Devices and methods for removing target agents from a sample

Abstract

DEVICES AND METHODS FOR REMOVING TARGET AGENTS FROM A SAMPLE. The invention provides devices, test kits and methods for removing target agents from a sample. The device contains one or more porous matrices that have pore sizes greater than 10 µm, and a plurality of particles impregnated therein. The target agents attach to the device and are removed from the sample.

Description

Descriptive report of the invention patent for: "DEVICES AND METHODS FOR REMOVING TARGET AGENTS FROM A SAMPLE". CROSS-REFERENCE TO RELATED DEPOSITS This filing claims benefit under 37 U.S.C. § 119(e) based on U.S. Provisional Filing No. 60 / 617,669, filed October 13, 2004, which claims benefit under 37 U.S.C. § 119(e) for U.S. Provisional Filing No. 60 / 618,118, filed October 6, 2004, which claims benefit under 37 U.S.C. § 119(e) for U.S. Provisional Filing No. 60 / 578,061, filed June 9, 2004, the entire contents of which are incorporated herein by reference. I. FIELD OF THE INVENTION This invention relates to devices and methods for removing target agents from a sample. In particular, the invention relates to the removal of pathogens from biological samples. II. BACKGROUND OF THE INVENTION The process of adsorption of biological species to Solid supports find various practical applications in Purification, detection, and removal of target molecules from multicomponent streams. For example, ion-exchange, hydrophobic, and affinity ligands are capable of preferentially adsorbing many agents onto chromatographic supports to affect their separation from aqueous solutions. Once adsorbed, the biological agent can either be eluted as a product or detected by ELISA and other analytical approaches. In some examples, the solution that also contains the target biological agent contains large entities, such as red blood cells, viruses, bacteria, liposomes, leukocytes, and aggregates of various sizes. In many of these examples, it is desirable to allow the large aggregates to flow through the solid matrix or support without interfering with the ability of the target biological agents to bind to the support. This requires pore spaces in the solid matrix that are large enough to accommodate the flow of the large entities. Unfortunately, large pore spaces may have a low surface area, which limits the ability of the solid matrix to bind to the target agents. In other cases, it is desirable to filter the particles. large enough to facilitate adsorptive separation of smaller target agents. An example is the removal of cells from a culture medium to recover an extracellular product. Furthermore, there are many instances where it is desirable to bind, rather than filter, biological entities that are very large. For example, it is specifically important to adsorb many pathogens, including infectious prions, viruses, bacteria, and toxins from mixtures of biological agents. These entities often have difficulty accessing the small pores that are required for binding within currently available sample separation and purification devices. Nonwoven nets or fibers, also called melt-blown polymer fibers or spunbonded nets, are well known and are used for filtration and separation of fine particles from air and aqueous solutions. (e.g., see U.S. Patent Nos. 4,011,067 and 4,604,203, each of which is incorporated herein by reference in its entirety.) Loading aerated particulates onto nonwoven nets is also well known in the art (see, for example, U.S. Patent Nos. 4,433,024; 4,797,318; and 4,957,943, each of which is incorporated herein). (through reference in its entirety). Applications These include face respirators for removing particulate and gaseous contaminants, protective clothing, fluid-containing items, and oil rags. More recently, methods for manufacturing particle-saturated nonwoven fabrics for separation and purification have been reported. For example, see U.S. Patent No. 5,328,758, incorporated herein by reference in its entirety. The patent describes functionalized particles for attaching affinity binders. It is described that the particles are blown into the polymer fibers during the blow spinning phase. The nonwoven fabric includes pores having pore sizes in the range of 0.24 to 10 µm, preferably 0.5 to 5 µm. It is also specified that the saturated fabric material must have a Gurley Time of at least 2 seconds. W093 / 01880 describes a leukocyte-removing nonwoven tissue filtering material produced by dispersing in a medium a mass of a large number of small fiber pieces having a fiber diameter of not more than 0.01 µm and a length of about 1 to 50 µm, along with short rotatable and braidable fibers having a common length of 3 to 15 mm. US Patents Nos. 4,550,123 and 4,342,811 each of which are here Incorporated through reference in their entirety, they describe microporous polymeric fibers and films containing particles capable of sorbing vapors, liquids, and solutes. Typical sorbent particles include activated carbon, silica gel, and molecular filter-type materials. The invention as described herein provides devices and methods for sample purification, and detection and removal of target agents from a sample with increased efficiency and specificity and significant reductions in time and cost over prior art devices. III. SUMMARY OF THE INVENTION The invention, as described and disclosed herein, provides methods, devices, and equipment for removing target agents from a sample. In one aspect, the invention provides a device for separating a target agent from at least one sample. The device contains two compartments, a porous matrix having pore sizes greater than 10 µm, and a plurality of particles impregnated therein. The particles are porous, non-porous, or a combination thereof. Targeted agents attach to the particles and are thus removed. of the sample. In one embodiment, the porous matrix, the particles, or both have uniform or variable pore sizes. In another embodiment, the particles have a pore size of about 0.001 µm to about 0.1 µm. In yet another embodiment, the particles comprise a porous resin having interconnected pores with surface areas in the range of about 1–2 µm. 2 / g of dry resin at approximately 300 m 2 / g of dry resin. In yet another embodiment, the porous matrix comprises natural fibers, synthetic fibers, or both. In a preferred embodiment, the porous matrix comprises at least one nonwoven fabric. In another embodiment, the porous matrix is ​​a blend of two or more of the same or different types of woven and / or nonwoven fabrics. In one embodiment, the particles, the porous matrix, or both are functionalized with one or more reactive groups. The target agents are attached to the particles, porous matrix, or both by absorption, adsorption, ion exchange, covalent bonds, hydrophobic interactions, dipole, quadrupole, hydrogen bonding, specific interactions, formation of charged species, or by affinity interaction for ligands. specific, or a combination thereof. In yet another In one embodiment, the particles are polymethacrylate or methacrylate resins, including, by way of example and not limited to, a FRACTOGEL™ EMD, TOYOPEARL™, or TSK-GEL™ polymer matrix. In yet another embodiment, the resin is TOYOPEARL™ Amino 650, including, for example, Amino 650 U, Amino 650 M, or a partially acetylated form of Amino 650M or Amino 650 U. Partially acetylated resin includes from about 5% to about 95% or more acetylated resins. In one embodiment, the partially acetylated resin includes from about 10% to about 85% acetylated resin. In another embodiment, the partially acetylated resin comprises from about 20% to about 75% acetylated resin. In yet another embodiment, the partially acetylated resin includes from about 30% to about 60% acetylated resin. In another embodiment, the partially acetylated resin comprises from about 40% to about 60% acetylated resin.It is planned here that by reciting such specific ranges, the ranges also recited include all whole quantities between the recited ranges. For example, in the range of approximately 40 and 60%, it is intended that it also includes 45%, 50%, 55%, 57%, etc., without actually reciting each specific range of this. In another embodiment, the resin comprises wet resins (i.e., fully pre-hydrated), dry resins (i.e., not pre-hydrated). before contact with the sample, and / or previously dried, but (hydrated before contact with the sample). The use of a partially dry and / or wet acetylated resin is also covered within the scope of the invention. In another aspect, the device contains a functionalized matrix of porous nonwoven or woven material that has the ability to adsorb the target agents. In one embodiment, the device contains a non-porous matrix as well as a porous matrix; one or both of these matrices may be functionalized. In another embodiment, the porous matrix contains uniform or variable pore sizes greater than 10⁻¹² / x². In yet another aspect, the invention provides methods for separating at least one target agent from a sample comprising: (a) providing a sample potentially containing one or more target agents; (b) providing a device comprising (i) a porous matrix having pore sizes greater than 10 µm, and (ii) a plurality of particles impregnated in the porous matrix, wherein the particles have the ability to bind at least one target agent; (c) subjecting the sample to the device; (d) binding at least one target agent to the particles; and (e) separating at least one target agent from the sample. In another aspect, the invention provides test kits for separation, detection and purification of target samples comprising one or more of the following: (i) a device containing a porous matrix having pore sizes greater than 10 µm, and a plurality of particles impregnated therein, (ii) a container containing one or more buffers, reagents, chemical agents, functionalizing reagents, enzymes, detection agents, control materials, (iii) instructions for use of the test kit, and (iv) packaging materials. Other preferred embodiments of the invention will be apparent to a person of ordinary skill in the art by taking into account what is known in the art, by considering the following drawings and description of the invention, and by considering the claims. IV. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a schematic representation for resin-impregnated nonwoven tissues (RINs). The nonwoven tissues have a pore size of approximately 12 µm and are impregnated with a porous resin support. The average pore size is large enough to allow red blood cells to flow freely through the device without showing any signs of infection. any sign of damage. Particles (10), fibers (20), impregnated particles (11), and nonwoven fabric (21) are shown here. Fig. 2 shows a schematic representation of a device composed of square sheets of a woven or nonwoven fabric. A zigzag arrangement of woven or nonwoven fabric sheets is covered with affinity binders on both sides. The sample flows in the tortuous path between the sheets. 0 pore size The size of the leaves is adjusted according to the desired application. Fig 3 shows a scanning electron micrograph of sample 13 inner / inner layer calendered at 150F, and 100 pounds per linear inch (PLI) with resin. The micrograph shows pocket areas of sample 13 at 50X magnification. Fig 4 shows a scanning electron micrograph of sample 11 inner / outer layer calendered at 180F, and 400 PLI, with resin. The micrograph shows sample 11 cavity areas at 50X magnification. Figo 5 shows a bar graph indicating the Results from a Micro BCA assay of 12 fractions collected for different numbers of membrane layers. Different concentrations of (3- Lactoglobulin fractions were shown to be present via flow of a solution through 1 layer of resin-embedded membrane ("labeled resin") or 1 layer of membrane ("labeled control"). Figo 6 shows a bar graph indicating the results of a Micro BCA assay of 12 fractions collected for different numbers of membrane layers. Different concentrations of [3- Lactoglobulins were shown in fractions via flow of a solution passing through 2 layers of resin-embedded ("labeled resin") membrane or 2 membrane layers ("control" marked). Fig. 7 shows a bar graph indicating the results of a Micro BCA assay of 12 fractions collected for different numbers of membrane layers. Different concentrations of (3- Lactoglobulins were shown in fractions via flow of a solution passing through 3 layers of resin-embedded membrane ("labeled resin") or 3 membrane layers ("control" marked). Fig. 8 shows a bar graph indicating the Results from a Micro BCA assay of 12 fractions collected for different numbers of membrane layers. Different concentrations of [3-Lactoglobulin] were shown in fractions via flow of a solution through 4 layers of resin-embedded membrane ("labeled resin") or 4 layers of membrane ("labeled control"). Fig. 9 shows the particle distribution in membrane rolls. Resin particles were uniformly dispersed over the background membrane without overflowing the membrane edges. V. DETAILED DESCRIPTION OF THE INVENTION This document describes methods, devices, and kits for the efficient separation of target molecules from a sample. The methods, kits, and devices of the invention are useful in a variety of applications including purification, separation, and processing of expressed gene products from cells, production and distribution of biopharmaceuticals, and prognostic, diagnostic, and / or detection applications, among others. The invention described herein defines novel devices that separate different components from a sample and allow the... flow of larger species through the device while They provide large surface areas for agents to bind to. target. Particular applications of this invention involve the removal of pathogens such as prions, viruses, bacteria and toxins from biological samples such as, for example, a blood sample including whole blood, red blood cell containing compositions, red blood cell concentrates, platelet concentrates, plasma, plasma derivatives, leukocytes, leukodepleted blood, mammalian cell culture, fermentation broths and other media used for the manufacture and distribution of biopharmaceuticals and the preparation of therapeutics. 1. Definitions The definitions used in this repository are for These are illustrative purposes and do not limit the scope of the invention. As used herein, "modified resins" are broadly defined within the scope of the invention and include analogs, variants, and functional derivatives of a resin with or without a functional group. Modification includes, for example, substitution, deletion, or addition of chemical entities (e.g., amino acids) to a resin. A particular resin, or its functional group, or both. For example, amino substitution, acetylation, and / or partial acetylation of resins are included in the definition of modified resins. Any modification to the functional group of a resin is included within the scope of modified resins according to the invention. As used herein, "target agents" are broadly defined within the scope of the invention and include chemical, biological, or physical agents that are captured by the device of the invention. Target agents include molecules, compounds, cell components, organelles, aggregates, toxins, prions, and microorganisms such as pathogens including viruses, bacteria, fungi, and protozoa, among others. Target molecules also include polymeric molecules such as polynucleotide molecules, for example, DNA, RNA, DNA-RNA hybrid, antisense RNA, cDNA, genomic DNA, mRNA, ribozyme, natural, synthetic, or recombinant nucleic acid molecules, oligopeptides, oligonucleotides, peptides, peptide-nucleic acid hybrids, antigens, antibodies, antibody fragments, large proteins and aggregates such as vWF:FVIIl, and HDL, among others. As used here, the term "pathogen" is intended. This means any replicable agent that can be found within or can infect a biological sample such as a blood sample. Such pathogens include the various viruses, bacteria, protozoa, and parasites known to those skilled in the art to generally be found within or infect all blood or blood components, and other pathogenic contaminants not yet known. Illustrative examples of such pathogens include, but are not limited to, bacteria such as Streptococcus species, Escherichia species, and Bacillus species; viruses such as human immunodeficiency virus and other retroviruses, herpesvirus, paramyxovirus, cytomegalovirus, hepatitis viruses (including hepatitis A, hepatitis B, and hepatitis C viruses), smallpox virus, and toga virus; and parasites such as malarial parasites, including Plasmodium species, and trypanosomal parasites. As used herein, "sample" includes any sample containing a target agent that can be captured by the device and method of the invention. Samples can be obtained from any source that potentially contains a target agent. Such sources include animals, plants, soil, air, water, fungi, bacteria, and viruses, among others. Animal samples obtained, for example, from tissue biopsies, blood, hair, buccal scrapings, plasma, serum, skin, ascites, plural effusion, thoracentesis fluid, spinal fluid, lymph fluid, bone marrow, respiratory fluid, genital fluid, intestinal fluid, feces, urine, sputum, tears, saliva, tumors, organs, tissues, in vitro samples, cell culture components, fetal cells, placental cells or amniotic cells and / or fluid, among others. As used herein, "cell culture medium" includes any prokaryotic or eukaryotic culture medium such as, for example, bacterial, fungal and other microbiological cell culture media, mammalian cell culture media, plant cell culture, and insect culture, fermentation broths and other media used for the production and distribution of biopharmaceuticals and the preparation of therapeutics. As used herein, "blood sample" includes, for example, and not as a limitation, whole blood, compositions containing red blood cells (e.g., red blood cell concentrates and platelet concentrates), leukocytes, and leukodepleted blood, blood proteins such as blood factors, Blood coagulation enzymes, albumin, plasminogen, and immunoglobulins; and liquid blood components such as plasma, plasma derivatives, and plasma-containing compositions, among other blood samples. As used herein, the term "composition containing red blood cells" means whole blood, red blood cell concentrates, and any other composition containing red blood cells. In addition to red blood cells, the composition may also contain a biologically compatible solution, such as ARC-8, Nutricell (AS-3), ADSOL (AS-1), Optisol (AS-5), or RAS-2 (Erythrosol), and one or more cellular blood components, one or more blood proteins, or a mixture of one or more cellular blood components and / or one or more blood proteins. Such compositions may also contain a liquid blood component, such as plasma. As used herein, "particle" means porous or non-porous organic or inorganic forms having a diameter of about 1 to about 200 µm; these include, for example, and not as a limiting factor, fibers with a length-to-diameter ratio of about 20 to about 1 µm, as well as sorbent particles such as granules, spheres, resins, or powders, among others. As used here, "sorbent," "sorbent," or "sorption" means capable of adsorbing and holding by absorption or adsorption. As used herein, "attachment" is broadly defined within the scope of the invention and comprises any type of physical, chemical, or biological bonding processes between two entities and includes, for example, and not as a form of limitation, absorption, adsorption, covalent bonding, ion exchange, hydrophobic, hydrogen bonding, dipole, quadrupole or affinity interaction, formation of charged species, attachment of affinity ligands (for example, including peptide, oligonucleotide, proteins, spacing arms, hydrophobic moieties, fluorinated packing materials), among others. As used here, "spiked solution" refers to a solution that has received a certain quantity of the target protein, toxin, virus, bacteria, or other organism, in its pure, partially purified, or crude form. 2. Porous matrix The devices of the present invention comprise a porous matrix that has particles impregnated in it. Selection The use of a porous matrix can vary widely within the scope of the invention. Useful matrices include woven and nonwoven fabrics (such as fibrous networks), microporous fibers, and microporous membranes. These fibers can be made from any material and by any method known to the art, including blow spinning, spin bonding, and electrostatic spinning. Fibrous networks are particularly desirable because such networks provide large surface areas, with nonwoven fibrous networks being preferred due to ease of manufacture, low material cost, and the possibility of variation in fiber texture and fiber density. A wide variety of fiber diameters, for example, 0.05 to 50 µm, is used in the preparation of the device of the present invention. Matrix densities are varied to adjust the desired utility of the device, for example, from about 0.1 µm to about 100 cm thickness or more. The matrix can be used in the form of a single sheet or can be stacked as desired to achieve the desired adsorption capacity. In one embodiment, calendering or pressurization of the porous matrix is ​​required to achieve the desired densities and pore size. The porous matrix of the invention's devices is made from a wide variety of natural and synthetic fibers, according to the precise physical and chemical properties of the porous matrix targeted to the intended application. The porous matrix of the invention is selected from natural or synthetic sources including, for example, polyester, polypropylene, rayon, aramid, and / or cotton, among others. Also included within the scope of the present invention is the use of two or more different matrices with different chemical or physical characteristics. In one embodiment of the present invention, the porous matrix is ​​a mixture of two or more of the same or different types of woven and / or nonwoven fabrics. In another embodiment, a hybrid of two or more porous matrices with different pore sizes is used, one matrix having smaller pore sizes that act in capturing smaller materials while the other matrix, which has larger pore sizes, acts as a filter for larger materials (leukocytes, for example). In another embodiment, a porous matrix functionalized for affinity separations that has a predetermined pore size is placed inside another membrane as a support. 2.1. Nonwoven fabrics Nonwoven fabrics are random fibrous networks. Nonwoven fabrics are typically flat, porous sheets made directly from separate fibers or from molten plastic or plastic film. These fabrics are broadly defined as sheets or network structures bonded together by, for example, interlacing fibers or filaments or perforating films mechanically, thermally, or chemically using various techniques including adhesive bonding, mechanical threading by needling or fluid jetting, interlacing, thermal consolidation, and stitching. They are formed by mechanical means, wet processing, or airflow, and have open areas of interconnection across the cross-section. Typically, nonwoven fabrics have mean pore flow diameters (MPF) ranging from about 1 to about 500 / xm. In one embodiment, the porous matrix has a pore size of at least 10 / xm. In a preferred embodiment, the porous matrix has a pore size of more than 10 / xm. In yet another embodiment, the porous matrix has a pore size of more than 15 / xm. It is planned here that by reciting such specific numerical values, the values ​​also recited comprise all those specific integer quantities between the recited values. For example, it is desired that more than 10 / xm It also covers 12, 20, 30, 45, 70, 100, 200, 300, 400 and 500 gm, etc., without actually reciting each specific range here. The average pore diameter of the tissue can be chosen to match the desired pore diameter for the flow of large aggregates in the biological mixture. For example, in the case of red blood cells, the pore flow diameter would be on the order of about 12 gm. In this case, any porous or non-porous particles with diameters much greater than 12 gm would be retained in the spaces between the fibers, and the particles would still be available for adsorption by the target species. As a result, significantly smaller diameter particles can be used for adsorption while allowing the flow of larger species through the pore spaces. Nonwoven fabrics are made from fibers that, depending on the manufacturing method, have diameters ranging from, for example, about 0.01 to about 10 g / m². The fibers consist of a wide variety of materials comprising both natural and synthetic fibers. For example, natural fibers include cellulose, cotton, and wool, among others. Synthetic fibers include common polymers such as polypropylene and polyester (PET, polyethylene terephthalate). polyethylene). Polymers satisfactory include polyalkylenes such as polyethylene and polypropylene, Polyvinyl chloride, polyamides such as various nylons, polystyrenes, polyaryl sulfones, polyvinyl alcohol, polybutylene, ethyl vinyl acetate, polyacrylates such as polymethyl methacrylate, polycarbonate, cellulosics such as cellulose acetate butyrate, polyesters such as poly(ethylene terephthalate), polyimides, and polyurethanes such as polyether polyurethanes, and combinations thereof. Nonwoven fabrics can also be prepared from combinations of co-extruded polymers such as polyester and polyalkylenes. Copolymers of monomers of the polymers described above are also included within the scope of the present invention. Additionally, nonwoven fabrics are combined into nets that are a particular mixture of fine fibers and commonly used crimped fibers. In one embodiment, the nonwoven fabric of the device of the invention also includes a permeable support fabric laminated to one or both sides of the fabric, as described in U.S. Patent No. 4,433,024 (incorporated herein by reference in its entirety), or additionally contains reinforcing fibers. Nonwoven fabrics are made by different methods, including blow spinning and spin consolidation. There are Several mechanical approaches exist for consolidating nonwoven fabrics together; for example, membranes are welded together using an ultrasonic cutter / sealant or by using a press to apply heat and press simultaneously. Dry-process nonwovens contain layers of fibers, each layer containing randomly positioned or parallel fibers. Consolidation with an adhesive or heat is required for dry-process nonwoven fabric. Wet-process nonwovens are paper-like fabrics containing a random order of fibers in layers, with layer formation resulting from the deposition of fibers from a water paste. Needle-punched nonwovens are characterized by the interlaced condition of the fibers of which they are composed, with the interlacing being the result of applying heat, moisture, and agitation to a fibrous network.Spunlaced nonwoven fabrics have fibers intertwined by the action of high-speed water jets (a process also called hydroentanglement). 3. Particles In one aspect, the device of the present invention comprises particles as well as the porous matrix. Particles have an ability to trap the target agents. The particles are either porous, non-porous, or both. In a In this modality, porous particles are sorbent particles capable of adsorbing or absorbing the target agent. The particles are made of one material or a combination of two or more materials, which are either swollen or non-swellable in organic or aqueous fluids and are substantially insoluble in water or fluids. It has been shown to be advantageous in some examples to employ the particles in two or more particle size ranges, depending on the size range falling within the broad range. The size and shape of the particles can vary widely within the scope of the invention and may depend to some extent on the type of porous matrix support in which such particles are embedded. For example, the particles have a spherical shape, a regular shape, or an irregular shape, or a combination thereof. Particles used in the device of the invention have an apparent size within the range of about 1-2 µm to about 200-300 µm. In general, differences in useful particle sizes are dictated by the type of porous matrix in which the particles are embedded, the processes and equipment used to form the porous matrix, and the porosity of the matrix thus formed. For example, networks Fibrous nonwovens and fibrillated polymer matrices can be formulated with the full range of particle sizes. Preferably, particles of approximately 40-200 µm are used for nonwovens, while particles of 1-100 µm are preferred for fibrillated polytetrafluoroethylene (PTFE) matrices. Also included within the scope of the present invention are particles having a wide range of pore sizes. Particles with a relatively large pore size are used for the efficient capture of larger target molecules, such as proteins, while particles with smaller pore sizes are used for the efficient capture of smaller target molecules. The range of available pore sizes is, for example, from about 0.001 µm to about 0.1 µm. In one embodiment, the pore sizes are about 0.1-0.55 µm. In another embodiment, the pore sizes are about 0.6-2 µm. In yet another embodiment, the pore sizes are about 0.25-5 µm or more. It is here planned that by reciting such specific ranges, the ranges also recited comprise all those whole quantities between the recited ranges. For example, in the range of about 0.1-0.55 µm, it is intended which also includes 0.2, 0.3, 0.4, 0.5 gm etc., without actually reciting each specific range with these. The particles are made of carbon or an organic compound that can be a polymer or copolymer. For example, the particles are made of a copolymer of styrene and divinylbenzene and derivatives thereof, polymethacrylate ester, azlactone-derived polymer or copolymer, organic-coated inorganic oxide particles such as silica, alumina, aluminum oxide, titania, titanium oxide, zirconia, and other ceramic materials, cup, cellulose, agarose, and a wide variety of different polymers, including polystyrene and polymethyl methacrylate, acrylic resins and other types of gels used for electrophoresis, among others. Other satisfactory particles for the purpose of this invention comprise any particle that can be coated with insoluble, swellable, or non-swellable sorbent materials on its external and / or internal surfaces. In one embodiment, the particles swell to a limiting volume 2-5 times or more when compared to their original dry weight. The function of coating is to provide functionality. specific and physical properties that can be sewn according to the intended specific separation assay. These functions include sorption, ion exchange, chelation, steric exclusion, chiral, affinity, etc. Preferred particle material for such layers includes inorganic oxide particles, preferably silica particles. Such particles having coated surfaces are well known in the art, see, for example, Snyder and Kirkland, "Introduction to Modern Liquid Chromatography", 2 aEd., John Wiley & Sons, Inc. (1979) and H. Figge et al., Journal of Chromatography 351 (1986) 393-408 and include modified silica particles, silica particles having covalently linked organic groups including cyano, cyclohexyl, C8 (octyl), and C18 (octadecyl) groups. Coatings can be applied mechanically by in situ cross-linking of polymers or can be functional groups covalently bonded to the surface of the particles. The amount of particles incorporated into the porous matrix can vary widely within the scope of the present invention. Generally, the amount of particles ranges from about 1% to about 99% by volume of the device. Preferably, the amount is greater than 20% by volume, and more preferably greater than 50% by volume. Thus, a The device of the present invention may contain up to 95% or more by weight of particles, thus providing a potentially high target-engaging capacity. The particles of the invention generally resist a wide range of pH values, for example, pH values ​​of about 4 or below to pH values ​​of about 12 or higher. The particles of the invention are versatile and are used to conduct a variety of chromatographic or non-chromatographic separation assays. Examples of separation methods contemplated within the scope of the present invention include reverse phase separations, affinity separations, expanded bed separations, ion exchange chromatography, gel filtration, chromatographic component separation, solid phase extraction, among other methods of separating, measuring or collecting chemical or biological target agents from other components of a sample. The particles are also used to bind and thus separate target agents from nucleic acid and / or polypeptide molecules in a sample. A preferred particle for the device of the invention is a porous resin. Porous resins for adsorption separations are available in a wide variety of... different materials, including silica, glass, cellulose, Agarose, and a wide variety of different polymers, including polystyrene polymethyl methacrylate, polyacrylamide, agarose, hydrogel, acrylic resins, and other types of gels used for electrophoresis. Many of the porous adsorption resins such as silica, glass, and polymers can be dried and have interconnected pores with surface areas in the range of about 1-2 m². 2 / g of dry resin for more than 300 m 2 / g of dry resin. Other types of resins are cross-linked gels that cannot be dried without damaging the structure. These types of resins regularly do not have a specific surface area since the materials can diffuse uniformly throughout the cross-linked matrix. Also included within the scope of the invention is the use of modified resins, including analogues, variants, and functional derivatives of a natural or modified resin, or its functional groups. Modification comprises, for example, substitution, deletion, or addition of chemical entities (e.g., amino acids) to a particular resin, or its functional group, or both. For example, amino substitution, acetylation, and / or partial acetylation of resins are included within the scope of the invention. Any modification to the functional group of a resin It is also included within the scope of the modified resins according to the invention. Other types of natural or modified resins useful within the scope of the invention include, but are not limited to, phenyl sepharose, butyl sepharose, octyl sepharose, divinylbenzene crosslinked polystyrene, C3 polystyrene-divinylbenzene hydrocell, C4 polystyrene-divinylbenzene hydrocell, phenyl polystyrene-divinylbenzene hydrocell, methyl HIC methacrylate, butyl HIC methacrylate, high-pore-high-phenyl phenyl, Fractogel END, hydrophobic resin-propyl methacrylate copolymer, Fractogel EMD, hydrophobic resin-phenyl octyl sepharose methacrylate copolymer, phenyl sepharose, Toyopearl HIC, Toyopearl amino-65OS, Toyopearl amino-65OM, Toyopearl amino-650C, Toyopearl amino-650EC, Toyopearl butyl-650S, Toyopearl butyl-650C, Toyopearl butyl-650M, Toyopearl ether-650S, Toyopearl ether-650C, Toyopearl ether-650M, Toyopearl hexyl-650S, Toyopearl hexyl-650C, Toyopearl hexyl-650M, Toyopearl phenyl-650S,Toyopearl phenyl-650C (PRDT), Toyopearl phenyl-650M, and Toyopearl 659 CU (PRDT) among others. All Toyopearl resins are, commercially available of Tosoh Montgomeryville, PA., Sepharose resins are available from GE Healthcare, Piscataway, NJ. Fractogel resins are available from Merck, Darmstadt, Germany. Hydrocell resins are available from BioChrom Labs, Inc., Terre Haute, IN. The remaining resins are generic names for a variety of publicly available resin base materials. If porous resins are placed in a column, the hydrodynamic diameter available for flow is determined by the particle diameter and the void fraction of the bed: in which D h is the equivalent hydraulic diameter for flow between particles, d p λ is the particle diameter, and μ is the fraction of empty space. As a result, allowing large species to flow through the column requires using large particles which, in turn, increase diffusion resistance by adsorption onto the resin. For example, allowing red blood cells to flow through the column, around 65 µm diameter particles are needed to provide a pore diameter of 14 µm in the interparticle space if the bed porosity is... approximately 0.4. (Kidneys) The incorporation of particles into the matrix can be achieved in a variety of ways. Since nonwoven fabrics with a controlled average pore diameter can be made, it is possible to impregnate porous resin particles, such as those described above, within the fibers that make up the nonwoven fabric. These impregnated nonwoven fibers can be made in a variety of ways. For example, dry particles can be hydroentangled between two previously formed nonwovens. Alternatively, dry particles can be introduced while the fibers are being formed during blow spinning or continuous filament spinning. It is also possible to interlace resin particles while wet using wet processes. In one embodiment, the particles are impregnated into an already formed porous matrix through hydroentanglement and there would be no fusion consolidation of the particles with the polymer fiber matrix. A preferred manufacturing method is the direct calendering of pre-prepared nonwoven fabrics, which could be blown web or continuous filament. In a The membrane is uniformly distributed with particles at a fixed mass rate through direct calendering, ensuring a specific particle mass per unit area. Once the particles are spread, a second membrane is placed on top to create a sandwich, and the combination is passed through a calendering roller with a pattern capable of bonding the two membranes together at low temperature and pressure. Particle densities on the surface range from approximately 0.1 to approximately 10 g / m². 2 or more. The pore size of the membranes used for this device allows larger entities, such as red blood cells, to pass through as long as their pore size is greater than 10 pn. The particles in the membrane are attached to a ligand that facilitates the binding of the particles to target agents such as prion proteins from red blood cell concentrates and plasma. The calendering process typically requires a high temperature to join nonwovens, but the temperature is kept below the melting point of the particles and does not affect their performance. In one embodiment, larger particles or hydroentanglement. The density and weight of the nonwoven fabric can assume a wide range of values ​​to ensure high particle density in the fabric while maintaining the desired pore dimensions. Particle concentrations of approximately 60% w / w can be impregnated into the fabrics. All common methods of making nonwoven fabrics can be used for this procedure, including fabrics with two different polymer fibers as well as co-extruded fibers with two different polymers. Because of this flexibility, both wet and dry resins can be impregnated. If necessary, embedded fibers can be soaked into the fabric to facilitate particle capture while still allowing pore flow of the required dimensions. Chopped fibers are typically less than 1 / 2 inch in length, and they are prepared by cutting a single fiber that is wound around a spindle or roll. The cutting is achieved mechanically using rotating blades or other sharp cutting surfaces. Chopped fibers can be made from a variety of polymeric or carbon fibers that have a range of diameters from very small (less than about 1 µm) to large (>100 µm). Chemically or coated onto the fiber, the fiber is then cut to lengths of about 1 / 2 inch. Cut fibers can then be distributed over a single layer of nonwoven fabric (polypropylene or other polymer) with a pore size and fiber diameter sufficient to allow large entities such as red blood cells to pass through the membrane (pore sizes >10 µm / zm). Chopped fibers can be distributed to the membrane at a prescribed rate to ensure uniformity in fiber distribution. Once the chopped fibers are on the surface, a second layer of nonwoven fabric can be placed on top of the chopped fibers, and the combination can be passed through a calender to join the two nonwoven layers. In one embodiment, a porous matrix membrane or chopped fibers are functionalized with a binder and placed inside another membrane by, for example, aerodynamic technology. In another embodiment, the binders are attached to a polymer that is subsequently extruded into a fiber. The fiber can be chopped to make small segments that could be readily integrated between two membranes. 5. Surface modification Also included within the scope of the present invention are surface-modified woven or nonwoven fabrics (SMNs) and surface-modified particles that are functionalized on one or more internal and / or external surfaces with a reactive group. Functionalization is achieved by adding one or more reactive groups to a surface of the porous matrix (e.g., woven or nonwoven fabrics), particles, or both. The reactive group interacts with and binds to the target agent. The interaction between the reactive group and the target agent is a chemical, physical, and / or biological interaction. In one embodiment, the porous matrix, the particles, or both have their surface modified with a functional group capable of forming a covalent chemical bond with a target agent. Useful functional groups within the scope of the invention include, but are not limited to, one or more of the following groups: epoxy, formyl, tresyl, hydroxysuccinimide ester, among others. Other useful groups within the scope of the invention include, but are not limited to, one or more of the following groups: sulfonic acid, quaternary amines, carboxylic groups, primary amines, cyano, cyclohexyl, octyl, and octadecyl groups. Oxirane, N-hydroxysuccinimide ester, sulfonyl esters, imidazolyl carbamate, quaternary amines, carboxylic groups, dye ligands, affinity ligands, antigen-antibody, nucleic acid molecules, ion exchange groups, chelation, oxidation / reduction reactions, steric exclusion reactions, catalysis reactions, hydrophobic reactions, reverse phase, and other reactions commonly found in chromatographic separations. The functional group, for example, ligands, are chemically conjugated to the support or can be attached by connectors such as streptavidin, beta-alanine, glycine, polymers containing glycine-serine, short-chain hydrocarbon of the formula --(CH2)--, polyethylene glycol, epsilon amino acid caproic acid, and connectors comprising --O(CH2)n, where n has 1-30. If desired, the ligand(s) can be attached by one or through several different cleavable connectors, for example, photolabile or acid-labile moieties, enabling the selective separation of a population of ligands for analysis. Separation ligands can be used, for example, as an affinity purification medium for proteins and enantiomeric separation (e.g., to concentrate, isolate, detect, characterize, to quantify, or identify targets in a sample), such as Therapeutic diagnostic tools, catalysts and enhancers of chemical reactions, and as selective protein stabilizers. In one embodiment, nonwoven membranes are coated with affinity ligands as a functional group whose affinity ligands have specific affinity for prions on their surface. Examples of affinity ligands include a primary amine with a hydrophilic spacer containing polyethylene glycol units. The ligands can be placed on the membrane (e.g., Macopharma polypropylene-treated plasma) by chemical grafting or by a latex emulsion coating method (padding). 5.1. Polymerization of Binders in a Porous Matrix Polymerization of monomers in a porous matrix introduces epoxy groups to the surface of these matrices, which in turn facilitate the chemical attachment of the binder to the matrix surface. In one embodiment, a monomer emulsion is applied to cotton, polypropylene, polyester, and synthetic fiber fabrics by padding. Padding is a continuous process used in the textile industry for dyeing, bleaching, and coating fabrics. Additional information on padding can be found on the Celanese LLC website: www.vectranfiber.com, incorporated here by reference. Padding generally consists of a set of squeeze-type rollers used to impregnate a fabric with a liquid by continuously passing the fabric through the liquid and then between the rollers to extract the excess solution. A single-dip padding technique is possible. Habeish et al. ("Improving cotton dyeing and other properties by emulsion polymerization with glycidyl methacrylate," American Dyesuff Reporter, April 26-34, 1986, incorporated here by (Reference) applied glycidyl methacrylate (GMA) emulsions to cotton fibers using padding techniques. After padding, excess water is evaporated and polymerization is conducted at elevated temperatures. The amount of polymer on the fiber surface is in the range of about 1 to about 10% or more. Polymerization can also be conducted on nonwoven networks of PET, PP, etc. with the desired pore size (> 10 µm). 5.2. Latex layers in fabrics Latex emulsions are synthesized through conventional emulsion polymerization in water to make small particles of the desired polymer. Various free radical and soluble initiators can be used. Non-ionic and anionic surfactants are used to create the emulsions. In one embodiment, the latex emulsions are coated onto the porous matrix by padding as described above on either single fibers or nonwoven networks of PP, PET, and other polymers. An example of this type of approach is provided by De Boos and Jedlinek, Application of epoxy functional POLYACRYLATE EMULSION TO TEXTILES, J. Macromol. Sci-Chem. A17(2), 311-235 (1982), incorporated herein by reference. 6. Methods of use The methods, kits, and devices of the invention are useful in a variety of applications including prognosis, diagnosis, detection, purification, separation, processing of gene-expressed products in vitro, and production and distribution of biopharmaceuticals. This invention is applicable to any device that is generally available for membrane operations, from flat plates, spiral wound, or even hollow fiber cartridge devices. Flow can be induced by the device by any common means, from gravity to pumps, depending of the pressure droplet and desired flow rate. of the invention The purification and extraction techniques offer advantages over purification techniques conventional methods, reducing the number of purification steps, Improving yields, increasing purity, and overcoming limitations associated with traditional methods. The devices of the invention are highly sensitive and capable of separating minute quantities of pathogen from a sample. In one embodiment, the devices of the present invention are used for the removal of pathogens such as prions, including PrP. c PrP sc PrP res Viruses, bacteria, and toxins from whole blood, red blood cell concentrates, platelet concentrates, plasma, plasma derivatives, leukocytes, leukodepleted blood, mammalian cell cultures, fermentation broths, and other media used for the production and distribution of biopharmaceuticals and the preparation of therapeutics. Multiple pathogens can be separated from the sample concomitantly and rapidly by the devices of the present invention from any stream in the plasma processing industry aimed at the production of therapeutic and / or pharmaceutical products. In particular, the methods and devices of the present invention improve the protein purification process and enhance the industrial process of biopharmaceuticals by increasing efficiency and purity. Biopharmaceuticals are drugs that are proteins, peptides, or other complexes of... Polynucleotides or protein-based macromolecules (collectively "gene products"). Their manufacturing process involves recovering the desired gene product from its host biomass, such as plasma or other human and non-human biological sources (e.g., recombinant or non-recombinant cell cultures, milk from transgenic animals, or recombinant or non-recombinant plant extracts). Commercially viable yields of separating the desired protein from biomass are challenging since the latter contains unwanted host proteins, nucleic acid molecules, and other naturally occurring chemical entities. Protein separation and purification processes present unparalleled challenges due to the variety of proteins, the different nature of potential contaminants and impurities, and the amount of product to be separated from the media. Conventional purification technologies typically involve a series of purification steps. With each step, yield decreases and industrial costs increase. Protein separation and purification costs typically account for more than 50% of total manufacturing costs. In another modality, the devices are designed for The invention describes devices that perform two simultaneous operations: filtration and adsorption. In this embodiment, the pore size of the tissue is sufficiently reduced to reject large particulate matter while simultaneously maintaining a pore size large enough to allow passage of the sample containing the desired molecule. This technology achieves simultaneous filtration and adsorption steps in a single device and replaces membrane filtration followed by adsorptive column chromatography. For example, the devices of the invention make it possible to adsorb a desired or undesired molecule secreted extracellularly directly from a culture medium. In another embodiment, the devices of the invention are used as an alternative to columns for adsorptive removal techniques in the biotechnology industry. These techniques utilize biochemical interactions such as, for example, ion exchange, chelation, oxidation / reduction reactions, stearic exclusion, catalysis, hydrophobic interaction, reverse phase, dye ligand, affinity ligand, antigen-antibody and other interactions regularly found in chromatographic separation techniques and / or others. 7. Test kits Also included within the scope of the invention are test kits for sample purification by separating target agents from the sample. Complete test kits contain solutions and devices for targeted separation and purification of biological samples. For example, the test kit contains plates with 96, 384, or 1536 wells for high-throughput sample purification, and / or solutions for attaching ligands to particles within the device of the invention to customize individual proteins, antibodies, and solutions required for protein separations in a plate format. Generally, the test kits of the invention contain one or more of the following: (1) one or more containers containing the devices as described herein; (2) instructions for practicing the methods described herein; (3) one or more test components; and (4) packaging materials. The devices described herein are packaged to include many, if not all, of the components necessary to perform the separation methods of the invention. For example, test kits comprise the device containing the porous matrix and particles in addition to one or more of the following: buffers, reagents, chemical agents, functionalizing reagents, enzymes, detection agents, control materials, or similar, among others. In one embodiment, the kit additionally contains the functional groups in separate containers, and the functional groups would have to be attached to the particles and / or porous matrix before performing an assay. Alternatively, the device may be provided in the kit without functional groups, in which case the porous matrix, particles, or both are preferably pre-functionalized. The devices of the invention can be of any desired size and shape. Preferably, the device is a sheet-like material that, for example, is in a disc or strip form. Other articles that may be provided as part of the test kit comprise solid surface syringes, pipettes, cuvettes, and containers. Coating the porous matrix or particles with monolayer materials or thickening materials provided by in situ cross-linking of polymers or functional molecules covalently linked to the surfaces of the porous matrix or particles allows for optimization of chromatographic selectivity and separation efficiency. Detection can be facilitated by combining the porous matrix, particles, or both, constitute a detectable agent. Examples of detectable agents include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, disperse dyes, gold particles, or a combination thereof. EXAMPLES It will be understood by one with ordinary skill in the relevant techniques that other satisfactory modifications and adaptations to the methods and applications described herein are readily apparent from the description of the invention contained herein due to information known to a normally qualified technician, and can be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, it will be more clearly understood by reference to the following examples which are included herein for illustrative purposes only and are not intended to limit the invention. Example 1: Surface nonwoven fabrics modified (SMNs) Nonwoven fabrics with modified surface Specifically, they are useful when the target species to be adsorbed are large and cannot penetrate the pores of the resins. In this example, the surface of the fibers comprising the nonwoven fabric has been modified to affect the adsorption of the target agents. The adsorption step involves ion exchange, hydrophobic or affinity interactions, or any other common adsorption process. If SMN is used without the particles, the surface area per The volume of material available for attachment is controlled by the porosity of the fabric and the diameter of the fibers. v = d f (2) In which v is the specific surface area per unit volume of solid, d f is the fiber diameter, and es is the void fraction. Since fiber diameters anywhere in the range of 100 nm to 10 µm are available, and porosities are regularly in the range of 0.4–0.5, they can be Very large surface areas are achieved in these devices. For example, with a fiber diameter of 0.1 gm, the surface area per unit volume of tissue would be on the order of, the v =2xlO 7 m 2 / m 3 = 20m 2 / cm 3 (3) This compares very favorably with the surface area per unit volume of many porous chromatographic supports. However, since the average pore flow diameter of the tissue can be controlled independently, pore sizes can reach several microns in diameter. Techniques such as electrostatic rotation can produce even smaller diameters, resulting in much larger areas per volume. Any surface modification that facilitates the binding of a target agent to the device and is compatible with the chemistry of the specific porous matrices used in the device falls within the scope of the invention. Surface modification includes, for example, the formation of charged species, the attachment of affinity ligands, peptides, oligonucleotides, proteins, spacers, hydrophobic moieties, fluorinated materials, among others. Since the surface of fibers in nonwoven fabrics tends to be smooth, these surfaces present a preferred configuration for the exposure of affinity ligands for a particular large species such as prion proteins, a virus, or a bacterium. Example 2: Device configuration for removal of prion protein (PrP) This example demonstrates the possibility of designing different device configurations to remove infectivity from endogenous transmissible spongiform encephalopathy by allowing adsorption to non-porous surfaces of various geometries. Endogenous infectivity of red blood cell concentrates involves the removal of PrP. sc infectious (shredded form of prion protein) or PrP res (resistant form of prion protein) to a total concentration of approximately 200 ng / ml. In a bag of red blood cell concentrate (RBCC) containing 350 ml, there was a total of 7 x 10' 5 g of PrP. Given that a protein monolayer covers a surface with a monolayer density of approximately 2 mg / m² 2 The total surface area required to bind all endogenous PrP into rbcc is calculated as follows. 7xl0~ 5 g 2xl0" 3 ^ / zn 2 = 3.5xl(T 2m 2 (4) Where A is the total area of ​​the device. As is evident from the equation above, the total surface area required to bind the prions is relatively small and accommodates several satisfactory geometric schemes. to expose affinity ligands to surface density Correct. A. Square sheets A set of N square sheets of nonwoven fabric having both sides covered with binders exposed to blood having a total surface area given by, 2NL 2 = 3.5xl0' 2 m 2 (5) Where N is the number of sheets, and L is the length / width of a square sheet. In the case of 10 sheets (N = 10), the required width of each sheet would be, L = 0.042m = 4.2cm (6) A device of this type consists of a zigzag arrangement of leaves with the fluid flowing in the tortuous path between the leaves, as shown in Figure 2. B. Coated fibers A set of N nonwoven coated fibers with affinity binders on the outside has a total surface area that is determined by, N2nRL = 3.5x10' 2 m 2 NRL = 5.57 xl0“ 3 m 2 (7) Where R is the radius of a fiber. For example, the number of fibers with a radius of 5 gm and a length of 5 cm would be, N= 22,280 (8) The volume of these fibers would be, V f =NjiR 2 L = 8.74x1 O' 8 m 3 x 1^-^- = 0.087^ / (9) 7 m In what V f It is the volume of fibers. As is evident from the equation above, the volume of the fibers is relatively small, which is mainly due to the very small fiber diameter resulting in a very high surface area per unit volume. To allow adequate flow of red blood cells through such a fiber mat, the porosity would have to be quite high, for example, around 50%. In this case, the volume of the device would be about twice the fiber volume or 0.17 ml. This is a very small volume, again indicating that a device for this type of capture does not need to be large to meet capacity requirements. For example, a fiber mat with a 2 cm radius would only need to be about 0.135 mm thick to provide this volume. One or more coverings on the outside with C. Particles Fiber sheets can be affinity binders. Small non-porous particles also exhibit an area very high surface area per unit volume. The number and volume of particles required to have the area The surface area stated above was calculated in a way similar to that used in the case of cylindrical structures, 3.5xl0" 2 zn 2 4nR 2 V = -7tR 3 N (10) In which, V s It is the volume of a particle. Certain particles with a radius of 10 ptm, the numbers and The particle volumes given by equation 5 are, N = 2.79x10 7 7 3 (11) V s =1.17xl0' 7 zn 3 = 0.117» / / The equations show an extremely small volume of small particles. The small particles could be dispersed with larger particles or suspended in a cross-linked gel (such as agarose) with large pores. to allow the easy flow of red blood cells through system. Example 3: Bonding of two membrane layers by calendering with or without resin. In order to develop a prion removal device, two layers of polypropylene membranes were successfully calendered at room temperature / 400 PLI and 150F / 100 PLI, with a resin density of 1 mg / cm³. 2 The calendered membranes were sealed using an ultrasonic sealer. The percentage of hemolysis in samples of the calendered membranes was well within the acceptable limit. Calendering was used for impregnation of Amino 650M resin between two membrane layers. Toyopearl Amino 650M resin particles were impregnated between two layers of nonwoven fabric membranes. Polypropylene (inner layer) and polyester (outer layer) membranes, which are commonly used in MacoPharma leucofilters, were good candidates for these membranes since they have already been approved for processing human blood. To investigate whether the particles could be immobilized without impeding the flow of red blood cells through the device, the inner layer membranes were examined. and / or external parts were calendered with or without particles. Calendering was achieved by pressing membranes between two rollers onto the sheets. Materials and methods A roll of polypropylene (PP) membrane and a roll of polyester (PET) membrane were wound onto a plastic spindle with a 3-inch inner diameter. The width of the rolls was approximately 0.5 meters. The membranes were 22.5 cm wide and 800 m long. The membranes were cut into 22.5 cm x 22.5 cm square sheets. The dried resin was spread at 1 mg / cm². 2On one side of the membrane, and then covered with another membrane. Solitary membrane samples did not require the resin to be spread. The previous samples were passed through two calender rolls, one shaping roll and one smooth roll. Both rolls could be heated to increase the temperature for calendering. The pressure between the rolls was also controlled. The calendered samples were tested through visual examination, weight measurement, cross-sectional examination via SEM (scanning electron microscopy) of the pore size test, and testing of the percentage of mainstream hemolysis after passing all the blood through the calendering device. Procedure for measuring the percentage of hemolysis. Membrane samples were cut into 25 mm circles and placed in 25 mm Millipore Swinnex filter holders. Each sample was tested in duplicate in mains flow, and then triplicated in 96-well plates. Each sample was rinsed with 2 mL of working buffer (working buffer is 20 mM citrate and 140 mM NaCl, pH 7.0), then all blood was pumped through the top membranes at 0.5 mL / min. Five mL of the mains flow was collected from each sample. The mains flow or untreated blood was centrifuged at 12000 rpm for 10 min at 4°C to collect the supernatant. Three 100 mL aliquots of each sample were placed in three wells of a 96-well plate. The UV absorbance of each plate was read at 415 nm. The average A4i value was calculated. 5nm It was divided by the 100% lysis value of the same blood. The percentage of hemolysis is considered acceptable if it is below 1%. Results TABLE 1. Conditions used for calendering and examination visual Samples made by calendering: Sample Membranes Temperature (F) Pressure No. Calendered Resin Main Roll Smooth Roll (PLI)* Results 1 Internal / Inner Layer Yes Cold Cold 100 with large cavity area 2 Internal / Inner Layer Yes Cold Cold 200 with medium cavity area 3 Internal / Inner Layer Yes Cold Cold 300 with small cavity area 4 Internal / Inner Layer Yes Cold Cold 400 with small isolated cavity area 5 Internal / Outer Layer Yes Cold Cold 300 poorly consolidated, large cavities 6 Internal / Outer Layer Yes 120 120 100 with small cavity area 7 Internal / Outer Layer Yes 140 140 100 with small isolated cavity area 8 Internal / Outer Layer Yes 150 150 100 Not well consolidated, large cavities 9 Internal / Outer Layer Yes 150 150 200 poorly consolidated, some cavities 10 Internal / External layer Yes 180 180 200 Poorly consolidated, some cavities 11 Internal / Yes 180 180 400 Loosely Sample Membranes Temperature (F) Pressure Outer layer consolidated 12 Inner / Outer layer Yes cold cold 400 slightly consolidated 13 Inner / inner layer Yes 150 150 100 Very good, with small cavity 14 Inner / inner layer None cold cold 100 loosely consolidated, with cavities 15 Inner / inner layer None cold cold 400 tightly consolidated without cavities 16 Inner / inner layer None 157 153 100 Very well consolidated 17 Inner / inner layer None 157 153 400 tightly consolidated 18 Inner / Outer layer None 157 153 400 loosely consolidated 19 Outer / Outer layer None 180 180 400 slightly consolidated, large cavities 20 Outer / Outer layer None 220 220 400 consolidated with some cavities 21 External / External layer None 220 220 600 Very well consolidated *PLI = pounds per linear inch From visual inspection, samples Nos. 4, 7, and 13 They proved to be the best for particle fitting. When the temperature of the rollers is increased, a higher pressure is applied. Low pressure can be used to join the membranes, as shown. For samples 4 and 13, the outer layer membrane was made of polyester, which was much thicker and harder than the inner layer. To calender the outer layer with either the outer or inner layer membrane, higher temperatures and roller pressures were required, as shown for sample 21. TABLE 2. Weight measurement Weight of calendered samples: Samples Resin Weight (gsm) Single inner layer 41 Single outer layer 67 Inner / calendered inner layer 82 Inner / calendered inner layer 1 mg / cm2 92 Inner / calendered outer layer 107 Inner / calendered outer layer 1 mg / cm2 2 117 External / calendered outer layer 1 mg / cm 2 129 *PLI = pounds per linear inch For resin density, 1 mg / cm³ 2 is equal to 10 g / m³ 2 (1 mg / cm 2 x 104 cm 2 / m 2 = 10 g / m 2 ) . The weight of the single layer, The properties of the double layer and double layer embedded with resin were relatively proportional. The results showed that the resin was well retained between the two membrane layers. Scanning electron microscopy (SEM) of samples 11 and 13 revealed that most resin particles were intact after calendering, although some were cracked. Sample 2 was also examined by SEM and revealed similar results. On the patterned calender roll, there were 2 mm x 2 mm square grid spaces. During calendering, the membranes were highly compressed where the grids touched. This area is referred to as the bonding area. The cavity area refers to an area that is further away from the bonding area. Sample 11 is an example of resin immobilization particles between an inner and an outer layer. Figure 3 shows cavity areas of sample 11 at 50X magnification. Sample 13 is an example of resin immobilization particles between two inner layers. Figure 4 shows cavity areas of sample 13 at 50X magnification. Pore ​​size distribution The results of the pore size distribution of Calendered samples are shown in Table 3 below. For Calendered samples with the smallest, medium, and largest pore sizes decreased by 30% to 50% compared to the single layer. To determine if the decrease in pore size would impede the passage of red blood cells through the device, additional tests were performed on whole-blood flow hemolysis. TABLE 3 Pore ​​size distribution of calendered samples: Sample No. Membrane Resin Temperature Pressure (PLI) Pore Size Distribution (gm) of Rolls (°F) Molded Smooth Smaller Medium Larger 1 Inner / inner layer Yes Cold Cold 100 2.28 3.62 7.71 2 Inner / inner layer Yes Cold Cold 200 2.10 4.52 8.80 3 Inner / inner layer Yes Cold Cold 300 1.91 3.89 7.80 4 Inner / inner layer Yes Cold Cold 400 1.99 4.69 9.62 15 Inner / inner layer No Cold Cold 400 2.06 3.90 9.26 7 Inner / inner layer Yes 140 140 100 1.82 4.05 8.00 13 Inner / inner layer Yes 150 150 100 1.99 4.88 8.73 16 Inner / inner layer No. 157 153 100 2.11 4.04 8.79 PP layer 4.15 7.03 13.91 Sample No. Membrane Resin Temperature Pressure (PLI) Pore Size Distribution (gm) Rolls (°F) Patterned Smooth Smaller Medium Larger Single Inner 11 Inner / Outer Layer Yes 180 180 400 1.12 3.32 7.99 18 Inner / Outer Layer No. 157 153 400 1.64 3.45 8.65 21 Outer / Outer Layer No. 220 220 600 N / A 17.76 47.35 PET Single Outer Layer 23.61 35.81 79.04 Example 4: Optimization of calendering using particles with high densities. The calender roll used in this sorting process was purchased. by ProMetic of BF Perkins. The roller is made of steel. 5 stainless steel, engraved with a honeycomb pattern, and covered with a Teflon release layer. The back roller used was covered with rubber (600 to 1000 cm thick). Samples of four grams of dry resin were spread manually in a 30 cm x 20 cm (600 cm²) area. 2 of 10 samples of plasma-treated polypropylene membrane which corresponds to a particle density of 6.6 mg of resin / cm³ 2 A second membrane sample was placed in on top of the resin layer, and the sandwich passed through the rollers from the calender at 10 m / min. The following table contains the results obtained during the tests. Table 4: Calendering optimization results Screening Temperature set (°F) Temperature measured (°F) Opening 1 (mm) Results 1 212 198 203 No bond with or without particles 2 230 215 0 Membranes were weakly fused 3 245 236 0 Better than before, but still very weak 4 255 245 0 Good bond without resin, but less efficient than with resin 5 260 248 0 Good results with and without resin 6 275 - 0 Temperature was too high, upper membrane did not fuse to the lower one, but adhered to the roller 7 265 - 0 Good results were achieved at this temperature with and without resin 1) A zero opening indicates that the pattern penetrates the bottom roller before about 1 / 1000 of an inch. The samples were observed under a microscope and showed no pinholes. One sample from each screening was kept (only without resin) for future reference. Example 5: Binding of an O-Lactoglobulin and flow characteristics of resin-saturated calendered membranes. This experiment was conducted to determine the breakthrough curves for the binding of a model protein (β-lactoglobulin) to calendered membrane materials containing resin dried to a density of 4 mg / cm². 2 . Polypropylene membrane material was calendered to 170 °F and 150 pounds per linear inch (PLI) containing dry resin at a density of 4 mg / cm³ 2This membrane material was cut and assembled into Millipore Swinnex filter units. Each filter unit contained a stack of 1 to 4 membrane layers plus an additional uncalendered membrane layer at the outlet side of the filter unit. A 0.5 mg / mL β-lactoglobulin solution in IX PBS was passed through the filter unit at 1.5 mL / min using a peristaltic pump. 0.5 mL fractions were collected over 4 minutes and analyzed for their protein concentration using the Pierce Micro BCA assay kit (Pierce, Rockford, IL). Figures 5 to 7 show the results of the Micro BCA assay of the 12 fractions collected for the different number of membrane layers. The resin-free calendered membrane was used as a control. The results of this experiment show the difference in binding between the resin-coated membrane and the control. All runs exhibited a similar initial slope of non-binding concentration; however, layers 2-4 were not run long enough to show the saturated condition. Table 5 Total amount of bound proteins and the quantity of proteins linked by resin weight. Total bound proteins mg Membrane Control Resin mg bound / g of resin 1 layer 0.805 1.095 4.5 2 layers 0.792 1.493 5.4 3 layers 0.731 2.085 6.9 4 layers 0.776 1.859 4.2 The amount of bound proteins generally increased for each additional membrane layer, peaking at three membrane layers followed by a very slight decrease at four layers (Table 5). Since the filters Containing 2 to 4 layers of membrane, they were not run. Long enough to exhibit saturated conditions doesn't necessarily mean they've terminated the connection. Example 6 Particle distribution in membrane rolls A particle spreading unit was developed to replace the previously used manual droplet distribution. The equipment was tested and calibrated. The powder applicator was manually adjusted to 60%, which was equivalent to a distribution rate of 6.52 to 6.99 kg / hour (shown). The weight-determined distribution rate measurement was 6.65 kg / h (average of three determinations), within 5% of the designated value of 6.96 kg / hour. The distributed powder appeared to be uniformly distributed (visual assessment) and did not overflow the edges of the membrane. Figure 9 shows the particle distribution on the lower membrane after running the line for approximately 1 hour. The sharp edges formed by the area containing the particles can be seen on both sides of the membrane. Another notable characteristic It's the lack of dust on the conveyor belt, even after some time in production. Example 7: Prion binders 1. Prion binders A list of resins used to bind prions is revealed below. a) Amino 650M - basic resin for joining peptides and other ligands. This basic resin has demonstrated usefulness in binding prion proteins, PrP. c normal and PrP 50 infectious (or PrPres). The resin was used in column chromatography and we demonstrated PrP removal / binding.sc (hamster, rat vCJD, Fukuoka rat, human spCJD and human vCJD) from red blood cell concentrate, plasma, whole blood to the detection limit by in vitro techniques (Western Blot) and a reduction in hamster infectivity in flaps, i.e., in vivo model. (red blood cell concentrate) of about 4 logs. b) Toyopearl - SYA - This tripeptide has demonstrated utility in binding the prion protein, PrP. c normal and PrP sc infectious (or PrPres). The resin was used in column chromatography and we demonstrated PrP removal / binding. sc (hamster, rat vCJD, Fukuoka rat, human spCJD and human vCJD from red blood cell concentrate, plasma, whole blood to the detection limit by in vitro techniques (Western Blot) and a reduction in hamster 263K infectivity in scraps, that is, in vivo model (red blood cell concentrate) of about 4 logs. c) Toyopearl. DVR - This tripeptide has demonstrated utility in binding the prion protein, PrP. c normal and PrP 50 infectious (or PrPres). The resin was used in column chromatography and we demonstrated PrP removal / binding. sc (hamster, mouse vCJD, Fukuoka mouse, human spCJD and human vCJD from red blood cell concentrate, plasma, whole blood to the detection limit by in vitro techniques (Western Blot) and a reduction in hamster infectivity in flaps, i.e., in vivo model) (red blood cell concentrate) of about 4 logs. Amino 650M, SYA, and DVR were used at full scale, i.e., 1 total unit of red blood cell concentrate passed over the resin (approximately 350 ml). The column size was 10 ml of swollen resin. SYA, DVR, and amino function at 400 µmol / g (dry resin). Example 8. Comparison of PrP binder sc Amino 650M and Amino 650U of SBH traced in buffer, filtered plasma and whole blood Amino 65 OU is a mixture of account sizes Different variants include Amino 650M, which is less expensive to produce than 650M. Amino 650U has been tested for endogenous PrP and its ability to bind PrP. sc In all matrices—buffer, filtered plasma, and whole blood—currently used, the binding of Amino 650M challenged with traced whole blood was compared. The experiment was designed to compare the binding of PrP. sc Buffer, plasma, and whole blood traced for Amino 650U and establishing PrP binding. c Endogenous plasma and whole blood for Amino 65OU. Additionally, the experiment was designed to determine the effect of leukofiltration on PrP removal. cThe traced buffer refers to the addition of brain homogenate to working buffer. Whole blood traced is the addition of brain homogenate to human or hamster whole blood. No difference in signal was found for prion removal by 650 U or 650 M when present in plasma or whole blood. In conclusion, 650 U and 650 M amino acids had the same performance. The amount of PrP c The amount removed by leukocyte filtration was greater than that calculated to be in platelets and leukocytes combined. Therefore, it was possible that leukocyte filtration also captured some of the PrP. c derived from plasma. Leukofilters have been shown to behave unlike with respect to PrP capture c derived from Human and hamster plasma. It is possible that while PrP c Hamster plasma was not captured by the filter, PrP chuman plasma was. Finally, it is also likely that the difference between the two results is due to a lack of correlation between PrP. c and infectivity. The amount of PrP c The amount removed by leukocyte filtration was greater than that calculated to be in platelets and leukocytes combined. Therefore, it was possible that leukocyte filtration also captured some of the PrP. c derived from plasma. For this, it was shown that leukofilters behaved differently with respect to PrP capture. c derived from human and hamster plasma. It is possible that while PrP c Hamster plasma was not captured by the filter, PrP c human plasma was. Finally, it is also likely that the difference between the two results is due to a lack of correlation between PrP. c and infectivity. Example 9: Binding of PrPsc hamster brain to AMN resins. Comparative linkage experiments were conducted for a series of resins (e.g., AMN-13, 14, 15, 16, and 17, amino 650M and amino 650U). AMN series related to 650U samples (recently designated as 650C-prdt) with variation in the level of amino substitution as follows: AMN-13; 0.094 eq / L AMN-14; 0.078 eq / L AMN-15; 0.072 eq / L AMN-16; 0.063 eq / L AMN-17; 0.098 eq / L PrP-bonded resins scThe results demonstrated that all AMN resins bound equally well when challenged with traced buffer, plasma, and whole blood. Furthermore, the signal with AMN resins was equal to that with amino 650M and 650U. Comparing the PrP binding resin to traced plasma, there was a slightly stronger signal from Amino 650M compared to all other resins. Among the AMN resins, #13 appeared to have a weak PrP signal, but very comparable to amino 650U, while #15, 16, and 17 all... They performed better than amino 650 U. No notable difference was observed between the AMN 14, 15, 16, and 17 resins. In conclusion, the study demonstrated greater similarity. among the resins and importantly showed a closer correlation with amino 650U than with 650M. The differences observed with plasma suggested that at least with that The challenge of reducing the level of substitution may be... beneficial and the resin performed more closely at 650 M. amino Example 10 Extraction of proteins bound to resin-embedded membranes and determination of PrP binding. c of normal hamster brain homogenate The development of the novel device using calendered resin-embedded membranes necessitates the development of new procedures for extracting the bound proteins from the resins. Changes had to be made to the material handling, as well as the composition, concentration, and volume of the extraction solution. The experiment was also designed to perform binding assessments in the novel format, using both Toyopearl Amino 650M resin-embedded membranes and their fully acetylated form. Normal hamster brain homogenate (HaBH) was treated with sarkosyl and centrifuged. The resulting supernatant was diluted to a final concentration of 1% using working buffer or human whole blood. Fifty milliliters of traced solution were passed through a 47 mm Swinnex filter support (Millipore) containing 4 sandwiches of calendered membranes soaked with 4 mg / cm² 2 chromatographic resin to full capacity, or a form of capacity reduced replacement of the same resin as a control of Toyopearl Amino 650M or its fully acetylated form. The flow rate used was 0.5 mL / min, using a peristaltic pump. Ten 5 mL aliquots were collected for each of the traced solutions and membrane types. Mainstream samples from both traced buffer-challenged membranes were analyzed by ELISA. Membranes containing fully acetylated resins and those challenged with traced whole blood were rinsed using working buffer. Membrane sections (in some cases the entire stack) were treated with either SDS-PAGE sample buffer or 99% formic acid. The formic acid treatment consisted of adding 0.5 mL of 99% formic acid and 10 g / L of 20% SDS to one-quarter of a membrane sandwich, followed by incubation for 1 hour, removal of the liquid, and evaporation using a SpeedVac. Samples had their volumes adjusted to 15 µL using water, followed by the addition of 15 µL of 2X sample buffer. The sample buffer treatment consisted of adding 3 mL of IX sample buffer to the entire membrane stack, followed by incubation for 30 minutes and boiling for 7 minutes. The solution was collected without pressing the membranes and The sample was briefly centrifuged to remove all resin. A variation of the previous treatment was also tested. This consisted of adding 1 mL of 2X sample buffer to two separate stacks of membranes corresponding to % of a filter, incubating for 1 hour, followed by boiling only one of them. Elution with sample buffer without boiling can be used if disassembling the filter holders becomes too risky when infectivity is used. A final condition tested was the incubation of sections (1 / 4) of the membranes with sample buffer to verify binding to the first, second, third, and fourth membranes to contact the challenge solution. Samples were then run on SDS-PAGE gels and stained for total protein. Western blots were also performed. The gap volume of the filter support was approximately 7 mL. After passing 50 mL of challenge solution through each of the filters, followed by air, the recovered volumes were 45 and 47 mL for whole blood. When using plotted buffer, the recovered volumes were 46 and 46 mL. No significant difference was noted when using the different challenge solutions. The first filter support to be opened was the one containing the fully acetylated Toyopearl membrane, which was challenged with traced whole blood. It was noted that Despite air passage and rinsing with buffer, some blood remained inside the filter. During screening to rinse the membranes with buffer, there was a significant loss of resin, and the membrane was discarded. The filter holder with Toyopearl amino 650M challenged with whole blood was rinsed with an extra 200 mL of buffer. The flow rate was higher than the maximum (99.9 on the dial). Upon opening the holder, it was noted that there was still some blood inside, especially between layers. It was also noted that some pair sections delineated by the radial distributor were avoided during washing. The membrane stack was cut into 4 quarters. One of the pieces had its four layers separated and treated with sample buffer to investigate whether the different layers had different bonding. Another quarter was also separated into pieces and subjected to formic acid treatment. The two remaining quarters were used to compare the treatments with and without heating. The two filters challenged with traced buffer were rinsed with 200 mL of working buffer each. The filters were opened and the entire stack was transferred to a small glass vial to which 3 mL of buffer was added. A sample was added. The resin embedded in the calendered membranes appeared to maintain the same PrP binding properties characteristic of the resin in column form. The fully acetylated amino showed a weaker PrP membrane binding signal compared to the amino signal, supporting the conclusions that fully acetylated amino may not bind PrP effectively. Overall, the results indicated that 50% acetylation, either in a mixture form or through chemical synthesis, reduced PrP binding. res . Example 11 PrP Bonding c homogenate of normal hamster brain to filters containing particle-impregnated membranes The following experiment demonstrates the binding of normal PrP (PrPc ) of normal hamster brain homogenate (NBH) through membranes containing resin particles. The elution method described in the previous example was applied to these samples. The filter was opened, the membranes were placed in a glass vial and incubated with shaking using 2 ml of NuPage sample buffer (Invitrogen Corporation, Carlsbad, CA). The vial was then heated and the resin that came out of the filter was collected. Western blot results of the eluted proteins indicate that the method eluted PrP from the membrane. The results also They indicate that the filters with the resins bound more PrP than The filter without the resin. Example 12 PrP connection c of Homogenate of pieces of the brain to filters containing membranes impregnated with particles This experiment demonstrates the performance of filters in binding PrP.sc Infectious hamster brain homogenate (HBBM) was applied to whole blood and buffer. The filters contained membranes saturated with full-capacity resins, as well as reduced-capacity resin and no resin as a control. Elution was performed by injecting 2 ml of NuPage buffer sample (according to the extraction described in Example 4). Western blotting of the eluted proteins showed a strong signal without PK (protein kinase), but weak signals with PK. Considering that the proteins were eluted with 2% SDS, PK digestion was conducted under a 2% detergent concentration instead of 0.2% SDS (standard procedure). The weak PrP signal is likely due to this. res with PK possibly due to excess SDS in the reaction mixture. The results indicate a weaker signal with the resin-free membranes, but with similar signal intensities. For all other resins tested, no difference was observed between SBH in buffer and in whole blood for full-capacity resin and no resin. Reduced-capacity resin showed a stronger signal with traced buffer compared to traced blood. Example 13 Testing the filter prototype In this example, the filters were grouped in plastic cases and welded together in a configuration similar to an end device. The performance of such a device was evaluated using various challenges, both traced and untraced. In one embodiment, the end device consisted of a rigid or flexible plastic package containing layers of non-calendered membrane (from 1 to about 25 or more), followed by several layers (between 1 and about 50, depending on the desired capacity of the device) of resin-embedded membranes, and between 1 and about 25 layers of non-calendered membranes. The filters were welded together using an ultrasonic cutter / sealer or by using a press to apply heat and pressure simultaneously. In this example, both heat and pressure were used. Hamster brain homogenate was treated with 0.5% Sarkosyl and diluted 100 times in buffer, plasma. Filtered whole blood was used as a challenge for the resins. The resins were challenged with 80 ml of sample at 0.5 ml / min using a peristaltic pump. Western blots of the resin were then linked with PrP samples. res The tests were conducted without PK digestion. The results indicated that the membranes performed well with the tracer in buffer and in plasma, but the same tracer in blood appeared to be reduced. Example 13 Removal of PrP from RBCC traced by a series of filter devices This experiment demonstrates the removal of PrP from traced red blood cell concentrate (RBCC) using a device containing a particle-impregnated device. Given that the device was challenged with an excess of target protein, a series of devices was used. The total volume of RBCC used was equivalent to one unit. All filtrations were without any obvious problems, and one pump was used for all filtrations. All filters had soft covers and were pre-tested for leakage. None of the filters leaked in the pre-test or in the present experiment. The filtration time was approximately 10 minutes each for one unit of RBCC (~300 ml). The filters were washed with approximately 460 ml of buffer. work (citrate). The efficiency of the washing step was evaluated empirically by examining the color of the filter after washing. After washing, the filters were injected with air to remove all liquid from the filter. The filters were treated with approximately 4.6 ml of sample buffer for elution (injected into one side of the filter). The sample buffer was collected and injected into the other side of the filter. This step was conducted three times. The results indicated that the filter was... rp res esca p OUThe signal was captured by filter 1 and then by filter 2, which was expected since the applied challenge was higher than the capacity of a single filter set. PrP res It could also be present in the eluate from filter 3, but it might be below the Western blot detection limit. These results, compared to those with RBCC in the first PRDT infectivity study, indicate that the filters performed as well as the same resins used in a column format. As with the previous study, a large excess of PrP was observed. res It passed through the filters, and it's not surprising that all PrP res do not get caught in the first filter. Example 15: Glycidyl methacrylate graft on polypropylene, polyethylene terephthalate, and cotton substrates. and synthetic fiber The graft is one efficient method for change Polymer surface properties. Grafted polymers stitched with specific binders in combination with superior mechanical properties of the substrates make grafting the most versatile method for protein ligation, whose applications often require changing binders as well as considerable mechanical strength. In this example, glycidyl methacrylate (GMA) grafting onto polypropylene (PP) was performed on polyethylene terephthalate (PET), cotton, and synthetic fiber substrates to show the possibility of applying this approach for PrP removal. c of human blood. Procedure: The substrates used for the tests include woven cotton fabric, woven synthetic fiber fabric, PP nonwoven fabric, PP nonwoven membrane (Macopharma PP175), PP fibers, and PET fibers from the College of Textiles. at NCSU. The grafting was carried out in the following steps: 1. The samples were washed three times with acetone; 2. The samples were dried in a vacuum oven for more than three hours; 3. The samples were treated with argon plasma for 15 s at 750W; 4. The samples were left to aerate for 30 minutes; 5. The samples were then immersed in a 10% GMA solution in a UV chamber for 6 hours. The UV intensity was 1.1 W / m². 2 The chamber temperature was 30 °C and the temperature of the sample shelf was above 60 °C; 6. After UV polymerization, the samples were then washed with acetone three times; 7. The samples were dried in a vacuum oven. At this stage, the samples were only analyzed for their weight gain and IR spectrum. After analysis, the nonwoven PP tissue graft and the nonwoven PP membrane were additionally aminated in ammonium hydroxide solution at 60°C overnight. These samples were then washed and dried for elemental analysis. Results: Based on gravimetric measurement and IR spectroscopy results, PP substrates show significantly better grafting results than the other substrates: cotton, synthetic fiber, and PET. Three Types of PP substrates were tested, PP fiber, PP Non-woven and PP non-woven membrane (Macopharma). The weight gain after grafting is 85%, 154% and 57%. respectively (Table 6). These values ​​are much higher than those (-3% to 4%) of the other substrates. These results are further confirmed by the IR spectra where the grafted PP systems show strong peaks at 1720 cm⁻¹. 1 related to the carbonyl group and at 845 and 910 cm⁻¹ 1 related to the epoxide group, the signature for GMA. Other substrates did not show much change to Compare the blank sample with the samples after grafting. The reasons for the differences in grafting on the various substrates are not yet clear. One possible reason is that The CH bond might be easier to break in a PP environment than in another environment. Another possible reason is that the synthetic fiber and cotton fabric samples might have had surface treatments unknown to us. Simply washing with acetone may not be sufficient to remove the finish. It is also interesting to note that for the original PP fiber and non-woven membrane samples (Macopharma PP175) peaks are shown in the range of 840 to 920 cm⁻¹. However, no PP fabric showed such peaks. The peaks for the The preceding findings could be results of oxidation of the sample surfaces, which is due to processing at high temperatures. From the IR spectrum, the Macopharma amine samples show a broader peak at 3400 cm⁻¹. 1 to which the -OH and -NH2 groups are attributed, the amination results. Determination of surface area of ​​nonwoven materials Determining the surface area of ​​nonwoven materials is no easy task due to the complex interweaving of the fibers that form the nonwovens. However, the surface area of ​​a single fiber can be accurately determined by measuring microscopic images of the fiber and its length. Therefore, it is theoretically possible to measure the surface area of ​​nonwoven materials through fibers of the same materials using a grafting method, provided that the surface properties of the fibers are the same. that of non-woven materials. This method can be expressed by the following equation: where W t ; it is the graft weight gain, S E is fiber surface area and Sfxivj and the surface area of nonwoven material. There are only two unknowns in the equation: D and Snw, which can be determined through two independent experiments. The area determined by this method is the effective surface area corresponding to each reaction type. In fact, any reaction whose extent depends on the surface area of ​​substrates can be used for this method. Conclusions: Primary GMA grafting tests on various polymer substrates show that PP is an ideal substrate for such grafting. The weight gain of GMA grafting on PP varies from 60% to 160%, depending on the shape of the PP materials. The grafting effects are also confirmed by FTIR spectra. Furthermore, based on grafting, a simple method was proposed to measure the surface area of ​​nonwoven materials (Table 7). Table 7 Effects of grafting measured on weight gain Sample Name Substrate Time Weight Initial UV Gain (h) (g) Weight (%) Co-g-GMA-032905 Cotton fabric 0.6 0.1283 -1.8 Sample Name Substrate UV Time (h) Initial Weight (g) Weight Gain (%) Ny-g-GMA-032905 Woven Synthetic Fiber Fabric 0.6 0.1323 -0.7 PP-g-GMA-032905 Nonwoven PP 0.6 0.1179 1.7 Co-g-GMA-033105 Woven Cotton Fabric 6 0.0954 -2.8 Ny-g-GMA-033105 Woven Synthetic Fiber Fabric 6 0.1446 -1.4 PP-g-GMA-033105 Nonwoven PP 6 0.0537 91. Mac-PP-g-GMA-040805 Macopharma Nonwoven PP Membrane 6 0.0668 57 NW-PP-g-GMA-040805 Nonwoven PP Fabric 6 0.0738 154 FB-PP-g-GMA-040805 PP Fibers 6 0.0789 85 FB-PET-g-GMA-040805 PET Fibers 6 0.0517 4 Example 16 PrP Bonding c through PGMA fibers, networks electrostatic rotation, PGMA graft and Purpose: To determine the PrP binding c through PGMA fibers, electrostatic spin netting and grafting of GMA and non-woven padded polypropylene. Procedure: The materials tested were polyglycidyl methacrylate (PGMA) polymer melt casting fibers, PGMA electrostatic spin nets, PGMA graft, and quilted polypropylene nonwoven fabric (GMA-g-PP). The substrates are samples from the College of Textiles at North Carolina State University, MacoPharma PP175, and Macopharma PP 235. For each material, two replicates were prepared for protein binding. The samples were prepared by weight and apparent area (the measured area of ​​tissues). The second method only applied to samples with regular shapes, such as the quilted MacoPharma membranes. All samples were cut into small pieces. Each sample was then immersed in a conical tube (50 ml) containing 5 ml of 1% normal hamster brain homogenate (HaBH). The samples were then incubated on a shaker platform for 30 minutes. After that, the samples were washed with 10 ml of sample buffer. Three 10-minute sessions on the platform with agitation. Results: 0 weight, area and level of animation of the samples that were determined by analysis elementary are shown in 5 next in Table 8 below: Table 8 Elemental Analysis of the samples. Name Weight (g) N% PGMA-fiber A 0.1009 3.8% PGMA-fiber B 0.1011 3.8% PGMA-g-PP A 0.1007 2.6% PGMA-g-PP B 0.1009 2.6% PGMA-g-PP 0.1011 1.5% (Macol75) A PGMA-g-PP 0.1010 1.5% (Macol75) B PGMA-p-PPIA 0.1015 Not available PGMA-p-PPIB 0.1007 Not available PGMA-p-PP2A 0.1007 Not available PGMA-P-PP2B 0.1012 Not available Name Weight (g) N% PGMA-p-PP3A 0.1012 Not available PGMA-p-PP3B 0.1007 Not available PP nonwoven in 0.1009 None white A PP nonwoven in 0.1011 None white B Macol75A in white 0.1011 None Macol75B in white 0.1006 None 650MA 0.1016 0.6% 650MB 0.1011 0.6% Rotation 0.1001 Not available electrostatic A Rotation 0.1005 Not available electrostatic B Area (cm2) PGMA-p-PP 4x4 Not available (Maco235) 6A PGMA-p-PP 4x4 Not available (Maco235) 6B Name Weight (g) N% PGMA-p-PP (Maco235) 7A 4x4 Not available PGMA-p-PP (Maco235) 7B 4x4 Not available PGMA-p-PP (Maco235) 9A 4x4 Not available PGMA-p-PP (Maco235) 9b 4x4 Not available PGMA-p-PP (Maco235) 10 a 4x4 Not available PGMA-p-PP (Maco235) 10B 4x4 Not available Maco235A in white 4x4 Not available Maco235B in white 4x4 Not available Based on the western blot results, both PGMA PP graft and padded PGMA PP bind prion. EQUIVALENTS The invention is illustratively described here. 5 can be properly practiced in the absence of any element or elements, limitation or limitations that The specific features are not disclosed herein. The conditions and expressions employed are used as descriptive conditions and not limiting conditions, and there is no intention in the use of such conditions and expressions to exclude any equivalents of the features shown and described or parts thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed, optional features, modifications and variations of the concepts disclosed herein may be made by a person skilled in the art, and such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. All references discussed herein are incorporated by reference. A person skilled in the art will readily appreciate that the present invention is well adapted for carrying out the objects and achieving the ends and advantages mentioned, as well as those inherent herein. The present invention can be materialized in other specific forms without departing from its spirit or essential attributes. Conversely, it is to be clearly understood that the device may have various other embodiments, modifications and equivalents which, after reading the description herein, may be suggested by a person skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. SEQUENCE LISTING SEQ ID NO: 1 Amino acid 5 LAFGAA. SEQ ID NO: 2 YRVTWG Amino Acid SEQ ID NO: 3 Amino acid FLQGQ

Claims

CLAIMS 1. Device for separating at least one target agent from a sample, characterized in that it comprises one or more porous matrices having pore sizes greater than 10 g / m², and a plurality of particles impregnated therein, wherein the at least one target agent attaches to one or more porous matrices, particles, or both, and is removed from the sample.

2. Device according to claim 1, characterized in that the particles are porous, non-porous, or both.

3. Device according to claim 1, characterized by the fact that in one or more porous matrices, the particles, or both, have a pore size uniform or variable.

4. Device according to claim 2. characterized by the fact that the particles have a size pore size of about 0.001 gm to about 0.1 gm.

5. Device according to claim 1, characterized in that one or more porous matrices comprise at least one nonwoven fabric.

6. Device according to claim 1, characterized in that one or more porous matrices comprise natural fibers, synthetic fibers, or both.

7. Device according to claim 2, characterized in that the particles comprise a porous resin having interconnected pores with surface areas in the range of about 1-2 m². 2 / g of dry resin at approximately 300 m 2 / g of dry resin.

8. Device according to claim 1, characterized in that at least one target agent attaches to particles, to one or more porous matrices, or both, by absorption, ion exchange, covalent bonding, hydrophobic interactions, affinity interactions, formation of charged species, affinity ligand attachment, or a combination thereof.

9. Device according to claim 1, characterized in that one or more porous matrices, the particles, or both are surface modified by at least one reactive group comprising a chemical group, a biological group, or both.

10. Device according to claim 9, characterized in that at least one reactive group comprises a functional group comprising epoxy, formyl, tresyl, hydroxysuccinimide ester, sulfonic acid, quaternary amines, carboxylic groups, primary amines, cyano, cyclohexyl, octyl, and octadecyl groups, epoxide, oxirane, N-hydroxysuccinimide ester, sulfonyl ester, imidazolyl carbamate, quaternary amines, carboxylic groups, dye ligand, affinity ligand, antigen-antibody, nucleic acid molecules, reactive groups for ion exchange, chelation, oxidation / reduction reactions, stearic exclusion reactions, catalysis reactions, hydrophobic reactions, or reverse phase, or a combination thereof.

11. Device according to claim 1, characterized in that the sample is a blood sample and at least one target agent comprises prions, viruses, bacteria, protozoa, and toxins or a combination thereof.

12. Device according to claim 1, characterized in that the particles comprise a polymethacrylate resin or a methacrylate resin, a modified resin, or both.

13. Device according to claim 12, characterized by the fact that the modified resin comprises TOYOPEARL™ AMINO 650.

14. Device according to claim 7, characterized in that the resin comprises a wet resin, a dry resin, or a combination thereof.

15. Device according to claim 10, characterized in that the particles comprise a modified resin, one or more porous matrices comprise plasma-treated polypropylene, and the reactive group comprises a linker having a primary amine and a hydrophilic spacer containing polyethylene glycol units.

16. Device according to claim 1, characterized in that the particles are intercalated between one or more porous matrices.

17. Device for separating at least one target agent from a sample, characterized in that it comprises one or more porous matrices having pore sizes greater than 10⁻¹² m², wherein one or more porous matrices are surface modified by at least one reactive group comprising a chemical group, a group biological, or both.

18. Method of separating at least one target agent of a sample characterized by the fact that it comprises; (a) provide a sample that contains one or more potentially target agents; (b) provide a device comprising (i) one or more porous matrices having pore sizes greater than 10 gm, and (ii) a plurality of particles impregnated in one or more porous matrices; (c) subject the sample to the device; (d) arrest at least one target agent for the particles; and (e) separate at least one target agent from the sample.

19. Test kit for target separation and sample purification characterized in that it comprises (i) a device containing one or more porous matrices having pore sizes greater than 10 g / m², and a plurality of particles impregnated therein, (ii) a container containing one or more buffers, reagents, chemical agents, functionalizing reagents, enzymes, detection agents, control materials, (iii) instructions for use of the test kit, (iv) packaging materials, or any combination of (ii), (iii) and (iv).

20. Test kit, according to claim 20, characterized in that the detection agents comprise various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, dyes. dispersed particles of gold, or a combination thereof.