HK1101596B - Particles embedded ina porous substrate for removing target analyte from a sample - Google Patents

Description

Particles embedded in porous substrates for removing target analytes from samples

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 37u.s.c.119(e) based on U.S. provisional application 60/617,669 filed on 13/10/2004, the latter claims priority under 37u.s.c.119(e) based on U.S. provisional application 60/616,118 filed on 6/10/2004, the latter claims priority under 37u.s.c.119(e) based on U.S. provisional application 60/578,061 filed on 9/6/2004, the entire contents of which are incorporated herein by reference.

I. Field of the invention

The present 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.

Background of the invention

Methods of adsorbing biological species onto solid supports find many practical applications in purifying, detecting or removing target molecules from multi-component streams. For example, ion exchange, hydrophobic and affinity ligands can preferentially adsorb many reagents into a chromatographic stationary phase, affecting their separation from aqueous solutions. If adsorbed, the biological agent may be eluted into the product or detected by ELISA or other analytical means.

In some examples, the solution containing the target biological agent also contains larger entities such as erythrocytes, viruses, bacteria, liposomes, leukocytes, and aggregates of varying sizes. In many such instances, it is desirable to allow larger aggregates to flow through the solid matrix or support without affecting the ability of the target biological agent to bind to the support. This requires a sufficiently large pore area in the solid matrix to accommodate the flow of the larger entities. Unfortunately, larger pore domains may have only a small surface area, which limits the ability of the solid substrate to bind to the target agent.

In other cases, it may be desirable to actually filter out larger particles to facilitate adsorptive separation of smaller target agents. One example is the removal of cells from the culture medium to recover extracellular products.

In addition, there are many examples of the need to combine rather than filter out very large biological entities. For example, it is important to specifically adsorb many pathogens, including infectious prion proteins, viruses, bacteria, and toxins, from a mixture of biological agents. In currently used sample purification and separation devices, these entities are often difficult to access in the wells where binding is desired.

Nonwoven fibers or webs, also known as melt blown polymer fibers or spunbond webs, are well known and can be used for filtering and separating fine particles from air and aqueous solutions (see, e.g., U.S. Pat. nos. 4,011,067 and 4,604,203, which are incorporated herein by reference in their entirety). The loading of sorbosote particles onto nonwoven webs is also well known in the art (see, e.g., U.S.4,433,024; 4,797,318; and 4,957,943, which are all incorporated herein by reference in their entirety). Applications include the removal of particulate or gaseous contaminants in facial respirators, protective garments, fluid retaining articles, and wipes for wiping oily soils.

Recently, a method of manufacturing particles impregnated in a nonwoven fabric for separation and purification has been reported. See, e.g., U.S.5,328,758, which is incorporated herein by reference in its entirety. This patent teaches functional particles attached to affinity ligands. It discloses blowing the particles into the polymer fibers during the melt blowing stage. The nonwoven fabric contains pores having a pore diameter of 0.24 to 10 μm, preferably 0.5 to 5 μm. In particular, the impregnated textile material must also have a Gurley time of at least 2 seconds.

WO93/01880 discloses a leukocyte-depleted nonwoven filter material which is prepared by dispersing a plurality of small fibrous sheets having a fiber diameter of not more than 0.01 μm and a length of about 1 to 50 μm in a medium together with a short, spinnable and knittable fiber having an average length of 3 to 15 mm. U.S.4,550,123 and 4,342,811 describe microporous polymeric fibers and films comprising particles capable of adsorbing water vapor, liquids and solutes, all of which are incorporated herein by reference in their entirety. Typical sorbent particles include activated carbon, silica gel, and molecular filtration type materials.

As described, the present invention provides a device and method for purification and detection of a sample, and removal of target reagents from a sample, which has greater efficiency and specificity and can save a great deal of time and cost compared to prior art devices.

Brief description of the invention

As disclosed and described herein, the present invention provides methods, devices, and kits for removing a target agent from a sample.

In one aspect, the present invention provides a device for separating at least one target agent from a sample. The device comprises one or more porous matrices having pore sizes greater than 10 μm, and a plurality of particles impregnated therein, wherein at least one target agent is attached to the one or more porous matrices, the particles, or both, and removed from the sample. In one embodiment, the porous matrix, the particles, or both have uniform or varying pore sizes. In another embodiment, the pore size of the particles is about 0.001 μm to 0.1 μm. And in another embodimentThe particles comprise a porous resin having interconnected pores and have a surface area of about 1-2m²Per g of dry resin to about 300m²Per g of dry resin.

In 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 mixture of two or more woven and/or non-woven fabrics of the same or different types.

In another embodiment, the device of claim 1, wherein the particles comprise polymethacrylate, methacrylic resins, modified resins, or combinations thereof.

In one embodiment, the device comprises a modified resin, and the one or more porous matrices comprise plasma-treated polypropylene functionalized with reactive groups comprising ligands having a primary amine and a hydrophilic spacer comprising polyethylene glycol units.

In another embodiment, the particles are sandwiched between one or more porous matrices.

In one embodiment, the particles, the porous matrix, or both are functionalized with one or more reactive groups. The target agent is attached to the particle, the porous matrix, or both, by adsorption, absorption, ion exchange, covalent binding, hydrophobicity, dipole, quadrupole, hydrogen bonding, specific interaction, formation of a charged species, by affinity interaction with a particular ligand, or a combination thereof. In another embodiment, the particles are polymethacrylate or methacrylate resins, by way of example only, including but not limited to FRACTOGEL^TMEMD、TOYOPEARL^TMOr TSK-GEL^TMA polymer matrix. In another embodiment, the resin is TOYOPEARL^TMAmino650, including for example Amino650U, Amino650M, or Amino650M or Amino650U in partially acetylated form. Partially acetylated resins include from about 5% to about 95% or more acetylated resins. At one endIn one embodiment, the partially acetylated resin comprises 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 another embodiment, the partially acetylated resin comprises 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 intended herein that, through the description of these ranges, these recited ranges also include all specific integer values between these ranges. For example, in the range of about 40 to 60%, it is contemplated that 45%, 50%, 55%, 57%, etc. are also included without actually citing each particular range. In another embodiment, the resin comprises a wet resin (i.e., fully pre-hydrated), a dry resin (i.e., not pre-hydrated prior to contact with the sample, and/or previously dried but hydrated prior to contact with the sample). The use of partially acetylated dry and/or wet resins is also included within the scope of the invention.

In another aspect, the device comprises a functionalized porous non-woven or woven substrate having the ability to adsorb a target agent. In one embodiment, the device comprises a non-porous matrix and a porous matrix, one or both of which may be functionalized. In another embodiment, the porous matrix comprises uniform or varying pore sizes greater than 10 μm.

In yet another aspect, the present invention provides a method of isolating at least one target agent from a sample, comprising: (a) providing a sample that may comprise one or more target agents; (b) providing a device comprising (i) one or more porous matrices having pore sizes greater than 10 μm, and (ii) a plurality of particles impregnated in the porous matrices, wherein the particles have the ability to attach to at least one target agent; (c) placing a sample into the device; (d) attaching at least one target agent to the particles, to the one or more porous matrices, or to both; and (e) isolating at least one target agent from the sample.

In another aspect, the invention provides a kit for target isolation, detection and sample purification comprising one or more of (i) a device comprising a porous matrix having pore sizes greater than 10 μm and a plurality of particles immersed therein, (ii) a container comprising one or more buffers, reagents, chemical reagents, functionalising reagents, enzymes, detection agents, control substances, (iii) instructions for use of the kit, and (iv) packaging materials.

Other preferred embodiments of the invention will be apparent to those skilled in the art from the following drawings and description of the invention, and from the claims, in view of knowledge known in the art.

Brief description of the drawings

Fig. 1 depicts a representation of resin impregnated nonwoven fabrics (RINs). The nonwoven fabric had a pore size of about 12 μm and was impregnated with a porous resin carrier. The average pore size is large enough to allow red blood cells to flow freely through the device without showing any signs of damage. The particles (10), fibers (20), impregnated particles (11) and nonwoven fabric (21) are shown.

Figure 2 depicts a schematic representation of a device comprising a square piece of non-woven or woven fabric. The interlaced array of sheets of non-woven or woven fibers are coated on both sides with an affinity ligand. The sample flows in a tortuous path between the sheets. The pore size of these plates is adjusted according to the desired application.

FIG. 3 depicts a scanning electron micrograph of the inner/inner layer of sample 13 laminated with resin at 150F Per Linear Inch (PLI) and 100 pounds. The micrograph shows the pocket area of sample 13 at 50X magnification.

FIG. 4 depicts a scanning electron micrograph of the inner/outer layer of sample 11 laminated with resin at 180F and 400 pounds Per Linear Inch (PLI). The micrograph shows the pocket area of sample 11 at 50X magnification.

FIG. 5 depicts a histogram showing the results of 12 fractional Micro BCA analyses collected from different numbers of membrane (porous matrix) layers. Different β -lactoglobulin concentrations were expressed as the flow-through fraction of the solution through one layer of the resin-embedded membrane (labeled "resin") or one layer of the membrane (labeled "control").

FIG. 6 depicts a histogram showing the results of 12 fractional Micro BCA analyses collected from different numbers of membrane layers. The different β -lactoglobulin concentrations were expressed as the flow-through fraction of the solution through both layers of the resin-embedded membrane (labeled "resin") or both layers of the membrane (labeled "control").

FIG. 7 depicts a histogram showing the results of 12 fractional Micro BCA analyses collected from different numbers of membrane layers. The different β -lactoglobulin concentrations are represented by the flow-through fractions of the solutions through three layers of the resin-embedded membrane (labeled "resin") or three layers of the membrane (labeled "control").

FIG. 8 depicts a histogram showing the results of 12 fractional Micro BCA analyses collected from different numbers of membrane layers. The different β -lactoglobulin concentrations are represented by the flow-through fractions of the solutions through four layers of resin-embedded membranes (labeled "resin") or four layers of membranes (labeled "control").

Figure 9 depicts the distribution of particles in a roll of film. The resin particles are uniformly dispersed on the bottom film without overflowing the edges of the film.

Detailed description of the invention

Methods, devices and kits for efficiently separating target molecules from a sample are described below. The methods, kits and devices of the invention are useful in a variety of applications, including, inter alia, purification, isolation and manipulation of expressed gene products from cells, production and delivery of biopharmaceuticals, and prophylactic, diagnostic and/or detection applications. Described below are novel devices defined by the present invention that can separate different components from a sample and allow larger substances to flow through the device while providing a larger surface area for binding a target agent.

Particular applications of the present invention include the removal of pathogens such as prion proteins, viruses, fungi, bacteria and toxins from biological samples such as blood samples, including whole blood, compositions comprising red blood cells, red blood cell concentrates, platelet concentrates, plasma derivatives, white blood cells, depleted white blood cells, mammalian cell cultures, fermentation broths, and other media used in the preparation and delivery of biopharmaceuticals and in the preparation of therapeutics.

1. Definition of

The definitions used in this application are for illustrative purposes and do not limit the scope of the present invention.

As used herein, "modified resins" are defined broadly within the scope of the invention to include analogs, variants and functionalized derivatives of the resins with or without functional groups. Modifications include, for example, substitution, deletion, or addition of chemical entities (e.g., amino acids) to a particular resin or functional group thereof, or both. For example, amino substituted, acetylated, and/or partially acetylated resins are included within the definition of modified resin.

In this context, "target agent" is a general definition within the scope of the present invention, including chemical, biological or physical agents captured by the device of the present invention. In particular, target agents include molecules, compounds, cellular components, organelles, aggregates, toxins, prion proteins, and microorganisms such as pathogens, including viruses, bacteria, fungi, and protozoa. Target molecules also include, inter alia, polymer molecules such as polynucleotide molecules such as DNA, RNA, DNA-RNA hybrids, antisense RNA, cDNA, genomic DNA, mRNA, ribozymes, natural, synthetic or recombinant nucleic acid molecules, oligopeptides, oligonucleotides, peptides, peptide-nucleotide hybrids, antigens, antibodies, antibody fragments, large proteins and aggregates such as vWF: FVIII, and HDL.

As used herein, the term "pathogen" refers to any replicable material that can be found in or infect a biological sample, such as a blood sample. These pathogens include various viruses, bacteria, protozoa, and parasites known to those skilled in the art to be generally found in or infected with whole blood or blood components, and other unknown pathogenic contaminants. Illustrative examples of such pathogens include, but are not limited to, bacteria, such as streptococcal species, Escherichia coli species, and Bacillus species; viruses such as human immunodeficiency virus and other retroviruses, herpes viruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis a, hepatitis b and hepatitis c), poxviruses and togaviruses; and parasites, such as plasmodium, including proplastid species, and trypanosomatid parasites.

As used herein, "sample" includes any sample containing a target agent that can be captured using the devices and methods of the invention. The sample may be from any source that may contain the target agent. In particular, these sources include animals, plants, soil, air, water, fungi, bacteria and viruses. In particular, the animal sample is derived from, for example, a biopsy, blood, hair, buccal swab, plasma, serum, skin, ascites, various exudates, pleural effusion, spinal fluid, lymph fluid, bone marrow, fluids of respiratory organs, intestinal fluid, fluids of genitalia, feces, urine, sputum, tears, saliva, tumor, organ, tissue, sample of in vitro cell culture components, fetal cells, placental cells, or amniotic fluid cells and/or fluids.

As used herein, "cell culture medium" includes any prokaryotic or eukaryotic medium, for example, bacterial, yeast, and other microbial cell culture media, mammalian cell culture media, plant cell cultures, and insect cultures, fermentation broths, and other media used for the production and delivery of biopharmaceuticals and for the preparation of therapeutic agents.

As used herein, a "blood sample" includes, but is not limited to, for example, whole blood, compositions comprising red blood cells (e.g., red blood cell concentrates and platelet concentrates), white blood cells and depleted blood of white blood cells, blood proteins such as clotting factors, enzymes, albumin, plasminogen, and immunoglobulins; liquid blood components, such as plasma, plasma derivatives and plasma-containing compositions are also included in other blood samples.

As used herein, the term "red blood cell-containing composition" refers to whole blood, red blood cell concentrates, and any other red blood cell-containing composition. In addition to red blood cells, the combination may also comprise a physiologically compatible solution, such AS ARC-8, Nutricell (AS-3), ADSOL (AS-1), Optisol (AS-5) or RAS-2(Erythrosol), and one or more blood cell components, one or more blood proteins or a mixture of one or more blood cell components and/or one or more blood proteins. These compositions may also comprise a liquid blood component, such as plasma.

As used herein, "particles" refers to organic or inorganic porous or non-porous forms having a diameter of from about 1 to about 200 μm or more, and in particular, include, for example, but not limited to, fibers having a length to diameter ratio of from about 1 μm to about 20 μm or more, with the exception of sorbant particles such as particles, beads, resins, or powders.

As used herein, "sorbent," "sorbate," or "sorption" refers to a material that is capable of adsorbing and being retained by adsorption or absorption.

Herein, "attached" is a general definition within the scope of the present invention, in particular including any type of physical, chemical or biological binding process between two entities, including, but not limited to, attachment of, for example, adsorption, absorption, covalent binding, ion exchange, hydrophobicity, hydrogen bonding, dipole, quadrupole or affinity, formation of charged charge, affinity ligands (e.g., including peptides, oligonucleotides, proteins, spacer arms, hydrophobic moieties, fluorides).

As used herein, a "spiking solution" refers to a solution that has received a quantity of a target protein, toxin, virus, bacterium, or other organism in pure, partially purified, or crude form.

2. Porous matrix

The device of the present invention comprises a porous matrix into which the particles are immersed. The choice of porous matrix can vary widely within the scope of the present invention. Useful substrates include woven or nonwoven fabrics (e.g., fibrous webs), microporous fibers, and microporous membranes. These fibers are made from any material and by any method known in the art, including melt blowing, spunbond, and electro-spinning.

Fibrous webs are particularly desirable because these provide a large surface area, while non-woven webs are preferred due to ease of manufacture, lower material cost, and the ability to vary fiber texture and fiber density. The range of fiber diameters used in making the devices of the present invention may be large, for example 0.05 to 50 μm. The thickness of the matrix is varied to suit the desired application of the device, for example from about 0.1 μm to about 100cm thick or more. The substrate may be used in a monolithic or stacked form as needed to achieve the desired adsorption capacity. In one embodiment, it is desirable to roll or press the porous matrix to achieve the desired thickness and pore size. The porous matrix of the device of the present invention is made from a wide range of natural and synthetic fibers depending on the precise physical and chemical properties of the porous matrix of the end use application. In particular, the porous matrix of the invention is selected from natural or synthetic sources, including, for example, polyester, polypropylene, rayon, aramid and/or cotton.

Also, it is within the scope of the invention to use two or more different substrates having different chemical or physical properties. In one embodiment of the invention, the porous matrix is two or more woven and/or non-woven fabrics of the same or different types. In another embodiment, hybrids of two or more porous matrices of different pore sizes are used, one matrix having a smaller pore size for capturing smaller substances and the other matrix having a larger pore size for use as a filter for larger substances (e.g., leukocytes). In one embodiment, a functionalized porous matrix having a predetermined pore size for affinity separation is placed within another membrane as a support.

2.1. Nonwoven fabric

Nonwoven fabrics are any fibrous webs formed by mechanical, wet or air laying means and having interconnected open areas throughout the cross-section. Nonwoven fabrics are typically flat, porous sheets made directly from discrete fibers or from molten plastic or plastic films. These fabrics are broadly defined as sheet-like or web-like structures that are mechanically, thermally, or chemically bonded together by, for example, entangling fibers or filaments or perforated films using various techniques such as adhesive bonding, interlocking by mechanical concentrated needling or liquid jet, entanglement, thermal bonding, and sewing.

Typically, the nonwoven fabric has a Mean Pore Flow (MPF) diameter in the range of about 1 to about 500 μm. In one embodiment, the pore size of the porous matrix is at least 10 μm. In a preferred embodiment, the pore size of the porous matrix is greater than 10 μm. In another embodiment, the pore size of the porous matrix is greater than 15 μm. These specific numerical values are intended to be recited herein and include all specific integer values between the stated values. For example, greater than 10 μm is also intended to include 12, 20, 30, 45, 70, 100, 200, 300, 400, and 500 μm, and so forth, without actually reciting each particular range.

The average pore diameter of the fabric may be selected to correspond to the pore diameter required for the flow of larger aggregates in the biological mixture. For example, in the case of red blood cells, the pore flow diameter should be around 12 μm. In this case, when any porous or non-porous particles with a diameter greater than 12 μm are trapped in the spaces between the fibers, the particles can still be used to adsorb target substances. Thus, particles of significantly smaller diameter can be used for adsorption while allowing larger species to flow through the pore space.

Nonwoven fabrics are made from fibers having diameters ranging, for example, from about 0.01 to about 10 μm, depending on the method of manufacture. Fibers include a wide variety of materials including natural and synthetic fibers. In particular, natural fibers include, for example, cellulose, cotton, and wool. Synthetic fibers include common polymers such as polypropylene and polyester (PET, polyethylene terephthalate). Suitable polymers include polyolefins such as polyethylene and polypropylene, polyvinyl chloride, polyamides such as various nylons, polystyrene, polyarylsulfone, polyvinyl alcohol, polybutylene, ethyl vinyl acetate, polyacrylates such as polymethyl methacrylate, polycarbonate, cellulose such as cellulose acetate butyrate, polyesters such as poly (vinyl terephthalate), polyimides, and polyurethanes such as polyether polyurethanes, and combinations thereof.

The nonwoven fabric may also be made from co-extruded polymers such as polyesters and polyolefins. Copolymers of the above-mentioned polymeric monomers are also included in the scope of the present invention. Additionally, nonwoven is a combined web that is an intimate mixture of fine fibers and crimped staple fibers. In one embodiment, the nonwoven fabric of the device of the present invention also includes a permeable carrier fabric laminated to one or both sides of the fabric, as described in U.S.4,433,024 (which is incorporated herein by reference in its entirety), or further comprises reinforcing fibers.

Nonwoven fabrics can be made by different processes, including melt blowing and spunbond. There are several mechanical approaches to bonding nonwoven fabrics together, for example, films are welded together with an ultrasonic cutter/sealer or pressed together by the application of heat and pressure simultaneously. The dry laid nonwoven comprises multiple layers of fibers, each layer comprising randomly laid or parallel fibers. The use of adhesives or thermal bonding is necessary for drying the laid nonwoven fabric. Wet laid nonwoven fabrics are paper-like nonwovens comprising an arbitrary array of layered fibers, the layering of which is caused by the deposition of fibers from an aqueous slurry. A needle-punched nonwoven fabric is characterized by entanglement of the fibers that make up it, the entanglement being caused by the application of heat, moisture and agitation to the fibrous web. Woven (Spunlaced) nonwoven fabrics contain fibers entangled by the action of high velocity water jets, a process also known as hydroentanglement.

3. Granules

In one aspect, the device of the present invention comprises particles and a porous matrix. The particles have the ability to adhere to the target agent. The particles are porous, non-porous, or both. In one embodiment, the porous particles are sorbent particles capable of adsorbing or absorbing the target agent. The particles are made of a material or a combination of two or more materials, wherein the material is non-swelling or swelling in an organic or aqueous liquid, and is substantially insoluble in water or liquid. We have found that it is advantageous in some cases to use particles having a wide range of two or more particle sizes.

The particle size and shape of the particles may vary widely within the scope of the present invention, depending to some extent on the type of porous matrix support into which the particles are incorporated. For example, the particles are spherical, regular, or irregular in shape, or a combination thereof.

The particles used in the device of the present invention have an apparent size of from about 1-2 μm to about 200 and 300 μm. Generally, the difference in useful particle size is dictated by the type of porous matrix into which the particles are incorporated, the method and apparatus used to form the porous matrix, and the porosity of the matrix formed. For example, nonwoven fibrous webs and fibril containing polymer matrices can be made or have the full range of particle sizes of the particles. Preferably, particles of about 40-200 μm size are used for the nonwoven, while particles of 1-100 μm size are preferably used for the fibril-containing Polytetrafluoroethylene (PTFE) matrix.

Particles having a wide range of pore sizes are also included within the scope of the invention. Particles with relatively large pore sizes are used to efficiently capture larger target molecules, such as proteins, while particles with relatively small pore sizes are used to efficiently capture smaller target molecules. Useful pore sizes range, for example, from about 0.001 μm to about 0.1 μm. In one embodiment, the pore size is 0.1-0.55 μm. In another embodiment, the pore size is about 0.6-2 μm. In another embodiment, the pore size is about 0.25 to 5 μm or greater. It is intended herein that the description of these specific ranges also include all the specific integer values between the stated ranges. For example, in the range of about 0.1 to 0.55 μm, it is also intended to include 0.2, 0.3, 0.4, 0.5 μm, etc., without actually reciting each particular range.

The particles are composed of carbon or organic compounds which may be polymers or copolymers. For example, the particles are composed of, inter alia: copolymers of styrene and divinylbenzene and derivatives thereof, polymethacrylates, derivatized azlactone polymers or copolymers, organic-coated inorganic oxide particles such as silica, aluminum, alumina, titanium oxide, zirconium, and other ceramic-like substances, glass, cellulose, agarose, and a wide variety of different polymers including polystyrene and polymethylmethacrylate, acrylics, and other types of gels used for electrophoresis.

Other particles suitable for the purposes of the present invention include any particle that may be coated on its outer and/or inner surface with an insoluble, water-swellable or non-water-swellable sorbent material. In one embodiment, the particles swell to a volume of about 2 to 5 times or more compared to their original dry weight.

The function of the coating is to provide specific functions and physical properties that can be tailored to the particular separation assay desired. These functions include adsorption, ion exchange, chelation, steric exclusion, chirality, affinity, and the like. Preferred particulate materials for the coating include inorganic oxide particles, most preferably silica particles. Such particles having a coated surface are well known in the art, see, e.g., Snyder and Kirkland, "Introduction to model Liquid Chromatography", 2d ed, John Wiley&Sons, Inc. (1979) and H.Figge et al, journal of chromatography351(1986)393-408, comprising modified silica particles having covalently bound organic groups including cyano, cyclohexyl, C₈(octyl) and C₁₈(octadecyl). The coating may be applied mechanically by in situ cross-linking of the polymer or may be a functional group covalently bound to the particle surface.

The amount of particles incorporated into the porous matrix may 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, more preferably greater than 50% by volume. The device of the invention may therefore contain up to 95% by weight or more particles, thus providing potentially high capacity for targeted attachment. The particles of the present invention can generally withstand a wide range of pH values, for example, from a pH of about 4 or less to a pH of about 12 or more.

The particles of the present invention are versatile and can be used to perform a variety of chromatographic and non-chromatographic separation assays. Examples of separation methods contemplated within the scope of the present invention include reverse phase separation, affinity separation, expanded bed separation, ion exchange chromatography, gel filtration, chromatographic component separation, solid phase extraction among other methods of separating, assaying or collecting chemical or biological target agents from other components of a sample. The particles may also be used for binding, thus allowing the isolation of nucleic acid molecules and/or polypeptide target agents from a sample.

The preferred particles of the device of the present invention are porous resins. Porous resins for adsorptive separation are effective in many different types of materials, including silica, glass, fiber, agarose, and many different types of polymers, including polystyrene polymethylmethacrylate, polyacrylamide, agarose, hydrogels, acrylics, and other types of gels for electrophoresis. Many porous adsorbent resins such as silica, glass and polymers can be dry and have interconnected pores with surface areas of about 1-2m²Per g of dry resin to about 300m²Per g of dry resin. Other types of resins are crosslinked gels, which cannot be dried without damaging the structure. These types of resins generally do not have a specific surface area because these materials can be uniformly dispersed through the crosslinked matrix.

It is also within the scope of the invention to use modified resins, including natural or modified resins, or functional analogues, variants and functionalized derivatives thereof. Modifications include, for example, substitution, deletion, or addition of chemical entities (e.g., amino acids) to a particular resin or functional group thereof, or both. For example, amino substituted, acetylated, and/or partially acetylated resins are included within the definition of modified resin. Any modification of the functional groups of the resin is also included in the scope of modifying the resin according to the invention.

Other types of natural or modified resins useful within the scope of the present invention include, but are not limited to, phenyl sepharose, butyl sepharose, octyl sepharose, styrene crosslinked with divinylbenzene, hydrogel C3 styrene-divinylbenzene, hydrogel C4 styrene-divinylbenzene, hydrogel phenylstyrene-divinylbenzene, methyl HIC methacrylate, butyl HIC methacrylate, wide-pore-hi-phenyl, fractalEMD, hydrophobic resin-propyl methacrylate copolymer, tacteleEMD, hydrophobic resin-phenyl methacrylate copolymer octyl sepharose, phenyl sepharose, Toyopearl HIC, Toyopearlamino-6505, Toyopearlamino-650M, Toyopearlamino-650C, Toyopearlamino-650EC, Toyopearlbutyl-650S, Toyopearlbutyl-650C, Toyopearlbutyl-650M, Toyopearlether-650S, Toyopearlether-650C, styrene-divinylbenzene, styrene-divinylbenzene, methyl-methacrylate copolymer, styrene-divinylbenzene, styrene-methacrylate copolymer, styrene-divinylbenzene, styrene-methacrylate copolymer, styrene-styrene, Toyopearlether-650M, _ toyopearhexyl-650S, Toyopearlhexyl-650C, Toyopearlhexyl-650M, Toyopearlphenyl-6505, toyopearphenyl-650C (PRDT), toyopearphenyl-650M, and Toyopearl659CU (PRDT). All Toyopearl resins are commercially available from Tosoh Biosep, Montgomeryville, PA. Agarose resins were purchased from GE Healthcare, Piscataway, NJ. Fractogel resin was purchased from Merck, Darmstadt, Germany. Hydrocell resins were purchased from BioChrom Labs, Inc., Terre Haute, IN. The remaining resins are common names for various basic materials of publicly sold resins.

If the porous resin is packed into a column, the effective fluid diameter of the flow is determined by the particle diameter and bed void fraction:

wherein D_hIs the equivalent fluid diameter flowing between the particles, d_pIs the particle diameter and e is the void fraction.

Thus, to allow larger species to flow through the column, larger particles must be used, which in turn increases the diffusion resistance to adsorption into the resin. For example, if the bed voidage is about 0.4, particles of about 65 μm in diameter are necessary to allow red blood cells to flow through the column to provide a 14 μm pore size in the spaces between the particles.

4. Impregnated resin nonwoven fabrics (RINs)

The particles can be incorporated into the matrix by various methods. Since the nonwoven fabric can be prepared with a controlled average pore size, it is possible to impregnate porous resin particles such as those described above into fibers made of the nonwoven fabric.

These impregnated nonwoven fabrics can be prepared by various methods. For example, dry particles may be water entangled between two previously formed nonwoven fabrics. Alternatively, the dry particles may be introduced while forming fibers during melt blowing or spunbond. It is also possible that the resin particles may be wound while being wetted by the wet laying method. In one embodiment, the particles are impregnated into the already formed fibers by hydroentanglement without melt bonding of the particles to the polymer fiber matrix.

A preferred method of manufacture is direct lamination of a ready-made nonwoven fabric, wherein the nonwoven fabric may be melt blown or spun bonded. In one embodiment, the film is covered with a given mass of particles per unit area by spreading the particles uniformly onto the non-woven fabric by direct rolling of the particles delivered at a fixed mass rate. Once the particles are spread, a second film is placed overThe sandwich is made on top of the first film and the combination is passed over a laminating roller in a pattern that can bond the two films together at low temperature and low pressure. The density of the particles on the surface ranges from about 0.1 to about 10gm/m²Or larger. The pore size of the membrane used in the device allows larger entities, e.g. red blood cells, to pass through since the pore size of the membrane is larger than 10 μm. The particles attach to ligands in the membrane, which can facilitate binding of the particles to target reagents such as prion proteins in red blood cell concentrates and plasma.

For nonwoven bonding, the operation of the calendering process generally requires higher temperatures, but the temperatures are kept below the melting temperature of the particles and cannot affect their properties. In one embodiment, larger or denser particles may be placed between the nonwoven membranes by hydroentanglement.

The density and weight of the nonwoven fabric may take a wide range of values to ensure a higher particle density on the fabric while maintaining the desired pore size. The particles may be impregnated into the fabric at a concentration of about 60% w/w. All of the usual methods for making nonwoven fabrics can be used in this process, including fabrics comprising fibers of two different polymers and coextruded fibers comprising two different polymers. Due to its flexibility, both wet and dry resins can be impregnated. If necessary, chopped fibers may be embedded in the fabric to facilitate particle capture while still allowing flow holes of a desired size.

Chopped fibers are typically less than 1/2 inches long and can be prepared by cutting individual fibers that are wound around a spindle or roller. The cut is done mechanically with rotating blades or other tip facets. Chopped fibers can be prepared from a variety of polymeric or carbon fibers of very small (less than about 1 μm) to larger (>100 μm) diameters. In one embodiment, the specific ligand is chemically implanted or coated onto the fiber, and the fiber is then cut to a length of about 1/2 inches. The chopped fibers can then be distributed onto a single layer of nonwoven fabric (polypropylene or other polymer) of appropriate pore size and fiber diameter to allow larger entities such as red blood cells to pass through the membrane (pore size >10 μm).

The chopped fibers may be delivered to the film at a specified rate to ensure uniform distribution over the fibers. A second layer of nonwoven fabric may be placed on top of the chopped fibers while the chopped fibers are on the surface, and the combination may be passed through a roller to bond the two nonwoven layers. In one embodiment, the porous matrix membrane or chopped fibers are functionalized with a ligand and placed within another membrane by, for example, air laying techniques. In another embodiment, the ligand is attached to a polymer, which is then extruded into a fiber. The fibers can be cut to make small segments that can be easily fused between the two membranes.

5. Surface modification

Surface-modified non-woven or woven fabrics (SMNs) and surface-modified particles functionalized with reactive groups in one or more of their inner and/or outer surfaces are also included within the scope of the present invention. Functionalization is achieved by adding one or more reactive groups to the surface of a porous matrix (e.g., woven or non-woven fabric), a particle, 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 particle, or both are surface modified with a functional group, wherein the functional group is capable of forming a covalent chemical bond with the target agent. Functional groups useful within the scope of the present invention include, but are not limited to, one or more of the following, in particular epoxy, formyl, 2, 2, 2-trifluoroethanesulfonyl, hydroxysuccinimide ester. Functional groups useful within the scope of the present invention include, but are not limited to, one or more of the following groups, sulfonic, quaternary amine, carboxylic acid groups, primary amine, cyano, cyclohexyl, octyl and octadecyl, ethylene oxide, N-hydroxysuccinimide ester, sulfonyl ester, imidazolylcarbamate, quaternary amine, carboxylic acid groups, dye ligands, affinity ligands, antigen-antibodies, nucleic acid molecules, groups for ion exchange, chelation, oxidation/reduction, steric exclusion, catalysis, hydrophobic reactions, reverse phase, and other reactions commonly used in chromatographic separations.

Functional groups such as ligands are chemically bound to the support or may be bound by linkers such as streptavidin, beta alanine, glycine-serine containing polymers of the general formula- - (CH)₂) - -short chain hydrocarbons, polyethylene glycols,. epsilon.aminocaproic acid and compositions comprising- -O (CH)₂) n, wherein n is a linker of 1 to 30. If desired, the ligands may be attached via one or several different detachable linkers, e.g., moieties that are not light-resistant or acid-sensitive, to allow selective detachment of all ligands for analysis. The isolated ligands can be used, for example, as affinity purification media for protein and enantiomer separations (e.g., concentration, separation, detection, characterization, quantification, or identification of target substances from samples), as diagnostic therapeutics, catalysts and enhancers of chemical reactions, and as selective stabilizers for proteins.

In one embodiment, the non-woven membrane is coated with an affinity ligand that is a functional group, wherein the affinity ligand has a specific affinity for prion protein on the surface of the membrane. Examples of affinity ligands include primary amines with hydrophilic spacers comprising polyethylene glycol units. The ligand may be placed on a membrane, such as plasma treated polypropylene from macropharma, by chemical implantation or by latex emulsion coating (padding).

5.1. Polymerization of ligands on porous matrices

The polymerization of monomers on porous substrates introduces epoxy groups on the surface of these substrates, which in turn promotes the chemical attachment of ligands to the surface of the substrates. In one embodiment, the monomer emulsion is applied to cotton, polypropylene, polyester, and nylon fabrics by padding. Exhaust dyeing is a continuous process used in the textile industry to dry, bleach and coat fabrics. Additional information on the dip can be found in the website of Celanese LLC: www.vectranfiber.com, incorporated herein by reference. A padding process, which typically includes a set of squeeze rolls, may be used to impregnate the fabric with a liquid by passing the fabric continuously through the liquid, and then passing it over rollers to squeeze out excess solution. Single-dip (single-dip) techniques are also possible. Habeish et al, IMPROVING COTTON DYING AND DOTHER PROPERTIES BY EMULSION POLYMERIZATION WITH GLYCIDYLMETHACRYLATE, Ameri can Dyestuff Reporter, April, 26-34(1986), herein incorporated BY reference, use a dip DYEING technique to apply a Glycidyl Methacrylate (GMA) EMULSION to COTTON fibers. After the padding, the excess water is evaporated off and the polymerization is carried out at higher temperatures. The amount of polymer on the surface of the fibers ranges from about 1 to about 10% or more. The polymerization can also be carried out on nonwoven webs of PET, PP, etc. with the desired pore size (>10 μm).

5.2. Latex coating on fabrics

Latex emulsions can be synthesized by conventional emulsion polymerization in water to obtain small particles of the desired polymer. The emulsion can be created with a variety of soluble and free radical initiators and non-anionic and anionic surfactants. In one embodiment, the latex emulsion is coated on a porous substrate by padding as described above on monofilaments or nonwoven webs of PP, PET and other polymers. An example OF this type OF process is provided by De Boos and Jedlinek, Polymer OF EPOXY FUNCTIONAL polymers, J Macromol. Sci-chem. A17(2), 311-235(1982), which is incorporated herein by reference.

6. Application method

The methods, kits and devices of the invention are useful in a variety of applications, including prevention, diagnosis, detection, purification, isolation, treatment of gene products expressed in vitro, and production and delivery of biological agents. The present invention is applicable to any device that can be used for membrane operation of flat sheet, spiral wound or even hollow fiber cartridge devices in general. Depending on the desired pressure differential and flow rate, flow through the device may be induced by any conventional means-from gravity to a pump.

The purification and extraction techniques of the present invention have the advantages over conventional purification techniques of reducing the number of purification steps, improving yield, increasing purity, and overcoming limitations associated with conventional methods.

The device of the present invention is highly sensitive and is capable of isolating minute amounts of pathogens from a sample. In one embodiment, the device of the present invention is used to remove pathogens, including, for example, PrP, from whole blood, red blood cell concentrates, platelet concentrates, plasma derivatives, leukocytes, leukocyte depleted blood, mammalian cell cultures, fermentation broths, and other media used in the production and delivery of biopharmaceuticals and in the preparation of therapeutics^c，PrP^sc，PrP^resPrion proteins, viruses, bacteria and toxins. By means of the device of the invention it is possible to simultaneously and rapidly isolate a plurality of pathogens from a sample intended for any liquid stream of the plasma processing industry for the production of therapeutic and/or pharmaceutical products.

In particular, by increasing efficiency and purity, the methods and apparatus of the present invention optimize protein purification methods and improve methods of manufacture of biopharmaceuticals. Biopharmaceuticals are drugs based on proteins, peptides or other complex polynucleotides or macromolecules of proteins (collectively "gene products"). Their preparation involves recovery of the desired gene product from their host biomass, e.g., plasma or other biological sources both human and non-human (e.g., recombinant or non-recombinant cell cultures, emulsions of transgenic animals, or recombinant or non-recombinant plant extracts). The yield of commercially viable proteins of interest from biomass is challenging to isolate because the biomass contains undesirable host proteins, nucleic acid molecules, and other naturally occurring chemical entities.

Protein isolation and purification processes present unique challenges due to the variety of proteins, the different nature of possible contaminants and impurities, and the amount of product isolated from the culture medium. Conventional purification techniques typically involve a series of purification steps. As the respective steps increase, the yield decreases and the manufacturing cost increases. Protein separation and purification costs typically account for over 50% of the total manufacturing cost.

In another embodiment, the devices of the present invention are designed so that they can perform two operations simultaneously: filtering and adsorbing. In this embodiment, the pore size of the fabric is significantly reduced to reject larger particulate matter while maintaining the pore size large enough to allow passage of a sample containing the desired molecule. This technique achieves both filtration and adsorption steps in one apparatus, and then replaces membrane filtration with adsorption column chromatography. For example, the device of the invention makes it possible to adsorb the desired or undesired molecules secreted extracellularly directly from the culture medium.

In another embodiment, the device of the present invention can be used as a replacement for columns used in the biotechnology industry for adsorption removal technology. These techniques utilize biochemical interactions such as ion exchange, chelation, oxidation/reduction reactions, steric exclusion reactions, catalysis, hydrophobic reactions, reverse phases, dye ligands, affinity ligands, antigen-antibodies, and other interactions commonly used in chromatographic and/or other separation techniques.

7. Reagent kit

Also included within the scope of the invention are kits for sample purification by isolating a target agent from a sample.

The complete kit contains solutions and devices for target isolation and purification of biological samples. For example, the kit comprises a 96, 384 or 1536 well plate for high throughput sample purification and/or a solution for attaching ligands to particles in the device of the invention, in order to tailor the solid proteins, antibodies and solutions required for protein isolation in a flat format.

Generally, the kits of the invention comprise one or more of: (1) one or more vessels containing the above-described apparatus; (2) instructions for carrying out the method; (3) one or more assay elements; and (4) a packaging material. The packaging of the device includes many, if not all, elements necessary to carry out the separation process of the present invention. In particular, for example, the kit comprises a device comprising a porous matrix and particles, in addition to one or more buffers, reagents, chemical reagents, functionalizing reagents, enzymes, detection agents, control substances, and the like.

In one embodiment, the kit further comprises a functional group in the separation vessel, which functional group must be attached to the particle and/or porous matrix prior to the assay. Alternatively, the device may be provided in a kit without functional groups, in which case the porous matrix, the particles, or both are preferably pre-functionalized.

The device of the present invention may be of any desired size and shape. Preferably the device is a sheet-like material, for example in the form of a disc or a belt. Other items that may be provided as part of the kit include solid surface syringes, pipettes, test tubes, and containers. The porous matrix or particles may be coated with a monolayer or thickening substance by in situ cross-linking of polymers or covalent bonding of functionalized molecules on the surface of the porous matrix or particles, which allows optimization of the selectivity and separation efficiency of chromatography.

Detection may be facilitated by coupling the porous matrix, the particles, or both to 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 combinations thereof.

Detailed description of the preferred embodiments

It will be understood by those of ordinary skill in the relevant art that other suitable variations and modifications of the methods and applications will be apparent from the description of the invention, with the knowledge of those of ordinary skill, and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the invention in detail, the same will be more clearly understood by reference to the following examples, which are given for purposes of illustration only and are not intended to limit the invention.

Example 1: surface-modified nonwoven fabrics (SMNs)

The surface-modified nonwoven fabric is particularly useful when the adsorbed target substance is large, and it cannot penetrate the pores of the resin. In this case, the surface of the fiber comprising the nonwoven fabric is modified to affect the adsorption of the target agent. The adsorption step may include ion exchange, hydrophobic or affinity interaction or any other commonly used adsorption method. If SMN is used without particles, the surface area available for attachment per unit volume of substance can be controlled by the porosity of the fabric and the diameter of the fibers,

wherein a is_vIs the specific surface area per unit volume of the solid, d_fIs the diameter of the fiber and epsilon is the void fraction.

Since fiber diameters in the range of 100nm to 10 μm are available and the porosity is typically in the range of 0.4-0.5, very large surface areas can be achieved in these devices. For example, when the fiber diameter is 0.1 μm, the surface area per unit volume of the fabric is approximately

a_v＝2x10⁷m²/m³＝20m²/cm³ (3)

This comparison is highly advantageous for the surface area of many porous chromatographic stationary phases per unit volume. However, since the average pore flow diameter of the fabric can be independently controlled, the pore size can be up to several microns in diameter. Techniques such as electrospinning (electrospining) can produce even smaller diameters, resulting in greater surface area per volume.

Any surface modification that facilitates binding of the target agent to the device and that is compatible with the chemistry of the particular porous matrix used in the device is within the scope of the present invention. In particular, surface modifications include, for example, the formation of charged species, attachment of affinity ligands, peptides, oligonucleotides, proteins, spacer arms, hydrophobic moieties, fluorides.

Since the surfaces of the fibers in the nonwoven tend to be smooth, these surfaces assume a preferred configuration to expose the affinity ligand to particularly large materials such as prion proteins, viruses, or bacteria.

Example 2: device configuration for prion protein (PrP) removal

This example demonstrates the possibility of designing different device configurations to remove endogenously transmissible spongiform encephalopathy agents by allowing adsorption onto non-porous surfaces of various geometries. Endogenous infectives of erythrocyte concentrates include infectious PrP removed at a total concentration of about 200ng/ml^sc(prion protein in scrapie form of sheep) or PrP^res(resistant forms of prion protein). In total, there was 7X10 in a bag containing 350ml of red blood cell concentrate (rbcc)^-5g PrP. Assuming a monolayer of protein coated on the surface, the monolayer density is about 2mg/m²The total surface area required to bind all endogenous PrP in rbcc was evaluated as follows.

Where a is the total area of the device.

As is evident from the above equation, the total surface area required to bind the prion protein is relatively small, and several shapes of the device are adapted to expose the affinity ligand at a suitable surface density.

A. Square sheet

The total surface area of a set of N square pieces of nonwoven fabric coated on both sides with blood-exposed ligands was,

2NL²＝3.5x10^-2m² (5)

where N is the number of tiles and L is the length/width of the square tiles. When 10 sheets (N ═ 10), the width required for each sheet is,

L＝0.042m＝4.2cm (6)

this type of device comprises staggered rows of needles of the sheets and a liquid flowing in tortuous channels between the sheets, as shown in figure 2.

B. Coated fibers

A set of N nonwoven fibers externally coated with an affinity ligand has a total surface area of

N2πRL＝3.5x10^-2m² (7)

NRL＝5.57x10^-3m²

Where R is the radius of the fiber.

For example, the number of fibers having a radius of 5 μm and a length of 5cm is

N＝22，280 (8)

The volume of these fibers is

V_f＝NπR²L＝8.74x10^-8m³x10⁶ml/m³＝0.087ml

Wherein V_fIs the volume of the fiber.

It is evident from the above equation that the volume of the fibers is relatively small, mainly due to the very small diameter of the fibers, resulting in a very high surface area per unit volume. To allow the red blood cells to flow properly through the fiber mat, the porosity should be fairly high, e.g., about 50%. In this case, the volume of the device is about 2 times the volume of the fibers or 0.17 ml. The volume is very small, again proving that the trapping type device does not need to be large to support capacity requirements. For example, a 2cm radius fiber mat need only be about 0.135mm thick to achieve this volume. The fibers of one or more sheets may be coated externally with an affinity ligand.

C. Granules

Smaller non-porous particles also exhibit very high surface area per unit volume.

The number and volume of particles required to have the above-mentioned surface area are calculated in a similar manner as used in the case of the cylindrical structure,

wherein V_sIs the volume of the particle.

Assuming that the radius of the particles is 10 μm, the number and volume of the particles obtained by equation 5 are

N＝2.79x10⁷

V_s＝1.17x10^-7m³＝0.117ml (11)

This equation shows very small volumes of small particles. Small particles can be dispersed with larger particles or suspended in a cross-linked gel (e.g., agarose) with larger pores to allow red blood cells to flow easily through the system.

Example 3: bonding two films by rolling, with or without resin

To develop a device for removing prion protein, at room temperature/40At 0PLI and 150F/100PLI, at 1mg/cm²The resin density of (a) successfully laminates two layers of polypropylene film. The rolled film was sealed with an ultrasonic sealer. The percentage of hemolysis in the rolled film samples was good, within acceptable limits. The Amino650M resin was impregnated between the two layers of the film by roller compaction.

The Toyopearl Amino650M resin particles were impregnated between two layers of the nonwoven fabric film. Polypropylene (inner) and polyester (outer) films currently used in macoparma leucofilms are good candidates for these films, as they have been approved for use in the treatment of human blood.

To investigate whether particles could be immobilized without impeding the flow of red blood cells through the device, the inner and/or outer membranes were crushed with or without particles. Lamination is accomplished by pressing the film into the sheet by applying pressure between two rollers.

Materials and methods

A roll of polypropylene film (PP) and a roll of polyester film (PET) were wound on a 3-inch inner diameter plastic spindle. The width of these rolls is about 0.5 meters. The film was 22.5cm wide and 800m long. The film was cut into 22.5cmx22.5cm square pieces. The dry resin is added at a rate of 1mg/cm²Spread on one side of the film and then covered with another film. The single film sample need not be coated with resin. The sample was passed through two rollers, one embossing roller and one smoothing roller. The two rollers may be heated to increase the temperature of the nip. The pressure between the rollers can also be controlled. The roller compacted samples were tested by visual inspection, gravimetric determination, cross-sectional inspection by SEM (scanning electron microscopy) pore size determination, and percentage of hemolysis test run after whole blood flowed through the roller compacted device.

Method for determining percent hemolysis

The membrane samples were cut into 25mm loops and placed in Millipore Swinnex25mm filter holders. Each sample flow through was assayed in duplicate and then assayed in triplicate in a 96-well plateEach sample of (a). Each sample was rinsed with 2mL of running buffer (running buffer 20mM citrate and 140mM NaCl, pH7.0) and whole blood was pumped through the membrane from the top at a rate of 0.5 mL/min. 5ml of flow-through was collected for each sample. The flow-through or untreated blood was centrifuged at 12000rpm for 10 minutes at 4 ℃ to collect the supernatant. 3 100 μ l aliquots of each sample were placed in 3 wells of a 96-well plate. The UV-absorbance of each plate was read at 415 nm. A. the_415nmDivided by the value of 100% hemolysis of the same blood. If it is less than 1%, the percentage of hemolysis is acceptable.

Results

TABLE 1 conditions of grinding and appearance inspection

From the visual inspection, it was determined that sample numbers 4,7 and 13 were the best for pellet implantation. As shown in samples 4 and 13, lower pressure can be used for film bonding when the temperature of the roller is increased. The outer film is composed of polyester, which is much thicker and stiffer than the inner layer. In order to laminate the outer layer with an outer or inner layer film, as shown in sample 21, higher roller temperatures and pressures are required.

TABLE 2 weight measurement

For resin density, 1mg/cm²Corresponding to 10g/m²(1mg/cm²x10⁴cm²/m²＝10g/cm²). The weight of the monolayer, bilayer and resin-implanted bilayer is relatively proportional. The results show that the resin is well retained between the two films.

Scanning Electron Micrographs (SEM) of samples 11 and 13 show that most of the resin particles were intact after crushing, although some were still fractured. Examination of sample 2 with SEM also showed similar results. There is a 2mmx2mm square grid space on the embossing roller of the roller. During lamination, the film is subjected to a high degree of lamination when in contact with the grid. This region is also referred to as the binding region. And the pocket area is the area furthest from the bonding area.

Sample 11 is an example of resin particles immobilized between an inner layer and an outer layer. Figure 3 shows the pocket area of sample 11 at 50X magnification. Sample 13 is an example of resin particles immobilized between two inner layers. Figure 4 shows the pocket area of sample 13 at 50X magnification.

Pore size distribution

The results of the pore size distribution of the crushed samples are shown in table 3 below. For the crushed samples, the minimum, medium and maximum pore sizes were reduced by 30% to 50% compared to the monolayer. To determine whether the decrease in pore size prevents red blood cells from passing through the device, a further hemolysis assay of the whole blood flowing through is performed.

TABLE 3

Pore size distribution of the rolled samples:

example 4 optimization of compaction with high particle density

The laminating rollers used for this test were ordered by ProMetic from BF Perkins. The roller is constructed of stainless steel, is engraved with a honeycomb pattern of threads, and is coated with a polytetrafluoroethylene resin release coating. The counter-rotating rollers used were coated with rubber (3/4 "to 1" thick).

A4 gram sample of dry resin was hand spread to 30cmX20cm (600 cm)²) A sample of plasma treated polypropylene film. It corresponds to 6.6mg resin/cm²The particle density of (a). A second sample of the film was placed on top of the resin layer and the interlayer was passed through the calender at a speed of 10 m/min, the table below containing the results obtained in this assay.

Table 4: optimization results of rolling

Test of	Set temperature (F)	Measurement temperature (F.)	Gap1(μm)	Results
					1	212	198	203	When there are or are no particles, they are not bonded
2	230	215	0	The membrane weakly fusing
					3	245	236	0	Better than before, but still too weak
4	255	245	0	Good bonding without resin, but less efficient with resin
					5	260	248	0	The results were good with or without resin
6	275	-	0	The temperature was too high and the top film did not fuse to the bottom but adhered to the roller
					7	265	-	0	Good results were obtained at this temperature, both with and without resin

1) A gap of 0 indicates that the pattern penetrated the bottom roller 1/1000 inches.

The sample was observed under a microscope and showed no pinholes. Samples (only without resin) from each test were saved for future reference.

EXAMPLE 5 binding of beta-lactoglobulin and flow Properties of resin impregnated rolled films

This experiment was performed to determine the penetration curve of a model protein (. beta. -lactoglobulin) in combination with a rolled membrane material comprising a density of 4mg/cm²The dry resin of (4).

The compaction at 170F and 150 pounds per linear inch comprises a density of 4mg/cm²Polypropylene film material of the dry resin of (1). The membrane material was cut and assembled into Millipore Swinnex filtration units. Each filtration unit contains a stack of 1 to 4 membrane layers and a non-laminated membrane on the outlet side of the filtration unit. A1 XPBS solution of 0.5mg/mL beta-lactoglobulin was flowed through the filtration unit at 1.5 mL/min using a peristaltic pump, and 0.5mL fractions were collected over 4 minutes and analyzed for protein concentration using a Pierce Micro BCA assay kit (Pierce, Rockford, IL.).

FIGS. 5-7 show the results of the 12 fractions of the Micro BCA assay collected on different numbers of membrane layers. A roller compacted film without resin was used as a control. The results of this test indicate the difference in binding between the resin-impregnated membrane and the control. All runs showed similar initial slopes of unbound concentration, however, layers 2-4 did not run long enough to show saturated concentrations.

Table 5: total binding protein and amount of protein bound per weight of resin

The amount of bound protein generally increased with each additional membrane layer, peaking at three layers of membrane and very little decrease at 4 layers (table 5). Since filters containing 2-4 membranes do not operate long enough to show saturation concentrations, it is not possible to determine whether they are bound.

Example 6 particle distribution on film rolls

The particle spreading unit was developed to replace the manual distribution of the beads used previously. The apparatus has been tested and calibrated.

The powder applicator was set manually to 60%, which corresponds to a dispensing rate of 6.52 to 6.99 Kg/hour (shown). The dispensing rate by weight was 6.65 Kg/hour (average of three determinations) to within 5% of the target value of 6.96 Kg/hour.

The dispensed powder showed a uniform distribution (appearance evaluation) without overflowing the edges of the film. Figure 9 shows the distribution of particles on the bottom film after running the line for about 1 hour. Sharp edges formed by the particle-containing regions on both sides of the film can be noted. Another notable feature is that even after a certain time of preparation, there is no powder on the conveyor belt.

Example 7: prion protein linker

A group of resins for binding prion proteins is disclosed below.

a) Amino 650M-base resin coupled to peptides and other ligands. This base resin proved to be useful for binding prion proteins, whether normal PrPc or infected PrPsc (or PrPres). The resin can be used in a column chromatography format, and we have demonstrated that by removing/binding PrPsc (empty rat, mouse vCJD, mouse Fukuoka, human spCJD and human vCJD) from erythrocyte concentrate, plasma, whole blood by in vitro techniques (Western Blot) to the limit of detection, the infectious in vivo model (erythrocyte concentrate) of empty rat 263K scrapie is reduced by about 4 logs.

b) Toyopearl-SYA-this tripeptide has proven useful for binding prion proteins, whether normal PrPc or infected PrPsc (or PrPres). This resin can be used in a column chromatography format, we have demonstrated that by removing/binding PrPsc (empty rat, mouse vCJD, mouse Fukuoka, human spCJD and human vCJD) from erythrocyte concentrate by in vitro techniques (Western Blot) to the limit of detection, empty rat 263K ovine scrapie infectivity, i.e. the in vivo model (erythrocyte concentrate) reduces by about 4 logs.

c) Toyopearl-DVR-this tripeptide has been shown to be useful for binding prion proteins, whether normal PrPc or infected PrPsc (or PrPres). This resin can be used in a column chromatography format, we have demonstrated that by removing/binding PrPsc (empty rat, mouse vCJD, mouse Fukuoka, human spCJD and human vCJD) from erythrocyte concentrate by in vitro techniques (Western Blot) to the limit of detection, empty rat 263K ovine scrapie infectivity, i.e. the in vivo model (erythrocyte concentrate) reduces by about 4 logs.

amino650M, SYA and DVR have been used on full scale, i.e. 1 complete unit of erythrocyte concentrate is passed through the resin (about 350 ml). The size of the column was 10ml of swollen resin. SYA, DVR and amino functions were 400. mu. mol/g (dry resin).

Example 8 PrP from SBH spiked buffer, filtered plasma and Whole blood^scComparison of binding Amino650M and Amino650U

Amino650U is a mixture of beads of different sizes, including Amino650M, which is less expensive to manufacture than 650M. Determination of the endogenous PrP of Amino650U and all the matrices, buffers currently usedPrP neutralization in filtered plasma and whole blood^scAbility to bind and compare it to binding of spiked whole blood challenged Amino 650M. The assay was designed to compare PrP in spiked buffer, plasma and whole blood^scBinding to Amino650U and determination of endogenous PrP in plasma and whole blood^cBinding to Amino 650U. In addition, the assay was designed to determine that leukocyte filtration is removing PrP^cThe function of (1). Spiking buffer involves adding brain homogenate to the working buffer. Spiked whole blood is obtained by adding brain homogenate to human or hamster whole blood.

No difference in signal was found by removal of prion protein by 650U or 650M when present in plasma or whole blood. Thus, amino650U and 650M perform the same function. PrP removed by leukocyte filtration^cThe amount is greater than the amount estimated in the platelets and leukocytes together. Therefore, leukocyte filtration may also capture some plasma-derived PrP^cThis shows that the leukocyte filter pair captures PrP from human or hamster plasma^cHave different effects. It is possible that the filter will not capture PrP of Lenza plasma^cWhile capturing human plasma PrP^c. Finally, it is also possible that the difference between the two results is due to PrP^cAnd infectivity are irrelevant.

Leukocyte filtered PrP^cThe amount is greater than the amount determined in the combination of platelets and granulocytes. Therefore, leukocyte filtration may also capture some plasma-derived PrP^cThis shows that the leukocyte filter pair captures PrP from human or hamster plasma^cHave different effects. It is possible that the filter will not capture PrP of Lenza plasma^cWhile capturing human plasma PrP^c. Finally, it is also possible that the difference between the two results is due to PrP^cAnd infectivity are irrelevant.

Example 9 Lung murine brain PrP^scBonding with AMN resin

A series of comparative binding assays were performed on resins (e.g., AMN-13, 14, 15, 16, and 17, Amino650M, and Amino 650U). The AMN series involves 650U (recently referred to as 650C-prdt) samples with different levels of amino substitution as follows:

AMN-13；0.094eq/L

AMN-14；0.078eq/L

AMN-15；0.072eq/L

AMN-16；0.063eq/L

AMN-17；0.098eq/L

resin with PrP in spiking buffer, plasma and whole blood^scAnd (4) combining. The results show that all the AMN resins bind equally well when challenged with spiking buffer and spiked whole blood. In addition, signals of the AMN resin are the same as amino650M and 650U. When compared to binding of resin to PrP in spiked plasma, Amino650M gave a slightly stronger signal than all other resins. In the AMN resin, #13 showed a weaker PrP signal, but was very similar to amino650U, while #15, 16, 17 all performed better than amino 650U. No significant differences were observed between the AMN14, 15, 16, 17 resins.

Thus, studies have shown more similarity between resins and most importantly, it shows a tighter relationship to 650U than to 650M. The observed differences in plasma indicate that at least with that challenge it is beneficial to reduce the level of substitution, the resin is made more compact with amino 650M.

Example 10 extraction of protein bound to resin-implanted Membrane and detection of PrP in Normal hamster brain homogenates^cIn combination with

The development of new devices using resin-embedded rolled membranes has led to a need for the development of new methods for extracting binding proteins from resins. Some changes have been made to the processing of the material and the composition, concentration and volume of the extraction solution. This test was also designed for binding evaluation in a new format using membranes implanted with Toyopearl Amino650M resin and its fully acetylated form.

Normal hamster brain homogenate (HaBH) was treated with sarcosyl and spun. The resulting supernatant was diluted to a final concentration of 1% with either working buffer or human whole blood. 50ml of the spiking solution was passed through a 47mm Swinnex filter cartridge (Millipore) containing 4 compartments implanted with 4mg/cm at full capacity²A rolled film of the chromatography resin, or a reduced displacement capacity form of the same resin, was used as a control for Toyopearl Amino650M or its fully acetylated form. The flow rate used was 0.5 mL/min, using a peristaltic pump. 5mL of 10 equal portions of each spiked solution and membrane type were collected. Flow-through samples of both membranes challenged with spiking buffer were analyzed by ELISA. The membrane containing the fully acetylated resin and challenged with spiked whole blood was washed with running buffer.

Portions of the membrane (in some cases whole stacks) were treated with SDS-PAGE sample buffer or 99% formic acid. Treatment with formic acid involved adding 0.5mL of 99% formic acid and 10L of 20% SDS to the membrane sandwich of 1/4, followed by incubation for 1 hour to remove the liquid and evaporation with a speedVac. The volume of the sample was adjusted to 15L with water, and then 15. mu.L of 2 Xsample buffer was added. Treatment with sample buffer included adding 3mL of 1X sample buffer to the entire stack of membranes, followed by incubation for 30 minutes and cooking for 7 minutes. The solution was collected without compacting the membrane and simply centrifuged to remove all resin. Variations of the above treatments can also be tested. Comprising adding 1mL of 2X sample buffer to two separate stacks of membranes corresponding to 1/4 filters, incubating for one hour, and then boiling only one of them. When infectivity is used, it may be possible to elute with sample buffer without boiling if it becomes too dangerous to disassemble the filter holder.

The final case of the assay is to incubate the membrane portion in sample buffer (1/4) to verify binding to the first, second, third and fourth membranes for contact with the challenge solution. The samples were then run on SDS-PAGE gels and total protein stained. Western blotting may also be carried out. The empty volume of the filter cartridge was about 7 mL. After 50mL of challenge solution was passed through each filter, followed by air, the volumes recovered were 45 and 47mL for whole blood. When spiking buffer was used, the volumes recovered were 46 and 46 mL. No significant differences were observed when different challenge solutions were used.

The first filter holder to be opened is the one comprising a fully acetylated Toyopearl membrane, which is challenged with spiked whole blood. We note that there is still some blood in the filter despite passing air and washing with buffer. During the attempt to rinse the membrane with buffer, no significant loss of resin occurred, and the membrane was discarded.

The filter cartridge with Toyopearl amino650M challenged with whole blood was washed with an additional 200mL of buffer. The flow rate is higher than the maximum (999 in the dial). When opening the clip we notice that there is still some blood inside, especially between the layers. We also note that during washing, several portions defined by the radial distributor are bypassed.

This stack was cut into 4 aliquots with one of the pieces having four separate layers and treated with sample buffer to investigate if the different layers had different binding. The other aliquot was also divided into tablets and treated with formic acid. The remaining two aliquots were used to compare treatments with and without heating.

The two filters challenged with spiking buffer were washed separately with 200mL of running buffer. The filter was opened, the entire stack was transferred to a small glass vial, and 3mL of sample buffer was added to the vial.

The resin implanted into the crushed membrane maintained the same PrP binding properties in the resin column. The fully acetylated amino group shows a weaker membrane-bound PrP signal compared to the amino signal, supporting the following conclusions: fully acetylated amino groups do not bind efficiently to PrP. In summary, the results show that 50% acetylation reduces PrP, whether in mixture form or chemically synthesized^resIn combination with (1).

Example 11 Pr in Normal hamster brain homogenatesP^cBonded to a filter comprising a membrane impregnated with particles

The following experiment demonstrates that membranes containing resin particles bind to normal PrP (PrP) in normal hamster brain homogenate (NBH)^c)。

The elution method described in the previous examples was applied to these samples. The filter was opened and the membrane was placed in a glass vial and incubated with 2ml of NuPage sample buffer (Invitrogencorporation, Carlsbad, CA.). The vial was then heated and the resin from the filter collected. Western blot results of the eluted proteins showed that this method eluted PrP from the membrane. The results also indicate that the filter with resin bound more PrP than the filter without resin.

Example 12 PrP in scrapie brain homogenate^cBonded to a filter comprising a membrane impregnated with particles

This experiment shows PrP in brain homogenate (SBH) in mice with infectious agents added to whole blood and buffer^SCPerformance of the combined filter. The filter contained a membrane impregnated with full capacity resin, reduced capacity resin, and no resin as a control.

Elution was done with injection of 2ml of NuPage sample buffer (extraction according to example 10). Western blotting of the eluted protein showed a strong signal without PK (protein kinase), but a weaker signal with PK. Since the protein was eluted with 2% SDS, PK digestion was performed at 2% detergent concentration, rather than using 2% SDS (standard method). It is possible that PrP is present in the presence of PK^resThe weaker signal is due to the excess SDS in the reaction mixture. The results show that the signal is weaker when the film contains no resin, but all other tested resins have similar signal strengths. No significant difference was observed between SBH in buffer and in whole blood between full capacity resin and no resin. The reduced capacity resin in spiked buffer showed a stronger signal than spiked blood.

EXAMPLE 13 measurement model Filter

In this embodiment, the filter is fitted into a plastic sleeve and welded together in a configuration similar to the final device. Various aggressors, spikes, and non-spikes were used to evaluate the performance of these devices. In one embodiment, the final device comprises a rigid or flexible plastic sleeve containing multiple layers of non-laminated film (1 to about 25 or more), followed by several layers (between 1 to about 50 depending on the desired capacity of the device) of resin-impregnated film, and between 1 to about 25 layers of non-laminated film. The filters are welded together with an ultrasonic cutter/sealer or by using pressure to apply heat and pressure simultaneously. In this embodiment, heat and pressure are used.

The leucineole brain homogenate was treated with 0.5% sarcosyl and diluted 100-fold in buffer, filtered plasma and whole blood and used as a challenge to the resin. The resin was challenged with 80ml sample at 0.5 ml/min with a peristaltic pump. Resin bound PrP without PK digestion^resWestern blotting of samples. The results indicate that the membrane performs well with spiking in buffer and plasma. But reduced for the same spiking in blood.

Example 14 removal of PrP from spiked RBCC by a series of filter devices

This experiment shows the removal of PrP from spiked Red Blood Cell Concentrate (RBCC) using an apparatus comprising a device for immersing the particles. Since the device is attacked with an excess of target protein, a series of devices are used. The total volume of RBCCs used is equal to one cell.

All filtration can be performed without significant problems, all using pumps. All filters had soft sleeves and were pre-tested for leakage. All filters did not leak in the preliminary experiments or in the actual experiments. The filtration time of the RBCC (. about.300 ml) for each cell was about 10 minutes. The filter was washed with about 460ml of running buffer (citrate). The efficiency of the washing step can be evaluated empirically by examining the color of the filter after washing.

After washing, the filter was injected with air to remove all liquid in the filter. The filter was treated with-4.6 ml of sample buffer to elute (injection on one side of the filter). Sample buffer was collected and injected on the other side of the filter. This step was performed three times. The results show PrP^resEscape from filter 1 and be captured by filter 2, which is desirable because the applied aggressors are higher than the capacity of one filter combination. PrP^resMay also be present in the eluate from filter 3, but below the detection limit of western blotting. Comparison of this result with the results of the RBCC in the first PRDT infectivity study shows that the filter functions equally to the resin used in the column format. According to the previous studies, a large excess of PrP^resPassing through the filter, not surprisingly, not all PrP in the first filter^resAre trapped.

EXAMPLE 15 Implantation of glycidyl methacrylate onto Polypropylene, polyethylene, terephthalate, Cotton and Nylon substrates

Implantation is an effective method of modifying the surface properties of polymers. The customized implant polymers containing specific ligands and substrates with good mechanical properties make implantation the most energetic method of protein binding, the application of which often requires modification of the ligands and sufficient mechanical strength. In this example, implantation of Glycidyl Methacrylate (GMA) was performed on polypropylene (PP), polyethylene, terephthalate (PET), cotton and nylon substrates to demonstrate the use of this pathway to remove PrP from human blood^cThe possibility of (a).

The method comprises the following steps:

substrates used for this assay include cotton woven fabrics, nylon woven fabrics, PP non-woven films (MacopharmaPP175), PP fibers and PET fibers from the textile institute of NCSU.

The implantation is performed by the following steps:

1. the sample was washed 3 times with acetone;

2. drying the sample in a vacuum drying oven for more than 3 hours;

3. the sample was treated with an argon plasma (plasma) at 750W for 15 seconds

4. The sample was exposed to air for 30 minutes;

5. the sample was then immersed in a 10% GMA solution in the UV chamber for 6 hours. UV intensity was 1.1W/m². The temperature of the chamber is 30 ℃ and the temperature of the sample holder is higher than 60 ℃;

6. after UV polymerization, the sample was then washed 3 times with acetone;

7. the samples were dried in a vacuum oven.

In this stage, only the weight gain and the IR spectrum of the sample were analyzed. After analysis, the implanted PP nonwoven and PP nonwoven membrane were further aminated overnight in an aqueous ammonia solution at 60 ℃. These samples were then washed and dried for elemental analysis.

As a result:

from the results of gravimetric and IR spectroscopy, it can be seen that PP substrates show significantly better implantation results compared to other substrates, cotton, nylon and PET. These three types of PP substrates, PP fibers, PP nonwoven fabrics and PP nonwoven films (macropharma), were tested. The weight gain after implantation was 85%, 154% and 57%, respectively (table 6). These values are much higher than those of other substrates (-3% to 4%). These results were further confirmed by IR spectroscopy, in which the implanted PP system was at 1720cm^-1The strong peaks shown are associated with carbonyl groups at 845 and 910cm^-1In relation to epoxides, this is the signal of the GMA. The other substrates did not show much change when comparing the blank sample and the post-implantation sample.

The reason for the implantation differences on the various substrates remains unclear. One possible reason is that the C-H bond is more prone to break in the PP environment than in other environments. Another possible reason is that nylon and cotton fabric samples may have a surface treatment that is not known to us. Simple washing with acetone is not sufficient to remove the polish.

It is also interesting to note that the original PP fiber and nonwoven film (Macopharma PP175) samples ranged from 840 to 920cm^-1Peaks are shown in the range. However, PP nonwoven fabrics did not show such peaks. The former peak is a result of oxidation of the sample surface, which has been treated at high temperatures.

In the IR spectrum, the aminated Macopharma sample was at 3400cm^-1Shows a broader peak, which is-OH and-NH produced by amination₂The radical is generated.

Determining the surface area of a nonwoven material

Determining the surface area of the nonwoven is not an easy task due to the complex interlocking of the fibers forming the nonwoven. However, the surface area of the individual fibers can be accurately determined by measuring the microscopic image and length of the fibers. Thus, if the surface properties of the fibers are the same as the surface properties of the nonwoven material, it is theoretically possible to determine the surface area of the nonwoven material by determining the fibers of the same material implanted by implantation. The method can be expressed by the following equation:

Wt＝D(S_f+S_NW)

where Wt is the weight gain of the implant, S_fIs the surface area of the fiber, S_NWIs the surface area of the nonwoven material. Only two terms are unknown in this equation: d and S_NWThey can be determined by two independent experiments.

The area determined by this method is the effective surface area corresponding to each type of reaction. In fact, any reaction whose extent depends on the surface area of the substrate can be used with this method.

And (4) conclusion:

the main trials of implanting GMA on several polymeric substrates indicate that PP is an ideal substrate for these implants. The weight gain of the GMA implanted on PP varies from 60% to 160% depending on the shape of the PP material. The effect of the implantation can also be confirmed by FTIR spectroscopy. Furthermore, based on the implantation, a simple method can be used to determine the surface area of the nonwoven (table 6).

TABLE 6 Effect of implantation measured as weight gain

Sample name	Substrate	UV time (hours)	Initial weight (g)	Weight gain (%)
					Co-g-GMA-032905	Cotton textile	0.6	0.1283	-1.8
Ny-g-GMA-032905	Nylon textile	0.6	0.1323	-0.7
					PP-g-GMA-032905	PP non-woven fabric	0.6	0.1179	1.7
Co-g-GMA-033105	Cotton textile	6	0.0954	-2.8
					Ny-g-GMA-033105	Nylon textile	6	0.1446	-1.4
PP-g-GMA-033105	PP non-woven fabricFabric	6	0.0537	91
					Mac-PP-g-GMA-040805	Macopharma PP non-woven film	6	0.0668	57
NW-PP-g-GMA-040805	PP non-woven fabric	6	0.0738	154
					FB-PP-g-GMA-040805	PP fiber	6	0.0789	85
FB-PP-g-GMA-040805	PET fiber	6	0.0517	4

[0263] Example 16 PrP through PGMA fibers, electrospun mesh, PGMA implants and impregnated PP substrate^cIn combination with

The purpose is as follows:

determination of PrP by PGMA fibers, electrospun mesh, PGMA implants and impregnated Polypropylene nonwoven^cIn combination with (1).

The method comprises the following steps:

the materials tested were polyglycidyl methacrylate (PGMA) melt-spun fibers, PGMA electro-woven mesh, PGMA implants, and dip-dyed polypropylene (GMA-g-PP) nonwoven fabrics. The substrates were samples from the textile institute at university of north carolina, macoparmap 175 and Macopharma PP 235. For each material, two replicates were prepared for protein binding.

Samples were prepared based on weight and apparent area (measured area of fabric). The second method is applied only to samples having regular shapes, such as a dip-dyed Maco Pharma film.

All samples were cut into small pieces. Each sample was then immersed in a conical tube (50ml) containing 5ml of 1% normal rat brain homogenate (HaBH). The samples were then incubated on the shaken plates for 30 minutes. Thereafter, the samples were washed 3 times with 10ml of sample buffer on the shaken plates, 10 minutes each.

As a result:

sample weights, areas and amination levels determined by elemental analysis are shown in table 7 below.

Elemental analysis of the samples in Table 7

Name (R)	Weight (g)	N％
			PGMA fiber A	0.1009	3.8％
PGMA fibre B	0.1011	3.8％
			PGMA-g-PPA	0.1007	2.6％
PGMA-g-PPB	0.1009	2.6％
			PGMA-g-PP(Maco175)A	0.1011	1.5％
PGMA-g-PP(Maco175)B	0.1010	1.5％
			PGMA-p-PP1A	0.1015	Invalidation
PGMA-p-PP1B	0.1007	Invalidation
			PGMA-p-PP2A	0.1007	Invalidation
PGMA-p-PP2B	0.1012	Invalidation
			PGMA-p-PP3A	0.1012	Invalidation
PGMA-p-PP3B	0.1007	Invalidation
			BLANK-NONWOVEN-PPA	0.1009	Is free of
blank-nonwoven-PPB	0.1011	Is free of
			blank-Macro 175A	0.1011	Is free of
blank-Macro 175B	0.1006	Is free of
			650MA	0.1016	0.6％
650MB	0.1011	0.6％
			Electric textile A	0.1001	Invalidation
Electric textile B	0.1005	Invalidation
				Area (cm)2)
PGMA-p-PP(Maco235)6A	4x4	Invalidation
			PGMA-p-PP(Maco235)6B	4x4	Invalidation
PGMA-p-PP(Maco235)7A	4x4	Invalidation
			PGMA-p-PP(Maco235)7B	4x4	Invalidation
PGMA-p-PP(Maco235)9A	4x4	Invalidation
			PGMA-p-PP(Maco235)9B	4x4	Invalidation
PGMA-p-PP(Maco235)10A	4x4	Invalidation
			PGMA-p-PP(Maco235)10B	4x4	Invalidation
blank-Macro 235A	4x4	Invalidation
			blank-Macro 235B	4x4	Invalidation

Both PGMA-implanted PP and PGMA-impregnated PP bind prion protein according to western blot results.

Equivalent means

The invention illustratively described herein suitably may be practiced in the absence of any component or components, limitation or limitations, not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of indicating and describing any equivalent features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. It will thus be appreciated that although the present invention has been specifically disclosed herein, those skilled in the art may devise variations of any of the features, variations and concepts herein disclosed which will be understood to be within the scope of the invention as defined by the appended claims.

All documents discussed herein are incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Claims (26)

1. A device for filtering and separating at least one target agent from a sample flowing through the device in use, the device comprising a stack of resin-embedded membranes comprising first and second layers of porous non-woven fabric bonded together with a plurality of particles impregnated therein or interposed therebetween, the porous non-woven fabric having a pore size greater than 10 μm, the particles having a particle size of 40-200 μm, wherein the at least one target agent is attached to the porous non-woven fabric, the particles, or both, and removed from the sample.

2. The device of claim 1, wherein flow through the device is induced by gravity or a pump.

3. The device of claim 1 comprising 1-25 monolayers of non-rolled film, followed by 1-50 layers of resin embedded film, followed by 1-25 monolayers of non-rolled film.

4. The apparatus of claim 1, wherein the laminations are welded together.

5. The device of claim 3, wherein the laminate is assembled in a plastic sleeve.

6. The device of claim 1, wherein the porous nonwoven fabric is comprised of natural fibers, synthetic fibers, or both.

7. The device of claim 6, wherein said porous nonwoven fabric is comprised of polypropylene or polyester fibers.

8. The device of claim 1, wherein the particles are porous, non-porous, or both.

9. The device of claim 1, wherein the porous nonwoven fabric, the particles, or both have uniform or varying pore sizes.

10. The device of claim 9, wherein the particles have a pore size of 0.001 μm to 0.1 μm.

11. The device of claim 8, the particles comprising a porous resin having interconnected pores and having a surface area of 1-2m²Dry resin per g to 300m²Per g of dry resin.

12. The device of claim 1, wherein the particles comprise a polymethacrylate resin, a methacrylate resin, a modified resin, or a combination thereof.

13. The device of claim 12, wherein the modifying resin comprises TOYOPEARL^TMAMINO650。

14. The apparatus of claim 1, wherein the particles are chopped fibers.

15. The device of claim 1, wherein at least one target agent is attached to the particles, the porous nonwoven fabric, or both by: adsorption, absorption, ion exchange, covalent binding, hydrophobic interaction, affinity interaction, formation of a charged species, attachment to an affinity ligand, or a combination thereof.

16. The device of claim 1, wherein one or more of the inner and/or outer surfaces of the porous nonwoven fabric, the particles, or both are functionalized with reactive groups that interact with and bind to the target agent.

17. The device of claim 16, wherein the reactive group comprises a functional group selected from the group consisting of: epoxy, formate, 2, 2, 2-trifluoroethanesulfonyl, hydroxysuccinimide ester, sulfonic acid, quaternary amine, carboxylic acid group, primary amine, cyano, cyclohexyl, octyl, and octadecyl, epoxide, ethylene oxide, N-hydroxysuccinimide ester, sulfonyl ester, imidazolyl carbamate, quaternary amine, carboxylic acid group, dye ligand, affinity ligand, antigen-antibody, nucleic acid molecule, or a combination thereof.

18. The device of claim 16, wherein the reactive group is a reactive group selected from the group consisting of reactive groups for ion exchange, chelation, oxidation/reduction, steric exclusion, catalysis, hydrophobic reactions, reverse phase.

19. The device of claim 17 or 18, wherein the particles comprise a modified resin, the porous non-woven fabric comprises plasma treated polypropylene, and the reactive groups comprise ligands having primary amines and hydrophilic spacers comprising polyethylene glycol units.

20. The device of claim 1, wherein the sample is a blood sample and the at least one target agent comprises prion proteins, viruses, bacteria, protozoa, and toxins, or combinations thereof.

21. A method of making the device of claim 1, the method comprising the steps of: (a) spreading the particles into a first layer of porous nonwoven fabric; (b) placing a second layer of porous nonwoven fabric onto the first layer; and (c) passing the combination through a laminating roller.

22. A method of isolating at least one target agent from a sample, comprising:

(a) providing a sample that may comprise one or more target agents;

(b) providing a device comprising a laminate of a resin-embedded film comprising first and second layers of porous nonwoven fabric bonded together with a plurality of particles impregnated therein or interposed therebetween, said porous nonwoven fabric having a pore size greater than 10 μm, said particles having a particle size of 40-200 μm;

(c) placing a sample into the device;

(d) attaching at least one target agent to the particle; and

(e) separating the at least one target agent from the sample.

23. A kit for isolating a target and purifying a sample comprising (i) the device of claim 1 and any combination of: (i) a container comprising one or more buffers or reagents, (ii) instructions for using the kit, and (iii) packaging materials.

24. The kit of claim 23, wherein the reagent comprises a chemical reagent, a functionalizing reagent, a detecting agent, or a control substance.

25. The kit of claim 24, wherein the detection agent comprises various enzymes, prosthetic groups, luminescent materials, radioactive materials, disperse dyes, gold particles, or combinations thereof.

26. The kit of claim 25, wherein the luminescent material is a fluorescent material or a bioluminescent material.