A medium for separating and storing plasma or serum, a method for producing the same, a device, a kit, and a method for measuring glycated proteins.

Abstract

One embodiment of the present invention provides a medium for separating and storing blood plasma. The medium comprises: a porous medium for separating blood plasma; and a substance that is loaded on the porous medium and suppresses the glycosylation of proteins by glucose.

Description

[Technical Field]

[0001] This disclosure relates to a medium for separating and storing plasma or serum, a method for producing the same, a device, a kit, and a method for measuring glycated proteins. [Background technology]

[0002] In mail-order diabetes screenings using blood samples, glycated hemoglobin (HbA1c) is generally measured. However, other glycated proteins in the blood are not measured in these mail-order diabetes screenings.

[0003] To avoid hemolysis, liquid blood should be mailed for a short period or at a low temperature. Alternatively, it may be necessary to centrifuge the blood beforehand to separate only the serum components. A separating agent may also be added to separate the serum from the blood clot before transporting the blood components. However, both methods require cumbersome work at the site.

[0004] Generally, in existing mail-order glycated hemoglobin (HbA1c) tests, blood is soaked into filter paper and dried. For the test, the dried blood is dissolved in a buffer and measured using immunoprecipitation. [Overview of the Initiative]

[0005] The inventors discovered that albumin in blood is prone to glycation, and that this glycation is further accelerated during the drying process of blood components before transport. Therefore, the need for a storage or transport medium that can preserve glycated proteins for a relatively long period of time and mail them, and that can subsequently enable proper measurement of glycated proteins, was recognized.

[0006] In some embodiments of the present disclosure, a medium for separating and storing plasma is provided. In some embodiments, the medium comprises a porous medium for plasma separation and a substance supported on the porous medium that inhibits glucose-mediated protein glycation.

[0007] According to the above embodiment, for example, plasma components can be stored and / or transported simply and stably.

[0008] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, other different embodiments are possible, and some of their details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not limiting in nature. [Brief explanation of the drawing]

[0009] [Figure 1] Figures 1A-1C are top views of porous media according to several embodiments. [Figure 2] 2A-2H are cross-sectional views of porous media according to several embodiments. [Figure 3] 3A-3D is a perspective view of a device comprising a porous medium according to several embodiments. [Figure 4] This is a schematic diagram illustrating the use of a device according to one embodiment. [Figure 5] This is a schematic diagram illustrating the use of a device according to one embodiment. [Figure 6] This is an optical photograph showing the usage of a filter according to one embodiment. [Figure 7] This graph shows the changes over time in GA concentration, ALB concentration, and GA% when a GOx-supported filter and a GOx-unsupported filter (for comparison) absorb the prepared HSA solution. [Modes for carrying out the invention]

[0010] In some embodiments, the subjects (test subjects) may include humans, or may be humans. In some embodiments, the subjects may include animals other than humans, or may be animals other than humans. Animals other than humans may include mammals, or may be mammals. Animals other than humans may, for example, be working animals, domesticated animals, pets, or wild animals, without limitation.

[0011] <medium> In some embodiments, the medium can separate plasma or serum from blood (whole blood, blood-derived solutions, etc.). In some embodiments, the medium can store blood cells, plasma, or serum, or a combination thereof. In some embodiments, medium The body, blood cell, It can be used for the transport of plasma or serum, or multiple thereof.

[0012] In some embodiments, the medium may be porous, a porous medium, or a porous material. The medium may have through holes or non-through holes. The holes may be continuous holes (multiple holes are fluid-connected) or discontinuous holes (multiple holes are substantially not fluid-connected).

[0013] In some embodiments, the porous medium may be a fibrous medium. The fibers may be natural fibers, synthetic fibers, or composite fibers.

[0014] In some embodiments, the porous medium may be made of materials such as cellulose, glass fibers, hollow fibers, cotton, nitrocellulose, or polysulfone. The hollow fibers may be made of materials such as polyvinyl alcohol copolymer, polymethyl methacrylate, or polymer alloy. The medium may also be a hollow fiber plasma separation membrane. The porous medium may be water-soluble. For example, water-soluble cellulose may be used.

[0015] In some embodiments, the porous medium may be a porous polymer (porous polymer body). The porous medium may be formed in a sponge-like shape.

[0016] In some embodiments, the porous medium may be formed in the shape of a membrane, a membrane, a test paper, or the like.

[0017] In some embodiments, the porous medium may have the ability to substantially separate plasma. In some embodiments, the porous medium may have the ability to capture blood cell components such as red blood cells. The porous medium may have a roughness (pore size) smaller than the size of deformed red blood cells in part or in whole. In some embodiments, the porous medium may be formed of a plasma separation filter, a plasma separation membrane, or the like.

[0018] The pore size or minimum pore size of the porous medium may be equal to or larger than 3 μm, 2 μm, 1 μm, 0.5 μm, 0.3 μm, 0.1 μm, etc.

[0019] In some embodiments, the pore size may be uniform throughout the medium. In some embodiments, the pore size may vary within the medium and may be asymmetric. For example, the roughness may change in the direction in which the solution flows. For example, the minimum pore size is larger than about 0.1 μm, and the pore size may gradually increase and the maximum pore size may be about 100 μm.

[0020] <Protein> In some embodiments, the target substance contained in the plasma or serum component may be a protein. The protein may be albumin, globulin, or hemoglobin. The glycated protein may be glycated albumin, glycated globulin, or glycated hemoglobin.

[0021] <Enzymes, etc.> The substance that inhibits glucose-induced protein glycation may be a glucose oxidoreductase. The substance may be glucose oxidase (GOx), glucose dehydrogenase, or glucokinase. Glucose dehydrogenase may, for example, not limited to, UDP-glucose-6-dehydrogenase, glucose-1-dehydrogenase, glucose-6-phosphate dehydrogenase (G6PD), glucose / galactose-1-dehydrogenase, glucose-6-phosphate 3-dehydrogenase, quinoprotein glucose dehydrogenase, etc.

[0022] The medium may further support enzyme cofactors (or coenzymes). For glucose oxidase, electron acceptors such as flavin adenine dinucleotide (FAD) or quinones can be used as cofactors. For glucose dehydrogenase, electron acceptors such as NAD+, NADP+, FAD, PQQ, PMS, coenzyme 420, or quinones can be used as cofactors. For glucokinase, for example, ATP or a Mg-ATP complex can be used as a cofactor. These are examples, and the combination of enzymes and cofactors is not limited to these.

[0023] Substances that inhibit the glycation of proteins by glucose, such as glucose oxidase (hereinafter also simply referred to as "supporting substances"), can be supported on a medium by various methods.

[0024] In some embodiments, a substance that inhibits glucose-mediated protein glycation (a supported substance) may be supported on the inner surface of a porous medium. Microscopically, the supported substance may be supported on the inner surface of fibers or porous materials. Plasma components containing proteins are absorbed into the porous medium and adsorbed onto the inner surface of the porous medium (e.g., the surface of fibers, the surface of pores) as water evaporates. In this process, the concentration of components in the solution increases. As a result, the probability of contact between proteins and glucose increases. Therefore, proteins become unnecessarily susceptible to glycation. Glucose oxidase, etc., supported on the inner surface of the porous medium decomposes glucose in its vicinity. Therefore, unnecessary glucose-mediated glycation of proteins in the porous medium can be efficiently prevented or reduced.

[0025] Various methods can be used to support the supported material on the inner surface of a porous medium. For example, a solution containing the supported material may be absorbed into the porous medium and then dried.

[0026] In some embodiments, a substance that inhibits glucose-induced protein glycation (a supported substance) may be supported on the external surface of a porous medium. The supported substance may be supported on the external surface of a porous medium formed in the shape of a membrane or stripe. Microscopically, the supported substance does not have to be supported on the internal surface of fibers or porous materials. For example, the supported substance may not be supported on the internal surface of the porous medium, but only on the external surface. For example, the supported substance may be supported only on the surface where blood is dropped. The supported substance may be placed at locations where blood or plasma passes. This allows the supported substance to efficiently come into contact with a liquid containing blood or plasma components. Alternatively, the application of the supported substance can be easily carried out.

[0027] Various methods can be used to support the supported material on the outer surface of a porous medium. For example, a solution containing the supported material may be applied to the surface of the porous medium using methods such as spin coating or inkjet printing, and then dried.

[0028] <Other carrier substances: Ketoamine oxidase> In some embodiments, the porous medium may be used for measuring or testing hemoglobin A1c. In some embodiments, the porous medium may support a ketoamine oxidase, such as fructosyl amino acid oxidase (FAOD).

[0029] Ketoamine oxidase breaks down glycated lysine in the blood. Free glycated amino acids such as glycated lysine and glycated valine are present in the blood. Ketoamine oxidase breaks down not only the glycated amino acids derived from the glycated protein to be measured (e.g., glycated albumin, HbA1c, etc.), but also these naturally free glycated amino acids. As a result, the measured value may be higher than the concentration of glycated amino acids derived from the glycated protein. Therefore, in some embodiments, ketoamine oxidase may be added before the glycated protein is broken down by protease to pre-decompose glycated amino acids such as glycated lysine and glycated valine present in the blood. This allows for a more accurate determination of the glycated protein concentration.

[0030] In some embodiments, the porous medium may further support catalase. Hydrogen peroxide may be generated by the reaction with glucose. Hydrogen peroxide has the effect of inactivating ketoamine oxidase. Hydrogen peroxide also has the effect of decomposing or denaturing blood components. Catalase can decompose hydrogen peroxide and prevent or reduce the inactivation of ketoamine oxidase.

[0031] <Blood coagulation inhibitors> In some embodiments, the anticoagulant (also known as a blood coagulation inhibitor; hereinafter the same) may be supported on a porous medium. In some embodiments, the anticoagulant may be supported or encapsulated within a blood collection tube. For example, if blood absorption, flow, or drying is slow, the blood may coagulate. Coagulation can make the extraction of plasma components inefficient. The anticoagulant can avoid or reduce such blood coagulation. In some embodiments, the anticoagulant may be supported on a medium for storing or mailing plasma.

[0032] <Dye> In some embodiments, the dye may be supported on a porous medium. For example, the dye may be a water-sensitive substance. For example, the porous medium containing the dye may be water-sensitive test paper. The dye may contain cobalt chloride. The use of the dye makes it easier to identify the location or amount of liquid, particularly plasma components with low contrast, reached or retained within the porous medium. In other words, the amount of blood introduced into the porous medium can be confirmed. The dye may be a component that does not substantially affect the subsequent measurement of blood components.

[0033] <Other configurations> In some embodiments, the porous medium may be configured to be cut after blood absorption so as to separate the areas containing blood cell components from the areas containing plasma components. For example, the porous medium may be cut to substantially completely include the areas that hold blood cell components. It may also be cut within the areas that hold plasma components. The porous medium may be cut with scissors or a cutter. The porous medium may also be cut by the structure of the housing that holds the porous medium (e.g., a built-in cutter). In some embodiments, the porous medium may have mechanically weak structures at the locations to be cut beforehand. For example, the porous medium may have perforations at such locations.

[0034] In some embodiments, the porous medium may be configured to have a portion for separating plasma components and a portion for carrying a substance that inhibits glucose-mediated glycation. The portion for separating plasma components may be composed of a plasma separation filter. The portion for carrying the substance that inhibits glucose-mediated glycation does not necessarily have the ability to separate plasma. The portion for carrying plasma components and the portion for carrying the substance that inhibits glucose-mediated glycation may each be fluidly connected in part. Blood may be dropped onto the portion for carrying plasma components, and only the plasma components may be absorbed into the portion for carrying the substance that inhibits glucose-mediated glycation, where they may be dried. The portion for carrying plasma components and the portion for carrying the substance that inhibits glucose-mediated glycation may each be formed in the form of a membrane or sheet. They may be arranged in a planar direction and partially overlapped (horizontal flow). They may be stacked in a direction perpendicular to the planar direction (vertical flow).

[0035] <Example of a porous medium - 1> Figures 1A to 1C show top views of porous media according to several embodiments.

[0036] Figure 1A shows a top view of a porous medium 100 according to one embodiment. The porous medium 100 is formed in a striped pattern. The entire porous medium 100 carries a substance (hereinafter sometimes simply referred to as "enzyme") that has the ability to separate plasma and suppress the glycation of proteins by glucose.

[0037] Figure 1B shows a top view of a porous medium 110 according to one embodiment. The porous medium 110 is formed in a stripe shape. The porous medium 110 has a portion (plasma separation portion) 111 at its end or edge that has the ability to separate plasma, and a portion (enzyme carrying portion) 112 that carries enzymes in contact with the plasma separation portion 111. Blood is dropped onto the plasma separation portion 111 and flows in the direction of the length of the stripe. Blood cell components are captured in the plasma separation portion 111, and plasma components are absorbed and retained in the enzyme carrying portion 112.

[0038] Figure 1C shows a top view of a porous medium 120 according to one embodiment. The porous medium 120 is formed in a circular shape. The porous medium 120 has a plasma separation section 121 in its center and an enzyme carrying section 122 surrounding the plasma separation section 121. Blood is dropped onto the central plasma separation section 121 and flows radially. Blood cell components are captured in the plasma separation section 121, and plasma components flow radially, are absorbed and retained in the enzyme carrying section 122.

[0039] <Example of a porous medium - 2> Figures 2A to 2H show cross-sectional views of porous media according to several embodiments. The top view shape of these media is not limited to striped or circular shapes, as long as it is consistent with other shapes.

[0040] Figure 2A shows a cross-sectional view of a porous medium 210 according to one embodiment. The entire porous medium 210 has the ability to separate plasma and supports enzymes.

[0041] Figure 2B shows a cross-sectional view of a porous medium 220 according to one embodiment. The porous medium 220 has a plasma separation portion 221 at its end or edge, and an enzyme carrying portion 222 that is arranged substantially coplanar with the plasma separation portion 221 and in contact with the plasma separation portion 221 at its end face.

[0042] Figure 2C shows a cross-sectional view of a porous medium 230 according to one embodiment. The porous medium 230 has a plasma separation section 231 at its end or edge, and an enzyme carrying section 232 arranged substantially coplanar with the plasma separation section 231. The plasma separation section 231 partially overlaps with the enzyme carrying section 232. The plasma separation section 231 and the enzyme carrying section 232 are in contact over a relatively wide surface area. As a result, blood components flow efficiently from the plasma separation section 231 to the enzyme carrying section 232.

[0043] Figure 2D shows a cross-sectional view of a porous medium 240 according to one embodiment. The porous medium 240 has an enzyme-carrying section 242, and a plasma separation section 241 is superimposed on its end or edge. Blood is dropped onto the plasma separation section 241, and the plasma components reach the enzyme-carrying section 242 in a vertical flow. The plasma components flow through the enzyme-carrying section 242 in a horizontal flow.

[0044] Figure 2E shows a cross-sectional view of a porous medium 250 according to one embodiment. The porous medium 250 has an enzyme-carrying section 252, and a plasma separation section 251 is superimposed on its center (or at a position spaced apart from the edge). Blood is dropped onto the plasma separation section 251, and the plasma components reach the enzyme-carrying section 252 in a vertical flow. The plasma components flow horizontally through the enzyme-carrying section 252, either to the sides or in the radial direction within the plane.

[0045] Figure 2F shows a cross-sectional view of a porous medium 260 according to one embodiment. The porous medium 260 has an enzyme-carrying portion 262, and a plasma separation portion 261 is arranged in the center of the enzyme-carrying portion 262 (or at a position spaced apart from the edge) and on substantially the same plane as the enzyme-carrying portion 262. The plasma separation portion 261 and the enzyme-carrying portion 262 are arranged so as to be in contact with each other at their end faces.

[0046] Figure 2G shows a cross-sectional view of a porous medium 270 according to one embodiment. The porous medium 270 has an enzyme-carrying portion 272, and a plasma separation portion 271 is located in the center (or spaced apart from the edge) of the enzyme-carrying portion 272, and substantially coplanar with the enzyme-carrying portion 272. The plasma separation portion 271 does not penetrate or is non-penetrating in the depth direction of the enzyme-carrying portion 272. Blood is dropped onto the plasma separation portion 271 and flows both horizontally and vertically, with the plasma components reaching the enzyme-carrying portion 272. The plasma components flow through the enzyme-carrying portion 272 mainly in a horizontal flow, either to the sides or in the in-plane diameter direction. The plasma components may also flow partially in a vertical flow.

[0047] Figure 2H shows a cross-sectional view of a porous medium 280 according to one embodiment. The plasma separation section 281 and the enzyme carrying section 282 are stacked. Blood is dropped onto the upper surface of the plasma separation section 281 and flows vertically, with the plasma components reaching the enzyme carrying section 282. The plasma components may further flow vertically through the enzyme carrying section 282. enzyme The material may flow horizontally through the support portion 282 in a direction parallel to the lamination surface.

[0048] <Manufacturing method> In some embodiments, a medium for separating and storing plasma can be produced by coating or absorbing a solution containing a substance that inhibits glucose-induced protein glycation (e.g., glucose oxidase solution) onto a porous medium having plasma separation ability (e.g., a plasma separation filter), and then drying it.

[0049] In some embodiments, a porous medium may be impregnated with a solution containing a substance that inhibits glucose-induced protein glycation. In some embodiments, a solution containing a substance that inhibits glucose-induced protein glycation may be applied to the surface of the porous medium by methods such as spin coating.

[0050] A porous medium may be impregnated with a predetermined amount (enzyme titer) of solution. This allows control over the enzyme titer (total, per unit area, per unit volume) of the porous medium. If the absorption capacity of the porous medium is known, the amount of enzyme or enzyme titer supported by the porous medium is determined by that absorption capacity. In some embodiments, a predetermined amount of solution may be prepared and absorbed entirely into the porous medium. The amount of enzyme or enzyme titer supported by the porous medium can be determined by the amount of solution prepared.

[0051] The amount of enzyme supported by the porous medium is 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2 units / mm³. 3The values ​​may be greater than or equal to these values. The amount of enzyme supported by the porous medium may be determined based on the enzyme titer. The medium needs to support the minimum amount of enzyme necessary to respond to a certain blood glucose level. Therefore, the amount of enzyme supported may be determined based on the enzyme titer of the supported enzyme.

[0052] The amount of enzyme supported by the porous medium is 0.2, 1, 2, 6, 10, 20, 40, 60, 70, 80 μg / mm³. 3 Values ​​such as below Or it may be smaller. The value of the amount of enzyme supported by the porous medium may be determined based on the mass to be supported. As the amount of enzyme supported increases, the hydrophobicity of the medium increases, making it more difficult for blood to be absorbed. Therefore, the amount of enzyme supported may be determined based on the actual physical quantity (number of moles, number of grams, etc.).

[0053] The solution may already contain components other than substances that inhibit glucose-induced protein glycation, such as cofactors and dyes.

[0054] In some embodiments, the porous medium may be dried at room temperature. The drying step may include heating. The drying step may be a non-heating step. The porous medium may be dried under reduced pressure.

[0055] <device> This disclosure also provides a device for separating and storing plasma. The device comprises a porous medium for separating and storing plasma and a housing for supporting or fixing it. Several embodiments are described below with reference to the figures. Figures 3A to 3D show perspective views of devices according to several embodiments.

[0056] Figure 3A shows a perspective view of a device 300 according to one embodiment. The device 300 comprises a housing 310 and a porous medium 320 supported therein. The housing 310 has an upper housing 311 and a lower housing 312, which are combined together and configured so that the porous medium 320 is fitted between them. The upper housing 311 is provided with an opening 331 for blood dripping (introduction). The upper housing 311 is further provided with a window 332 for observing the leading edge of blood or plasma components flowing through the porous medium 320.

[0057] The window 332 may be a through-hole and may be covered with a transparent material. Inside the window 332, a determination line 340 is provided to indicate the position of the end of the flowing liquid (distance from the opening 331, which is the dripping position). The determination line 340 may be fixed to the window 332 or fixed to the surface of the porous medium 320.

[0058] Figure 3B shows a perspective view of a device 400 according to one embodiment. The device 400 comprises a housing 410 and a porous medium 420 supported therein. The housing 410 has an upper housing 411 and a lower housing 412, which are combined together and configured so that the porous medium 420 is fitted between them. The upper housing 411 is provided with an opening 431 for blood dripping (introduction). The upper housing 411 is made of a transparent material. Therefore, the leading edge of the blood or plasma component flowing through the porous medium 420 can be observed in a wide field of view.

[0059] The upper housing 411 is provided with a determination line 440 that indicates the position of the end of the flowing liquid (distance from the opening 431, which is the dripping position). The determination line 440 may be fixed to the upper housing 411 or fixed to the surface of the porous medium 420.

[0060] Figure 3C shows a perspective view of a device 500 according to one embodiment. The device 500 comprises a housing 510 and a porous medium 520 supported therein. The housing 510 has an upper housing 511 and a lower housing 512, which are combined together and configured so that the porous medium 520 is fitted between them. The upper housing 511 has striped slits 531. The leading edge of blood or plasma components flowing through the porous medium 520 can be observed in a wide field of view. The upper housing 511 has a mark 542 on its upper surface indicating the position of blood dripping (introduction) and a judgment line 541 indicating the distance from the mark 542.

[0061] Using Figure 4, the usage methods of devices 300, 400, and 500 shown in Figures 3A to 3C will be briefly explained. Figure 4 uses device 300, as shown in Figure 3A, as an example. Blood 351 is drawn from the body surface 350 of the subject (user) using a lancet or other puncture device (not shown). This blood 351 is collected using a blood collection device 360. The collected blood 351 is dripped from the blood collection device 360 ​​into the opening 331 of device 300. The dripped blood 351 is absorbed into the porous medium 320 at the location of the opening 331 and flows through the porous medium 320 by capillary action. When the red component reaches the judgment line 340, it is confirmed that a sufficient amount of blood 351 has been introduced into the porous medium 320.

[0062] Figure 3D shows a perspective view of a device 600 according to one embodiment. The device 600 comprises a housing 610 and a porous medium 620 supported therein. The housing 610 has an upper housing 611 and a lower housing 612, which are combined together and configured so that the porous medium 620 is fitted between them. The porous medium 620 has projections 621. One end of the projection 621 protrudes outward from the end of the housing 610. The upper housing 611 is provided with a window 632 for observing the leading edge of blood or plasma components flowing through the porous medium 620. Window 632 for A judgment line 640 indicating the distance from the projection 621 is positioned.

[0063] Figure 5 briefly illustrates how to use device 600. Blood 651 is drawn from the body surface 650 of the subject (user) using a lancet or other puncture device (not shown). The projection 621 of the porous medium 620 of device 600 is brought close to the blood and brought into contact with it. The blood 651 is absorbed by the projection 621 and flows through the porous medium 620 by capillary action. When the red component reaches the judgment line 640, it is confirmed that a sufficient amount of blood 651 has been introduced into the porous medium 620.

[0064] This disclosure is not limited to the embodiments described above. The edges of the opening for dripping blood may be tapered or vertical. A transparent or opaque cover may be attached to the top surface in a way that allows it to be opened and closed or removed. The upper housing or porous medium may be configured to slide into place along the longitudinal or lateral direction of the device.

[0065] The housing may have grooves or protrusions (dot-like, linear, wall-like protrusions, etc.) on the surface that comes into contact with the medium. This can, for example, reduce the adhesion of blood components absorbed by the medium to the inner surface of the housing, or it can facilitate the flow of blood components within the medium.

[0066] Figure 6 shows an optical photograph of the filter 700 in actual use. Whole blood was dropped onto the left end of the filter 700. On the left side of the filter 700, region 701 was observed where blood cell components were captured. To the right of this, a region 702 was observed that was not red but had a color (yellow) relative to the filter 700. This region 702 is where the plasma component, after the blood cell components have been removed, is captured.

[0067] <Transportation Kit> This disclosure also provides kits for storing and transporting plasma components. The kits have a porous medium and a housing that supports it, or a device formed by combining them. This disclosure also provides methods for storing or transporting the porous medium or the device.

[0068] In some embodiments, the kit may include a package configured to enclose a device or porous medium to be stored or transported. The package may be configured to seal and enclose the device or porous medium.

[0069] In some embodiments, the kit may include a drying component. The drying component can absorb moisture in the package and reduce or maintain low humidity. The drying component may be silica gel or a fibrous material such as dry paper. The porous medium may be dried during storage or transport.

[0070] The packaging may be designed to prevent blood from adhering to the envelope or lid of the package during mailing. This can prevent contamination of the porous medium. The porous medium may be completely dried before being sealed in the package.

[0071] In some embodiments, the kit may include blood collection equipment. For example, a lancet or other puncture device, blood collection tubes, etc., may be provided.

[0072] <Post-transport processing> In some embodiments, a method is provided for extracting plasma components from a porous medium. A porous medium that holds plasma components is provided through storage or transport. The porous medium may be immersed in a solution. This allows the plasma components held by the porous medium to be dissolved or eluted into the solution.

[0073] <Mechanical pretreatment> In some embodiments, a porous medium containing plasma components may be separated into a portion containing plasma and a portion containing other parts (blood cell components). For example, Identify the red areas using visual inspection or a camera. The red portion may be separated from the rest of the sample (the portion containing plasma). Scissors, a cutter, or other cutting device can be used.

[0074] To efficiently elute the plasma components in the subsequent processing solution, the portion of the porous medium containing the plasma components may be further broken down. Scissors, cutters, or other cutting devices can be used. The porous medium may be crushed to some extent using a rod-shaped member. The porous medium may also be ground using a masher.

[0075] In some embodiments, the separated porous media portion may be impregnated in the processing solution and then shaken. In some embodiments, the porous media portion may be impregnated in the processing solution and then heated.

[0076] <Processing solution> In some embodiments, the processing solution may be a diluent. The diluent may be used to dilute the acquired liquid. The processing solution may be a liquid (pretreatment solution) used to perform a predetermined treatment (pretreatment) on the acquired liquid before any measurements are taken. The processing solution may be water or an aqueous solution. The processing solution may be a buffer solution. The processing solution may be, for example, Good's buffer. The processing solution may be physiological saline. The processing solution may be an organic solvent.

[0077] For example, if the substance being measured is sensitive to pH or salt concentration, such as a protein, a buffer solution such as physiological saline or Good's Buffer may be used. For example, if the substance being processed is a small molecule such as an amino acid, buffering capacity is not necessarily required. In that case, water or other aqueous solutions, or organic solvents, may be used. The processing solution may contain additives such as stabilizers and preservatives. Additives to maintain the structure of proteins and other substances may also be used.

[0078] In some embodiments, the processing solution may contain a stabilizer for the target substance. For example, a protein structure stabilizer may be used. The protein may, for example, be albumin, not limited to that example. For example, the structure of albumin can be stabilized using a stabilizer. By using a stabilizer, an indicator molecule such as BCP (bromocresol purple) can be specifically bound to a predetermined site. If the charge at or near the binding site and the environment of the amino acid side chain are suitable, it becomes easier to bind the indicator molecule to the protein, and specificity is maintained. Protein stabilizers include, for example, sugars, polysaccharides, salts, etc., not limited to that example.

[0079] The processing solution may contain a substance (coagulant) that coagulates substances contained in the target liquid. For example, the target liquid may contain a coagulant that coagulates specific substances such as fine dust, blood cells, membrane proteins, and oils. These substances may be formed into larger clumps. The clumps formed by coagulation are easier to remove in subsequent filtering.

[0080] The treatment solution decomposes, solubilizes, coagulates, or inhibits the reaction of substances. or It may contain substances that cause [something].

[0081] The components of the treatment solution may be selected from the group consisting of physiological saline, HEPES, TES, MES, tricine, sodium carbonate buffer, TBS, and PBS. The components of the treatment solution may include Good's buffer (e.g., HEPES, TES, MES, tricine). The treatment solution may contain standard substances to be used in subsequent measurements. The treatment solution may be pure water or ultrapure water.

[0082] This disclosure provides a method for measuring protein properties such as glycated albumin (GA) values. In some embodiments, the albumin concentration and / or glycated albumin concentration contained in the eluate from a porous medium that has been stored or transported as described in this disclosure may be measured. In some embodiments, the GA value may be determined based on the measured albumin concentration and glycated albumin concentration.

[0083] A glass fiber filter (LF1 / F487-14, Cytiva) with blood cell separation capabilities was shredded into pieces measuring 4 mm vertically and 30 mm horizontally.

[0084] AMANO enzyme glucose oxidase (GO“AMANO”AM, 226 U / mg) was weighed and dissolved in ultrapure water to prepare a 2.5 mg / mL aqueous solution (GOx solution). 27 μL of the GOx solution was added to a 4 mm × 30 mm LF1 filter and dried overnight at room temperature. This was used as a glycation inhibition filter (GOx-supported filter). The filter prepared in this way produced approximately 0.5 u nit / mm 3 , about 2.25μg / mm 3 This filter is loaded with GOx. On the other hand, as a control, a 4mm x 30mm LF1 filter (GOx-free filter) without GOx loading was used as is.

[0085] HSA (Human Serum Albumin, Wako Pure Chemical Industries, Ltd.) High concentration By reacting glucose, high A GA-HSA solution was prepared. This was mixed with HSA that had not undergone the glycation reaction to prepare a modified HSA solution with an ALB concentration of 4.43 g / dL, a GA concentration of 0.83 g / dL, a GA% of 19.29%, and a glucose concentration of 200 mg / dL.

[0086] 5 μL of prepared HSA solution was absorbed into the ends of a GOx-supported filter and a GOx-unsupported filter using a micropipette. Each filter was left to dry at room temperature.

[0087] Subsequently, albumin was eluted from each filter on days 1, 2, 3, 5, and 10 using the following method. First, a buffer solution containing 150 mM NaCl and 10 mM HEPES was prepared as the processing solution. The portion of each filter that did not contain blood cells (region 702 in Figure 6) was shredded to a width of 2 mm and placed in a 1.5 mL tube. 100 μL of HEPES buffer was added, and the mixture was shaken at 1800 rpm for 30 minutes at room temperature to prepare the sample for measurement.

[0088] The concentrations of glycoalbumin (GA) and total albumin (ALB) in each of the above samples were measured using the glycated albumin measurement reagent Lusica® GA-L (Asahi Kasei Pharma Corporation) and an automated analyzer (DM-JACKEx+, Minaris Medical Co., Ltd.).

[0089] Figure 7 shows the changes in ALB concentration (coarse dashed line), GA concentration (fine dashed line), and GA% (solid line) captured by a GOx-supported filter ("GOx15U") and a GOx-unsupported filter ("LF") from the capture date to day 10. The ALB concentration remained almost constant until day 10 in both cases: GOx-supported filter ("GOx15U") (black square) and GOx-unsupported filter ("LF") (white square). [Effective time course of ALB concentration] It was confirmed that it was not present.

[0090] On the other hand, a significant difference was observed in GA concentration between the two methods. When using the GOx-free filter ("LF") (white triangle), the measured GA concentration increased day by day. This indicates that the concentrations of albumin and glucose present in the prepared HSA solution increased with drying, increasing the probability of contact between the two, and consequently, the glycation of albumin by glucose progressed. In contrast, when using the GOx-supported filter ("GOx15U") (black triangle), the measured GA concentration remained almost constant. This indicates that GOx digested the glucose in the prepared HSA solution, and therefore, even though the albumin concentration increased with drying, the glycation of albumin by glucose did not progress.

[0091] GA% is obtained by dividing the GA concentration by the ALB concentration. When using a GOx-unsupported filter ("LF") (white circle), the GA% increased daily, exceeding 100% on the third day. One possible reason for this is as follows: Albumin has multiple glycation sites, some of which are more easily glycated than others. When the glucose concentration is relatively low, the most easily glycated site (said to be Lys525) is glycated, and as the glucose concentration increases, other sites are also thought to be glycated. In other words, as the glucose concentration increases, the number of glycated amino acids in a single albumin molecule increases. Since this measurement was performed with a relatively low GA% (e.g., 40% or less), if multiple glycation sites were glycated, their counts increased, and as a result, the GA% showed a falsely high value. However, this is only one consideration, and other interpretations may be possible. In any case, GA% is 100 % The reason for exceeding the limit is thought to be a significant increase in the degree of saccharification due to drying and the passage of time. Thus, it was found that with GOx-free filters, GA% is not accurately measured over time.

[0092] On the other hand, when a GOx-supported filter ("GOx15U") was used (black circle), GA% remained almost constant until day 10. In other words, the GOx-supported filter suppressed the promotion of glycation after blood absorption. Thus, it was shown that by using a GOx-supported filter, GA% can be measured relatively accurately even when blood is collected via mail or transport to a remote location or when a significant amount of time has passed.

[0093] This disclosure also provides the following embodiments. A001 A medium for separating and storing serum or plasma, Porous media for plasma separation, A substance that suppresses glucose-induced protein glycation, supported on the porous medium, A medium equipped with these features. A011 The porous medium is a fibrous medium or a porous polymer (polymer porous body). The medium described in Embodiment A001. A012 The porous medium is composed of a substance selected from the group consisting of cellulose, glass fiber, hollow fiber, cotton, nitrocellulose, and polysulfone. The medium described in Embodiment A001. A013 The porous medium consists substantially of a glass fiber medium. The medium described in Embodiment A012. A014 The porous medium has the ability to substantially separate plasma. A medium according to any one of embodiments A001 to A013. A015 It has the ability to filter substances larger than 3 μm. A medium according to any one of embodiments A001 to A014. A016 It has the ability to filter substances larger than 1 μm. A medium according to any one of embodiments A001 to A015. A017 It has the ability to filter substances larger than 0.1 μm. A medium according to any one of embodiments A001 to A016. A021 The substance that inhibits the glycation of proteins by glucose includes glucose oxidoreductase, A medium according to any one of embodiments A001 to A017. A022 The substance that inhibits the glucose-induced glycation of proteins includes an enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase, and glucokinase. A medium according to any one of embodiments A001 to A021. A023 The substance that suppresses the glucose-induced glycation of proteins further contains a cofactor. The medium according to Embodiment A021 or A022. A031 The substance that suppresses glycation of the protein by the glucose is supported on the inner surface of the porous medium. The medium according to any one of Embodiments A001 to A023. A032 The substance that suppresses glycation of the protein by the glucose is supported on the outer surface of the porous medium. The medium according to any one of Embodiments A001 to A023. A033 0.04 unit / mm 3 Glucose oxidase of 0.04 unit / mm or more is supported on the porous medium. The medium according to any one of Embodiments A001 to A032. A034 0.1 unit / mm 3 Glucose oxidase of 0.1 unit / mm or more is supported on the porous medium. The medium according to Embodiment A033. A035 0.2 unit / mm 3 Glucose oxidase of 0.2 unit / mm or more is supported on the porous medium. The medium according to Embodiment A034. A036 10 μg / mm 3 Glucose oxidase of 10 μg / mm or less is supported on the porous medium. The medium according to any one of Embodiments A001 to A035. A037 6 μg / mm 3 Glucose oxidase of 6 μg / mm or less is supported on the porous medium. The medium according to Embodiment A036. A038 2 μg / mm 3 Glucose oxidase of 2 μg / mm or less is supported on the porous medium. The medium according to Embodiment A037. A041 The porous medium further comprises catalase supported thereon. A medium according to any one of embodiments A001 to A038. A042 The porous medium further comprises a ketoamine oxidase (for example, fructosyl amino acid oxidase (FAOD)), A medium according to any one of embodiments A001 to A041. A043 The porous medium further comprises a blood coagulation inhibitor. A medium according to any one of embodiments A001 to A042. A051 The porous medium further comprises a dye supported thereon. A medium according to any one of embodiments A001 to A043. A061 The porous structure configured to retain separated plasma components medium Part one and The porous material configured to retain blood cell components medium Part two and It is stipulated that, The entire second part is configured to be separated from other parts, including at least a portion of the first part. A medium according to any one of embodiments A001 to A051. A101 A medium for separating and storing plasma, A first porous medium for plasma separation, A second porous medium is arranged in contact with the first porous medium and carries a substance that suppresses the glycation of proteins by glucose, A medium equipped with these features. B001 A method for producing a medium for separating and storing plasma, To provide a porous medium having plasma separation capability, To provide a solution containing a substance that inhibits the glycation of proteins by glucose (for example, a glucose oxidase solution), The above solution is applied to the porous medium, Drying the porous medium to which the solution has been applied, A method for providing this. B011 Applying the aforementioned solution to the porous medium comprises impregnating the porous medium with the aforementioned solution. The method described in Embodiment B001. B012 Applying the solution to the porous medium comprises applying the solution to the surface of the porous medium. The method described in Embodiment B001. B021 The substance that inhibits the glucose-induced glycation of proteins includes an enzyme selected from the group consisting of glucose oxidase, glucose dehydrogenase, and glucokinase. The method according to any one of embodiments B001 to B012. B022 The solution further comprises a cofactor of the enzyme, The method described in Embodiment B021. B031 Impregnating the porous medium with the (glucose oxidase) solution comprises absorbing a predetermined amount of the (glucose oxidase) solution into the porous medium. The method according to any one of embodiments B001 to B022. B032 The drying process includes heating the porous medium that has absorbed the enzyme solution. The method according to any one of embodiments B001 to B031. C001 A device for separating and storing plasma, A porous medium according to any one of embodiments A001 to A101, A housing that supports the porous medium, A device equipped with the following features. C011 The housing has an opening for dripping blood and a window for checking the amount of blood that has been dripped. Embodiment C The device described in 001. C021 A scale indicating the amount to be collected is provided in the window or on the inside of the window. Embodiment C001 A device as described in any one of the items C011. C022 The scale is one or more determination lines provided at predetermined positions from the opening. The device described in Embodiment C021. C023 The scale is printed on the surface of the porous medium or fixed to the window. The device described in Embodiment C021 or C022. D001 A kit for storing or transporting blood components, A device according to any one of Embodiments C001 to C023, A package configured to enclose the aforementioned device, A kit that includes the following: D011 The package is configured to seal and enclose the device, The kit described in Embodiment D001. D021 It also contains moisturizing ingredients, The kit described in Embodiment D011. D031 Further equipped with blood collection equipment, A kit according to any one of Embodiments D001 to D021. E001 A method for measuring glycated albumin levels (GA levels), To provide a porous medium in which plasma obtained from a sample is separated and stored, Eluting plasma components from the porous medium into a solution, The albumin concentration and the glycated albumin concentration contained in the eluate containing the plasma components are to be measured. Based on the measured albumin concentration and glycated albumin concentration, the GA value is determined. A method for providing this. E011 Dissolving plasma components from the porous medium into a solution is Separating the portion that holds the blood cell component from the porous medium, The remaining portion of the porous medium, at least the portion that holds the plasma component, is immersed in the solution. Equipped with, The method according to Embodiment E001.

[0094] While several embodiments and examples of the present disclosure have been described above, these embodiments and examples are illustrative in nature. For example, each of the above embodiments has been described in detail to make the present disclosure easier to understand, and dimensions, configurations, materials, and circuits may be added or modified as needed. Embodiments that arbitrarily combine one or more of the features of the present disclosure listed above are also included in the scope of the present disclosure. The claims encompass a number of variations on the embodiments, without departing from the technical idea of ​​the present disclosure. Therefore, the embodiments and examples disclosed herein are provided for illustrative purposes only and should not be considered as limiting the scope of the present disclosure.

Claims

1. A medium for separating and storing plasma, A porous medium for plasma separation, comprising a porous medium supporting catalase, A substance that suppresses glucose-induced protein glycation, supported on the porous medium, A medium equipped with these features.

2. The porous medium is a fibrous medium or a porous polymer. The medium according to claim 1.

3. The porous medium is composed of a substance selected from the group consisting of cellulose, glass fiber, hollow fiber, cotton, nitrocellulose, and polysulfone. The medium according to claim 2.

4. The porous medium has the ability to substantially separate plasma. The medium according to claim 1.

5. The substance that inhibits the glycation of proteins by glucose includes glucose oxidoreductase, The medium according to claim 1.

6. The substance that suppresses the glucose-induced glycation of proteins further contains a cofactor. The medium according to claim 5.

7. The substance that inhibits the glycation of proteins by glucose is supported on the inner surface of the porous medium. The medium according to claim 1.

8. The substance that inhibits the glycation of proteins by glucose is supported on the outer surface of the porous medium. The medium according to claim 1.

9. 0.04 unit / mm ３ The above and / or 10 μg / mm² ３ The following glucose oxidases are supported on the porous medium: The medium according to claim 1.

10. The porous medium further comprises ketoamine oxidase supported on the porous medium. The medium according to claim 1.

11. The porous medium further comprises a blood coagulation inhibitor. The medium according to claim 1.

12. A portion of the porous medium configured to hold the separated plasma components, The second part of the porous medium configured to hold blood cell components and It is stipulated that, The entire second part is configured to be separated from other parts, including at least a portion of the first part. The medium according to claim 1.

13. A device for separating and storing plasma, A porous medium according to any one of claims 1 to 12, A housing that supports the porous medium, A device equipped with the following features.

14. The housing has an opening for dripping blood and a window for checking the amount of blood that has been dripped. The device according to claim 13.

15. The window is provided with one or more determination lines positioned at predetermined locations from the opening. The device according to claim 14.

16. A kit for storing or transporting blood components, The device according to claim 13, A package configured to enclose the aforementioned device, A kit that includes the following:

17. Further comprising dried components and / or blood collection equipment, The kit according to claim 16.

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