereinafter "binding materials") are provided. In some embodiments, the binding materials are poiimeric particles, preferably chromatographic resins, which bind selectivity and specificity to the prion analytes. In other embodiments, the binding materials are inorganic materials that bind with selectivity and specificity to the prion analytes. The binding materials are capable of binding to one or more forms of prion protein including prion cellular protein (PrPc), infectious prion protein (PrPsc), and recombinant prion protein (PrPr). Prions from different species, including humans and hamsters, are joined by the binding materials. Also provided are compositions containing the binding materials on a support such as column chromatography. The binding materials are useful for detecting, binding, isolating, removing, removing, extracting or separating a prion protein in or from a sample, such as a biological fluid or an environmental sample. Binding materials are used to remove all forms of prion protein from a sample or can be selectively chosen to detect or remove a particular form of prion protein. Therefore, the binding materials can be used to distinguish between infectious prion protein and non-infectious prion protein in a sample from patients suffering from TSEs of humans and animals suffering from lumbar pruritus, BSE and CWD. . In one embodiment, one or more prion proteins are removed from a biological fluid using the binding materials described in the present invention and then the purified or decontaminated biological fluid is delivered, or returned, to an animal or human. Hemodialysis techniques can be used in this modality. The binding materials are also useful for the detection of one or more prion proteins in a sample.

Another aspect of the invention provides a method for identifying additional binding materials, particularly specific binding materials for conformationally altered forms of proteins, some of which are involved in the development of diseases. Other features and advantages of the invention will be apparent from the following detailed description and preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1 D are photographs of Western blots showing the binding of endogenous PrPc from human plasma samples to prion binding materials and appropriate controls. Figure 2 is a photograph of a Western blot showing the binding of PrPsc from a brain homogenate with lumbar pruritus to prion binding materials and appropriate controls. Figure 3 is a photograph of a Western blot demonstrating the capture of PrPc by prion binding materials in samples containing human serum albumin and appropriate controls. Figures 4A-4B are photographs of Western blots demonstrating removal with a resin comprising an outstanding functional amino group of PrPres in human serum albumin.

DETAILED DESCRIPTION OF THE INVENTION The materials that bind to the prion proteins and the methods for using the prion protein binding materials are described above. Binding materials are polymeric materials, such as chromatographic resins or beds, or inorganic materials, such as aluminum oxide, which bind with specificity and affinity to prion proteins. The polymeric materials contain one or more of the following functional groups: a negatively charged portion; a positively charged portion; an uncharged portion and a hydrophobic portion. Preferably, the polymeric bonding materials have a functional group attached to a base structure of methacrylate or polymethacrylate matrix. The binding materials form a complex with a prion protein in a sample and are useful in methods for the detection, attachment, isolation, removal, removal, extraction or separation of a prion protein in or from a sample, such as a tissue, organ, or biological fluid derived from human or animal or an environmental sample. Methods for diagnosis or monitoring of prion disease in a human or animal, or tissue, organ, or biological fluid thereof are also provided. For example, the binding materials described in the present invention may be useful for detection or diagnosis of pathologies such as CJD, vCJD, GSS, fatal insomnia, lumbar pruritus, BSE and CWD and other TSEs by testing a biological sample, such as as whole blood, compositions or components derived from blood, cells, serum, plasma, plasma derivatives, cerebrospinal fluid, urine, tears; tonsils, brain, appendix and others. The importance to detect prion infection in an animal or individual is readily understood before the donation of blood, tissue, or organs. The binding materials are particularly useful for the removal of prion protein from a sample or biological fluid, such as whole blood, blood components, serum, plasma, plasma derivatives, and the like. Removal of the prion is essential when the biological fluid is transmitted to another animal or human, such as in a blood transfusion or in the administration of a blood product such as a clotting factor. The binding materials are useful for removing or detecting all different forms of prion protein from the sample or they can be selectively chosen to remove or detect a particular form of prion protein and therefore can be used to distinguish between protein Infectious prion and non-infectious prion protein in the sample.

Definitions The terms "a", "an" and "the" as used in the present invention are defined to mean "one or more" and include the plural unless the context is inappropriate.

The term "3F4 antibody" refers to a monoclonal antibody specific to the native forms of PrPc, but not to the native forms of PrPsc or PrPres. The antibody has specificity for denatured forms of the PrPc, PrPsc and PrPres of hamster and human. As used in the present invention, the terms "blood-derived compositions", "blood components" and "blood compositions" are used interchangeably and are intended to include whole blood, erythrocyte concentrate, plasma, serum, platelet-rich fractions and platelet-poor fractions, platelet concentrates , leukocytes, blood plasma precipitates, precipitates and supernatants from the fractionation of blood plasma, immunoglobulin preparations including IgA, IgE, IgG and Ig, concentrates of the purified coagulation factor, fibrinogen concentrate, plasma fractionation intermediate, albumin preparation, or various other substances which are derived from human or animal blood. The terms also include proteins derived from purified blood prepared by any of several methods common in the art including ion exchange, affinity chromatography, gel permeation, and / or hydrophobic chromatography or by differential precipitation with alcohol or polyethylene glycol. The term "PrPc" refers to the native molecule of prion protein, which is naturally and widely expressed in the body of mammals. Its structure is highly conserved and is not associated with a disease state. The term "PrPsc" refers to the conformationally altered form of the PrPc molecule that is believed by those skilled in the art to be associated with diseases such as TSE / prion diseases, including vCJD, CJD, kuru, fatal insomnia, GSS, lumbar pruritus, BSE, CWD, and other TSEs, including rare TSEs of captive and experimental animals. PrPsc has the same amino acid sequence as normal, cellular PrPc, but has converted part of the a-helix to β-folded sheet and is associated with a disease state. The term "PrPres" refers to the proteinase-resistant derivatives of the PrPsc protein of molecular weight 27-30 kDa that remain after partial digestion of the PrPsc with proteinase K (PK). The term "PrPr" refers to the prion protein expressed by recombinant technology. The term "PrP" refers to prion protein in general. The term "bed" refers to a solid phase particle or granule to which a reactive group or linking components can be attached. Beds that have an irregular shape as well as beds that have spherical, oval, roller, or even angular shapes are included within the scope of this term. The term "resin" refers to a polymeric medium.

The term "polymeric" as used in the present invention describes a compound or molecule composed of various small, repetitive chemical or structural units (monomers).

Samples The term "sample" is used in the present invention to denote any solution, suspension, extract, composition, preparation, product, component, tissue, organ, cell, or other entity that comes into contact with the binding materials. prion in accordance with the methods of conformity with certain aspects and modalities of the present invention. Samples in accordance with certain aspects and embodiments of the present invention include, but are not limited to, biological samples, food products, environmental samples, or water samples. Biological samples include, but are not limited to: samples derived from blood; brain derived samples; body fluids, such as, but not limited to, blood, plasma, serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, or semen; biological extracts, such as collagen extracts, gland extracts, or homogenates or tissue extracts. Biological samples are derived from humans or animals, including but not limited to bovine, ovine, porcine, equine, murine, or Cervidae animals. Samples derived from blood include, but are not limited to, platelet concentrates, plasma protein preparations, immunoglobulin preparations, fibrinogen preparations, factor Xlll preparations, thrombin preparations, factor Vlll preparations, von Willebrand factor preparations , preparations of protein C, or preparation of activated protein C. Samples in accordance with certain aspects and embodiments of the present invention also include, but are not limited to, pharmaceutical compositions, therapeutic compositions, compositions and cosmetic products, food or food products, or nutritional supplement compositions. Examples of food product samples include, but are not limited to, gelatin, jelly, milk, milk products, collagen, or an infant formula. Samples, in accordance with certain aspects and preferred embodiments, include protein solutions comprising various proteins, including, but not limited to, human or animal serum albumin. For example, samples include, but are not limited to, therapeutic products containing human serum albumin; preparations of human or animal serum albumin; or preparations containing human or animal serum albumin as a stabilizer. Samples according to certain preferred embodiments of the present invention may contain a human or animal serum albumin at concentrations of up to about 50% (w / v), or from about 1% to about 50%, or about 5%. % to approximately 25%. In one aspect, the present invention, in its preferred embodiments, unexpectedly and advantageously allows one to remove, separate, or bind prion proteins from or in samples with high concentrations of proteins, particularly blood proteins, such as albumin. of serum. Environmental samples include but are not limited to soil, sewage or water, such as water from a source such as a stream, river, aquifer, well, facility for water treatment or recreational water. Samples include, but are not limited to, liquid samples, solid samples, or colloidal samples. A solid sample can be extracted with an aqueous solvent, an organic solvent or a critical fluid, and the resulting supernatant can be contacted with the binding materials. Examples of solid samples include, but are not limited to, animal products, particularly those that have been exposed to agents that transmit prions, for example, bone meal derived from bovine sources, brain tissue, corneal tissue , fecal matter, bone meal, beef byproducts, sheep, sheep byproducts, deer and elk, deer and elk byproducts, and other animals and products derived from animals.

Materials The binding materials provided in the present invention bind to the peptides or polypeptides derived from the prion protein, or the entire prion molecule and can be used in a variety of methods for separation, including but not limited to, chromatography, such as, but not limited to, thin layer, column and batch chromatography; separation in solid support and membrane; separation in reactor; magnetic separation; immunoseparation; and colloidal separation. In a preferred embodiment, the binding materials are contained in a column such as a column chromatography, and a sample is introduced and allowed to pass through the column so that the prion proteins in the sample bind to the materials of union and are retained in the column. The other components of the sample pass through the column and can be collected. It should be understood that the use of the binding materials described in the present invention is not limited to batch chromatography or column chromatography. A variety of configurations, modifications and variations of the use of the binding materials for the binding of the prion proteins are foreseen and fall within the scope of the present invention. Such variations and modifications include, but are not limited to: batch procedures, continuous procedures; mobile bed chromatography procedures; procedures at low, medium, or high pressure; or small, medium, or large-scale procedures. In alternative embodiments, the bonding materials are found in a membrane, fiber, bed, impregnated in a non-woven mesh, coating of a fiber, contained within a filter housing, and the like.

Inorganic Components In a first embodiment, the bonding materials comprise an inorganic compound or component, such as, but not limited to, aluminum or silica. Preferably, aluminum is an aluminum oxide and silica is fuming silica. More preferably, the organic compounds are AI2O3; o SiO2. These bonding materials can be provided in a variety of ways, including but not limited to, a bed or resin. The binding materials can be used in a variety of separation processes, and can be contained in, or adapted in, a column chromatography, a membrane, or any device or implement for suitable separation, or they can be used in a process in batch, or can be used in any separation procedure that allows contact of the material with a sample under conditions that allow the formation of the prion and prion binding material. Binding materials containing inorganic compounds may comprise a variety of functional groups. The functional groups are hydrophilic, such as positively charged, negatively charged, uncharged or neutral, hydrophobic, amphiphilic, or combinations thereof. The specific functional groups are described in greater detail below. It should be understood that the functional groups may be inherently present in an inorganic compound, or the inorganic compound may be further modified to include the functional groups. The functional groups include organic and inorganic functional groups.

Polymeric Components In a second embodiment, the bonding materials comprise polymeric materials or components and preferably include a polymeric matrix, also referred to as a polymer matrix base structure. Optionally, one or more functional groups are attached to the polymer matrix. In a preferred embodiment, the polymeric materials are resins, preferably, chromatographic resins. The polymer matrix base structure preferably is a methacrylate base structure, such as that found in, but not limited to, a commercially available TSK ™, and TOYOPEARL ™ or FRACTOGEL ™ resin (Tosoh Bioscience, Montgomeryville, PA). The s includes, but is not limited to, positively charged, negatively charged, uncharged, hydrophobic functional groups or combinations thereof. Specifically, preferred functional groups are described in greater detail below. The bonding materials take any form, or are processed, shaped, adapted into, or applied to any solid support including, but not limited to, a bed, membrane, cartridge, filter, rod, microtitre plate, tube test, solid powder, molded or extrusion molded module, mesh, magnetic particle composite, or any other solid material initially coated with a substance such as polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polyacrylate, polyethylene terephthalate, rayon, nylon, poly (vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Alternatively, substances that form gels are used, such as proteins (for example, gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Also suitable are polymers which form various aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain alkyl ammonium salts (12-24 carbon atoms) and the like. The bonding materials are optionally dispersed along these components. The binding materials are preferably in the form of a particle, in granular form or in the form of a globule. The particulate bonding materials preferably have a particle size range, or bed, of about 1 μm to 500 μm, and more preferably about 20 μm to 150 μm.

Functional Groups The prion-binding materials according to certain aspects and embodiments of the present invention comprise functional groups. The term "functional group" is used in the present invention to denote groups, subgroups, or chemical substructures that impart chemical, physical, or physicochemical characteristics to a molecule or a material. The functional groups described in the present invention include, but are not limited to, hydrophilic functional groups, such as positively, negatively or uncharged or neutral, or hydrophobic. Amphiphilic or multifunctional functional groups are also contemplated and fall within the scope of the present invention. The functional groups include organic and inorganic functional groups. Preferred functional groups contain amine, phenyl or sulfite groups. A preferred amine is a primary, secondary, tertiary, or quaternary ammonium ion such as dimethylaminoethyl (DMAE) or trimethylaminoethyl (TMAE). Other exemplary functional groups include, but are not limited to: -CH2-CHOH-CH2NH2; -C6H5; - (CH2) 3-CH3; -CH2-CH2-N + H (C2H5) 2; -SO2-CH2-CF3; -CH2-CH2-N + H (CH3) 2; -CH2-CH2-N + (CH3) 3; -SO32. "In addition, useful functional groups include sulfonyl groups and tresyl groups Although not intended to be limited by the present disclosure, it is believed that prion proteins have three different binding regions that bind to positively charged functional groups, functional groups negatively charged, and hydrophobic functional groups, respectively Therefore, the use of one or more binding materials, each including one or more types of functional groups, is provided for increased or more specific identification or removal of the prion. From a sample When two or more bonding materials are used, a sample is contacted with the two or more bonding materials simultaneously or in succession in any order, therefore, preferably the bonding materials are composed of two or more binding materials each containing either a positively charged functional group, a functional group and actively charged, an uncharged functional group or a hydrophobic functional group. When the binding materials are in particle form and column chromatography is used, each different type of binding material can be found in the same column or in different columns. In a more preferred embodiment, three binding materials are used, one having a positively charged functional group, one having a negatively charged functional group, and one having a hydrophobic functional group. As used in the present invention, the term "positively charged functional group" refers to any chemical moiety carrying a positive net charge. Non-limiting examples of positively charged functional groups include amino-containing groups such as primary amines, diethylaminoethyl, dimethylaminoethyl, trimethylaminoethyl, and quaternary amino groups. The term "negatively charged functional group" refers in the present invention to any chemical moiety carrying a negative net charge. The term "non-charged functional group" refers in the present invention to any chemical portion that is neutral or that carries no charge. Non-limiting examples of negatively charged functional groups include sulphite-containing groups. In addition, the term "hydrophobic functional group" refers to any group that resists being wetted by water, including alkyl, aromatic, siloxane and fluorinated functionalities. Non-limiting examples of hydrophobic functional groups are phenyl and butyl-containing groups. The term "amphiphilic functional group" refers to a group that is both hydrophobic and hydrophilic.

In certain aspects and embodiments of the present invention, the prion binding material contains a positively charged functional group, a negatively charged functional group, a non-charged or neutral functional group, a hydrophobic functional group, one or both. An example of a negatively charged functional group is a group containing sulfite. An example of a positively charged functional group is an amino group. An example of an uncharged functional group is a phenyl or butyl group. Examples of a hydrophobic functional group are a phenyl group or a butyl group. In accordance with certain aspects and embodiments of the present invention, the use of the amino groups, including primary, secondary, tertiary, or quaternary amino groups in the binding materials are particularly advantageous for binding to the prion. However, the use of various groups, depending on a particular prion protein, a sample, and conditions are provided under which a sample and a binding material are contacted, and fall within the scope of the present invention. Optionally, a plurality of different materials are used in the bonding materials, such as laminates, for example, to impart various desirable properties to the bonding materials. For example, protein coatings, such as gelatin, are used to prevent non-specific binding and improve detection of the signal or the like.

Functionalization of the surface and spacers In a prred embodiment, the bonding materials possess a variety of functional groups on their surface. It should be understood that the functional groups may be inherently present on the surface of a binding material, or may be added to the surface of the binding material by methods known to one skilled in the art. The manner of linking a wide variety of groups or compounds to various surfaces is well known and is widely illustrated in the literature. The functional groups are for the binding of prions, in accordance with the methods described in the present invention, for linking additional functional groups, or for any modification of any physical, chemical, or physical-chemical properties of a material, such as, but not limited to, their ionic or hydrophobic properties. Functional groups that may be present on the surface of prred binding materials include, but are not limited to, carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups, epoxy and the like. In a prred embodiment, the functional groups may include spacer groups. Spacers are groups to provide a space or a distance between the surface of a material, also rred to as a matrix or a support, and a functional group. The spacers are prably composed of carbon, nitrogen, or oxygen atoms. In one aspect, a spacer is used to advantageously alter the prion binding properties of a prion binding material. According to certain embodiments, the spacers are up to 20 atoms in length, or up to 15 atoms in length, or from 5 to 10 atoms in length. Prably the spacers are composed of, but are not limited to, alkyl groups, polyethylene glycol (PEG), carbohydrate groups, amino acids, peptides of up to 20 amino acids in length, or peptides of 1 to 10 amino acids in length or mixtures thereof. More prably, the spacers contain combinations of alkyl and PEG groups.

Commercially available chromatography resins Prably, the binding materials are one or more of the following commercially available chromatography resins: FRACTOGEL ™ EMD resins; TOYOPEARL ™ Amino, Butyl, Phenyl, or AF-Tresyl; or TSK-GEL ™ Amino, Phenyl or DEAE. More prably, the bonding materials include, but are not limited to: FRACTOGEL ™ EMD TMAE, FRACTOGEL ™ EMD SO32", FRACTOGEL ™ EMD DMAE, Toyopearl ™ Amino, TSK-GEL ™ Amino, TSK-GEL ™ Phenyl, TSK-GEL ™ DEAE, TOYOPEARL ™ Butyl, TOYOPEARL ™ Phenyl, aluminum oxide, TOYOPEARL ™ AF-Tresyl, and silica resins In a more prred embodiment, the bonding material is TOYOPEARL ™ Amino, TSK-GEL ™ Amino, TSK-GEL ™ Phenyl or FRACTOGEL ™ EMD SO32". The use of other commercially available resins and supports for chromatography, including inorganic supports, is contemplated and falls within the scope of the present invention. In a prred embodiment of the present invention, the binding material includes a polymethacrylate, a hydroxyl polymethacrylate, or an AMINO 650 ™ resin (Tosoh Biosciences), and an amino group, such as a primary amine, a secondary amine, or a tertiary amine. The binding material according to a prred embodiment may additionally include a spacer of a formula O-R-O-CH2-CHOH-CH2, wherein R is 1-10 carbons in length. The binding material is optionally applied to or adapted on a solid support, such as a bed, membrane or resin for chromatography.

Identification of bonding materials In addition to the bonding materials previously established, additional bonding materials can be identified as follows. The binding materials are selected for their ability to bind to the prion analytes. The terms "analyte" or "analytes" as used in the present invention r to a multitude of molecules, including, but not limited to, a protein, a polysaccharide, and any aggregate or combination thereof. The binding materials are incubated with a sample known to contain a prion protein, the unbound protein is removed, and the binding protein is detected using conventional methods such as by a specific antibody labeled for the prion protein.

The binding materials to which the analyte binds are identified as suitable binding materials. Controls without primary antibody or secondary antibody are also used to remove nonspecific binding materials. In a preferred embodiment, the analyte of the prion to which the identified binding materials bind is a prion protein that is found in blood or brain samples derived from a human or animal. More preferably, the analyte is found in blood or blood products. It is further preferred that the analyte be associated with, or a causative factor of, a TSE in the human or animal.

Use of binding materials to remove prions The binding materials that bind to prion proteins or fragments of prion proteins are useful for a variety of analytical, preparative, and diagnostic applications. In some embodiments, the binding materials contain a solid phase, or solid surface, in the form of a bed or membrane that can be used to bind and remove prion proteins or peptides from a sample. The binding material is allowed to come in contact with a sample, such as a biological fluid, under conditions suitable for causing the formation of a prion-binding material complex, and the prion protein in the sample is bound to the binding material. . The binding material is then separated from the sample, thereby removing the prion protein bound to the ligand from the sample.

Methods for using beds and membranes for the binding protein are well known in the art such as those described in the U.S. Patent. No. 5,834,318 to Baumbach et al. and PCT / US01 / 11150. In some embodiments of the present invention, substantially all of the prion proteins are removed from a sample. By "substantially all" it is understood that the concentration of prion protein was significantly reduced. In other words, the transfer of all or a portion of the sample to an otherwise healthy patient carries a risk of low prion infection, acceptable in public health guidelines. Substantially all the prion proteins can be removed from a sample using, simultaneously or sequentially, a particular binding material or multiple binding materials. When multiple binding materials are used, it is preferable, as described above, to use two or more binding materials, each containing a positively charged functional group, a negatively charged functional group, or a hydrophobic functional group. In a more preferred embodiment, two or more binding materials are used, each containing a negatively charged functional group or a hydrophobic functional group. A sample is contacted with the two or more bonding materials in succession in any order. In a preferred embodiment, three binding materials are used, each containing one of a positively charged functional group, a negatively charged functional group, and a hydrophobic functional group.

In other modalities, only the particular prion materials are removed from a sample. For example, only infectious prions (PrPsc) can be removed from a sample or only non-infectious prions (PrPc) can be removed from a sample. An important discovery described in the present invention is the identification of a multitude of binding materials having different prion specificities. Table 4 shows various binding materials and their specificities for non-infectious and infectious hamster and human prions. Preferred binding materials for the selective removal of human PrPsc contain an amino group such as that contained in the Toyopearl ™ Amino-650M or TSK-GEL ™ -Amino 750C chromatographic resin or functional equivalents thereof or contain a phenyl group such as that contained in TSK-GEL ™ Phenyl-5PW or functional equivalents. Preferably, the binding materials are beds packed in a column, such as a column chromatography. A solution, homogenized or suspension of the sample is then passed through the column, either due to the force of gravity or under pressure, such as in a high-pressure liquid column chromatography. The prion protein in the sample will bind to the binding materials described in the present invention in the column. The sample that passes through the column is collected and is free from contamination of the prion or at least has a reduced level of prion material.

Once the sample passes through the column, the bound prion protein can be eluted and collected for analysis, or if desired, for diagnostic or prognostic purposes. If it is desired to remove the prion protein from the binding material, the mobile phase of the column can be changed first to a pH regulator that barely removes the bound contaminants to rinse the column. Subsequently the prion protein was removed from the column by boiling or by the addition of a solution containing strong detergents such as Sarkosyl detergent (sodium lauryl sarcosinate, Shelton Scientific-IBI, Shelton, CT) or sodium dodecyl sulfate (SDS). ), chaotropic agents, such as guanidinium hydrochloride, or agents having a low pH, such as acetic acid or by chemical modification of the binding ligand, such as, for example, acetylation of an amino group. Examples of biological samples from which prions can be removed include, but are not limited to, whole blood, compositions or components derived from blood, serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, semen, or compositions derived from the brain from humans or animals. Other biological samples include those that contain collagen or gland extracts. In one embodiment, the prions are removed from the blood of a human or animal by the use of a hemodialysis circuit containing one or more binding materials described in the present invention. In this embodiment, the blood was removed from the human or animal, was directed to a device that contained one or more of the binding materials described in the present invention, wherein the prion proteins were removed from the blood according to they joined the binding materials, and the prion-free or reduced-prion blood went back to the human or animal again. The prion proteins can also be removed from a biological sample such as a food product (either for animal or human consumption) using the binding materials described in the present invention. For example, the sample may contain a derivative of animal material or obtained from an animal, including, but not limited to, a cow, a sheep, a pig, a horse, a mouse, a hamster, or a cervidae animal. Alternatively, the sample material can be referred to as a human; bovine; ovine; porcine; equine; murine, such as derived from material derived from mouse and hamster; and cervidae, such as deer and elk. The animal-derived materials from which the prion proteins can be removed in accordance with methods according to certain aspects and embodiments of the present invention include, but are not limited to, gelatin, jelly, milk, collagen, and formula for infant The sample from the current one can remove the prion proteins according to the methods according to certain aspects and embodiments of the present invention may also include, but are not limited to, pharmaceutical compositions, therapeutic compositions, nutritional supplement compositions , food, or cosmetic compositions.

The samples, in accordance with the preferred embodiments, are protein solutions and contain various proteins, including, but not limited to, human or animal serum albumin. For example, samples may be, but are not limited to, plasma protein preparation containing human serum albumin as a stabilizer, immunoglobulin preparations, fibrinogen preparations, factor Xlll preparations, thrombin preparations, factor preparations Vlll, preparations of von Willebrand factor, preparations of protein C, preparations of activated protein C, or preparations of any combination or variation of the preceding; Therapeutic products containing human serum albumin; preparations of human or animal serum albumin; and diluted protein preparations containing human or animal serum albumin as a stabilizer. Samples, in accordance with the preferred embodiments, contain a human or animal serum albumin at concentrations of up to about 50% (w / v), or from about 1% to about 50%, or from about 5% to about 25% In one aspect, the present invention, in its preferred embodiments, unexpectedly and advantageously allows one to remove, separate, or bind prion proteins from or in samples with high concentrations of proteins, particularly blood proteins, such as albumin. of serum. The binding materials described in the present invention are also useful for the removal of prion proteins from environmental samples such as water from a source such as a stream, river, aquifer, well, water treatment facility. or recreational water.

Use of binding materials to detect prions The binding materials described in the present invention are also useful in a method for detecting the presence of or for quantifying a prion protein or peptide in a biological or environmental sample. Biological samples in which prion proteins are detected include, but are not limited to, whole blood, compositions or components derived from blood, serum, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, semen, compositions derived from brain, feces, or extracts or homogenates of collagen, glands, tissues (such as an amygdala or appendix), or organs. Both qualitative and quantitative detection methods are envisaged and fall within the scope of certain aspects and modalities of the present invention. As described above with respect to the removal of the prion protein, the binding materials are also useful for the detection of prion proteins in materials derived from animals used as food products. For the purposes of detection, the term "animal-derived materials" refers to the materials described above as well as materials that contain animal parts such as muscle, connective tissue or organ tissue. Animal-derived materials additionally include, but are not limited to, bone meal, beef, beef byproducts, sheep, sheep by-products, elk, elk by-products, pork, pork by-products, sausages, hamburgers, and baby food . The binding materials described in the present invention are also useful for detecting prion proteins in environmental samples such as those described above and soil extracts. Due to the discovery of a multitude of binding materials with different prion-binding characteristics, the binding materials are useful in methods for distinguishing between infectious and non-infectious prions in a particular sample or between samples. Therefore, methods for the diagnosis and prognosis of prion diseases in a human or animal are provided. Prion diseases include, but are not limited to, transmissible spongiform encephalopathies (TSEs) such as lumbar pruritus, which affects sheep and goats; bovine spongiform encephalopathy (BSE), which affects cattle; transmissible mink encephalopathy, feline spongiform encephalopathy and chronic devastating disease (CWD) of cariacus, white-tailed deer, black-tailed deer and elk; kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal insomnia and a variant Creutzfeldt-Jakob disease (vCJD), which affects humans. In one embodiment, a sample is passed through a binding material that has a high specificity for a PrPsc compared to a PrPc and the bound PrPsc of the prion is detected using the methods described below. The same sample can then be passed through a binding material having a high specificity for the PrPc compared to the PrPsc and the bound PrPc is detected using the methods described below. The specificities of various binding materials for PrPc and PrPsc are given in Table 4. Preferred binding materials for the selective detection of human PrPsc contain an amino group similar to that contained in the compound TOYOPEARL ™ TSK-GEL ™ -Amino 750C Amino-650M or the compound TSK-GEL ™ -Amino 750C or contains a phenyl group similar to that contained in TSK-GEL ™ Phenyl-5PW (all resins from Tosoh Biosciences, Montgomeryville, PA). When the method provided in the present invention is used to detect a prion in a sample, the sample is contacted with a binding material under suitable conditions to cause the formation of a complex between the prion protein and the binding material. . The complex is then detected by conventional methods, thus detecting the presence of the prion in the sample. For example, the binding material (a first ligand) can be labeled with a detectable label. As an alternative example, the complex is detected by labeling a secondary ligand such as an antibody or other protein, combining the secondary ligand labeled with the sample in the presence of the binding material, and detecting the labeled secondary ligand-prion complex of Union.

The secondary ligand can be attached to the prion either covalently or non-covalently. A wide variety of brands and conjugation techniques are known and are reported extensively in both the scientific literature and the patent literature. In one embodiment, the secondary ligand is marked during its production. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent portions, magnetic particles, and the like. Included within the scope of certain aspects and embodiments of the present invention are methods of detection, qualitatively and quantitatively, of a prion protein linked to a prion protein binding material, or a protein complex of the prion-material for attachment to the prion. The prion-binding material that forms a complex can be packed or adapted into a column, a membrane, or a filter, or attached or adapted to, or immobilized on a solid support. Also included within the scope of certain aspects and embodiments of the present invention are methods for detecting, qualitatively and quantitatively, a bound prion protein and subsequently released from a material for binding to the prion. Detection can proceed by any method including immunoblot, Western analysis, gel mobility change assays, tracing radioactive or bioluminescent markers, nuclear magnetic resonance, electron paramagnetic resonance, stopped flow spectroscopy, column chromatography, capillary electrophoresis, or other methods that trace a molecule based on an alteration in size or charge, or both. The secondary-prion ligand complex may or may not be separated from the binding material before detection. Other assay formats include, but are not limited to, liposome immunoassays (LIAs), which are liposomes designed to bind to specific molecules (eg, secondary ligands) and release reagents or encapsulated labels. Subsequently, the chemicals released are detected in accordance with standard techniques. Non-radioactive brands are often linked by indirect methods. Generally, a secondary ligand molecule (eg, biotin) is covalently bound to the binding material (first ligand). The secondary ligand then binds to a tertiary ligand molecule (e.g., streptavidin) which is either inherently detectable or covalently linked to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Many secondary and tertiary ligates can be used. Where a secondary ligand has a natural tertiary ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled tertiary ligands, which occur naturally. Alternatively, any haptenic or antigenic compound may be used in combination with an antibody. The particular label or detectable group used to detect the materials of the binding-prion complex is not critical. The detectable group can be any material that has a detectable physical or chemical property. Said detectable marks have been well developed and, in general, any useful mark in said methods can be applied to the present method. Therefore, a brand is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. Useful labels include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, and the like), radiolabels (e.g., 3H, 125l, 35S, 14C, or 32P), enzymes (e.g., LacZ (beta galactosidase ), CAT (chloramphenicol acetyltransferase), horseradish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either in an EIA (enzyme immunoassay) or in an ELISA (enzyme-linked immunosorbent assay)), and colorimetric marks such as colloidal gold or colored glass or plastic beds (for example polystyrene, polypropylene, latex, etc.). The label can be coupled directly or indirectly to the desired component of the assay in accordance with methods well known in the art. As indicated above, a wide variety of brands can be used, with the choice of brand depending on the required sensitivity, ease of compound conjugation, stability requirements, available instrumentation, and disposal provisions. Secondary ligands can also be conjugated directly to the signal generating compounds, for example, by conjugation with an enzyme or fluorophore. The enzymes of interest as labels will be mainly hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, for example, luminol. Methods for detecting marks are well known to those skilled in the art. Therefore, for example, where the brand is a radioactive label, methods for detection include, but are not limited to, a scintillation counter or photographic film as in autoradiography. Where the tag is a fluorescent tag, it can be detected by exciting the fluorochrome with an appropriate wavelength of the light and detecting the resulting fluorescence, for example, by microscopy, visual inspection, via photographic film, by the use of electronic detectors, such as devices for charge coupling (CCDs) or photomultipliers, and the like. Similarly, enzymatic labels are detected by providing suitable substrates for the enzyme and detection of the resulting reaction product. Finally, simple colorimetric marks can be detected simply by observing the color associated with the brand. Therefore, in various rod assays, conjugated gold often appears pink, while various conjugated beds appear as bed color.

The binding materials of the invention can also be used to remove or detect prion proteins or peptides extracted in solution from a solid sample of the material. For example, a solid sample can be extracted with an aqueous solvent, an organic solvent or a critical fluid and the resulting supernatant can be contacted with the binding materials. Examples of solid samples include, but are not limited to, animal products, particularly those that have been exposed to agents that transmit prions, for example, bone meal derived from bovine sources. The binding materials in some embodiments can be used to detect the presence of prion protein in soil. Other solid samples include, but are not limited to, brain tissue, corneal tissue, fecal matter, bone meal, beef byproducts, sheep, sheep byproducts, deer and elk, deer and elk byproducts, and other animals and products. derived from animals. Alternatively, prions and prion-binding material complexes can be treated with proteinase K (PK). PrPc is highly sensitive to PK, while PrPsc is partially digested to form PrPres. The PrPres molecule itself is highly resistant to proteolysis. Therefore, the PK treatment will digest the PrPc, and convert the PK-sensitive PrPsc to PrPres. After the removal of PK, PrPres can be denatured and detected by antibodies such as 3F4.

In another embodiment, the binding materials according to the invention can be used for the selective concentration of PrPsc over PrPc.

Use of binding materials to quantitate prions A complex of the prion-binding material, or alternatively, an antibody to the prion or prion-binding material complex, can be detected and quantified by any of numerous methods well known to those skilled in the art. The technique. These include, but are not limited to, analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods, such as, but not limited to, precipitation reactions in fluid or gel, particular or double immunodiffusion), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.

Reduction of nonspecific binding When a solid support is used as a component of an assay for detecting a prion protein from a sample, one skilled in the art will appreciate that it is often desirable to reduce non-specific binding to the solid support. Methods for reducing such non-specific binding are well known to those skilled in the art. Typically, this involves coating the solid support with a proteinaceous composition. In particular, protein compositions, such as bovine and human serum albumin (BSA), and gelatin are widely used. The invention will be described in more detail by means of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit or define the invention in any way.

EXAMPLE 1 Identification of prion binding materials Eighty polymeric or inorganic particles were tested by the use of prion binding tests in beds by an NBT / BCIP chromogen (nitroblue tetrazolium / 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine salt), as described below, using a normal hamster brain homogenate. The results of the union are given in table 1 where "-" means without union and "+" means positive union. The greater "+" in a classification of the particle, the stronger the bond observed. Twelve particles that had at least "++" were further evaluated. Table 2 summarizes the twelve particles by their ability to bind to the prion of the normal hamster. A higher evaluation indicated an increased amount of binding to the prion. TABLE 1 1 DAP: diaminopropane 2 DAB: diaminobutane 3 TETA: triethylene tetramine 4 Amberchom ™ is a registered trademark of Rohm and Haas Company (Philadelphia, PA) * Non-specific: means that the negative control without the 3F4 antibody has the same signal as those evaluated with the 3F4 antibody.

TABLE 2 Capacity of the polymeric binding materials to bind to the normal hamster prion (HaPrPc) ** PMMA: polymeric methacrylate. The prion binding test on beds by NBT / BCIP was carried out as follows. When started with normal PrP from a 10% hamster brain homogenate, the sample was solubilized with 0. 5% sarcosil (200 μL from 10% to 4 ml brain) for 30 minutes at room temperature on a shaker. The sample was centrifuged at 14,000 rpm for five minutes. The supernatant was removed and a dilution of the supernatant was performed in a desired medium. The ninety-six microtiter well plates (Catalog No. 3075, Becton Dickinson, Franklin Lines, NJ) and Millipore MultiScreen-DV plates (Catalog No. MADV N65 10, Millipore Corporation, Bedford, MA) were blocked! Initially with 200 μL / well of 1% casein (w / v) from Pierce (Rockford, IL) at 65 ° C for one hour. 10 milligrams (10 mg) of dry beds were soaked in 1 ml of 10 mM PBS pH 7.4 and washed twice. The microtiter plate was emptied and 20-30 μL of a suspension of soaked beds was added to each well. The suspension was allowed to stand, and the excess water was removed. The normal hamster brain homogenate was diluted 1: 10 in 5% human serum albumin (Alpha Therapeutic Corp. Los Angeles, CA) which had already been heat treated at 60 ° C for ten hours. The suspension was added to a volume of 150 μL per well and incubated at room temperature for 1.5 hours with the beds. The unbound protein solution was removed, and 100 μL of the monoclonal antibody 3F4 (Signet Laboratories, Inc., Dedham, MA), diluted 1: 4000 in 1% casein, was added to the experimental wells. The control wells contained 100 μL of 1% casein. The beds were incubated with 3F4 overnight at 4 ° C with gentle shaking. The beads were then washed twice with 10 mM PBS + 10 μM CuCI2 at pH 7.4. The secondary antibody, mouse anti-IgG conjugated to alkaline phosphatase (# A3688, Sigma, St. Louis, MO), which was diluted 1: 1000 in 1% casein, was added to a volume of 100 μL / well. The samples were incubated for one hour at room temperature with shaking. All beds were transferred to Millipore (Bedford, MA) MultiScreen-DV plates to carry out the washings. Samples were washed three times with PBS + Cu2 ++ Tween 20 (0.05%) at pH 7.4, 3X with PBS + Cu2 +, twice with 1M NaCl and twice with 50 mM Tris-HCl + 5 mM MgCl2 at pH 9.5. In step 1 the NBT / BCIP substrate was mixed well and 100 μL was added directly to each well to develop the desired color (light purple). Typical incubations ranged from five to fifteen minutes. A filter paper (# 1703932, BioRad, Hercules, CA) was cut to shape and moistened with distilled water. The suspension of beds was added into the wells of the blot of the Dot-blot system of S &S Minifold I (Schleicher-Schuell Bioscience, Keene, NH) under vacuum. The wells were rinsed with water and the results recorded on a computer.

EXAMPLE 2 Identification of prion binding materials The identification of the prion binding materials was carried out using hamster brain homogenate in batch format, using two different detection systems. In the first, the amount of prion bound to a material was detected by staining the beds after incubation with the white material. The second method detected the amount of the prion present in the unbound fraction contained in the flow through and the washing of the samples using SDS-PAGE and western blots. A detailed description of each methodology is described below. As shown in Table 3 below, the Toyopearl ™ Amino-650M, TSK-GEL ™ Amino 750C and TSK-GEL ™ Phenyl-5PW provide the most specific binding to the PrPc of hamster brain.

TABLE 3 Results of secondary selection For the bed detection method, the 96-well microtiter plates (Catalog No. 3075, Falcon, Becton Dickinson, Franklin Lines, NJ) and Millipore Multiscreen-DV plates (Catalog No. MADV N65 10, Millipore, Bedford, MA) were blocked with 200 μL / well of 1% casein (w / v) at 65 ° C for 1 hour. The 10 mg aliquots of each polymer were soaked in 1 mL of 10 mM PBS pH 7.4 and washed twice. The microtiter plates were drained and 20-30 μL of a resin suspension was added to each well. The resin was allowed to stand and the excess of the solution was removed. To the resins were added 150 μL of a 1: 10 solution of the normal hamster brain homogenate in 5% human serum albumin. The mixture was incubated for 1.5 hours at room temperature. The wells were drained and 100 μL of the antibody 3F4 (1: 4,000) in 1% casein was added to each well and incubated overnight under cooling and gentle agitation. The beads were then washed twice with 10 mM PBS + 10 μM CuCI2, pH 7.4, followed by the addition of 100 μL / well of secondary antibody conjugated to alkaline phosphatase (Catalog No. A3688, Sigma, St. Louis, MO) and incubation for 1 hour at room temperature under gentle agitation.

All the wells were drained and the beds were transferred to the Multi-Screen plates, where they were washed three times with 10 mM PBS + CuC μM + 0.05% Tween 20 at pH 7.4, followed by 10 mM PBS + 10 μM CuCI2, twice with 1 M NaCl, twice with 50 mM Tris-HCl + 5 mM MgCl2 at pH 9. 5. Then the beds Washes were reacted with 100 μL of the NBT / BCIP substrate for 5-15 minutes for color development. The beds were transferred to filter paper using a Dot-Blot S &S Minifold system and the resulting color was evaluated. For the detection of the unbound fraction, 100 μL of each bed sample previously soaked with 10 mM PBS pH 7.4 at 4 ° C was placed overnight in microfuge tubes. After washing with PBS at least four times, the beads were transferred to Ultrafree-MC 0.45 μm filter units (UFC30HVNB, Millipore, Bedford, MA) and rinsed again with PBS. Ten percent of the hamster brain homogenate (HBH) was treated with 0.5% Sarcosil and diluted 1: 10 and 1: 20 in PBS. An aliquot of 200 μl of this was added to each sample bed and incubated for eight minutes under agitation followed by a two minute centrifugation at 10,000 rpm to recover the unbound reaction. The 26-μL aliquots of the flow through were placed in 0.7 mL microcentrifuge tubes and stored at -20 ° C for Western blot analysis. The samples were thawed before the Western blot, and 10 μL of sample pH regulator (NuPAGE, pH regulator sample SDS, NP0007, Invitrogen, Carisbad, CA) was added and 4 μL of the reducing agent (sample reducing agent) was added. NuPAGE, NP0004, Invitrogen) (DTT, 1M in H2O). The solution was incubated at 90-100 ° C for ten minutes. The samples were applied to a NuPAGE gel 4-12% Bis-Tris Gel (NP0323, Invitrogen) 15-well. To each well of a gel, 17 μL were applied for a Western blot analysis and 14 μL for a protein staining gel. The volume of the molecular weight marker (MultiMark Multi-Colored Standard, LC5725, Invitrogen) was 5 μL. The Western blots used PVDF membranes, 1% casein as a blocker, 1: 10,000 of 3F4 as primary antibody, 1: 3000 goat anti-mouse conjugated to horseradish peroxidase (HRP) as a secondary antibody and ECL plus as substrate. The films were exposed for six minutes. Samples with high binding of PrP to the binding materials produced no signal in the flow through and "5+" were evaluated. Those who do not have a union were evaluated as "-". The other samples were graduated between these values.

EXAMPLE 3 Determination of prion binding specificity Generally, the wetted beds composed of different bonding materials were placed quantitatively in individual disposable columns. The columns contained pores small enough to retain the beds but large enough to allow flow through the test solutions. The test solutions were homogenized from the brain containing protruding prions in Sarcosil (Sigma) in erythrocyte concentrate co-mixtures. More specifically, the test solutions contained infectious homogenates of human brain by TSE, infectious hamster brain homogenates, human brain infectious homogenates, or non-infectious hamster brain homogenates. The test solutions were allowed to pass through the white binding material for a defined period of time, while the flow through was collected. The beds were then rinsed and quantitatively transferred from their columns into collection vials from which known amounts were removed for subsequent processing to determine specific binding to the prion and specific binding to the protein. The solution of flow through and the reacted remnant of the beds were also stored for future potential analysis. Using the methods described in more detail below, the binding activity of eleven binding materials for prion proteins was determined as described in Table 4. The found materials are classified (with a classification of 1 being the material of binding that exhibits the greatest amount of binding to prion proteins) for its ability to bind either to the hamster prion protein or normal human. For example, the Fractogel ™ EMD TMAE 650 (S) binding material (which has a methacrylate base structure and the functional group -CH2-CH2-N + (CH3) 3) that binds the larger amounts of both prion infectious protein of hamster and human (PrPsc), and the Fractogel ™ EMD SO32_ 650 (S) binding material (which has a methacrylate base structure and the functional group -SO32-) that binds the larger amounts of both prion protein hamster like human. These amounts were measured by releasing the bound protein from the prion binding material, separating the proteins released by electrophoresis, and using Western blot to analyze the immunoreactivity of the protein released from the binding material with a specific monoclonal antibody. to the prion. The binding of the antibody to the prion proteins was detected by chemiluminescence. Quantification was achieved by comparing the darkness of the electrophoretic bands in the film (indicated by the antibody bound to the prion protein that had bound to the binding material) with control bands of 2 ng, 10 ng and 50 ng of the mouse IgG, and assigning the classification value to the binding materials. Evaluations were established for the bonding materials that had the same evaluation by comparing the bands of the sample with each other directly.

TABLE 4 Evaluation of the 11 polymeric compounds based on their ability to bind to normal hamster prion (HaPrPc), normal human prion (HuPrPc), infectious hamster prion (HaPrPsc) and human infectious prion (HuPrPsc) after selection high school.

Preparation of dry beds Dry beds were prepared in bulk by wetting the beds with a 20% methanol solution in water. The beds were left for at least 24 hours before using them. When the original amount of dry beds was between 0.5 g and 2.5 g, the pre-moistened bedded slurry was transferred to a 50 ml conical plastic tube. The excess liquid was removed and 25 ml of 20% methanol was added. Subsequently the sample was shaken gently or in cartwheels for 30 seconds. When the original bed weight was outside these aforementioned limits, the methanol volume was adjusted using 10 ml for less than 0.5 g of resin and 40 ml for 2.5 to 4.0 g. The beds were allowed to settle by gravity for approximately 12-15 minutes or until most of the total beds were in the lower part of the tube. The supernatant solution (including purifications) was carefully removed and discarded. The methanol rinse was repeated once more and then 20% methanol was added to make a 1: 1 (v / v) wet bedding paste. The beds were stored at 4 ° C.

Preparation of the slurry from the bed Four hundred forty microliters (440 μl) of the slurry from the bed were transferred to a conical plastic tube of 15 ml (220 uL of wet beds per column were used), and 10 ml of pH buffer for work (pH regulator of 20 mM citrate / 140 mM NaCl, pH 7.0). The sample was shaken gently or in cartwheels for 30 seconds. The beds were allowed to settle by gravity for approximately 12-15 minutes or until most of the total beds were in the lower part of the tube.

The supernatant solution (including purifications) was carefully removed and discarded. The rinsing with pH regulator for work was repeated twice more, and a sufficient volume of pH regulator was added for work to produce a 1: 1 volume ratio. The sample was gently shaken again or in cartwheels for 30 seconds, allowing it to settle and allow it to stand overnight at room temperature. The volume of pH regulator for work was maintained at 1: 1 by any necessary addition of the pH regulator for work, and the hydrated beds were kept at pH regulator at 4 ° C until use.

Preparation of Amino Toyopear ™ pre-hydrated beds and other beds were obtained as slurries wetted in 20% ethanol, and did not require additional steps of hydration. However, the equilibrium of the pH regulator for work was carried out as follows. The manufacturers estimate that the commercial slurries contain approximately 72% resin by volume, and 300 μL of slurry was used per column. The beds were transferred to a 15 ml plastic tube (for example Falcon) and 10 ml of the working pH buffer was added. The sample was shaken gently or in cartwheels for 30 seconds. The beds were allowed to settle by gravity for approximately 12-15 minutes or until most of the total beds were in the lower part of the tube. The supernatant solution (including purifications) was carefully removed and discarded. The rinsing with pH regulator for work was repeated twice more, and a sufficient volume of pH regulator was added for work to produce a 1: 1 volume ratio. The sample was gently shaken again or tumbled for 30 seconds, allowing it to settle and allowing it to stand overnight at room temperature. The volume of pH regulator for work was maintained at 1: 1 by any necessary addition of the pH regulator for work and the hydrated resin was kept in pH regulator at 4 ° C until its use.

Preparation and handling of erythrocytes A 110 ml bag of Adsol ™ (Baxter, Bloomington, IN) was added to approximately 250 ml packed erythrocyte concentrate (RBC) containing approximately 250-300 ml of RBC and residual leukocytes and platelets. The resulting hematocrit (erythrocyte volume (RBC) / total volume) was approximately 50-55%. The Adsol ™: CPD (phosphate dextrose citrate) and the RBCs were mixed by inversion. The mixture was then passed through a Pall leukoreduction filter (Pall Corporation, East Hills, NY) at room temperature within the first eight hours after collection and placed in a new bag. This procedure decreased the amount of leukocytes in the RBC mixture. The leucofiltrated RBCs were kept in a new bag at 4 ° C for up to 42 days. The final composition of the pH regulator mixture in this preparation was 30.6% Adsol / 8.5% CPD v: v. Before use, the percentage of hemolysis of the RBC preparation was monitored by measuring the absorbance at 415 nm of the supernatant after centrifugation. The preparations of RBC that had an increase greater than 2% of hemolysis in relation to the value of hemolysis obtained immediately after the preparation were not used.

Preparation and management of brain homogenate The normal hamster brain homogenate (10% w / v) was prepared by Dr. Robert Rohwer and colleagues at the University of Maryland in accordance with their established methods. The aliquots were prepared in 1.8 ml volumes and kept frozen at -80 ° C or on liquid nitrogen until use. Alternatively, they were thawed once for aliquot formation. Sixty microliters of the brain homogenate was used per column in each experiment. After thawing of a sample of brain homogenate, the sample was placed on ice with water. Then 6.6 μL of the 5% Sarcosil reagent (0.5 g of Sarcosil dissolved in 9.5 ml of CPD: pH regulator Adsol (8.5% CPD, 30.6% Adsol and 60.9% PBS v: v: v)) was added. each aliquot of 60 μL of the thawed brain homogenate. The sample was vortexed and gently rocked on ice with water for 30 minutes to allow denaturation. Then the sample was centrifuged at 14,000 rpm in a microcentrifuge for five minutes at 4 ° C. The supernatant was transferred to a new tube, and the concentrate was discarded. The supernatant was kept on ice with water for a maximum of one hour. The resulting supernatant was a 10% brain homogenate containing 0.5% Sarcosil reagent. The aliquots created were applied to the columns no more than one hour after their preparation and were kept on ice with water during their handling.

Quantitative transfer of hydrated beds to columns At each empty column, 750 μL of a 0.1% Tween ™ 20 solution was added. Then one milliliter of the 20% ethanol (v: v) was added to each column and allowed to flow under gravity. An additional 2 x 1 ml of deionized, degassed water was added to each column to wash the ethanol solution and remove any remaining air trapped in the pores. Using a quantitative pipette, 400 μL of a hydrated bed suspension was transferred to a column. The excess pH regulator for work was allowed to flow by gravity through the transferred wet beds, and then the column was washed three times with 1 ml of pH buffer for work before the introduction of the samples.

Preparation of a RBC co-mix (test solutions) Using a syringe and an 18-gauge needle (or larger), 540 μL of RBC / column was placed in a conical polypropylene tube. The tube was centrifuged at 3,000 rpm for ten minutes in order to separate a layer of Adsol ™ on the top. Then 60 μL of the 10% treated brain homogenate was added to the top of the Adsol ™ layer, thus reducing the direct contact between the RBC preparation and the highly concentrated protruding material, eg brain homogenate and Sarcosil detergent. . The co-mixture was mixed by inversion, kept on ice with water and used within the first four hours of preparation. Prior to use in the column assay, the mixture was brought to room temperature for ten minutes.

Addition of the test solutions to the columns Once all columns were filled with hydrated beds, the test solutions were mixed and placed very gently on the beds at a volume of 0.5 ml / column. The solutions were allowed to flow through the columns by gravity. The total flow time was between approximately five and twenty minutes. The first flow through 0.5 ml of test solution was collected in a 2 ml cryovial. An additional 0.5 ml of working pH buffer was added to each column, and the flow through was collected in the same cryovial. Subsequently, the bed columns were rinsed five times with 1 ml of pH buffer for work, during which time the beds were continuously resuspended by pipetting to ensure a general and uniform wash. Subsequently, the beds were recovered from the columns as described below.

Quantitative recovery of bound beds To each column, 0.75 ml of pH buffer for work was added. The column was subjected to pressure washing using a pipette to suspend the beds and the suspension was quickly transferred to a graduated tube. The bed suspension was allowed to settle in the tube and as much supernatant was removed as possible without disturbing the bed layer. This supernatant was then added back to the same column and the above-mentioned steps were repeated twice to transfer any remaining beds from the column into the tube. Then the beds were allowed to settle by gravity for ten minutes, and the volume of the bed layer in the graduated tube was recorded.

Preparation of bound beds for analysis First, the level of the pH regulator for work in each tube was adjusted to 1 ml, and a suspension of the beds was made by gently vortexing the tube. Using a pipette, 500 μL of the suspension was removed and transferred to a small Eppendorf ™ microfuge tube (Brinkmann instruments, Westbury, NY). The suspension was allowed to settle for ten minutes, and the settled volume of the beds was adjusted to 100 μL. Subsequently, the transferred beads were centrifuged, and the supernatant was removed. Aliquots of the bed were immediately prepared for electrophoresis and western blot analysis.

Quantification of dry beds versus hydrated beds Dry weight is calculated based on the volume of seated beds and inflation ratio as follows: Dry bed weight = set volume / inflation ratio Inflation rate = Volume of hydrated bed (μL) / dry weight (mg) For Toyopearl ™ 42.5 mg dry weight = 200 μL wet beds in 20% methanol Swelling ratio (in 20% methanol) = 200 / 42.5 = 4.71 i EXAMPLE 4 Analysis of Western Blot The following Western blot methods were designed to allow the evaluation of the infectious and non-infectious prion proteins recovered or diminished from solutions of the protruding brain homogenates in erythrocyte concentrates (RBC). These procedures were applied to the samples obtained from the column prion binding assay previously described in Example 3, including samples from beds exposed to the test solutions and samples from the test solutions flowing through the columns, and They collected.

Generally, samples were derived from the binding assay column of beds having white binding materials treated with solutions containing prions, or from flow through these reactions. Subsequently, the prepared samples were analyzed by Western blot for the presence of the prion protein. The immunodetection of the prion protein was carried out by the use of mouse primary monoclonal antibodies specific to prion proteins. These immunocomplexes of the prion were then detected with a secondary antibody conjugated to alkaline phosphatase and a chemiluminescent reaction was visualized on an X-ray film. The following steps were preferably carried out immediately following the column test in example 3. For each sample of bed prepared in column, 100 μL of well suspended beds mixed with 100 μL of pH regulator for 2X sample were mixed by vortexing. Controls were also prepared by mixing unused brain homogenate (human normal brain, sporadic CJD brain, normal hamster brain, brain with hamster lumbar pruritus) from the column pH-buffer assay for 2X Invitrogen sample. More specifically, an aliquot of 20 μL of the brain homogenate was added to 40 μL of the pH regulator for 2X sample. A mouse control IgG was also prepared as follows. A standard of 50 ng per line was prepared by mixing 20 μl of 2.5 mg / ml mouse IgG with 480 μl of PBS, which is equivalent to 100 μl / ml. Add 25 of this mixture to 475 of Invitrogen 2X pH regulator for sample to produce a solution of 5 ng / ml. 10 μl of this per line yielded 50 ng / line for the high concentration direct charge gel standard. Five microliters of a 100 μg / mL mouse IgG solution was mixed with 495 μL of the reduced pH 2X Invitrogen buffer for sample. This produced 1 ng / μL of mouse IgG; the charge of 10 μL of this per line yielded 10 ng / line (the high concentration direct charge gel standard) (10 ng / line). A standard mouse IgG for direct loading gel of low concentration (2 ng / line) was also prepared by diluting the standard of the medium concentration from the previous step by mixing 50 μL of 1 ng / μL of IgG of mouse in pH regulator for loading with 200 μL of pH regulator 2X Invitrogen for sample (which produced 0.2 ng / pL). The load of 10 μL of this per well produced 2 ng / line. Invitrogen See Blue Plus2 Pre-dyed molecular weight standards that were also prepared as directed by the manufacturer. All samples were heated in Invitrogen pH regulator for ten minutes at 90 ° C. The samples were then centrifuged briefly and stored overnight at -20 ° C. The heating procedure was repeated the next morning, before applying the samples to the SDS-PAGE gel.

Immunoreaction procedure After loading a 12% Bis Tris NuPAGE SDS-PAGE gel with the above-described samples, the gel was subjected to electrophoresis for 45 minutes at constant 200 V, and a transfer procedure was carried out. electroblot The membrane to which the protein was transferred was placed in a Fisher Square dish and incubated for one hour on a platform with room temperature movement in 25 ml of Western Breeze blocking agent (12.5 ml of water, 5 ml of Diluent A , and 7.5 ml of Diluent B). The blocking solution was discarded. The membrane was incubated in a 1: 5,000 dilution of the Signet 3F4 primary antibody solution in 20 ml of freshly prepared Western Breeze primary antibody diluent (14 ml of water, 4 ml of diluent A, 2 ml of diluent B). The primary antibody was previously diluted 1: 1 in glycerol, and therefore, the working dilution was 1: 10,000. The membrane was incubated under refrigeration on a moving platform. The primary antibody solution was discarded and the membrane was washed three times for ten minutes each in 20 ml of wash solution of the Western Breeze antibody (1.25 ml of solution for washing the antibody (16X) in 18.75 ml water) at room temperature on a platform with movement. The membrane was then incubated in 1: 10,000 of the AP3 secondary antibody (KPL, Gaithersburg, MD) in 20 ml of diluent for the Western Breeze primary antibody for 60 minutes at room temperature on a moving platform. The secondary antibody solution was discarded and the membrane washed in washing for Western Breeze antibody as described above. The membrane was then washed with 20 ml of 20 mM Tris-HCl, 1 mM MgCl 2 at pH 9.8 for ten minutes at room temperature.

Chemiluminescence Development Procedure The membrane was transferred to a dry tray and soaked with 5 ml of Western Breeze pre-mixed chemiluminescent substrate (CDP Star ™ substrate, Applied Biosystems, Foster City, CA) for five minutes with gentle shaking. The membrane was lightly blotted with a paper towel and then placed on a sheet protector. Subsequently the membrane was transferred in the film protector to a film cassette (without an intensifying screen) maintaining an ambient temperature for 30 minutes and exposing to autoradiography for five minutes at room temperature.

EXAMPLE 5 Binding of endogenous PrPc from human plasma To show the ability of the resins for prion binding to remove the PrPc from the endogenous human plasma, not outstanding in PrPc, the following experiments were carried out.

Undiluted, freshly obtained, pooled human plasma was used to induce endogenous PrPc by prion binding materials. The frozen plasma, pooled from human, was thawed at 37 ° C, filtered through a 0.45 μm filter, and sarcosil was added to a final concentration of 0.05%. The binding of the plasma to the columns and the detection of the prions was carried out as described elsewhere in the present specification. The results of the endogenous PrPc binding test from human plasma are illustrated in Figures 1A-1D. Figure 1A illustrates the results of detection by Western blot of prion protein in the eluate of the bed in the absence of sarcosil (line 1 is the molecular weight marker; line 2-Mo IgG low; line 3-Mo IgG medium; 4-Mo high IgG, line 5-human normal platelets, lines 6-7-resin a, lines 8-9-resin b, lines 10-11-Amino 650-M, lines 12-13-Amino acetylated 650. Figure 1 B illustrates the results of the detection by Western blot of the unbound fraction of the samples in Figure 1A (line 1 - molecular weight marker, line 2-Mo low IgG, line 3-Mo IgG medium, line 4 Mo IgG high, line 5-normal brain of hamster (nHB), line 6-normal platelets of human lines 7-8-resin a; lines 9-10-resin b; lines 11-12-Amino 650-M; lines 13-14-Acetylated Amino 650.). Figure 1C shows the results of detection by Western blot of the prion protein in the presence of sarcosil (line 1 - molecular weight marker, line 2-Mo IgG low, line 3-Mo IgG medium, line 4-Mo IgG high, line 5-normal hamster brain, line 6-human platelets, line 7-human normal plasma + sarcosil, 1:10, line 8-resin a, line 9-resin a, line 10-resin b; line 11 -resin b, line 12-Amino 650-1, line 13-Amino 650-2). Figure 1D shows the detection results of unbound fractions of the samples in Figure 1C by Western blot of the prion protein in the presence of sarcosil (line 1 - molecular weight marker, line 2-Mo IgG low, line 3 - Mo IgG Medium, line 4-Mo IgG high, line 5-normal brain of hamster, line 6-platelets of human, line 7-normal human plasma + sarcosil, line 8-resin a, line 9-resin a, line 10 -resin b, line 11-resin b, line 12-Amino 650-1, line 3-Amino 650-2). With reference to Figures 1A-1 D, in Figure 1A, lines 10 and 11, and in Figure 1C, lines 12 and 13, the binding of the PrPc resin to Toyopearl ™ Amino 650-M was detected; the union was avoided if the charge in the amino group was removed by acetylation (the results are shown in figure 1A, lines 12 and 13). The experimental results demonstrate the ability of the resins to bind endogenous PrPc from human plasma, thus providing evidence that the resins are useful for the removal of PrP samples obtained from humans or animals.

EXAMPLE 6 Requirements for the spacer to join the blood choices from the PrPsc As shown in Figures 1A-1 D, the Toyopearl ™ resin Amino 650-M binds endogenous PrPc from human plasma. At least a portion of this resin contains a spacer arm, or group, owned by Tosoh ™. The importance of the spacer for binding to PrPsc has been investigated. Four (4) equipment have been packaged for the use of protein isolation columns for the identification of the Sorbent (PIKSI ™) (0.5 ml / each packaged as follows: two columns each of an experimental sample of Toyopear ™ F Amino 650 M lacking a spacer, and a Toyopearl ™ Amino 650M commercial resin with a spacer Toyopearl ™ Amino 650C resin lacking a spacer was also evaluated Two milliliters (2 ml) of brain homogenate with 10% lumbar pruritus (SBH) were treated with 0.5% sarcosil.The columns were tested with diluted supernatant treated with sarcosil with working pH regulator (1: 100) by the addition of 3 ml of SBH in 297 ml of working pH buffer. The columns were tested in duplicate with 10 ml of SBH diluted in pH regulator by loading at the flow rate of 0.5 ml / minute.The solutions were collected through the flow, and the aliquots of resin were removed from each col. umna and washed with 10 ml of pH regulator for work. Half of each sample was subjected to digestion with proteinase K with each resin and the pH regulator tested. Samples were evaluated by Western Blots as described above elsewhere. The results as shown in figure 2 (line 1 -molecular weight markers; line 2-0.1% of SBH treated with sarcosil in pH + PK regulator; line 3-0.1% of SBH treated with sarcosil in pH regulator + PK, line 4- MWM, line 5-Amino 650M (commercial) -PK (1), line 6-Amino 650M (commercial) -PK (2), line 7-Amino 650M (commercial) + PK (1); 8-Amino 650M (commercial) + PK (2), line 9-Amino 650M (experimental) + PK (1), line 10-Amino 650M (experimental) + PK (2), line 11-Amino 650M (experimental) + PK (1), line 12-Amino 650M (experimental) + PK (2), line 13-Amino 650C-PK (1), line 14-Amino 650C-PK (2), line 15 - Amino 650C + PK (1) ) line 16-Amino 650C + PK (2)). The experimental results shown in Figure 2 clearly indicate that the presence of a spacer arm is necessary for the binding of the PrPsc by the Amino 650-M resin.

EXAMPLE 7 Capture of PrPc in the presence of high concentrations of human serum albumin (HSA) To demonstrate the ability of the neighbors to remove PrP from a therapeutic product comprising various proteins, the binding of PrPc in the presence of human serum albumin was investigated. Four Bio-Rad ™ columns were packed with Toyopearl ™ Amino 650 M amino resin. The height of the resin bed was 1 cm and the volume was 0.5 ml. The columns were rinsed abundantly with pH regulator for work. The samples loaded in the columns were as follows: Column I-15 of nHaBH (homogenized from normal hamster brain) in pH regulator for work; Column 11-1% HaBH, 25% HSA (Sigma) in pH regulator for work; Column 111-1% of HaBH, 25% of HSA (Sigma) and N-Ac-Trp 20 mM (Acros organics, Belgium) in pH regulator for work; Column IV- 1% of HaBH, preparation of the American Red Cross (ARC preparation). The 20 mM N-Ac-Trp was dissolved in 25% HSA in working pH buffer, with stirring and heated at 37 ° C, for 45 minutes. The 10% supernatant nHaBH was prepared as described above and diluted 1: 10 in the materials of choice (step 2) to obtain 1% nHaBH. The bottom of each column was connected to a 4-channel peristaltic pump. Five milliliters (5 ml) of 1% nHaBH prepared in the previous step on columns I-IV were processed at a flow rate of 0.5 ml / minute. The columns were washed with 10 ml of pH regulator for work / column, at a flow rate of 0.5 ml / minute. The resins were recovered, the samples were prepared as previously described and processed in 12% gels of Bis-Tris SDS-PAGE. Western blots using the primary antibody 3F4 were used to detect the PrPc that had been captured by the resins. The photograph of the blot is shown in figure 3 (line 1-low IgG mouse control; Line 2-Medium IgG mouse control; line 3-nHaBH control; line 4-1% HaBH column; line 5-1% of HaBH, 25% of HSA in column; line 6-1% HaBH, 25% HSA, N-AC-Trp 20 mM column; line 7-1% of HaBH, ARC preparation for column). Bands of approximately equal intensity were observed in each line, indicating that the Toyopearl ™ 650-M amino resin captured the PrPc from hamster brain homogenate in the presence of 25% human serum albumin obtained from a variety of sources. The experimental results as shown in Figure 3 show the ability of the resins to bind to a prion protein from a sample comprising HSA, thus providing evidence that the resins are useful for the binding proteins. prion in a variety of therapeutic products and verifying the safety of therapeutic products in which blood proteins are used, for example, as stabilizers or therapeutic agents, and which may be contaminated with PrP.

Binding of PrPsc to amino resin in human serum albumin The binding of albumin-labeled infectious PrPsc was demonstrated in the experiment described below. 12 columns PIKSI, 0.5 ml each, were packaged withToyopearl ™ Amino resin 650 M. 2 ml of 10% SBH (brain homogenate with lumbar pruritus) was treated with 0.5% sarcosil ». The following six changes were discussed as outlined below. 1. Test with SBH in pH regulator: dilute the supernatant of the pH regulator for work treated with sarcosil (1: 100); 0.22 ml of SBH was added to 22 ml of pH buffer for work. 2. Test with SBH in HSA (formulation of the American Red Cross (ARC)): dilute supernatant treated with sarcosil with 25% HSA (1: 100); 0.22 ml of SBH was added to 22 ml of HSA (American Red Cross formulation). 3. Test with SBH in HSA (Sigma) with N-acetyl-DL-tryptophan and caprylate: dilute sarcosil-treated supernatant with 25% HAS (1: 100) containing 20 mM N-acetyl Trp and 20 mM caprylate; Albumin was obtained from Sigma and contains no additives; 0.22 ml of SBH was added to 22 ml of HSA solution. 4. Test with SBH in HSA (Sigma) with N-acetyl Trp: dilute supernatant treated with sarcosil with 25% HSA (1: 100); 0.22 ml of SBH was added to 22 ml of HSA with 20 mM N-acetyl Trp. 5. Test with SBH in HSA (Sigma) with caprylate: dilute supernatant treated with sarcosil with 25% HAS (1: 100); They were added 0. 22 ml of SBH to 22 ml of HSA with 20 mM caprylate. 6. Test with SBH in HSA (Sigma) alone: dilute the supernatant treated with sarcosil with 25% HAS (1: 100); 0.22 ml of SBH was added to 22 ml of HSA (Sigma). Each resin was tested in duplicate with 10 ml of solution. The columns were added at a flow rate of 0.5 ml / minute controlled with a peristaltic pump. Solutions were collected from the flow through, as was the resin from each column. Digestion with Proteinase K was carried out with each of the resins and the pH regulator tested. The samples were subjected to Western blots according to the method described above. The blots are illustrated in Figures 4A-4B (Figure 4A: line 1-molecular weight standard; line 2-0.1% of SBH treated with sarcosil in pH-PK buffer; line 3-0.1% of SBH treated with sarcosil in pH + PK regulator, line 4- SBH in pH-PK regulator (I), line 5-SBH in pH-PK regulator (2), line 6-SBH in pH regulator + PK (I), line 7 -SBH pH regulator + PK (2), line 8-SBH in HSA (ARC formulation) -PK (I), line 9-SBH in HSA (ARC formulation) -PS (2), line 10- SBH in HSA (ARC formulation) + PK (I), line 11 -SBH in HSA (ARC formulation) + PK (2), line 12-SBH in HSA (Sigma) -PK (l), line 13-SBH in HSA (Sigma) -PK (2), line 14- SBH in HSA (Sigma) + PK (1), line 15- SBH in HSA (Sigma) + PK (2), line, figure 4B: line 1 - molecular weight standard; line 2-0.1% of SBH treated with sarcosil in pH-PK regulator, line 3- 0.1% SBH treated with sarcosil in pH + PK regulator, line 4-SBH in HSA (Sigma) with AcetiloTrp-PK (I); line 5-SBH in HSA (Sigma ) with Acetyl Trp-PK (2), line 6-SBH in HSA (Sigma) with Acetyl Trp + PK (I); line 7-SBH in HSA (Sigma) with Acetyl Trp + PK (2); line 8-SBH in HSA (Sigma) with caprylate-PK (1); line 9-SBH in HSA (Sigma) with caprylate-PK (2); line 10-SBH in HSA (Sigma) with caprylate + PK (1); line 11-SBH in HSA (Sigma) with caprylate + PK (2); line 12-SBH in HSA (Sigma) with Acetyl Trp and caprylate-PK (l); line 13-SBH in HSA (Sigma) with Acetyl Trp & caprylate-PK (2); line 14-SBH in HSA (Sigma) with Acetyl Trp and caprylate + PK (I); line 15-SBH in HSA (Sigma) with Acetyl Trp and caprylate + PK (2) The results shown in Figures 4A-4B demonstrate that the infectious PrPres was able to bind to an amino resin when combined with serum albumin of human, and that a variety of additives does not interfere with the union.

Although methods and materials similar or equivalent to those described above can be used in the practice or in the test of the present invention, suitable methods and materials were described above. All publications, patent applications, patents and other cited references mentioned in the present invention are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. The foregoing description is provided to describe various embodiments that relate to the invention. Various modifications, additions and deletions can be made to the modalities and structures without departing from the scope and spirit of the invention.

Claims (11)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for forming a complex between a prion protein and a prion protein binding material in a sample comprising contacting the sample with the prion protein binding material under conditions that allow complex formation between the prion protein and the prion protein binding material, wherein the prion-binding material comprises a functional group, wherein the functional group is a hydrophilic functional group, a hydrophobic functional group, or an amphiphilic functional group .

2. The method according to claim 1, further characterized in that it comprises separating the complex from the sample.

3. The method according to any of claims 1 or 2, further characterized in that it comprises detecting the complex.

4. The method according to any of claims 1-3, further characterized in that the functional group is a primary amine group, a secondary amine group, a tertiary amine group, a quaternary amine group, an amine group, a sulfite group , a sulfonyl group, a tresyl group, an alkyl group, an aromatic group, a phenyl group, a butyl group, a siloxane group, or a fluorinated group.

5. The method according to any of claims 1-4, further characterized in that the functional group is selected from the group consisting of: a) -OCH2-CHOH-CH2NH2; b) -C6H5; c) - (CH2) 3-CH3; d) -CH2-CH2-N + H (C2H5) 2; e) -SO2-CH2-CF3; f) -CH2-CH2-N + H (CH3) 2; g) -CH2-CH2-N + - (CH3) 3; h) -SO32 '; i) a diethylaminoethyl group; j) a dimethylaminoethyl group; and k) a trimethylaminoethyl group.

6. The method according to any of claims 1-5, further characterized in that the bonding material comprises aluminum or silica.

7. The method according to any of claims 1-6, further characterized in that the bonding material comprises a polymer matrix.

8. The method according to any of claims 1-7, further characterized in that the polymer matrix is a polymethacrylate, a methacrylate, a FRACTOGEL ™ EMD, a TOYOPEARL ™ or a polymer matrix TSK-GEL ™.

9. The method according to any of claims 1-8, further characterized in that the sample is a biological sample, a food product, an environmental sample, or a water sample.

10. - The method according to any of claims 1-9, further characterized in that the sample comprises up to about 50% serum albumin by weight.

11. The method according to any of claims 1-10, further characterized in that the prion binding material comprises a spacer attached to the functional group.