TW402688B - Method and apparatus for single step assays of whole blood - Google Patents

Abstract

This invention relates to the apparature which is used in the liquid testing sample wherein the sample contains or is predicted to contain the cell and cell composition. The apparature of this invention comprises the hydrophilic cell catcher, which is used to separate the cell portion from the single cell portion of the test sample. The latter-mentioned portion passes through the porous, absorptive or intake film of this device. The said film is filled with the reagent at least in three separate regions (including the dye region, the test region and the contrast region). In the control section, identifible eyeseeing pattern shows that whether the test is proceeding properly; and in the test section eyeseeing pattern with relative strength, shows whether there is desired substance. The apparatus of this invention is especially suitable for analyzing the ligand in the whole blood without separating the red blood cell and other cells from the blood and without measuring the reflective ratio of the liquid phase. This invention also discloses the method of the above-mentioned invention analysis process.

Description

a7 _B7_ V. Description of the invention (1) The scope of the invention (2 Read the notes on the back and then fill out this page) The present invention relates to a method and device for analyzing biological liquid samples. In particular, the present invention relates to the detection of whole blood samples. Analyte method and device, the sample does not need to separate the cell part from the sample before analysis. BACKGROUND OF THE INVENTION The development of reagent-filled capillary membranes for the analysis of a variety of body fluids has long led to the production of test devices that are simple to use and produce results quickly (i.e., 10 minutes or less). Perhaps the most common application of this type of device is to measure human chorionic gonadotropin, which is an indicator of human pregnancy. Examples of such devices are described in commonly cited US Patent No. 5,384,264, EPO Publication No. 056041 1A2, PCT Application No. PCT / GB / 00322, and US Patent No. 4,366,241. • The simplest version of this type of device allows the analysis to be performed and interprets the results in a single step; for example, placing a liquid sample (such as urine) on an absorbent membrane, and any desired analyte is matched with the corresponding Combination (printed by a consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and the consumer cooperative), and the results (that is, the formation of a specific complex) are evident in the test area separated from the sample placement area. Although these devices work well for analyzing liquid samples. These liquid samples traditionally do not contain large cells, so they can quickly pass through the membrane filled with reagents by capillary action. However, these devices have not been suitable for detecting liquids containing large cells ( For example, in whole blood) • In other words, the analysis of analytes present in whole blood is traditionally performed using substantially cell-free blood. The paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297). (Mm) B7 V. Description of the invention (2) Plasma or serum part is performed, so this kind of analysis needs to remove red blood cells and other cells from the sample before performing the analysis method. (Please read the precautions on the reverse side before filling out this page) The device described in US Patent No. 5,304,4,68 avoids the analysis of analytes (especially glucose) on the surface. Need for whole blood separation. The device, a test strip, includes a control cell introduction site and a test surface, the latter being filled with reagents. Positive or negative results are shown by changes in the refractive index on the test surface, which are determined visually. According to this disclosure, the change in the measured refractive index is not affected by the presence of red blood cells in the test sample, so a whole blood sample can be applied to the surface where the sample is introduced. However, because the result is a measurement of the change in refractive index, an optical measurement device is required to perform the analysis method using the disclosed device * Brief description of the invention Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economics The present invention is made by a single step analysis method The device and method are configured to detect the analyte in a liquid sample containing cells, but it is not necessary to separate the cell-free liquid portion from the sample cells before performing the analysis. Furthermore, the present invention does not require the use of a water-repellent film to block the cellular components in the sample or a subsequent washing step, thereby removing the blocked components from the analysis device. In addition, the results of the analysis according to the present invention can be visually interpreted without the need for a separate measuring device and independent of the refractive index of the test sample. Therefore, performing the analysis method according to the present invention only requires the user to introduce the required amount of a test sample (preferably whole blood) into the device of the present invention, and then observe any subsequent color changes in the test area of the device, etc., which are then quickly visible. So far, the device of the present invention includes a cell trap at one end of the inlet. The paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) A7 _B7_____ V. 晷 _ ^ 船 3) It is used to test Sample introduction hydrophilic sample introduction membrane. The cell trap is preferably a multi-layered hydrophilic net having holes of a smaller effective diameter than the expected diameter of the cells in the analyte sample. The hydrophilic net is placed in liquid communication with the sample introduction membrane. Therefore, only the liquid portion of the test sample will enter the sample introduction membrane through the cell trap during use. Sequentially, the liquid entering the sample introduction membrane will enter the dye-filled dye zone through this introduction membrane, and then enter the test zone and control. Area. In the best implementation system of the device of the present invention, a visually detectable special dye (for example, a sol composed of an inorganic compound containing gold tincture) * is used to obtain the positive or negative test results. The device of the invention may be in any suitable shape or form, including test strips and test cartridges containing the various components of the device. According to the method of the present invention, a small amount of a test sample (preferably whole blood) is pipetted or added dropwise, or is added to the cell trap in another way (for example, by directly contacting a blood-punching finger). Any analyte present in the sample (not bound to the membrane with the cell component being separated) that binds to one or more specific ligands will form a specific visible pattern that shows the test result. The present invention can be used to test any analyte present in a liquid sample (such as soluble antigens in whole blood), of which at least one specific ligand (ie, binding partner) is known (such as multiple or monoclonal antibodies). The present invention is particularly useful for detecting single epitope or multiple epitope antigens and antibodies, and the antigen and antibody system are related to infectious or non-infectious pathological and physiological compounds and drugs. Although the device of the present invention is particularly suitable for analysis, the paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) | Read the precautions on the back and fill in this page)

-IIII J- II ^ · 1! 111111 I Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 A7 Printed by the Shell ’s Consumers’ Cooperative of the Intellectual Property Bureau of the Ministry of Economy ___B7___ V. 4) Whole blood samples, which can also be used for analysis Other liquid samples, especially cells of similar size or other interfering particles, are known to be present. Detailed description of the present invention 1. Definition of terms In order to facilitate understanding, the following definitions apply to the entire text of this specification: a) An analyte contains a molecule or compound with one or more binding positions (such as epitopes), the binding position is another Where a molecule or compound will bind to it b) A ligand or binding partner can bind to a specific binding site of the analyte to form a molecule or compound that binds to the target. Any desired analyte / ligand binding pair can be used in the present invention. Structurally, ligands can include proteins, peptides, carbohydrates, polysaccharides, nucleic acids, oligonucleotides, haptens and combinations thereof. Functionally, ligands can include (but are not limited to) antibodies, Antigens (eg, microbial antigens, prostate-specific antigens), hormones (eg, thyroid-stimulating hormones), drugs, enzymes, allergens, and fragments, and combinations thereof with modified ones. Those skilled in the art are familiar with or can easily identify such ligands and their corresponding binding partners. c) The test sample is suspected of containing a liquid that is intended to be an analyte, and a specific analysis method will be performed for this liquid. d) The object of this paper shall be in accordance with China National Standard (CNS) A4 (210 X 297 mm) (Please first Read the notes on the back and fill in this page) Order --------- line — 7 Printed by the Shellfish Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs A7 402 & SS --- 5. Description of invention (5) Direct or Molecules or compounds that indirectly contribute to signal formation (such as a color change) * are used in analytical methods to indicate the presence or absence of a desired analyte in a test sample, or its concentration range. Labels can include enzymes, luciferin, Lipid microparticles, red blood cell ghost cells, polymer microcapsules, color-developing polymer particles (tree milk), and preferably include a sol containing gold tincture compounds. E) Gold tincture markers Gold tincture-containing sol markers; that is, gold tinctures Or gold rhenium compounds, such as gold rhenium oxide, gold rhenium hydroxide, metal salts mixed with or coated on the polymer core, gold rhenium or metal-containing compounds. These gold tincture markers may include the anhydrous form of the gold tincture or the gold tin compound sol, and preferably include the colloidal gold in the anhydrous form. f) Complexes Complex # shall mean any multi-molecular complex formed by the analyte and one or more ligands, labeled ligands, or immobilized ligands, depending on the content used. In the sandwich immunoassay, for example, the following complexes are formed: the analyte / labeled ligand duplex (* first complex ^) first produced in the assay, and the second one formed in the assay Analyte / labeled ligand / cured ligand triplet (^ Second complex "). G) Cell components represent cell membranes and intracellular structures, such as mitochondria and nucleus. h) Non-cellular fraction means the liquid phase of the test sample, including any analytes present in the sample, and cellular components not captured by the cell trap of the device of the invention. The size of this paper is applicable to China National Standard (CNS) A4 (210 X 297 mm) --------: ---! Ί [---------- order ----- ---- Line (Please read the note on the back of 1M and fill in this page again) 8 A7 A7 B7 V. Description of the invention (6) 2. Preferred implementation system of the device of the present invention Although the device of the present invention can adopt any shape or structure, It includes the material elements of the present invention. In terms of function, it is suggested that the preferred structure of the device is a test strip and a test box with the internal components of the device of the present invention. Therefore, the device of the present invention is described in this specification as a test strip and cassette type device, and it is understood that the present invention is not limited by these special configurations. The test strip constructed according to the present invention is shown in FIG. 1. On the left-hand side of the figure, the cell trap 1 is shown as a layered net, which covers the sample introduction membrane 3. The application of the test sample to the cell trap is achieved through the window 4 in the cover 5. The cover 5 is a thin layer or film, which is an hydrophobic material that covers the entire upper surface of the test strip by extension or covers part of the test strip. Composition • Cover 5 is incorporated into window 4. Although the cover 5 can be omitted from the test strip, to prevent the cell trap 1 from being contaminated and to prevent the cell trap 1 from being separated from the sample introduction membrane 3, the cover 5 helps to correctly add the sample through the window 4. Therefore, the test strip of the present invention including the cover 5 is preferable. Opposed to the cover 5 is a thin and hydrophobic substrate 6. The substrate 6 is a fixed support for all the functional components of the test strip described above, and a barrier that prevents liquid from flowing out of the test strip through the bottom surface of the device. Substances suitable for use as the cover 5 and the substrate 6 include cellulose derivatives, polyethylene and polypropylene compounds, and other solid polymers, which can be used to form the cover 5 and the substrate 6, or as coating agents. When the cover 5 and the substrate 6 are made of different materials (for example, paper). The preferred placement of the cover 5 and the substrate 6 on the test strip is shown in FIG. 2. The structure of the cell trap 1 is further shown in FIG. 3. As shown in Figure 3, this paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) < Please read the notes on the back before filling this page) ----- IT --- Order! I · Line · Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 9 A7 A7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. The description of the invention (7) shows that the cell trap is composed of multiple layers of fiber (from fiber 6 a And 6b), these 繊 dimensions are arranged in overlapping crosses to form a network (the lower layers of the overlapping dimensions are shown in the internal structure). The holes formed by overlapping dimensions must not be larger than, preferably smaller than in the test sample. Estimated diameter of red blood cells or other cells. In order to facilitate filtration, the hole size of the upper layer of the suspected layer of the cell trap may be different from that of the lower layer of the suspected layer. In the present invention, the smallest hole size of some layers in the cell trap is expressed as the effective hole size of the cell trap. Taking human blood cells as an example, the average diameter is 7.7 microns and the average thickness is about 2 microns. For ease of understanding, in the present specification, the cells to be separated in the cell trap means red blood cells, but it is clear that the present invention is not limited to the use of a test sample containing red blood cells. As shown in Figure 3 (not shown), the effective size of any hole in the cell trap (represented by hole 7) is slightly smaller than the size of the captured cell (represented by cell 10 in Figure 3). The weaving dimension that forms the boundary of each hole is densely woven so as not to deform when receiving the test sample; in other words, the size of the hole will not be enlarged. For effective cell separation, the effective pore size of the general cell trap 1 is preferably at least 10% smaller than the expected diameter of the cells to be captured. Smaller holes can also be used to enhance the ability of the device of the invention to capture cellular components and intact cells. The size of the pores should never be so small that the non-cellular liquid portion of the test sample is blocked from passing so that it cannot flow through the pores to the sample introduction membrane. Those skilled in the art will understand that if the red blood cells or other cells present in the test sample are not the expected size, they will pass through the holes of the cell trap 1 when they are smaller than the expected size. Therefore, in order to improve the size of the cell capture paper, the Chinese National Standard (CNS) A4 specification (210 X 297 public love) is applicable. --------; --- r ί4 ----- 1 —Order- ------- Line · (Please read the precautions on the back before filling out this page) -10 _ Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 --- 、 Description of Invention (8) Capture of Catcher 1 For cell potency, at least 2 layers, preferably more than 3 layers of overlapping fibers will form a cell trap in a layered manner in a horizontal arrangement. Furthermore, although the neatly overlapped fibers are shown in Figure 3, those skilled in the art—the more casually placed fibers can also be used in cell traps. 1 * In this configuration, not all holes have a uniform size. . Therefore, it is possible for red blood cells in the analyte sample applied to the cell trap 1 to pass through one or more overlapping fibers. Each layer of overlapping fibers in the cell trap should be placed laterally so that cells that are not captured by the surface layer can be captured deeper in the cell trap. Therefore, it is preferred to include at least three layers of overlapping fibers in the cell trap. In order for the non-cell liquid portion of the test sample to quickly penetrate the cell trap and penetrate into the sample introduction membrane, the cell trap preferably has a total thickness of 0.1 to 3 microns. The surface of the sample addition area of the cell trap (that is, the surface on which the test sample is applied through Figure 1, window 4) will be changed according to the test sample volume and application method. When testing most whole blood samples according to the present invention, It is appropriate to expect a surface area of about 3 mm 2 to 25 mm 2. The fiber of a cell trap does not have to have a specific electricity price or a unique thickness to capture red blood cells. However, in order to assist the non-cellular portion of the test sample to pass through the cell trap to the sample introduction membrane, the cell trap is preferably somewhat hydrophobic, so that the liquid is not repelled and cannot penetrate the holes of the cell trap. Any fibrous material with the above characteristics can be used as a cell trap. Pre-formed mats or strips with multiple layers of nylon or cellulose membrane material (such as nitrocellulose) are suitable. Returning to Figure 1, the sample introduction membrane 3 is under the cell trap 1 and ------------ Ί ^ --------- Order -------- -Lines (please read the notes on the back before filling this page) This paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) -11-A7 ___B7___ V. Invention age 9) Liquid communication. In this manual, liquid communication ^ • means that they are connected to each other. Preferably, the sample introduction membrane is more hydrophobic than the cell trap to facilitate liquid capture by the cell. The material used to form the cell trap is used as the other layer under the cell trap. The membrane 3 is completely located in the cell trap 1. The entire structure is explained. This technical product introduction membrane can extend in the direction of 20 below the cell trap. Similarly, the size of the sample trap is slightly different. In either trap, they should communicate with each other by liquid. The Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs printed the contact, but the structure that does not need to be fixed has more holes, and similarly, the army has moved in the direction. In other words, a cell-introducing membrane can also be formed, and its surface is shown in the first circle below the sample guide. However, once a person of ordinary skill in the field of test strips of Fig. 1 will perceive the sample, the size of the membrane introduced into the membrane with the flow of reagents can be in the same size as the cell capture configuration. The film can be formed using any absorbent material. This type includes tightly woven paper and nitrocellulose, such as polypropylene and acrylic with high molecular weight. It is enough to absorb blood in a few seconds. The next step is explained below. It is also used when the analysis method is performed. When a small amount of analyte passes through the zone 13 of the test strip, it is fictitiously shown in Figure 1. The cell trap captures some devices within the cell. Liquid push from the test sample. (Please read the notes on the back before filling this page) Another example of more porous, absorbent or hydrophilic materials for cell traps is known in the art, vitamins, glass fiber and porous plastic, Acrylonitrile * is a liquid group of about 20 to 40 microliters in the porosity of the selected material. In the method of the test strip of the present invention, the buffer solution is introduced to the proximal end of the membrane than the sample to assist the advancement of various membranes. Thus, the buffer can be a dye-filled membrane and / or an area extending proximally of the organ. According to the present invention, the liquid flow of all the liquids is only for this purpose. In order to make the required test samples, the paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -12-A7 B7 (10). It is better to minimize the size and increase the rate of analysis, providing an implementation using buffers. The sample introduction membrane is in liquid communication with a membrane 15 filled with a dye, which is a ligand labeled with a marker. More specifically, one or more labeled ligands (such as antigens or antibodies) are bound to a porous, absorbing form by using soluble aminosilanes or other suitable bonding means known to those skilled in the art. Sexual or absorbent film. The preferred film material is glass fiber. For example, Lyda 1 1 is a product marketed under the brand name of MANN IWEB or MANN I GLAS #. Other suitable materials include polyethylene or nitrobenzidine pads and test strips; methods for binding ligands to these materials are well known in the art. Alternatively, the dye-filled membrane may be part of the sample introduction membrane. However, in order to minimize the backwashing of the labeled ligand to the sample introduction membrane, it is preferable that the membrane filled with dye is a separate structure, and it is best to place a woven fabric with a dimensional texture (such as the glass fiber described above). Place the reagent-filled membrane in the same direction as described below. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The ligands labeled with the marker can be prepared according to methods known in the art. For a clearly visible response, the preferred markers are those containing gold sol, and the ones with colloidal gold or selenium are the best. Examples of suitable products are colloidal gold from Janssen Life Science Products. These colloidal metals produce a visual pattern that can be distinguished without the addition of other reagents; however, fluoresceins (such as luciferin) and enzymes (such as U.S. Patent No. 4,275,149 appraiser, which (Incorporated in this manual). In order to make the test sample and the labeled paper conform to the Chinese National Standard (CNS) A4 (210 X 297 mm) -13-5: ί »Α7 Β7 11 the most labeled contact, the latter The occupied area (dye area) should span the membrane surface, that is, the dye area should extend from one end of the film to the other. The first immobilized ligand is a ligand immobilized in a reagent-filled membrane 20 (immediately next to a dye-filled membrane) in the test zone 21. It will be the location of the sample test area. The reagent-filled membrane 20 is preferably a gelatin-coated porous test strip. Gelatin helps extend the life of the test strip and increases the clarity of any visible reactions that occur during the test. The first immobilized ligand can be immovably bonded to the reagent-filled membrane 20 by methods known in the art, including covalent linkage or attachment to an insoluble protein-coated surface (see U.S. Patent No. 4 No. 200, 690, etc., the disclosure of this case is also incorporated into this article. The Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs printed better. The area occupied by the first fixed ligand has a rod shape or a round shape. One end of the membrane 20 filled with reagents in zone 21 extends to the other end. The simple and one-way configuration, such as a rod, prevents the user from deciding more complex shapes (such as 'ten' or ~-whether it is sufficient It is sufficient to express a specific result. Furthermore, the simple shape overcomes the squeezing of the fine boundary effect and makes the visual interpretation of the reaction result easier for the user. The far end of the membrane filled with dye 15 The second immobilized ligand is located in the control region 22 of the membrane filled with the reagent. The second immobilized ligand should have a specific affinity for at least one ligand labeled with the label. The curing reaction of the second immobilized ligand can be used The same method for bonding the first immobilized ligand is described. For comparison, the shape and orientation of the control area 22 should be similar to the shape and orientation of the test area. Those skilled in the art can know that on the membrane filled with reagent < 2 Read the notes on the back and fill out this page)

This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm). -14-Printed by the Sheller Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7-Hide 2 Dian 8-Bar-V. Description of Invention (12) The positions of the test area 21 and the control area 22 can be interchanged, so that the former is at the distal end of the dye-filled membrane 15 and the latter is at the proximal end. The absorption pad 23 is in liquid communication with the reagent-filled membrane, and serves as a storage place for excess liquid, and a pump that provides a unidirectional flow of liquid along the reagent-filled membrane 20. For the purposes described below, the absorbent pad is preferably at its distal end and away from the control area, overlapping the membrane filled with the reagent. Those skilled in the art will appreciate that some implementation systems of the present invention may not include an absorbent pad. The function of the absorbent pad can be accomplished, for example, by the extended distal end of a reagent-filled membrane 20. However, for best performance, the application system incorporated into the absorbent pad is better * Instructions for use of the test strip can be printed on the cover or on the test strip packaging, and / or on the instructions packaged with the test strip on. A better test strip is part of a set. This set can consist of test strips, instructions for use, dry packing bags, capillary devices for measuring test samples, buffers, straws for measuring buffers, and reactors. The container is, for example, It is a cup. After the analyte sample is applied to the test sample, the cup contains a buffer and a buffer introduction area for the test strip. Components of this type for analysis (for example, excluding printed instructions) are preferably sealed in one or more air-tight packages * such as aluminum boxes. 3. Another implementation system: test cassette device. Alternatively, all the components of the test strip of the present invention (except the cover 5 and the substrate 6) can be packed in a protective sheath made of a solid plastic. The cover is composed of 2 5 and it is tightly fixed on the solid plastic base 2 6 shown in Fig. 4 • Add the test sample to the inlet above the cell trap 2 7 This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) (Please read the note on the back ^ before filling this page) Order --------- line. -15-A7 —— ^ 〇2 & ββ-- 纪-V. Invention Note (13) runs through the cover 25. In a preferred implementation system, the application of a buffer solution to the inlet 28 of the device also penetrates the cover 25. However, in another implementation system, the test sample and the buffer are added through the same inlet, which is also within the scope of the present invention. The display port 29 also penetrates the cover 25, which is used to view the film 20 filled with the reagent. A particularly good test case design for use in the present invention is disclosed in commonly cited U.S. Patent No. 5,384,264, the disclosure of which is incorporated herein by reference. In short, the test cartridge device assembled according to Patent No. 5,384,264 is shown in the expanded view of FIG. 5. In this figure, it can be seen that the base 26 is divided into two different regions. The first area is a low-lying area 30, which is bounded by a bottom surface 31, a side wall 32, and an inclined surface 33. In a preferred implementation system, a vertical rod (not shown) extends downward from the cover 25 to the low-lying area 30, just next to the inclined surface 3 3, so that the dye-filled film 15 can be positioned along the inclined surface 3 3. The second area of the base 26 starts from the top of the bevel 33, and consists of an extended surface forming a platform 36, which is parallel to the low-lying area 30 and extends outward from the area. The liquid ditch 40 is preferably included at the proximal end of the platform 36, which serves as another reservoir for the excess liquid. The platform 36 can extend the length of the substrate 26 from the inclined surface 36, which can be terminated slightly shorter than the distal end of the substrate 26, thus leaving a space for the second liquid groove 41 to collect the superfluous liquid. The reagent-filled membrane is placed along most of the length of the platform 36 (below the display port 29 in the opposite position), and the absorption pad 23 is located at the distal end of the platform 36, which can be extended to cover Liquid ditch 4 1. The dried ingot can be placed in the liquid groove 41. Desiccant can provide low-humidity conditions The paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) Order --------- line · Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description (14), which is necessary for the storage of reagents during the exhibition period of the device. To change the package, dry tablets or dry packaging can be included in the airtight protective bag with the device. 4. The test sample using the device of the present invention can represent any body fluid, including blood, urine, lymph fluid, ascites, crude Tissue extract or homogenate, these body fluids are derived from fetuses, newborns, adolescents or adults, but preferably microvascular or venous whole blood. For most applications, about 5 microliters to 50 microliters of test sample That is, it is sufficient for the present invention, preferably 20 microliters to 30 microliters. The method of the present invention is performed by adding a test sample to a cell trap of a device (test strip or cassette) constructed according to the present invention. To determine a test sample that contains (or is suspected of containing) cellular components, allow sufficient time for the cells to separate from the non-cellular fraction, typically about 60 to 120 seconds. After the above steps are completed, add buffer through the buffer zone of the device or its inlet. The volume added by buffers printed by employees of the Intellectual Property Bureau of the Ministry of Economic Affairs must be limited to avoid washing the test samples. Generally, less than 5 drops (preferably 3 large drops) of buffer is sufficient to push the test sample along the membrane of the test device. Suitable buffers include any pharmaceutically acceptable buffered aqueous solution that does not react with the test sample or its components. Those with ordinary skills in this skill are equally familiar with such buffers, including physiological saline, Ringer's solution, etc., and can be easily determined. Buffer can be added dropwise to the buffer inlet (for a cassette-type device), or the required body buffer is placed in a reaction container (cup), and the buffer zone of the test strip is immersed (for the test of the present invention). For the hip system). The paper size of the test sample applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling out this page) -17-17 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 — 働68S --- 5. Description of the invention (15) Both the product and the buffer solution should be added to the device at room temperature. After adding the test sample and buffer solution, the results can be interpreted in about 10 minutes. • No buffer is required. In the liquid implementation system, the results are slower, which depends on the flow rate of the non-cellular part of the test sample through the membrane of the test device. Without buffer, the results were interpreted at about the same time as a band pattern appeared in its control area. If no band pattern appears, or the control band pattern cannot be distinguished or is not completely formed, the analysis should be regarded as not indicating the presence or absence of analytes in the test sample, and therefore analysis should be performed. · More detailed description, sandwich analysis In the method, the intended analyte in the test sample, if present, will bind to the labeled ligand in the dye region to form a first complex. The first complex and the unbound melancholic ligand will be mixed with the test sample and will be capillarized by the core effect #) Through the membrane filled with dye (dye zone), it will be carried to the device filled with reagent membrane * The sample is carried The first complex (if present) is contacted with the unmarked ligand immobilized on the reagent-filled membrane through the reagent-filled membrane, and the ligand system is used to bind the first complex to form the recorded ligand. Analysis Once the object has passed through the second complex of the immobilized ligand. If a second complex is formed, a visually visible colored pattern will appear in the test area. Labeled ligands that are not bound to the analyte in the test sample will continue to swim to the control area by contact with the immobilized ligand. The scripture will be combined with the fixed ligand in the control area to form a third complex, which will be captured in the control area. Within the scope of the present invention, the labeled ligand forming the complex in the control region may be the same as the labeled ligand forming the first and second complexes, or it may be another medicinal ligand. Matching fixed in the control area This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)

-18-Printed A7 by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs — it seems like 6 — — V. Description of the invention (16) The body should have a specific affinity, in order to combine with the intention of forming a third complex body. The formation of the third complex is shown by a visual pattern in the control area. In addition to the sandwich immunoassay, other assays can also apply the device of the present invention. These methods can include competition analysis and inhibition analysis. In competition analysis, the analyte and the labeled ligand have similar affinity characteristics. Therefore, it competes for binding with the fixed ligand. In this way, patterns (such as bands) in the test area will have the highest intensity without the analyte. If an analyte is present, it binds to the immobilized ligand, thus preventing the labeled ligand from being captured in the test zone. For this reason, the strength of the test strip pattern decreases with the degree of analyte in the test sample. In the inhibition assay, the analyte and the immobilized ligand in the test zone have an affinity for the labeled ligand. Without the analyte, the imprinted ligand is captured by the immobilized ligand and forms a visual pattern in the test area. If there is an analyte, it will bind to the labeled ligand, thus preventing the analyte from binding to the fixed ligand in the test zone. The strength of the generated test strip pattern decreases with the concentration of the analyte in the test sample. All analytes within the scope of the invention can be designed to interpret the test results in one of two modes: visual inspection and identification. Mode and comparison mode. In the visual identification mode, the positive or negative result (with or without the analyte at a specific concentration) is determined by the specific pattern or color in the test area. The pattern in the control area is only used as an internal control of the device function. In comparison mode, the 'positive or negative' result is the visual comparison of the pattern strength formed by the test area and the control area (please read the precautions on the back before filling this page)

This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 gong) -19-Consumption Cooperation by Employees of the Intellectual Property Bureau of the Ministry of Economic Affairs Du printed A7 --- 5. Description of the invention (17) obtained in the situation described below In the comparison, the pattern of the comparison area serves as an internal quality control, and also serves as a reference standard for judging the results of the comparison mode. Examples of the results of the visual inspection mode are shown in Figures 6 and 7. As shown in Figure 6, the positive result was identified by the formation of a similar parallel rod-like pattern of test area 21 and control ® 2 2. In contrast, as shown in Fig. 7, the negative result is recognized by a distinguishable parallel bar pattern that appears only in the control area 22. Other results signals for comparison or comparison can be provided, including signals indicating whether an invalid result was obtained, which is achieved by similar methods known to those skilled in the art (see, European Patent Application No. 8611367. 0 [publication number 0217 403 92) The signal system described above, etc.) β The present invention can be applied to devices and procedures for the determination of a wide variety of analytes. For example, the types of analytes can be mentioned as follows: proteins and protein derivatives, including antibodies, Immunoglobulins, stimulus, enzymes, peptides; infectious pathogens, including bacteria, viruses, fungi, mold, parasites and their products and components; drugs, including therapeutic drugs and drugs; cancer flags. Specific examples are anti-ΗIV antibodies, antibodies against Helicobacter pylori (Η. Py 1 Ο Γ i), antibodies against hepatitis C virus, human chorionic gonadotropin, estradiol, thymus stimulation, Prostate-specific antigen, hepatitis B surface antigen, myoglobin, immunoglobulin E. An example of explaining the present invention is shown below. This embodiment should not be used to limit the scope of the present invention. The scope of the present invention is limited by the scope of the attached patent application. • Standard abbreviations (such as hour abbreviations) and measurement units (examples- ---------- ,! {^ --------- Order --------- line · (Please read the precautions on the back before filling this page) This paper The scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 g t) -20-A7 ___ Β7 ____ V. Description of the invention (18) (in milliliter) is used in the examples. (Please read first? ^ Notes before filling in this page)

Example I Determination of Antibacterial Bacteria in Human Blood Test samples of microvascular whole blood were obtained from adults using the conventional sampling technique of punching with a pointer. Approximately 20 microliters of blood was brought into direct contact with the surface of the cell trap of the test strip constructed according to the present invention, which contained a multi-epitope microbial antigen reagent (a reagent for Helicobacter pylori), which was All isotype reactions of human antibodies · After 90 seconds * Add three large drops of buffer (physiological saline) to the buffer zone of the test strip by immersing the test strip in a cup containing three drops of buffer • The test sample and buffer solution added to the test strip are at a temperature of about 15-3 Ot when added. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs After adding the buffer solution, let the test strips stand in a buffer cup at room temperature for 10 minutes. See two colored round cases in the test area and control area of the test strip, indicating that it is a competent test (ascertained by the appearance of the control tape in the control area) and that it is resistant to pylorus in the test sample The Spirulina antibody showed a positive reaction (identified by the colored band pattern in the test area). Then according to the conventional biological waste treatment technology, discard the test strip and all unused test samples.

Example II The test sample for the determination of venous whole blood of the whole prostate TJH is obtained from an adult 'borrowed a conventional blood sample. The paper size is applicable to the Chinese National Standard (CNS) A4 (210 X 297 mm) B7. 2. Description of the invention (19) (please fill in this page before filling in this page) Set technology, namely intravenous catheter. About 20 microliters of a test sample was drawn and placed on a cell trap of a container device constructed according to the present invention, which contained a monoclonal antibody reagent specific for a human prostate-specific antigen. After 90 seconds, three large drops of buffer solution (physiological saline) are added dropwise to the cell trap via a pipette. The test sample and buffer solution added to the test strip are at a temperature of about 15-30 ° C when added. . After the buffer was added, the device was allowed to stand on a flat horizontal surface and left at room temperature for 10 minutes. Two colored ribbon patterns appear in the test and control areas of the test strip, indicating that they are competent tests (identified by the appearance of the control ribbon pattern in the control area) and that they respond positively to the human prostate-specific antigen in the test sample (Identified by the colored band pattern in the test area). The device and all unused test samples are then discarded according to conventional biological waste treatment techniques. The present invention has fully explained that the improvement of the above implementation system is obvious to those skilled in the art. Such improvements are considered part of the present invention and are included in the scope of the attached patent application. Brief description of the plaque printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Figure 1 is an expanded view of the test strip of the present invention. Figure 2 is a perspective view of the test strip of the present invention. An enlarged disassembly view of 3, which is viewed through a plane containing the cell trap 1. Fig. 4 is a perspective view of a cassette device of the present invention. Fig. 5 is a development view of a cassette device of the present invention.

This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) II 4G2688 5. Description of the invention (2Q) Figure 6 is a perspective view of the test strip (without cover) of the present invention, which shows the test area The visual result indicates the presence of the analyte in the sample. Fig. 7 is a perspective view of a test strip (without cover) of the present invention, which shows: in the test area: a visual result of a species, indicating the presence of an analyte in the sample. ------ Ίίν ------ I Order --------- Line- (Please read the note on the back before filling out this page) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) -23-

Claims (1)

402666 —— 、 Applicable patent scope A8 B8 mM date month and month. 5 · 19 supplement (please read the precautions on the back before filling this page) 1. A device for analyzing blood test samples, the sample contains cells The cell component and the desired analyte that it is expected to contain. The device includes: (1) a cell trap with a total thickness between 0.1 mm and 3-0 mm, with sufficient porosity and capacity to accommodate the desired The blood sample to be tested is composed of at least three layers of overlapping fibers, and the effective pores formed by the overlapping weaves are at least 10% smaller than the minimum expected diameter of any cell in the test sample; (2) the sample is introduced into a membrane, which is made of porous It is composed of absorbent material, which is located under the cell trap and is in liquid communication with the cell trap; (3) A membrane filled with dye, located at the catching end of the cell trap, is composed of a porous absorbent material and presents with the sample introduction membrane In liquid communication, the membrane contains a dye, which is a ligand labeled with a label; (4) A reagent-filled membrane, which is composed of a porous absorbent material, is located far away from the dye-filled membrane. It is in liquid communication with the dye-filled membrane, and has a control area and at least one test area. The control area and the test area each contain a reagent with a set pattern. This reagent is a fixed ligand; the wisdom of the Ministry of Economic Affairs The property bureau's industrial consumer cooperative prints at least one labeled ligand, which is located in a dye-filled membrane, which can bind to the analyte or compete with the analyte to bind to the fixed ligand in the test area; Fixed ligands in the test zone that can bind analytes or imprinted ligands; and fixed ligands in the control zone that can bind at least one labeled ligand, which is not used on this paper scale China National Standards (CNS) A4 specification (210X297mm) -24-402666——, patent application scope A8 B8 mM year and month assistance. 5. 19 supplement (please read the precautions on the back before filling this page) 1. A device for analyzing a blood test sample, the sample containing cells, cell components, and a desired analyte that is expected to be contained, the device includes: (1) a cell trap with a total thickness Between 0.1 mm and 3-0 mm, with sufficient porosity and capacity to contain the blood sample to be tested, and composed of at least three layers of overlapping fibers, the effective pores formed by the overlapping weaves are smaller than the test The minimum expected diameter of any cell in the sample is at least 10%; (2) the sample introduction membrane is composed of a porous absorbent material, which is located below the cell trap and is in liquid communication with the cell trap; (3) a membrane filled with dye Located at the catching end of the cell trap, it is composed of porous absorbent material and is in liquid communication with the sample introduction membrane. The membrane contains a dye, which is a ligand labeled with a marker; (4) a membrane filled with reagents, It is composed of porous absorbent material, which is located at the distal end of the dye-filled membrane and is in liquid communication with the dye-filled membrane. It has a control area and at least one test area, and the control area and the test area contain distributions, respectively. A reagent with a set pattern. This reagent is a fixed ligand; at least one labeled ligand is printed by the Industrial and Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, It is located in a dye-filled membrane, and the ligand can bind to the analyte or compete with the analyte for binding to the fixed ligand in the test zone; the fixed ligand in the test zone can bind to the analyte or be labeled Ligands; and fixed ligands in the control area, capable of binding at least one labeled ligand, this paper uses China National Standard (CNS) A4 size (210X297 mm) -24-A8 B8 C8 -403688 --- 6. Scope of patent application In which the sample is introduced into the membrane, the dye-filled membrane and the reagent-filled membrane are located on the anaerobic matrix. (Please read the precautions on the back before filling this page) 2. For the device in the scope of patent application, the effective hole size between the weaving dimension and the weaving dimension of at least one layer should not exceed 8 microns. 3. If applying The device in the scope of the patent item 1, wherein the effective hole size between the suspected dimension and the suspected dimension in at least one layer does not exceed 2 micrometers. 4. For the device in the scope of the patent application, the cell trap is located on the sample introduction membrane Above. 5. The device according to item 1 of the patent application, wherein the sample introduction membrane has an effective hole size equal to or larger than the cell trap * 6. The device according to item 1 of the patent application, wherein the cell trap is made of a hydrophilic material selected from the following Sexual material composition, including vitamin derivatives, nylon, glass dimensional and their combinations. 7. For the device under the scope of patent application, the sample introduction membrane is composed of a hydrophilic material selected from the following, including anthocyanin derivatives, nylon, fiberglass and combinations thereof. • Employee Cooperative of Intellectual Property Bureau, Ministry of Economic Affairs Printed 8. The device according to item 1 of the scope of patent application, further comprising an anaerobic cover covering the cell trap and membrane of the device, wherein the cover has at least one window penetrating the cover and is located in the cell trap. Top · 9. For the device in the scope of patent application, the substrate is a test strip. 10. The device according to item 8 of the patent application, wherein the matrix and the cover form a protective sheath comprising a cell trap and a membrane of the device. 1 1. If the device in the scope of patent application No. 10, it further contains the paper size, using China's national rate (CNS > A4 Xiege (210XM7 mm), printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A8 B8 C8 6. The scope of the patent application is that the absorbent material is in liquid communication with the membrane filled with reagents ^ 1 2 · If the device in the scope of patent application No. 1 is used, the arrangement of the cell trap and membrane of the device in the test strip can make the The test sample flows from the cell trap through the sample introduction membrane, the dye-filled membrane, and the reagent-filled membrane. 1 3. The device according to item 1 of the patent application scope, wherein the marker in the dye-filled membrane is selected from enzymes , Luciferin, lipid particles, red blood cell ghost cells, polymer microcapsules or colored polymer particles (tree milk), preferably a metal-containing compound sol. , Where the marker is colloidal gold · 15. The device according to item 1 of the patent application scope, wherein the ligand system is selected from the group consisting of antigens, antibodies, hormones, enzymes, peptides, proteins, nucleic acids, oligonucleotides, sugars Proteins, carbohydrates, polysaccharides or combinations thereof 16. 16. The device according to any of claims 1 to 15 of the patent application scope, which further comprises an absorption pad made of an absorbent material, which is located on the end of a membrane filled with reagents and It is in liquid communication with the membrane, so that the absorption force of the absorption pad causes the sample to move from the cell trap area to the membrane filled with reagents. 1 7. If the device according to any one of claims 1 to 15, It further contains a buffer zone, which is composed of a porous absorbent material, and is a region filled with dye and / or a cell trap extending to the proximal end of the cell trap, respectively. 18, such as the 16th scope of the patent application The device further comprises a buffer zone, which is composed of a porous absorbent material, and is filled with dyed paper standards using the Chinese National Standard (CNS) A4 (210X297 mm) --- ί [- --- ^ ----- ^-I Order ------ line- * (Please read the precautions on the back before filling this page) -26-A8 B8 C8 D8 Six, hide the i range Membrane and / or cell trap area extending towards the proximal end of the cell trap, respectively * 1 9. An analysis A method for testing a liquid sample. The sample contains cells and cell components, and the desired analyte is expected to be contained in the sample. The method includes the following steps: adding a blood sample to a cell trap of a device such as the first patent application逋 After the reaction time, observe the visual pattern in the control area and the visual pattern in the test area; or compare the intensity of the pattern in the test area and the control area; where the identification pattern in the control area indicates that a competent analysis has been performed The identification pattern or its intensity in the test area indicates the presence or absence of the analyte in the test sample. 20. If the method in the scope of patent application No. 19, after adding a blood sample, add additional buffer to the device's buffer Liquid zone, or immerse the buffer zone of the device in the buffer solution. --- ί ---- 社, -------- ^-I 打 ------ line. (Please note on the back Please fill in this page again) The paper size printed by the 8th Industrial Cooperative Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, using the Chinese National Standard (CNS) A4 (210 × 2!) 7mm) -27-