Test paper, test card and kit

Abstract

The utility model relates to the field of biological detection, and particularly discloses detection test paper, a test card and a kit. The detection test paper comprises a sample pad, a detection film and a water absorption film, the sample pad and the water absorption film are respectively arranged on two sides of the detection film along a first direction; the detection film is provided with a plurality of detection lines and a plurality of grooves; the plurality of detection lines and the plurality of grooves are arranged at intervals along the first direction. The antibody arranged on the detection line can be used for rapid and convenient clinical detection; the groove has a certain depth and width, so that the flowing speed of a sample can be slowed down, the detection sensitivity is improved, the requirement of clinical rapid detection is met, and the problem that the clinical rapid detection cannot be realized by an existing blood and peptide detection method is effectively solved.

Description

Technical Field

[0001] This application relates to the field of biological detection, and particularly to a test strip, a test card, and a test kit. Background Art

[0002] Copeptin (CPP) is a carboxy-terminal peptide of pro-pressophysin, that is, the precursor of the pituitary hormone vasopressin. Its chemical structure is relatively stable. In the human body, it mainly synthesizes the pituitary hormone vasopressin (AVP), which is the main regulatory hormone for sodium and water metabolism. When Copeptin (CPP) exerts its corresponding biological effects, it can produce effects such as antidiuresis, immunomodulation, and vasoconstriction. Copeptin (CPP) has become a stable and easily measurable alternative marker for AVP, showing great potential in clinical practice. Compared with the unstable and difficult-to-measure vasopressin hormone (AVP), Copeptin (CPP) is easy to handle, stable at room temperature, and can provide a reliable measurement of the bioactive hormone. Existing studies have shown that Copeptin (CPP) can better reflect an individual's stress level than circulating cortisol. This new biomarker enables early decision-making in clinical practice and also provides reliable correlations for various acute disease states, such as cerebrovascular events, myocardial infarction, or pneumonia, kidney diseases and hypertension, osmotic changes, human obesity, bipolar disorder, major depressive disorder, and child abuse, etc. During the acute disease onset period, such as acute myocardial infarction (AMI), stroke, and sepsis, etc., the blood CPP concentration increases significantly. Among these diseases, the diagnosis and management of CVD can benefit the most from the introduction of CPP measurement. It has important diagnostic value in quickly excluding AMI, a cardiovascular disease, and can also predict the mortality of heart failure (HF) and stroke. When used in combination with other traditional cardiac biomarkers, it can be used as a prognostic predictor for heart failure (HF).

[0003] In the classification of patients with chest pain, in addition to troponin, the determination of CPP can improve the early diagnostic performance, especially after the onset of chest pain. During an acute myocardial infarction attack (AMI), CPP is rapidly released from the pituitary gland, especially significantly within the 2-hour golden treatment time, and begins to return to normal levels within a few hours, while the concentration of troponin T remains normal. In AMI patients, myocardial cell damage or myocardial cell death occurs, so specific cardiac troponin I (cTnI) is gradually released into the blood, with a low initial concentration and then gradually increasing. Therefore, the combined use of cTnI and CPP can quickly screen out negative AMI patients and enable immediate diagnosis of the patients.

[0004] Existing methods for detecting blood copeptin include chemiluminescence immunoassay, electrochemiluminescence immunoassay, time-resolved fluorescence immunoassay, and enzyme-linked immunosorbent assay (ELISA) by hand, etc. The first three use relatively large detection instruments and are difficult to achieve bedside detection, while the ELISA by hand takes a long time and the operation is cumbersome. Therefore, none of these four methods can achieve the purpose of rapid detection. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a test strip, a test card and a test kit, which are used to solve the problem that the existing methods for detecting blood and copeptin cannot achieve rapid clinical detection.

[0006] To achieve the above technical purpose, the first aspect of this application provides a test strip, which includes: a sample pad, a detection membrane and a water absorption membrane;

[0007] The sample pad and the water absorption membrane are respectively arranged on both sides of the detection membrane along the first direction;

[0008] A plurality of detection lines and a plurality of grooves are arranged on the detection membrane;

[0009] The plurality of detection lines and the plurality of grooves are arranged at intervals along the first direction.

[0010] Further, the plurality of detection lines include: a troponin I monoclonal antibody line and / or a copeptin monoclonal antibody line.

[0011] Further, a quality control line is arranged on the detection membrane;

[0012] The quality control line is coated with: goat anti-mouse IgG antibody.

[0013] Further, the groove penetrates through the detection membrane along the second direction;

[0014] The second direction is perpendicular to the first direction.

[0015] Further, the size of the detection membrane covered by the sample pad is 0.8 to 1.2 mm.

[0016] Further, the size of the detection membrane covered by the water absorption membrane is 1.8 to 2.2 mm.

[0017] Further, the depth of the groove is 1 to 100 μm;

[0018] The size of the groove along the first direction is 1 to 500 μm.

[0019] Further, it further includes a substrate;

[0020] The detection membrane is attached to the substrate.

[0021] Further, the substrate is a PCV rubber plate.

[0022] Further, the sample pad is a glass cellulose gasket or a polyester film gasket.

[0023] The second aspect of the present application provides a test card, which includes the test strip described in any one of the above and a card box;

[0024] The test strip is arranged in the card box;

[0025] A sample adding port and an observation window are arranged on the card box.

[0026] The third aspect of the present application provides a kit, which includes: fluorescent microspheres and a test card;

[0027] The fluorescent microspheres include: fluorescent microspheres conjugated with mouse anti-human cardiac troponin I monoclonal secondary antibody and / or fluorescent microspheres conjugated with mouse anti-copeptin monoclonal secondary antibody.

[0028] Furthermore, it further includes: a freeze-dried tube for fluorescent microspheres;

[0029] The fluorescent microspheres are placed in the freeze-dried tube for fluorescent microspheres.

[0030] It can be seen from the above technical solutions that the present application provides a test strip, a test card and a kit. A plurality of test lines and a plurality of grooves are arranged on the test membrane of the test strip; antibodies are arranged on the test lines, which can be used for rapid and convenient clinical detection; the grooves have a certain depth and width, which can slow down the flow rate of the sample and improve the detection sensitivity, so as to meet the requirements of clinical rapid detection and effectively solve the problem that the existing methods for detecting blood copeptin cannot achieve clinical rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a front view frame schematic diagram of a test strip provided by an embodiment of the present application;

[0033] Figure 2 It is a top view of a test strip provided by another embodiment of the present application;

[0034] Figure 3 It is a side view of a test strip provided by another embodiment of the present application;

[0035] Figure 4 It is a cTnI standard curve graph corresponding to the test strip (cTnI + copeptin product) provided by an embodiment of the present application;

[0036] Figure 5It is the standard curve graph of CPP corresponding to the test strip (cTnI + CPP product) provided by the embodiment of the present application;

[0037] Figure 6 It is the comparison graph of cTnI results of the cTnI + CPP dip card product of the test strip provided by the embodiment of the present application using chemiluminescence method;

[0038] Figure 7 It is the comparison graph of CPP results of the cTnI + CPP dip card product of the test strip provided by the embodiment of the present application using time-resolved fluorescence method.

[0039] In the figure: 1. Sample pad; 2. Detection membrane; 3. Absorbent membrane; 4. Substrate; 21. Detection line; 22. Groove; 23. Quality control line. Detailed implementation mode

[0040] Next, the technical solutions of the embodiments of the present application will be clearly and completely described in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in this specification of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the scope claimed by the present application.

[0041] In the description of the embodiments of the present application, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the embodiments of the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be understood as a limitation to the embodiments of the present application. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0042] In the description of the embodiments of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a replaceable connection, or an integral connection. It can be a mechanical connection or an electrical connection. It can be directly connected or indirectly connected through an intermediate medium. It can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific situations.

[0043] In the first aspect of the embodiments of the present application, a test strip is provided; please refer to Figure 1 , Figure 1 It is the front view line schematic diagram of the test strip provided for this embodiment.

[0044] The test strip provided in this embodiment includes: a sample pad 1, a test membrane 2, and a water-absorbing membrane 3. The sample pad 1 is used to contact the sample to be tested; the water-absorbing membrane 3 can be blotting paper, which is used to generate suction on the sample to be tested to drive the sample to flow along the test membrane 2.

[0045] The sample pad 1 and the water-absorbing membrane 3 are respectively arranged on both sides of the test membrane 2 along the first direction; wherein, the first direction can be Figure 1 the positive and negative directions indicated by the x arrow in the shown view.

[0046] A plurality of test lines 21 and a plurality of grooves 22 are arranged on the test membrane 2; the plurality of test lines 21 and the plurality of grooves 22 are arranged at intervals along the first direction. Antibodies are arranged on the test lines 21, which can bind to the sample to be tested for reaction. Among them, when there are multiple test lines 21, different antibodies or the same antibodies can be arranged on the multiple test lines 21.

[0047] The groove 22 has a certain depth, specifically, the depth of the groove 22 is less than the thickness of the test membrane 2. The groove 22 can be one or include multiple ones. The groove 22 can slow down the flow rate of the sample to be tested on the test membrane 2, improve the detection sensitivity, and thus ensure the accuracy of clinical detection.

[0048] In practical applications, both the depth and width of the groove 22 will affect the flow rate of the sample to be tested. Therefore, the staff can control the flow rate of the sample to be tested by adjusting the size of the groove 22. At the same time, because the groove 22 has the effect of being easy to process, the test strip provided in this embodiment also has the effects of convenient regulation and convenient processing.

[0049] The sample pad 1 can be a glass cellulose gasket or a polyester film gasket; the test membrane 2 can be a nitrocellulose membrane. The actual processing process of the groove 22 can be: according to the size of the nitrocellulose membrane large board, after designing the width, depth and position of the groove 22 in the channel (lateral engraving) using CAD software, input the designed graphic software into the operating system of a femtosecond or nanosecond laser engraving machine, and then input the cutting power and speed of the laser, and engrave the groove 22 on the front of the nitrocellulose membrane (test membrane 2) to make the designed isolation hydrophobic channel and groove 22 with the depth and width. The depth of the isolation channel and the groove 22 is related to the power and cutting speed of the laser used.

[0050] The depth of the groove 22 can be set to 1 - 100 μm, and the size of the groove 22 along the first direction can be set to 1 - 500 μm.

[0051] In one embodiment, the plurality of test lines 21 include: a troponin I monoclonal antibody line and / or a copeptin monoclonal antibody line.

[0052] Specifically, the troponin I monoclonal antibody line means that the mouse anti-human troponin I monoclonal antibody is diluted with an antibody diluent and then sprayed on the test line 21 of the test membrane 2. The copeptin monoclonal antibody line means that the mouse anti-human copeptin monoclonal antibody is diluted with an antibody diluent and then sprayed on the test line 21 of the test membrane 2.

[0053] In this embodiment, the test membrane 2 can be provided only with a troponin I monoclonal antibody line to serve as a high-sensitivity troponin test membrane, or only with a copeptin monoclonal antibody line to serve as a copeptin test membrane, or can be provided with both a copeptin monoclonal antibody line and a troponin I monoclonal antibody line so as to be able to simultaneously detect high-sensitivity troponin and copeptin. And in this solution, the application of the nitrocellulose membrane strip technology to the detection of blood copeptin can achieve rapid, convenient, and bedside quantitative determination, providing powerful laboratory data for the rapid diagnosis and screening of AMI patients.

[0054] As an implementation manner, in the above antibody diluent, every 100 ml can include: 5 - 10 g of NaCl, 0.001 - 0.01 g of ProclinTM 300, 1 - 10 g of sucrose, 0.5 - 1.5 g of bovine serum albumin, 1.0 - 5.0 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and 0.1 - 1.0 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and the pH value is 7.5 - 8.5.

[0055] In another embodiment, a quality control line 23 is provided on the test membrane 2; the quality control line 23 is coated with: goat anti-mouse IgG antibody.

[0056] Specifically, the quality control line 23 can be at a preset position on the test membrane 2. After the goat anti-mouse IgG antibody is diluted with a buffer solution, it is then sprayed on the above preset position and dried.

[0057] In practical applications, as Figure 1 shown, the position of the groove 22 can be set before the test line 21 and the quality control line 23, that is, between the sample pad 1 and the test line 21. In other implementation manners, the position of the groove 22 can be between the test line 21 and the quality control line 23, or located between the quality control line 23 and the water absorption membrane 3.

[0058] In one embodiment, the groove 22 penetrates the test membrane 2 along the second direction; the second direction is perpendicular to the first direction.

[0059] As Figure 2 shown, the second direction can refer to the width direction of the test membrane 2, that is, the y-axis direction shown in Figure 2 . The groove 22 penetrating the test membrane 2 along the second direction can ensure that its buffering effect on the width direction of the test membrane 2 is consistent.

[0060] In one embodiment, the size of the sample pad 1 covering the detection membrane 2 is 0.8 to 1.2 mm. The size of the water absorption membrane 3 covering the detection membrane 2 is 1.8 to 2.2 mm.

[0061] In practical applications, the size of the sample pad 1 covering the detection membrane 2 affects the rate at which the sample to be tested on the sample pad 1 flows into the detection membrane 2; the size of the water absorption membrane 2 covering the detection membrane 2 affects the flow rate of the sample to be tested within the detection membrane 2. For this reason, in this embodiment, the size of the sample pad 1 covering the detection membrane 2 is 0.8 to 1.2 mm, and the size of the water absorption membrane 3 covering the detection membrane 2 is 1.8 to 2.2 mm, which can make the flow rate of the sample to be tested on the detection membrane 2 moderate.

[0062] In one embodiment, the size of the sample pad 1 covering the detection membrane 2 is 1 mm; the size of the water absorption membrane 3 covering the detection membrane 2 is 2 mm.

[0063] Optionally, please refer to Figure 2 and Figure 3 , the test strip provided in this embodiment further includes a substrate 4; the detection membrane 2 is attached to the substrate 4.

[0064] The substrate 4 can be a PCV rubber plate. During the process of preparing the test strip, the non-spotting surface of the detection membrane 2 can be pasted on the substrate 4, and then the sample pad 1 and the water absorption membrane 3 are respectively pasted on both sides of the detection membrane 2.

[0065] In the second aspect of the embodiments of the present application, a test card is provided, including the test strip of any one of the above and a card box; the test strip is arranged in the card box; a sample addition port and an observation window are arranged on the card box.

[0066] Specifically, during preparation, first, the sample pad 1 is wetted with the sample pad treatment solution and then dried, and then the sample pad 1 is pasted above one end of the detection membrane 2; a water absorption membrane 3 is taken and pasted above the other end of the detection membrane 2 to obtain the test strip. After the test strip is prepared, the pasted test strip can be cut into a test strip with a width of 4 mm by an automatic cutting machine, and then installed in the card box to make a test card with a result observation window and a sample addition port.

[0067] In the above sample pad treatment solution, every 100 mL of the sample pad treatment solution includes the following components: 5 to 10 g of NaCl, 0.001 to 0.01 g of ProclinTM 300, 1 to 10 g of sucrose, 0.5 to 1.5 g of bovine serum albumin, 0.4 to 0.6 g of polyvinylpyrrolidone, 0.06 to 0.1 g of rabbit anti-human red blood cell antibody, and the balance is phosphate buffer solution.

[0068] In a third aspect of the embodiments of the present application, a kit is provided, comprising: fluorescent microspheres and a test card; the fluorescent microspheres include: fluorescent microspheres conjugated with mouse anti-human cardiac troponin I monoclonal secondary antibody and / or fluorescent microspheres conjugated with mouse anti-human copeptin monoclonal secondary antibody.

[0069] The kit provided in this embodiment may further include a sample diluent; the sample diluent includes: PBS, Tween-20, and ProclinTM300.

[0070] Optionally, it further includes: a fluorescent microsphere lyophilized tube; the fluorescent microspheres are placed inside the fluorescent microsphere lyophilized tube. During preparation, the fluorescent microspheres conjugated with mouse anti-human cardiac troponin I monoclonal secondary antibody and mouse anti-human copeptin monoclonal secondary antibody can be separately conjugated, then diluted with an antibody diluent and placed in a small test tube with a lid, and then freeze-dried to form the fluorescent microsphere lyophilized tube.

[0071] Moreover, an antibody protective solution is stored inside the fluorescent microsphere lyophilized tube; every 100 mL of the antibody protective solution includes the following components: 5 - 10 g of NaCl, 0.0001 - 0.01 g of ProclinTM300, 1 - 10 g of sucrose, 0.5 - 1.5 g of bovine serum albumin, and the balance is phosphate buffer solution.

[0072] The preparation method of the above-mentioned fluorescent microspheres includes the following steps:

[0073] Take carboxyl fluorescent microspheres and suspend them in MES buffer solution to obtain a microsphere solution;

[0074] After adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the microsphere solution, then add cardiac troponin I monoclonal antibody for reaction to obtain a reaction solution;

[0075] After adding bovine serum albumin blocking solution to the reaction solution for blocking, then centrifuge and resuspend to obtain the time-resolved fluorescent microspheres conjugated with the specific cardiac troponin I antibody.

[0076] The kit provided in the embodiments of the present application is based on the technical principle of immunochromatography, and adopts the double antibody sandwich method to capture copeptin antigen or cardiac troponin I antigen. It can detect picomolar copeptin or picogram-level cardiac troponin I antigen content in serum within 15 minutes. It can detect lower concentrations of copeptin and cardiac troponin I to achieve the purpose of detecting copeptin and ultrasensitive detection of cardiac troponin I.

[0077] English annotations in this solution:

[0078] CPP: copeptin

[0079] cTnI: Myocardial troponin I;

[0080] HscTnI: High sensitivity myocardial troponin I;

[0081] In the above embodiments, the materials and reagents used can be purchased from the market.

[0082] The following further elaborates on the solution of the present application in combination with other embodiments:

[0083] In one embodiment:

[0084] As shown in the appendix Figure 2 and 3 This embodiment provides a test kit, including a test card, a calibration card, and a fluorescent microsphere lyophilized tube. The test card includes a test strip, wherein the test strip includes:

[0085] Substrate 4;

[0086] The detection membrane 2 provided on the substrate 4 is respectively provided with a detection line 21, a groove 22, and a quality control line 23; there are multiple detection lines 21, and some of the detection lines 21 contain monoclonal antibodies against troponin I, and some of the detection lines 21 contain monoclonal antibodies against copeptin; the quality control line 23 contains goat anti-mouse IgG antibodies;

[0087] Sample pad 1, provided at the proximal end of the detection membrane 2;

[0088] Absorbent membrane 3 provided on the other side of the detection membrane 2 away from the sample pad 1.

[0089] The assembly method of the test strip is specifically as follows: paste the sample pad 1 above the detection membrane 2, covering a width of 1 mm of the detection membrane 2; paste the absorbent membrane 3 above the detection membrane 2, covering a width of 2 mm of the detection membrane 2; cut the pasted test strip into a test strip with a width of 4 mm in an automatic cutting machine, and install it in a plastic cartridge to make a test card with a result observation window and a sample addition port.

[0090] The fluorescent microsphere lyophilized tube is used to store fluorescent microspheres.

[0091] In one embodiment:

[0092] The test strip provided in the above embodiment can be prepared by the following steps:

[0093] S1. Take a PVC rubber plate as the substrate, cut the NC membrane into 25 mm * 300 mm, and paste the non-spotting surface of the NC membrane on the PVC rubber plate. Among them, the PVC rubber plate serves as the substrate 4, and the NC membrane serves as the detection membrane 2.

[0094] S2. Dilute the cTnI monoclonal antibody and the CPP antibody with the antibody diluent to a concentration of 2 mg / mL. Dilute the goat anti-mouse IgG antibody with 0.05 M phosphate buffer solution containing 1% sucrose and pH 8.0, and then spray it on the test line of the NC membrane (the first test line is 6 mm away from one end of the sample pad, the second test line is 4.5 mm away from the first test line, and the quality control line is 15 mm away from one end of the sample pad) at a speed of 100 mm / s and a concentration of 1 μL / cm under the condition that the indoor humidity is 45%, and dry it in an oven at 37°C for 2 h for standby. Among them, the antibody diluent includes sucrose and phosphate buffer solution; in the antibody diluent, the mass concentration of sucrose is 0.5%; 0.01 M phosphate buffer solution, pH = 7.5.

[0095] S3. The preparation method of the time-resolved fluorescence microspheres linked with the specific troponin I antibody and the CPP antibody includes the following steps, taking the specific troponin I antibody (the preparation method of the copeptin antibody is the same as the following, only replacing the antibody) as an example:

[0096] (1) Take 100 μL of carboxyl fluorescence microspheres (10 mg / mL), and suspend them in 800 μL of MES buffer solution to obtain a microsphere solution; among them, the MES buffer solution is prepared by the following method: weigh 0.976 g of MES, pour it into 98 mL of deionized water and dissolve it completely, detect the solution concentration with a pH meter, and when the pH is greater than 6.0, dropwise add 1 M dilute hydrochloric acid solution to neutralize excessive HO - , when the pH is less than 6.0, dropwise add 1 M sodium hydroxide solution to neutralize excessive H + , so that the pH is finally stabilized at 6.0, and finally make up the volume to 100 mL with deionized water.

[0097] (2) Freshly prepare NHS (5 mg / mL) and EDC (5 mg / mL) with MES buffer solution, take 20 μL of each and add them to the microsphere solution, and the final concentration is NHS (0.1 mg / mL) and EDC (0.1 mg / mL), and rotate and mix at room temperature (25°C) for 30 minutes for activation, with a rotation speed of 10 rpm. Then, after centrifugal washing (remove the supernatant after centrifugation at 17,500 rpm and resuspend it in 900 μL of MES buffer solution (wherein, NHS can be mixed with EDC first and then added to the microspheres. EDC cannot be added to the microspheres first, otherwise it will cause microsphere aggregation); then, take 0.1 mg of cTn I monoclonal antibody and add it to the above microsphere solution, oscillate and mix well, and react at room temperature (25°C) by rotation for 2 hours to obtain a reaction solution.

[0098] (3) Add 100 μL of BSA blocking solution (20 mg / mL) to the above reaction solution, and block the reaction at room temperature (25 °C) for 2 hours. Then, centrifuge at 17,500 rpm for 20 min, resuspend with 0.05 M Tris-HCl buffer (pH 8.0), and ultrasonically disperse for 30 seconds to 1 minute each time. (First, add 500 μL of buffer, place the centrifuge tube in the ultrasonic instrument, make the liquid level in the centrifuge tube form a 45-degree angle with the liquid level in the ultrasonic instrument, and ultrasonically disperse evenly. Then add another 500 μL of buffer and ultrasonically disperse again; the ultrasonic time each time shall not exceed 1 minute.) Repeat the washing twice. After the last centrifugation, discard the supernatant and resuspend with the microsphere preservation solution. Then, centrifuge at 2,000 rpm for 3 min, discard the precipitate, and store in the dark at 4 °C to obtain the time-resolved fluorescence microspheres conjugated with specific troponin I antibody. Among them, the preparation method of the BSA blocking solution is as follows: Weigh 0.2 g of BSA and 0.075 g of glycine, and make up the volume to 10 mL with water.

[0099] S4. Use glass cellulose SB06 (30 mm × 300 mm) as the sample pad material. Pour the sample pad treatment solution along the glass rod into the liquid tank of the dipping hydraulic pad machine to avoid foaming. After starting the machine, adjust the parameters of the micrometer screws at both ends so that the net weight of each glass fiber dipped in the liquid is 30 g ± 2 g. Put the glass fiber 8795 with a specification of 25 cm × 30 cm into the dipping hydraulic pad machine for liquid dipping preparation. Horizontally pick up the dipped sample pad, transfer it to the sieve, and place it horizontally at (37 ± 1) °C for drying for 12 to 24 hours. After drying, horizontally cut the sample pad along the 30-cm long side, discard the part of the glass fiber with a width of 0.8 to 1.2 cm at the edge, and cut the remaining part into a specification of 2.4 cm × 30 cm. Then, paste the sample pad at one end of the NC membrane. Among them, every 100 mL of the sample pad treatment solution contains the following components: 0.1 g of Tween-20, 0.8 g of bovine serum albumin, 0.6 g of polyvinylpyrrolidone, 0.04 g of rabbit anti-human red blood cell antibody, 0.6 mL of 10% ProclinTM 300, and the balance is 0.01 M PB buffer.

[0100] S5. Take a piece of absorbent paper with a size of 22 mm × 300 cm as the absorbent membrane 3, and paste it above the NC membrane to obtain the test strip.

[0101] Preparation of the freeze-dried tube of fluorescent microspheres:

[0102] S1. 100 mL of antibody-fluorescent microsphere freeze-dried dilution solution: 2.901 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 0.296 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), 8.5 g of NaCl, 0.1 mL of ProclinTM 300, 5 g of sucrose, 1 g of BSA, add purified water to 100 mL and mix well.

[0103] S2. The CPP antibody-fluorescent conjugate is taken out from the 2-8°C refrigerator, ultrasonically mixed, restored to room temperature, and diluted to the target concentration with a lyophilized diluent.

[0104] S3. Sub-packaging of the fluorescent-antibody lyophilized conjugate working solution: Place 600 μL centrifuge tubes on the plate rack, and use a continuous dispenser to sub-package the diluted lyophilized working solution into the centrifuge tubes, with 0.03 mL sub-packaged in each tube.

[0105] S4. Lyophilization of the fluorescent-antibody lyophilized conjugate working solution: Perform lyophilization according to the operating instructions of the vacuum freeze dryer. After the freeze dryer is ready, place the sub-packaged lyophilized working solution inside the freeze dryer and lyophilize for 12 - 24 hours.

[0106] S5. Post-lyophilization treatment - capping or film-sealing, and sealing: After the lyophilization process is completed, take out the lyophilized product from the freeze dryer and immediately cap or film-seal it to avoid the sample absorbing moisture. Finally, seal it and store it at room temperature with a validity period of 3 months.

[0107] In one embodiment:

[0108] A preparation method of the above-mentioned test strip is provided, which includes the following steps:

[0109] S1. Take a PVC rubber plate as the substrate, cut the NC membrane into 25 mm * 300 mm, and paste the non-spotting surface of the NC membrane on the PVC rubber plate. Among them, the PVC rubber plate serves as the substrate 4, and the NC membrane serves as the detection membrane 2.

[0110] S2. Dilute the cTn I monoclonal antibody and the CPP antibody respectively with an antibody diluent to a concentration of 2 mg / mL. Dilute the goat anti-mouse IgG antibody with a 0.05 M, pH 8.0 phosphate buffer containing 1% sucrose, and then spray it on the detection line of the NC membrane (the detection line 1 is 6 mm away from one end of the sample pad, the distance between the detection line 1 and the detection line 2 is 4.5 mm, and the quality control line is 15 mm away from one end of the sample pad)) at a speed of 100 mm / s and a concentration of 1 μL / cm under the condition of an indoor humidity of 45%, and dry it in an oven at 37°C for 2 h for standby. Among them, the antibody diluent includes sucrose and phosphate buffer; in the antibody diluent, the mass concentration of sucrose is 1.0%; 0.01 M phosphate buffer, pH = 8.0.

[0111] The preparation method of the time-resolved fluorescence microspheres conjugated with the specific troponin I antibody and the CPP antibody includes the following steps, taking the specific troponin I antibody as an example for illustration:

[0112] (1) Take 100 μL of carboxyl fluorescent microspheres (10 mg / mL) and suspend them in 800 μL of MES buffer to obtain a microsphere solution. Among them, the MES buffer is prepared by the following method: Weigh 0.976 g of MES, pour it into 98 mL of deionized water and dissolve it completely. Use a pH meter to detect the solution concentration. When the pH is greater than 6.0, gradually add 1 M dilute hydrochloric acid solution to neutralize the excessive HO - , and when the pH is less than 6.0, gradually add 1 M sodium hydroxide solution to neutralize the excessive H + , so that the pH is finally stabilized at 6.0, and finally make up the volume to 100 mL with deionized water.

[0113] (2) Freshly prepare NHS (5 mg / mL) and EDC (5 mg / mL) using MES buffer. Take 20 μL of each and add them to the microsphere solution. The final concentrations are NHS (0.1 mg / mL) and EDC (0.1 mg / mL). Rotate and mix at room temperature (25 °C) for 30 minutes at a speed of 10 rpm. Then, centrifuge and wash (remove the supernatant after centrifugation at 17,500 rpm and resuspend in 900 μL of MES buffer. (Among them, NHS can be mixed with EDC first and then added to the microspheres. EDC cannot be added to the microspheres first, otherwise it will cause microsphere aggregation); Then, take 0.1 mg of cTn I monoclonal antibody and add it to the above microsphere solution, oscillate and mix well, and react at room temperature (25 °C) by rotation for 2 hours to obtain a reaction solution.

[0114] (3) Add 100 μL of BSA blocking solution (20 mg / mL) to the above reaction solution. After blocking and reacting at room temperature (25 °C) for 2 hours, centrifuge at 17,500 rpm for 20 min, resuspend with 0.05 M Tris-HCl buffer (pH 8.0), and ultrasonically disperse for 30 seconds to 1 minute each time; (First add 500 μL of buffer, place the centrifuge tube in the ultrasonic instrument, make the liquid level in the centrifuge tube form a 45-degree angle with the liquid level in the ultrasonic instrument, and ultrasonically disperse evenly, then add another 500 μL of buffer and ultrasonically disperse again; The ultrasonic time each time shall not exceed 1 minute), repeat the washing twice. After the last centrifugation, remove the supernatant and resuspend with the microsphere preservation solution; Then centrifuge at 2,000 rpm for 3 min, remove the precipitate, and store in the dark at 4 °C to obtain time-resolved fluorescent microspheres conjugated with specific troponin I antibody. Among them, the preparation method of the BSA blocking solution is as follows: Weigh 0.2 g of BSA and 0.075 g of glycine, and make up the volume to 10 mL with water.

[0115] S4. Use glass cellulose SB06 (30 mm × 300 mm) as the sample pad material. Pour the sample pad treatment solution along a glass rod into the liquid tank of the dipping hydraulic pad machine, avoiding foaming. After starting the machine, adjust the parameters of the micrometer screws at both ends so that the net weight of each glass fiber impregnated with liquid is 30 g ± 2 g. Put the glass fiber 8795 with a specification of 25 cm × 30 cm into the dipping hydraulic pad machine for liquid impregnation preparation. Horizontally pick up the impregnated sample pad and transfer it to a sieve, and place it horizontally at (37 ± 1) °C for drying for 12 - 24 hours. After drying, the sample pad is horizontally cut along the 30 cm long side, discarding the part of the glass fiber with a width of 0.8 - 1.2 cm at the edge, and the remaining part is cut into a specification of 2.4 cm × 30 cm, and then the sample pad is pasted at one end of the NC membrane. Among them, every 100 mL of the sample pad treatment solution includes the following components: 0.1 g of Tween-20, 0.8 g of bovine serum albumin, 0.6 g of polyvinylpyrrolidone, 0.04 g of rabbit anti-human red blood cell antibody, 0.6 mL of 10% ProclinTM300, and the balance is 0.01 M PB buffer solution.

[0116] S5. Take a piece of absorbent paper of 22 mm × 300 cm and paste it above the NC membrane to obtain a test strip.

[0117] The preparation of the fluorescent microsphere freeze-dried tube is the same as that in the above-mentioned embodiment.

[0118] In one embodiment:

[0119] Provide a preparation method of the above-mentioned test strip, which includes the following steps:

[0120] S1. Take a PVC rubber plate as the substrate, cut the NC membrane into 25 mm * 300 mm, and paste the non-spotting surface of the NC membrane on the PVC rubber plate. Among them, the PVC rubber plate serves as the substrate 4, and the NC membrane serves as the detection membrane 2.

[0121] S2. Dilute the cTnI monoclonal antibody and the CPP antibody with an antibody dilution solution to a concentration of 2 mg / mL respectively. Dilute the goat anti-mouse IgG antibody with a 0.05 M, pH 8.0 phosphate buffer solution containing 1% sucrose, and then spray it on the detection line of the NC membrane (the detection line 1 is 6 mm away from one end of the sample pad, the detection line 1 is 4.5 mm away from the detection line 2, and the quality control line is 15 mm away from one end of the sample pad)) at a speed of 100 mm / s and a concentration of 1 μL / cm with a scribing and gold-spraying instrument under the condition that the indoor humidity is 45%, and dry it in an oven at 37 °C for 2 h for standby. Among them, the antibody dilution solution includes sucrose and phosphate buffer solution; in the antibody dilution solution, the mass concentration of sucrose is 1.5%; 0.01 M phosphate buffer solution, pH = 7.0.

[0122] S3. The preparation method of time-resolved fluorescence microspheres conjugated with cTnI antibody and CPP antibody includes the following steps, taking the cTnI antibody as an example for illustration:

[0123] (1) Take 100 μL of carboxyl fluorescence microspheres (10 mg / mL), and suspend them in 800 μL of MES buffer to obtain a microsphere solution. Among them, the MES buffer is prepared by the following method: Weigh 0.976 g of MES, pour it into 98 mL of deionized water and dissolve it completely. Use a pH meter to detect the solution concentration. When the pH is greater than 6.0, gradually add 1 M dilute hydrochloric acid solution to neutralize excessive HO-, and when the pH is less than 6.0, gradually add 1 M sodium hydroxide solution to neutralize excessive H+ until the pH is finally stabilized at 6.0. Finally, make up the volume to 100 mL with deionized water.

[0124] (2) Freshly prepare NHS (5 mg / mL) and EDC (5 mg / mL) using MES buffer. Take 20 μL of each and add them to the microsphere solution, with the final concentrations of NHS (0.1 mg / mL) and EDC (0.1 mg / mL). Then rotate and mix evenly at room temperature (25 °C) for 30 minutes at a rotation speed of 10 rpm. Next, centrifuge and wash (remove the supernatant after centrifugation at 17,500 rpm and resuspend in 900 μL of MES buffer. (Among them, NHS can be mixed with EDC first and then added to the microspheres. EDC cannot be added to the microspheres first, otherwise it will cause microsphere aggregation); Then, take 0.1 mg of cTnI monoclonal antibody and add it to the above microsphere solution, oscillate and mix evenly, and react by rotation at room temperature (25 °C) for 2 hours to obtain a reaction solution.

[0125] (3) Add 100 μL of BSA blocking solution (20 mg / mL) to the above reaction solution, block and react at room temperature (25 °C) for 2 hours, then centrifuge at 17,500 rpm for 20 minutes, resuspend with 0.05 M Tris-HCl buffer (pH 8.0), and ultrasonically disperse for 30 seconds to 1 minute each time; (First add 500 μL of buffer, put the centrifuge tube into the ultrasonic instrument, make the liquid level in the centrifuge tube form a 45-degree angle with the liquid level in the ultrasonic instrument, disperse evenly, and then add 500 μL of buffer and ultrasonically disperse again; The ultrasonic time each time shall not exceed 1 minute), repeat the washing twice. After the last centrifugation, remove the supernatant and resuspend with the microsphere preservation solution; Then centrifuge at 2,000 rpm for 3 minutes, remove the precipitate, and store in the dark at 4 °C to obtain time-resolved fluorescence microspheres conjugated with specific troponin I antibody. Among them, the preparation method of the BSA blocking solution is as follows: Weigh 0.2 g of BSA and 0.075 g of glycine, and make up the volume to 10 mL with water.

[0126] S4. Use glass cellulose SB06 (30 mm × 300 mm) as the sample pad material. Pour the sample pad treatment solution along a glass rod into the liquid tank of the dipping and pressing machine, avoiding foaming. After starting the machine, adjust the parameters of the micrometer screws at both ends so that the net weight of each glass fiber impregnated with liquid is 30 g ± 2 g. Put the glass fiber 8795 with a specification of 25 cm × 30 cm into the dipping and pressing machine for liquid impregnation preparation. Horizontally pick up the impregnated sample pad and transfer it to a sieve, and place it horizontally at (37 ± 1) °C for drying for 12 - 24 hours. After drying, the sample pad is horizontally cut along the 30 cm long side, and the part of the glass fiber with a width of 0.8 - 1.2 cm at the edge is discarded, and the remaining part is cut into a specification of 2.4 cm × 30 cm, and then the sample pad is pasted at one end of the NC membrane. Among them, every 100 mL of the sample pad treatment solution includes the following components: 0.1 g of Tween-20, 0.8 g of bovine serum albumin, 0.6 g of polyvinylpyrrolidone, 0.04 g of rabbit anti-human red blood cell antibody, 0.6 mL of 10% ProclinTM300, and the balance is 0.01 M PB buffer solution.

[0127] S5. Take a piece of absorbent paper of 22 mm × 300 cm and paste it above the NC membrane to obtain a test strip.

[0128] The preparation of the fluorescent microsphere freeze-drying tube is the same as that in the above-mentioned embodiment.

[0129] Verification example

[0130] The above-mentioned kit can be used in combination with a dry fluorescence immunoassay analyzer for ultra-sensitive detection of troponin I, and its calibration process is as follows:

[0131] 1. Preparation of calibration solution

[0132] Use cTnI negative serum / whole blood or antigen diluent as the buffer solution to prepare the following concentration groups of cTnI calibrators: 50 / 10 / 5 / 1 / 0.5 / 0 ng / L. Each concentration group is detected 4 times repeatedly.

[0133] Use CPP negative serum / whole blood or antigen diluent as the buffer solution to prepare the following concentration groups of CPP calibrators: 500 / 200 / 50 / 10 / 2.7 / 0 pmol / L. Each concentration group is detected 4 times repeatedly.

[0134] 2. Drawing of calibration curve

[0135] (1) Draw the reagents to be calibrated in batches. Each concentration group is detected 4 times repeatedly. Add 100 μL of sample to each test card. After waiting for 15 minutes of incubation time, use a dry fluorescence immunoassay analyzer to detect the T / C value.

[0136] Note: Serum and whole blood need to be detected separately. The sampling time interval for each test card should be consistent with the instrument detection time interval (for example, if the current instrument detection takes 15 seconds, then the sampling time interval for each test card is 15 seconds).

[0137] (2) Obtain calibration data: Based on the four-parameter fitting equation of the concentration and the average value of the corresponding T / C value, R should be greater than 0.99.

[0138] Among them, the standard curve graph of the high-sensitivity cTn I project corresponding to the test strip prepared in Example 2 is as Figure 4 and shown in Table 1 (compared with the membrane without grooves, its detection sensitivity is increased by 10 times); the standard curve graph of the CPP project of the test strip prepared in Example 2 is as Figure 5 and shown in Table 2 (compared with the membrane without grooves, its detection sensitivity is increased by 6 times); the comparison graph of the high-sensitivity cTnI+CPP dipstick product and the high-sensitivity cTn I results of a certain hospital is as attached Figure 6 shown; the comparison graph of the high-sensitivity cTnI+CPP dipstick product and the results of a certain hospital for CPP is as attached Figure 7 shown.

[0139] Table 1

[0140]

[0141] Table 2

[0142]

[0143] The above are the preferred embodiments of this application and are not used to limit the present utility model. Although this application has been described in detail with reference to the examples, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements for some of the technical features. However, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims

1. A test strip, characterized in that: include: A sample pad (1), a detection membrane (2) and a water-absorbing membrane (3); The sample pad (1) and the water absorption film (3) are respectively arranged on two sides of the detection film (2) along a first direction; The detection film (2) is provided with a plurality of detection lines (21) and a plurality of grooves (22); A plurality of the detection lines (21) and a plurality of the grooves (22) are arranged at intervals along the first direction.

2. The test paper according to claim 1, characterized in that The plurality of detection lines (21) include: a troponin I monoclonal antibody line and / or a copeptin monoclonal antibody line.

3. The test paper according to claim 1 or 2, characterized in that: The detection membrane (2) is provided with a quality control line (23); The quality control line (23) is coated with goat anti-mouse IgG antibody.

4. The test paper according to claim 1, characterized in that: The groove (22) penetrates the detection film (2) along the second direction; The second direction is perpendicular to the first direction.

5. The test paper according to claim 1, characterized in that: The size of the sample pad (1) covering the detection membrane (2) is 0.8 to 1.2 mm.

6. The test paper according to claim 1 or 5, characterized in that: The size of the water-absorbing film (3) covering the detection film (2) is 1.8 to 2.2 mm.

7. The test paper according to claim 1, characterized in that: The depth of the groove (22) is 1-100 μm; The dimension of the groove (22) along the first direction is 1-500 μm.

8. The test paper according to claim 1, characterized in that: Also includes a substrate (4); The detection film (2) is attached to the substrate (4).

9. The test paper according to claim 8, characterized in that: The substrate (4) is a PCV rubber sheet.

10. The test paper according to claim 1, characterized in that: The sample pad (1) is a glass cellulose pad or a polyester film pad.

11. A test card, characterized in that: Comprising the test strip and card box according to any one of claims 1 to 10; The test strip is arranged in the card box; The card box is provided with a sample adding port and an observation window.

12. A kit, characterized in that: include: Fluorescent microspheres and the test card according to claim 11; The fluorescent microspheres include: fluorescent microspheres linked to mouse anti-human troponin I monoclonal secondary antibodies and / or fluorescent microspheres linked to mouse anti-human and copeptin monoclonal secondary antibodies.

13. The kit according to claim 12, characterized in that Also includes: Fluorescent microsphere freeze-dried tube; The fluorescent microspheres are placed in the fluorescent microsphere freeze-drying tube.

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