JP5564051B2 - Method for separating red blood cells from a blood sample and use thereof - Google Patents

Description

  The present invention relates to a method and apparatus for separating red blood cells from a whole blood sample. More particularly, the present invention relates to a detection device that uses the method and apparatus to separate red blood cells by separating fibrin from a whole blood sample, thereby reducing the disturbing effect of red blood cells on detection.

  The latest clinical diagnostic decisions are routinely made on blood samples. Unfortunately, red blood cells interfere with many diagnostic decisions. Thus, before a test or analysis can be performed on a blood sample, red blood cells must first be removed from the whole blood sample. On the other hand, in chromatographic analyzers, specifically in chromatographic immunological analyzers, red blood cells can delay the fluid flow required for the reactions that occur in these devices. For these and other reasons, many analytical methodologies are performed on plasma or serum that must first be separated from a whole blood sample.

US Pat. No. 5,939,331 US Pat. No. 6,673,629 US Pat. No. 4,594,327 US Pat. No. 4,678,757 US Pat. No. 5,558,834 US Pat. No. 6,818,180 US Patent Application Publication No. 2006/0029923 US Patent Application Publication No. 2004/0202783

  There are many known techniques for separating red blood cells from whole blood samples. A conventional method is a centrifuge. U.S. Patent Nos. 5,939,331, 6,673,629, 4,594,327, 4,678,757, 5,558,834, and 6,818,180, and U.S. Patent Application Publication No. 2006/0029923. And methods disclosed in US 2004/0202783. In general, these methods primarily use physical and chemical processes to separate red blood cells from plasma, and most of these methods are costly and complex and can result in incomplete red blood cell separation. is there. In addition, existing techniques are not suitable for whole blood samples that have been placed for a very long time and have become clotted. Specifically, a clotted blood sample can result in non-specific binding or high background in a chromatographic immunoassay device, resulting in a loss of analytical sensitivity. In addition, existing techniques can block the chromatographic support and inhibit plasma or serum flow over the support, rendering the analysis ineffective. This can be fatal for devices that are specifically required to provide detection results in a very short period of time. Thus, there is a need to have devices and methods that include not only reagents but also reagents for separating red blood cells from a whole blood sample, but the devices and methods do not adversely affect the detection system.

  In order to solve the problems mentioned above, the present invention provides a reagent for separating red blood cells from a whole blood sample, and the application of the reagent. By using reagents, red blood cells can be effectively separated from whole blood samples.

  One aspect of the present invention relates to a method for separating red blood cells from a whole blood sample. Specifically, the method includes contacting the whole blood sample with a receptor capable of binding to fibrin in the blood sample, and then removing the fibrin binding receptor from the blood sample. The method may also include a receptor that can bind to red blood cells. The method further comprises providing a support comprising only fibrin-coupled receptors, or both fibrin-coupled receptors and erythrocyte-coupled receptors, and having a blood sample flowing through the support. The support is preferably a chromatographic support.

  Another aspect of the invention relates to an apparatus for separating red blood cells from a whole blood sample, comprising a support with a receptor capable of binding to fibrin in the blood sample. The support may also comprise a receptor that can bind to red blood cells in the blood sample. The support is preferably a chromatographic support.

  Another aspect of the invention includes a support having a sample receiving region and a detection region disposed downstream of the sample receiving region, wherein the support upstream of the detection region can bind to fibrin in a blood sample. The present invention relates to a detection apparatus for detecting an analyte in a whole blood sample, comprising a receptor. The sample receiving area may be an area where the fibrin-binding receptor is immobilized. The sample receiving area may also comprise a receptor that can bind to red blood cells. Further, the support upstream of the detection region may further comprise a labeling substance that can be carried away by the blood sample stream, but the detection region may comprise specific binding molecules. The support is preferably a chromatographic support.

  Another aspect of the invention relates to a method for detecting an analyte in a whole blood sample, the method comprising providing a chromatographic support comprising a sample receiving region comprising a receptor capable of binding fibrin; Allowing flow of the blood sample through the receiving area and measuring the content of the analyte in the blood sample. The support may further include a receptor that can bind to red blood cells, and may include a detection region in the downstream region of the support for detecting the analyte.

  In all of the above embodiments, the receptor capable of binding to fibrin comprises an anti-fibrin antibody, while the receptor capable of binding to erythrocytes comprises an anti-erythrocyte antibody, said antibody comprising a monoclonal antibody, a polyclonal antibody, and Includes antibody fragments.

  The invention disclosed herein provides a number of advantages. The present invention effectively removes red blood cells from a whole blood sample, no matter how fresh the sample is or if clotting has already occurred, thereby extending the usable life of the blood sample. Furthermore, devices that use receptors that can bind to fibrin to separate red blood cells from a blood sample or to detect an analyte in a blood sample also have an extended usable lifetime.

1 depicts a specific embodiment of the present invention. The detection device 10 includes a filtering layer 11 and a reaction layer 12 for receiving and passing a sample thereon. The reaction layer includes a substrate for a reduction reaction and a handle 13. The filtering layer comprises a polyclonal anti-fibrin antibody and a polyclonal anti-erythrocyte antibody. After applying the whole blood sample 01 on the sample receiving layer 11, the anti-fibrin and anti-erythrocyte antibodies deposited on the layer 11 act to block red blood cells in a coordinated manner, holding the red blood cells on the filtering layer, Allow plasma to pass to the reaction layer 12. When an analyte, such as blood glucose, is presented in the sample, a color change can be observed directly on the bottom surface of the reaction layer 12. 1 depicts a preferred specific embodiment of the present invention. In the detection device 30, the sample receiving area 35 is made of glass fiber. The detection area 36 is made of a nitrocellulose film and is located downstream of the sample receiving area. A label holding region 34 is located between the sample receiving pad 35 and the detection region 36. The detection region includes a test line 33 comprising specific molecules that can bind to the analyte, and a control line 32 used as a control to confirm detection. Glass fibers in the sample receiving area are treated with polyclonal anti-fibrin antibody and polyclonal anti-erythrocyte antibody. The sample receiving area 35, the label holding area 34, and the detection area 36 are all connected together so that the fluid sample flows from the sample receiving area 35 to the detection area 36 for further reaction.

  The following is a detailed description of the structures relevant to the present invention and definitions of technical terms used in the description.

Detection As used herein, “detection” is a substance or material, such as a chemical, organic compound, inorganic compound, metabolite, drug or drug metabolite, organic tissue or metabolite of an organic tissue, nucleic acid, protein Or alternatively, a test or analysis of the presence of a polymer but not limited thereto. Furthermore, detection also means testing or analysis of the content of a substance or material. Furthermore, the analysis used herein includes immunoassay, chemical analysis, enzyme analysis, and the like.

Fibrin and fibrin-coupled receptors Fibrinogen is the earliest discovered coagulation factor. Fibrinogen has an elongated elliptical shape and is composed of two sets of three polypeptides (ie, a pair of α chains, a pair of β chains, and a pair of γ, linked together via disulfide bonds). Chain) and has a molecular weight of 340,000 daltons. Fibrinogen is synthesized in the liver and then enters the plasma and exists in dissolved form. There is about 0.3 g fibrinogen per 100 ml human plasma. However, fibrin is a protein multimer that does not dissolve very much, and is a crystal with a thin needle shape. The conversion of fibrinogen to fibrin is a fundamental change in the blood clotting process, and the blood clotting process must go through three stages. (1) Hydrolysis of fibrinogen: each of the two alpha and two beta chains of the fibrinogen molecule cleaves the peptide bond under the influence of thrombin and releases two pairs of small polypeptides (ie, a total molecular weight of about Fibrinopeptide A and fibrinopeptide B) with 9000 daltons, and the formation of fibrin monomers. Therefore, the molecular weight of the fibrin monomer is smaller than the molecular weight of the fibrinogen molecule. (2) Aggregation of fibrin monomers: In the presence of Ca ²⁺ , many fibrin monomers aggregate and polymerize to form a soluble fibrin polymer. (3) Formation of a clot: in the presence of thrombin, factor IIIa, and Ca ²⁺ , the α chain of the fibrin monomer is cross-linked to form an intertwined fiber network, thereby making the fibrin monomer insoluble Instead of fibrin polymer, erythrocytes are trapped in the fiber network, and blood like the original sol is converted into blood clot like gel.

  Surprisingly, it has been found that removal of fibrin from a whole blood sample using a receptor capable of binding fibrin can effectively separate red blood cells in the blood sample. Specifically, when fibrin-coupled receptors are used in concert with receptors that can bind to red blood cells, the separation of red blood cells becomes more effective and complete. Furthermore, such techniques for removing red blood cells work well no matter how fresh the blood sample is or how much clotting has occurred.

Separation Method On the other hand, the present invention relates to a method for separating red blood cells from a whole blood sample. Specifically, the method includes contacting a whole blood sample with a receptor capable of binding to fibrin in the blood, and then removing the fibrin binding receptor from the blood sample. In a specific embodiment, an anti-fibrin antibody is used as a receptor to bind to or capture fibrin monomers or polymers in a blood sample, where the anti-fibrin antibody is a polyclonal antibody, a monoclonal antibody Or an antibody fragment. The receptor can also be another naturally synthesized or artificially synthesized receptor that can bind to fibrin. The binding can be “specific” or “non-specific” and the receptor can bind to fibrin as long as the receptor can bind to or capture fibrin in the blood sample. it is conceivable that. One relatively simple example involves contacting a whole blood sample with an anti-fibrin antibody and then separating the red blood cells in the blood sample by separating the anti-fibrin antibody in the blood sample. A more preferred embodiment is to first contact the anti-erythrocyte antibody with the whole blood sample, then contact the anti-fibrin antibody with the blood sample, and then separate the anti-fibrin antibody in the blood sample in the sample. Separating the red blood cells. Yet another embodiment includes separating red blood cells by simultaneously contacting the anti-fibrin and anti-erythrocyte antibodies with the whole blood sample and then simultaneously separating the anti-fibrin and anti-erythrocyte antibodies in the sample. As used herein, the phrase “separate” means that the anti-fibrin antibody is separated or removed directly or indirectly from the blood sample using physical or chemical means. For example, antibodies against fibrin-coupled receptors can be used directly as a means for separating fibrin-coupled receptors, or another method can be used.

In another embodiment, the method comprises providing a support comprising a receptor capable of binding fibrin and passing a whole blood sample through the support. Including. Furthermore, the present invention also relates to an apparatus for separating red blood cells from a whole blood sample, comprising a support comprising a receptor capable of binding fibrin. The support may consist of or consist of a water-absorbing material, also called a “chromatographic support” or non-absorbable material.

  Non-absorbable materials include, but are not limited to plastic, glass, ceramic, and other metallic materials. For example, a receptor capable of binding fibrin, eg, an antibody, can be first immobilized on a plastic surface, contacting the receptor on the plastic surface with a whole blood sample, and then fibrin in the blood sample is , Captured by a receptor immobilized on a plastic surface, and thus separated from the blood sample, so that red blood cells are also separated from the blood sample. In a preferred embodiment, such non-absorbable material is provided, both antibodies against red blood cells and fibrin are immobilized on the material, and then the whole blood sample can pass through the surface of the non-absorbable material. To be done.

  On the other hand, the support may be a “chromatography support”, in which case “chromatography support” refers to any suitable porous and absorbent material, or capillary material. These materials should be essentially absorbent so that fluid can flow by capillary action. These materials can be, for example, filter paper, glass cellulose, polyester membrane, nylon membrane, nitrocellulose membrane, cellulose acetate, natural material (eg cotton) fabric, and synthetic material (nylon) fabric, porous gel, and the like. In a preferred embodiment using a support that supports fluid flow, the fibrin-bound receptor is processed on the support so that it cannot be carried away by the blood sample due to fluid flow. When a whole blood sample is applied to a support, fibrin molecules in the blood sample are bound to the receptor and collected on the support, and thus separated from the blood sample for a continuous flow of blood sample. In yet another preferred embodiment, the support may also comprise one or several receptors that can bind to red blood cells. These receptors can collect red blood cells in a whole blood sample. The erythrocyte-binding receptor may be placed upstream of the fibrin-binding receptor, or may be placed at the same location as the fibrin-binding receptor or at a different location. Methods of treating the receptor on the support are known in the art. Furthermore, the support is made from a single material, or two or more materials, so long as multiple layers are in contact with each other in the fluid flow so that a fluid sample can pass between these materials. It will be appreciated by those skilled in the art that, for example, different portions, regions, or layers can be made from different materials. After receiving the fluid sample, these supports may be placed in different ways, for example vertically as shown in FIG. 1, horizontally as shown in FIG. 2, or any other way. Can pass through.

  The reason why the red blood cells in the sample can be separated or removed by separating the fibrin in the whole blood sample may be as follows. Fibrin-binding receptors, such as anti-fibrin antibodies, collect fibrin molecules by binding to fibrin molecules, thereby forming a fiber network that can cross-link fibrin molecules together and capture red blood cells in a sample . Thus, when the fibrin binding receptor in the sample is separated, the red blood cells in the sample are indirectly separated. In another preferred embodiment, if there are also receptors capable of binding to erythrocytes, such as antibodies to erythrocyte surface antigens, such erythrocyte-bound receptors collect individual erythrocytes to form a “hemagglutination”. If present, the fiber network formed by fibrin-coupled receptors and fibrin molecules facilitates these “hemagglutinations” so that red blood cells cannot be easily carried away by the flow of blood samples. Can be captured. As a result, red blood cells in the blood sample can be more easily separated. In a specific preferred embodiment, the receptor capable of binding fibrin is a polyclonal antibody that is processed and immobilized on a chromatographic support. In addition, the chromatographic support also comprises a polyclonal antibody against the erythrocyte surface antigen, to which the polyclonal antibody is immobilized. When a whole blood sample is applied to a chromatographic support, the anti-erythrocyte antibody provided on the support binds to or captures red blood cells in the sample to form a “hemagglutination” on the support The anti-fibrin antibody provided in 1 binds to or captures fibrin in the sample to form a fibrin molecular network. These fibrin molecular networks can easily capture “red blood cell agglutination” having a volume larger than the volume of individual red blood cells, thus delaying the red blood cells to flow more effectively and continuously from the blood sample Isolate more effectively. The method of separating red blood cells by separating fibrin-coupled receptors provided on a support is accomplished by directly immobilizing the receptors on the support or by another indirect means of separating fibrin. be able to. For example, an antibody against an anti-fibrin antibody (referred to as a second antibody against the anti-fibrin antibody) is immobilized on the support, and the whole blood sample and the anti-fibrin antibody are mixed and reacted, and then the mixed solution is mixed with the anti-fibrin antibody. To a support with a second antibody against. The second antibody captures the anti-fibrin antibody, thereby indirectly capturing fibrin. Similarly, an antibody against an anti-erythrocyte antibody (referred to as a second antibody against the anti-erythrocyte antibody) is immobilized on a support for indirectly capturing red blood cells in the blood sample. In a preferred embodiment, anti-fibrin antibody and anti-erythrocyte antibody are processed directly on the support.

Detection Device Another aspect of the present invention relates to a detection device for detecting an analyte in a whole blood sample. The apparatus includes a support including a sample receiving area and a detection area disposed downstream of the sample receiving area, the support upstream of the detection area including a receptor capable of binding to fibrin in the blood sample. . Preferably, the support is a chromatographic support and the sample receiving area comprises a receptor capable of binding to fibrin in the blood sample. The objects of the present invention can be achieved using any detection system, preferably a chromatographic immunological analysis system, including but not limited to horizontal flow systems, vertical flow systems, and test bars. A description of some known detection systems is given below.

  In general, there is at least one sample receiving area and one detection area on a chromatography reagent test strip, where the detection area determines whether the analyte is present in the sample or the content of the analyte in the sample. Including certain specific binding molecules or chemicals. The specific binding molecule can be an antibody or antibody fragment. The binding molecule can bind directly or indirectly to the analyte, or can bind to the moiety that the analyte is involved in. The moiety can be an immunological epitope of the analyte itself, or an immunological epitope of a reagent that binds to the analyte (eg, a reagent that binds to the analyte when the reagent passes through the label-carrying region). When the fluid sample to be detected is applied to the sample receiving area, the fluid sample moves along the chromatographic support by capillary action and thus is preliminarily formed by a binding reaction (eg, an immunological reaction) that occurs in the detection area. The accumulation of labeled material (labeled, conjugated using a label) that is processed and placed on the chromatographic support is allowed and can be carried away and flow with the fluid sample. Accumulation behavior is directly proportional to the presence or content of the analyte in the sample (eg, two-antibody sandwich) or inversely proportional (competitive method), and by measuring the presence or content of the labeled substance, The presence or content of light can be determined indirectly. Of course, the reaction that takes place in the detection region can also be a color reaction between substances having pure chemical properties, such as an oxidation-reduction reaction, followed by the presence or inclusion of an analyte in the sample. The quantity can be determined by measuring the intensity of the color shown in the detection area. Known detection devices applying this principle include those described in, for example, US Pat. No. 6,818,180, such as a blood glucose reagent test strip. In a specific embodiment, the sample receiving area is arranged at the same position as the place where the labeling substance is arranged, but is preferably arranged upstream of the labeling substance (the direction of movement of the sample caused by capillary action is Called “downstream” and the opposite direction is called “upstream”). When a fluid sample (which is likely to contain the analyte of interest) is brought into contact with the sample receiving area, the fluid sample flows downstream along with the analyte along the chromatographic support due to capillary action. The analyte is generally a compound that can bind to a detection molecule that is processed and immobilized on the detection region in a specific manner. For example, the analyte is an antigen, the labeling substance is an antibody against the antigen, and the detection molecule immobilized on the detection region is another antibody against the antigen. Devices and methods for detecting the presence of an analyte in a blood sample by applying the above principle are already prior art and not the center of the present invention. However, although the present invention is intended to be used with any blood sample including serum and plasma, it is preferably used with blood samples containing red blood cells, such as whole blood samples.

  As used herein, a labeling substance is a substance, such as an antibody or antigen, provided or conjugated with a label. The “label” can be any suitable label that provides a detectable signal. For example, the label can be a sol particle, a fluorescent molecule, a chemiluminescent molecule, a metal or alloy (eg, colloidal gold), or a capsule, specifically a liposome containing a visible dye. Also useful are hydrophobic sols where the hydrophobic organic dye or pigment does not dissolve in water or dissolves to a limited extent. The label can also be a polymer particle, such as a colored polystyrene particle (eg spherical), or a colored particle (eg dextran beads). Another useful specific label is ferritin, phycoerythrin or another phycobiliprotein, precipitated or insoluble metal or alloy, fungal, algal or bacterial pigments or derivatives such as bacterial chlorophyll, or another plant material including. In certain embodiments, a labeling substance disposed upstream of the detection region can bind (directly or indirectly) to the analyte of interest, whereby the analyte of interest passes through the support. When flowing, label the analyte of interest with a detectable label.

  In various embodiments, the analyte-bound reagent is an antibody, a fragment or portion of an antibody, an antibody (or antibody fragment) obtained from a different species than the antibody attached to the analyte-binding region, or specific. It can be avidin, streptavidin, or biotin, which can be bound to another member of the binding pair, such as the moiety itself bound to the analyte. As used herein, “antibody” refers to an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also includes derivatives of antibodies that retain specific binding ability. The term also encompasses any protein that has a binding domain that is similar or largely similar to an immunoglobulin binding domain. These proteins may be obtained from natural sources or may be partially or wholly synthetically produced. The antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes, ie, IgG, IgM, IgA, IgD, IgG, and IgE. An “antibody fragment” is any derivative or part of an antibody that is shorter than full length. Antibody fragments can retain at least a substantial portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (ab') 2, scFv, Fv, dsFv bispecific antibodies (diabody), and Fd fragments.

  Antibody fragments may be generated by any means. For example, antibody fragments may be produced enzymatically or by chemically fragmenting a complete antibody, or antibody fragments may be produced recombinantly from a gene encoding a partial antibody sequence. Alternatively, antibody fragments may be generated synthetically in whole or in part. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragments may comprise multiple chains linked together, for example by disulfide bonds. The fragment may also optionally be a multimolecular complex. Functional antibody fragments typically comprise at least about 50 amino acids and more typically comprise at least about 200 amino acids.

  Prior to detecting the analyte of interest in the blood sample, it is preferred that the red blood cells in the sample be removed if the detection should work with the desired sensitivity. Thus, according to the present invention, a receptor capable of binding to fibrin in a blood sample is processed on the chromatographic support, preferably in the sample receiving area of the support. It is preferred that the fibrin-coupled receptor is processed into the sample receiving area of the chromatography support because such a configuration is effective when red blood cells are effectively removed from the blood sample when the blood sample is applied over the sample receiving area. Is to be able to be removed. This is because such a configuration eliminates, or at least reduces, the blockage of red blood cells to a continuous flow of plasma or serum along the support so that the flow is not affected. On the other hand, such a configuration reduces background interference. For example, FIG. 2 illustrates a preferred specific embodiment of the present invention. In the detection device 30, the sample receiving area 35 is made of glass fiber. The detection area 36 is made of a nitrocellulose film and is arranged downstream of the sample receiving area. The label holding area 34 is disposed between the sample receiving area 35 and the detection area 36. The detection region includes a test line 33 with specific binding molecules and a control line 32 that is used as a control to confirm detection. Glass fibers in the sample receiving area are treated with polyclonal anti-fibrin antibody and polyclonal anti-erythrocyte antibody. The sample receiving area 35, label holding area 34, and detection area 36 are all connected such that fluid flows from the sample receiving area 35 to the detection area 36 for further reaction.

  FIG. 1 depicts another preferred specific embodiment of the present invention. The detection apparatus 10 includes a filtering layer 11 and a reaction layer 12 for sample reception and sample passage. The reaction layer includes a substrate for a reduction reaction and a handle 13. The filtering layer comprises a polyclonal anti-fibrin antibody and a polyclonal anti-erythrocyte antibody. After applying the whole blood sample 01 on the sample receiving layer 11, the anti-fibrin and anti-erythrocyte antibodies attached to the filtering layer 11 work in a coordinated manner to capture red blood cells in the sample, thereby filtering the red blood cells Hold on the bed and allow the plasma to pass to the reaction bed 12. When an analyte, such as blood glucose, is presented in the sample, a color change can be directly observed at the bottom surface of the reaction layer 12. The application of the method described in the present invention for removing red blood cells from a whole blood sample should not be limited to the specific embodiments described above. The method can be used with any other detection system. For example, the method can be used with the detection apparatus described in FIG. 1 of US Pat. No. 6,818,180. Specifically, the method can be applied to the surface layer 5 of the porous membrane 1 to separate the whole blood sample applied through the holes 21. One skilled in the art will appreciate that the methods or devices of the present invention can be used in combination with any other methods, structures, and reagents of the prior art for separating red blood cells. For example, the method can be combined with the structures and reagents described in US Pat. No. 6,818,180.

Detection Method Another aspect of the present invention relates to a method for detecting an analyte in a whole blood sample. The method is preferably applied to the chromatography support device. Specifically, the method includes providing a chromatographic support comprising a sample receiving area comprising a receptor capable of binding to fibrin in a blood sample; passing the blood sample through the sample receiving area; And then detecting whether the analyte is present in the blood sample. The sample receiving area preferably comprises a receptor capable of binding to red blood cells. It is preferable that the downstream of the sample receiving region includes a detection region to which specific binding molecules are immobilized, so that the sample that has passed through the sample receiving region can pass through the detection region.

  The following examples further illustrate the present invention but should not be construed as limiting the scope of the invention in any way.

Example 1: Application of erythrocyte separation from blood sample with anti-fibrin antibody in detection of CTI (cardiac troponin I) The polyclonal anti-fibrin antibody (Cat. , Ltd., Ltd. (Dongbeiwang South Rd., No. 26, Haidian District, Beijing). Monoclonal anti-erythrocyte antibody (Cat. No. ME060822-1-0419) and polyclonal anti-erythrocyte antibody (Cat. ). Except where otherwise noted, the CTI detection reagent test strips used in this experiment were prepared by methods known in the prior art. The preparation and assembly of the reagent test strip is further described herein with reference to FIG.

1. Configuration and preparation of sample receiving pad 35 The sample receiving pad 35 is made of glass fiber. Such a glass fiber pad was first treated with a Tris buffer. Thereafter, the glass fiber pads were subjected to different treatments according to the following description.

  1) Glass fiber pads treated with only polyclonal anti-fibrin antibody (total of 100 glass fiber pads, 20 for each treatment): 100 glass fiber pads were evenly divided into 5 groups. Polyclonal antifibrin antibodies prepared at different dilutions, ie 1:50, 1: 100, 1: 200, 1: 300, and 1: 400 were used to treat each of the five groups of glass fiber pads; As a result, 5 groups of glass fiber pads each having a separately diluted anti-fibrin antibody attached thereto were obtained. That is, they were processing A (1:50), processing B (1: 100), processing C (1: 200), processing D (1: 300), and processing E (1: 400).

  2) Glass fiber pads treated with both anti-erythrocyte polyclonal antibody and polyclonal anti-fibrin antibody (total 50 glass fiber pads, 10 in each group): each of treatments A, B, C, D, and E Evenly divided into two subgroups. Each subgroup therefore contained 10 glass fiber pads. In each treatment (ie A, B, C, D, and E) one of these two subgroups is further treated with a polyclonal anti-erythrocyte antibody (diluted 1:10), whereby two antibodies Five new groups of glass fiber pads (10 glass fiber pads in each group) were obtained, respectively. Five new groups were treated G = 1: 50 (anti-fibrin) +1: 10 (anti-RBC), treated H = 1: 100 (anti-fibrin) +1: 10 (anti-RBC), treated I = 1: 200 (anti-antibody). Fibrin) +1: 10 (anti-RBC), treatment J = 1: 300 (anti-fibrin) +1: 10 (anti-RBC), treatment K = 1: 400 (anti-fibrin) +1: 10 (anti-RBC).

  3) Glass fiber pads treated with only polyclonal anti-erythrocyte antibodies (diluted 1:10): Another group consisting of 10 untreated glass fiber pads (using only Tris buffer) was selected , Treated with a polyclonal anti-erythrocyte antibody (diluted 1:10), resulting in a new group designated treatment F (10 glass fiber pads).

  All treated glass fiber pads were then dried in a dryer at 37 ° C. and stored for further use.

2. Preparation of Label Retention Pad 34 Rabbit monoclonal anti-human CTI antibody and mouse polyclonal anti-human IgG antibody are conjugated with a common colloidal gold solution having an average particle size of about 20-40 nm, and then the polyester membrane Sprayed uniformly on top. The membrane was further dried at 37 ° C. in a dryer and stored for further use.

3. Preparation of Nitrocellulose Membrane 36 By using a micro-injector controlled by a microprocessor, the first polyclonal goat anti-mouse IgG antibody (1.3 mg / ml) was transferred to the control line (C line) 32 (for test confirmation). And then mouse anti-human CTI monoclonal antibody (4.0 mg / ml) was placed on the nitrocellulose membrane as test line (T line) 33 (for analyte binding). Dispensed and placed into. Both antibodies were dispensed at a rate of 1.1 μl / cm. Upon completion, the coated nitrocellulose membrane was immediately dried at 45 ° C. (2 hours) so that the attached antibody was immobilized on the membrane.

4). Reagent Test Strip Assembly A CTI detection reagent test strip was assembled according to the schematic shown in FIG. A sample receiving pad 35 (one of the aforementioned treated glass fiber pads) is placed overlying the label holding pad 34, and then the label holding pad 34 is placed on the other end of the label holding pad 34 (from the sample receiving pad). A nitrocellulose membrane 36 is overlaid at the remote end), after which the nitrocellulose membrane 36 is overlaid with an absorbent filter paper 31 disposed at the distal end of the test strip, thereby providing a reagent test strip as shown in FIG. Formed.

5. Preparation of blood samples Twenty whole blood samples (collected for more than 8 days and stored at 2-8 ° C) were prepared for further use.

6). Test Procedure 100 μl of whole blood sample was dropped onto the sample receiving pad of the reagent test strip and the time when the C line appeared on the reagent test strip was recorded. The time at which the C line appeared showed the flow rate of the blood sample liquid. The C line had to appear within 5 minutes of sample introduction, and the sample had to travel through the nitrocellulose membrane 36 to reach the absorbent filter paper 31 within 10 minutes of sample introduction. Otherwise, reagent test strips were considered inappropriate.

  Processes F and J were used to determine the sensitivity and specificity of the reagent test strip, respectively. A positive specimen is prepared from a CTI-negative (confirmed experimentally) whole blood sample and the test result of the positive specimen is recorded so that the CTI concentration in the specimen can be manipulated to 0.5 ng / ml It was done. To determine specificity, 20 standard CTI negative (experimental confirmed) whole blood samples were used as negative specimens in the test. Those strips that showed a red background were also considered inappropriate.

注：Ｃラインが出現する時間が５分を超えたため、または試料が１０分以内にニトロセルロース膜を通って進まなかったために、テストされた試薬テストストリップが不適当であると考えられた場合、結果に下線を引いた。 7). result

Note: If the tested reagent test strip is deemed unsuitable because the appearance time of the C-line has exceeded 5 minutes or because the sample has not advanced through the nitrocellulose membrane within 10 minutes, The results are underlined.

注：感度を検証するためにＣＴＩ陽性標本が使用された。「＋」は陽性のテスト結果が得られたことを示す。

Note: CTI positive specimens were used to verify sensitivity. “+” Indicates that a positive test result was obtained.

注：特異性をテストするためにＣＴＩ陰性全血試料が使用された。「−」は陰性の結果が得られたことを示す。

Note: CTI negative whole blood samples were used to test specificity. “-” Indicates that a negative result was obtained.

8). Conclusion The above results suggest that anti-fibrin antibodies can separate red blood cells from blood samples, thereby significantly reducing the probability that clotted blood samples will block the chromatographic support and greatly increase the efficiency of blood separation. To do. On the other hand, it is clear that anti-fibrin antibodies have no adverse effect on the detection itself. Furthermore, when anti-fibrin antibodies are used in combination with anti-erythrocyte antibodies, the beneficial effects of anti-fibrin antibodies on blood separation become more apparent.

Example 2 Application of red blood cell separation from blood samples with anti-fibrin antibody in PSA (prostate specific antigen) detection The antibody used in this experiment was the same as used in experiment 1. Compared to Experiment 1, the configuration of the reagent test strip used in Example 2 was used in Experiment 1, except that the target analyte was different and therefore the sample receiving pad treatment on the strip was different. It was similar to the composition of the strip. The preparation and assembly of the reagent test strips used in this experiment are described herein with reference again to FIG.

1. Configuration and preparation of sample receiving pad 35 The sample receiving pad 35 was made of glass fiber. Such glass fiber pads were first treated with Tris buffer. Thereafter, the glass fiber pads were subjected to the following different treatments.

  1) Treatment 1 (Glass fiber pad treated with both polyclonal anti-fibrin antibody and polyclonal anti-erythrocyte antibody): Glass fiber pad diluted polyclonal anti-fibrin antibody (diluted 1: 300), and diluted polyclonal Treated with anti-erythrocyte antibody (diluted 1:10).

  2) Treatment 2 (glass fiber pad treated with polyclonal anti-erythrocyte antibody): The glass fiber pad was treated with diluted polyclonal anti-erythrocyte antibody (diluted 1:10).

  3) Control treatment CK (glass fiber pad treated with monoclonal anti-red blood cell antibody): The glass fiber pad was treated with diluted monoclonal anti-red blood cell antibody (diluted 1:75).

  Next, any treated glass fiber pads were dried in a dryer at 37 ° C. and stored for further use.

2. Preparation of Label Holding Pad 34 Rabbit monoclonal anti-human PSA antibody and mouse polyclonal anti-human IgG antibody are conjugated with a common colloidal gold solution having an average particle size of about 20-40 nm, and then the apparatus is used to top the polyester membrane. Sprayed uniformly. The membrane was further dried at 37 ° C. in a dryer and stored for further use.

3. Preparation of Nitrocellulose Membrane 36 By using a microprocessor-controlled microinjector, the first polyclonal goat anti-mouse IgG (1.3 mg / ml) antibody was transferred to control line (C line) 32 (for test confirmation). And then monoclonal antibody anti-human PSA antibody (4.0 mg / ml) was placed on the nitrocellulose membrane as test line (T line) 33 (for analyte binding). Dispensed and placed into. Both antibodies were dispensed at a rate of 1.1 μl / cm. Upon completion, the coated nitrocellulose membrane was immediately dried at 45 ° C. (2 hours) so that the attached antibody was immobilized on the membrane.

4). Reagent Test Strip Assembly A PSA detection reagent test strip was assembled according to the schematic as shown in FIG. The sample receiving pad 35 is arranged to overlap the label holding pad 34, and then the label holding pad 34 overlaps the nitrocellulose membrane 36 on the other end of the label holding pad 34 (the end away from the sample receiving pad). Finally, a nitrocellulose membrane 36 was overlaid with an absorbent filter 31 disposed at the distal end of the test strip, thereby forming a reagent test strip as shown in FIG.

5. Preparation of blood samples Twenty whole blood samples (collected for more than 8 days and stored at 2-8 ° C) were prepared for further use.

6). Test Procedure 40 μl of whole blood sample was dropped onto the sample receiving pad along with 40 μl of whole blood phosphate buffered saline used to facilitate movement of the blood sample. The time when the C line appeared was recorded. The time at which the C line appeared showed the blood sample liquid flow rate. The C line had to appear within 3 minutes of sample introduction, and the sample had to travel through the nitrocellulose membrane 36 to reach the absorbent filter paper 31 within 5 minutes of sample introduction. Otherwise, reagent test strips were considered inappropriate.

  Control treatment CK and treatment 1, respectively, were used to determine the sensitivity and specificity of the product. Positive from PSA negative (confirmed experimental) whole blood sample so that the PSA concentration in the specimen can be manipulated to be 2 ng / ml, 4 ng / ml, 10 ng / ml and 20 ng / ml, respectively Samples were prepared and positive test results were recorded. To determine specificity, 20 standard PSA negative (experimental confirmed) whole blood samples were used as negative specimens in the test.

注：「−」は血液試料が３分以内にＣラインを通過することができなかったことを示す。「＊」は血液試料が５分以内にニトロセルロース膜を通って進むことができなかったことを示す。 7). result

Note: "-" indicates that the blood sample failed to pass the C line within 3 minutes. “*” Indicates that the blood sample could not travel through the nitrocellulose membrane within 5 minutes.

注：「−」は血液試料が３分以内にＣラインを通過することができなかったことを示す。

Note: "-" indicates that the blood sample failed to pass the C line within 3 minutes.

注：ＰＳＡ陽性全血試料が使用された。「Ｇ＋数字」はＴライン／テストラインの色強度を示す（より大きな数字がより強い色を示す）。

Note: PSA positive whole blood samples were used. “G + number” indicates the color intensity of the T line / test line (a larger number indicates a stronger color).

注：２種類の標本がこの実施例において使用された。標準ＰＳＡ陰性（実験で確認された）全血試料がテストされ、結果の１００％が、ＣＫグループでもＴ１グループでも陰性であった（テストラインの色強度はＧ１であった）。臨床的に収集された全血試料（実験でＰＳＡ陰性試料と確認されず、ＰＳＡを含む可能性があった）も使用され、すべての結果が、ＣＫグループでもＴ１グループでもすべて陰性（テストラインの色強度はＧ１であった）であることも明らかになった。

Note: Two types of specimens were used in this example. Standard PSA negative (experimental confirmed) whole blood samples were tested and 100% of the results were negative in both CK and T1 groups (test line color intensity was G1). A clinically collected whole blood sample (which was not confirmed as a PSA negative sample in the experiment and could contain PSA) was also used, and all results were all negative in the CK and T1 groups (test line It was also revealed that the color intensity was G1).

8). Conclusion The above results show that anti-fibrin antibodies can separate red blood cells from blood samples, thereby significantly reducing the probability that clotted blood samples will block the chromatographic support and greatly increase the efficiency of blood separation. Suggest. On the other hand, it is clear that anti-fibrin antibodies have no adverse effect on the detection itself. Furthermore, when anti-fibrin antibodies are used in combination with anti-erythrocyte antibodies, the beneficial effects of anti-fibrin antibodies on blood separation become more apparent.

Example 3: Accelerated Stability Test Processes F and J from Experiment 1 were also used to perform an accelerated stability test to determine the maximum shelf life of the product.

1. Method The products from treatments F and J were exposed at a temperature of 55 ° C. The product subjected to this high temperature treatment was then used to test sensitivity and specificity according to the data schedule listed in Table 3-1 below. Ten samples from each treatment (treatment F and treatment J) were used for each of the tests performed. For each test, 100 μl of whole blood sample was dropped on the sample receiving pad and the time after which the C line appeared was recorded.

注：実施例３では以後、「＋」は結果が陽性であったことを示す。「−」は結果が陰性であったことを示す。「＋」の前の数値は、結果として生じたテストラインの色強度を示す（すなわち、「３＋」の３は、結果として生じたテストラインの色強度がＧ３であったことを示す）。 Results on day 0:

Note: In Example 3, “+” indicates that the result was positive. “-” Indicates that the result was negative. The number before “+” indicates the color intensity of the resulting test line (ie, 3 of “3+” indicates that the color intensity of the resulting test line was G3).

２．結論
上記の結果から、試料受入パッドが抗フィブリン抗体を備えるとき、生成物の有効寿命が２年にもなり得ることは明白である。一方、血液試料がＣラインに到達する時間は低減される。

2. Conclusion From the above results, it is clear that when the sample receiving pad is equipped with anti-fibrin antibody, the useful life of the product can be as long as 2 years. On the other hand, the time for the blood sample to reach the C line is reduced.

Claims (8)

separating the red blood cells from the blood sample comprising: a) contacting the blood sample with a receptor capable of binding to fibrin in the blood sample; and b) subsequently separating the fibrin-binding receptor from the blood sample. A method,
Contacting the blood sample with a receptor capable of binding to red blood cells in the blood sample.   The method of claim 1, wherein the fibrin binding receptor and the red blood cell binding receptor are immobilized on a support that supports fluid flowing through the support.   3. The method of claim 1 or 2, wherein the receptor comprises a) a monoclonal antibody, or b) a polyclonal antibody, or c) a) or b) fragment.   A method for separating red blood cells from a blood sample, comprising: providing a chromatographic support comprising a receptor capable of binding fibrin in the blood sample; and allowing flow of the blood sample through the chromatographic support. The chromatographic support further comprises a receptor capable of binding to red blood cells in a blood sample.   5. The method of claim 4, wherein the receptor comprises a) a monoclonal antibody, or b) a polyclonal antibody, or c) a) or b) fragment.   An apparatus for separating red blood cells from a blood sample comprising a support comprising a receptor capable of binding to fibrin in the blood sample, wherein the support is capable of binding to red blood cells in the blood sample. The apparatus further comprising:   The apparatus of claim 6, wherein the support is a chromatography support.   7. The device of claim 6, wherein the receptor comprises a) a monoclonal antibody, or b) a polyclonal antibody, or c) a) or b) fragment.

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