Antigen detection test strip and preparation method thereof

Abstract

The invention relates to the technical field of rapid diagnosis, and particularly discloses an antigen detection test strip and a preparation method thereof. The test strip comprises a bottom plate, and a sample pad, a blank pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped on the bottom plate in a chromatography direction, a marking area, a detection line and a quality control line are arranged on the nitrocellulose membrane; the marking area, the detection line and the quality control line are sequentially positioned on the nitrocellulose membrane along the chromatography direction; the detection line is coated with a detection antibody, and the quality control line is coated with a quality control antigen or antibody; an NC membrane treatment component and a quantum dot conjugate are sequentially fixed to the labeling area, the quantum dot conjugate is stacked on the NC membrane treatment component, rapid release of the labeled conjugate can be achieved, detection can be achieved by using a small amount of peripheral blood / vein whole blood / serum / plasma samples, a report can be given within 5 min, and the detection sensitivity is high. The device has the advantages of convenience in use, simplicity in operation and convenience in popularization.

Description

Technical Field

[0001] This application relates to the field of rapid diagnostic technologies, and more specifically, to an antigen detection test strip and a preparation method thereof. Background Art

[0002] Diabetic nephropathy (DN) is proteinuria and a progressive decrease in glomerular filtration rate (GFR) caused by long-term diabetes. Diabetic nephropathy is one of the most important complications of diabetic patients. The incidence rate in China is also on the rise and has become the second leading cause of end-stage renal disease, second only to various glomerulonephritis. Due to its complex metabolic disorders, once it develops into end-stage renal disease, it is often more difficult to treat than other kidney diseases. Therefore, timely prevention and treatment are of great significance for delaying diabetic nephropathy.

[0003] Galectin-3 (Gal-3) is a cytoplasmic protein and belongs to one of the members of the Gal family. It can mediate immune responses, participate in regulating renal cell apoptosis and renal tissue fibrosis, and is correlated with various kidney damages. Research has found that the expression level of Galectin-3 in patients with diabetic nephropathy is significantly higher than that of diabetic patients without diabetic nephropathy, and it can be used as a biological marker for the progression and prognosis of diabetic nephropathy.

[0004] Secreted frizzled-related protein-1 (SFRP-1) belongs to one of the members of the SFRP family and is a secreted glycoprotein, which is correlated with the occurrence of renal interstitial fibrosis. As the condition of patients with diabetic nephropathy progresses, most of them can develop glomerular sclerosis and atrophy, and renal interstitial fibrosis.

[0005] Currently, the main detection methods for the two DN indicators of Galectin-3 and SFRP-1 on the market are enzyme-linked immunosorbent assays. The enzyme-linked immunosorbent assay is complex to operate, time-consuming for detection. Patients need to wait half a day or the next day to get the test report. Moreover, the enzyme-linked immunosorbent assay requires supporting instrument equipment that occupies a large amount of desktop space and can only detect a single indicator; the enzyme-linked immunosorbent assay can only detect serum or plasma samples and cannot detect them immediately after obtaining the samples. Summary of the Invention

[0006] This application provides an antigen detection test strip and a preparation method thereof. Through the NC membrane treatment component, the quantum dot conjugate is fixed on the nitrocellulose membrane, which can achieve the rapid release of the quantum dot conjugate. It can be detected using a small amount of fingertip blood / venous whole blood / serum / plasma samples, and a report can be issued within 5 minutes. It has the advantages of convenient use, simple operation, and easy promotion.

[0007] In a first aspect, the present application provides an antigen detection test strip, adopting the following technical solution: An antigen detection test strip includes a bottom plate and a sample pad, a blank pad, a nitrocellulose membrane, and a water absorption pad that are sequentially lapped on the bottom plate in the chromatography direction; the nitrocellulose membrane is provided with a marking area, a detection line, and a quality control line; the marking area, the detection line, and the quality control line are sequentially located on the nitrocellulose membrane along the chromatography direction; The detection line is coated with a detection antibody, and the quality control line is coated with a quality control antigen or antibody; the marking area is sequentially fixed with an NC membrane treatment component and a quantum dot conjugate, and the quantum dot conjugate is superimposed on the NC membrane treatment component; the NC membrane treatment component includes a macromolecular polymer, a protein, sucrose, and a surfactant, and the quantum dot conjugate is obtained by conjugating a quantum dot microsphere with a labeled antibody.

[0008] By adopting the above technical solution, after the NC membrane treatment component is fixed on the nitrocellulose membrane, the quantum dot conjugate is then fixed. Among them, the macromolecular polymer and the protein in the NC membrane treatment component jointly seal the internal pore diameter of the nitrocellulose membrane with small molecule sucrose, so that the quantum dot conjugate can float on the membrane surface of the nitrocellulose membrane. The surfactant increases hydrophilicity, and when the sample flows through, it can promote the NC membrane treatment component to synergistically release the quantum dot conjugate quickly. Then, rapid chromatography can be achieved using the nitrocellulose membrane, and thus the detection result can be obtained quickly and accurately. The NC membrane herein is the abbreviation of the nitrocellulose membrane.

[0009] Further, in the NC membrane treatment component, the mass ratio of the macromolecular polymer, the protein, the sucrose, and the surfactant is (30 - 60):(20 - 60):(20 - 60):(1 - 2).

[0010] By adopting the above technical solution, an appropriate content of the NC membrane treatment component can promote the more rapid and stable release of the quantum dot conjugate.

[0011] Further, the macromolecular polymer includes at least one of polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol.

[0012] Further, the protein includes at least one of BSA and Casein. Among them, BSA is bovine serum albumin, and Casein is casein.

[0013] Further, the surfactant is a Tween type or a Triton type. Among them, the Tween type surfactants include but are not limited to Tween-80 and Tween-20; the Triton type surfactants include but are not limited to Triton-114 and Triton-100.

[0014] Further, the labeled antibodies are Galectin-3 antibody 1 and SFRP-1 antibody 1; the detection antibodies are Galectin-3 antibody 2 and SFRP-1 antibody 2, and Galectin-3 antibody 2 and SFRP-1 antibody 2 are coated on different detection lines.

[0015] In a second aspect, the present application provides a method for preparing an antigen detection test strip, adopting the following technical solution: A method for preparing an antigen detection test strip, comprising: Sequentially lapping and pasting a sample pad, a blank pad, a nitrocellulose membrane, and a water absorption pad on a bottom plate in the chromatography direction; Among them, the preparation method of the nitrocellulose membrane includes: Coating the detection antibody on the nitrocellulose membrane to form a detection line on the nitrocellulose membrane; coating the quality control antigen or antibody on the nitrocellulose membrane to form a quality control line on the nitrocellulose membrane; using the dissolution and drying method to sequentially fix the NC membrane treatment component and the quantum dot conjugate on the labeling area to obtain the prepared labeling area.

[0016] Still further, the preparation method of the labeling area includes: Redissolving the quantum dot conjugate in a preservation solution to obtain a quantum dot conjugate working solution; adding the NC membrane treatment component to purified water to obtain an NC membrane treatment solution; drawing the NC membrane treatment solution on the specified labeling area of the nitrocellulose membrane, drying, and fixing the NC membrane treatment component on the nitrocellulose membrane; then drawing the quantum dot conjugate working solution on the labeling area where the NC membrane treatment component is fixed, drying, and fixing the quantum dot conjugate on the nitrocellulose membrane to obtain the prepared labeling area.

[0017] Still further, the blank pad includes a blank glass fiber or a blank polyester film, preferably glass fiber. Still further, the processing steps of the sample pad are: soaking the blank gasket in the sample pad treatment solution and taking it out to dry at 37°C.

[0018] Further, the quantum dot conjugate is obtained by conjugating quantum dot microspheres with labeled antibodies, the labeled antibodies are Galectin-3 antibody 1 and SFRP-1 antibody 1; the detection antibodies are Galectin-3 antibody 2 and SFRP-1 antibody 2; the quality control antibody is goat anti-mouse IgG; The preparation method of the labeling area includes: Add the NC membrane treatment component to pure water to obtain the NC membrane treatment solution. Draw the NC membrane treatment solution with a parameter of 1.2 - 1.5 μL / cm in the marked area and dry it to obtain the marked area containing the NC membrane treatment component. Reconstitute the quantum dot conjugate in the preservation solution to obtain the quantum dot conjugate working solution. Draw the quantum dot conjugate working solution with a parameter of 0.2 - 0.3 μL / cm in the marked area and dry it to obtain the prepared marked area.

[0019] Furthermore, at a position 3 - 4 mm upward from the lower edge (near the sample loading end) of the nitrocellulose membrane, draw the NC membrane treatment solution according to the parameter of 1.2 - 1.5 μL / cm and dry it at 37°C for 10 - 30 min. On the marked area where the NC membrane treatment component is fixed, draw the quantum dot conjugate working solution according to the parameter of 0.2 - 0.3 μL / cm and dry it at 37°C for 0.5 - 1 h.

[0020] Furthermore, dilute Galectin-3 antibody 2 to a working concentration of 0.8 - 1 mg / mL with the coating solution, dilute SFRP-1 antibody 2 to a working concentration of 0.8 - 1 mg / mL with the coating solution, and dilute goat anti-mouse IgG to a working concentration of 0.8 - 1 mg / mL with the coating solution. Coat Galectin-3 antibody 2 and SFRP-1 antibody 2 on the corresponding positions of the nitrocellulose membrane respectively and dry it to form two detection lines on the nitrocellulose membrane.

[0021] Furthermore, in the quantum dot conjugate working solution, calculated by quantum dot microspheres, the concentration of the quantum dot conjugate working solution is 2.0 - 3.0 mg / ml; in the NC membrane treatment solution, the concentration of the macromolecular polymer is 15 - 30 g / L, the protein concentration is 10 - 30 g / L, the sucrose concentration is 10 - 30 g / L, and the surfactant concentration is 0.5 - 1 g / L.

[0022] Furthermore, the preparation method of the quantum dot conjugate includes the following steps: Add quantum dot microspheres to the coupling buffer, add the activator, then add Galectin-3 antibody 1 and SFRP-1 antibody 1, react in the dark, centrifuge and discard the supernatant, add the blocking solution to reconstitute and block, centrifuge and discard the supernatant to obtain the quantum dot conjugate; the composition of the blocking solution is: 3.58 - 8.96 g / L tris(hydroxymethyl)methylglycine, 5 - 10 g / L BSA.

[0023] By adopting the above technical solutions, tris(hydroxymethyl)methylglycine is an zwitterionic buffer that can maintain the stability of the reaction system. At the same time, its hydroxyl and amino small molecule structures can cooperate to block the sites that BSA fails to block. The activator includes but is not limited to EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride).

[0024] In summary, the present application has the following beneficial effects: 1. In the present application, the quantum dot conjugate is processed on the nitrocellulose membrane, and the NC membrane treatment components are pre-fixed on the nitrocellulose membrane. Compared with the traditional process of treating on the conjugate pad, the reaction time is greatly shortened, and the product repeatability is better.

[0025] 2. The Galectin-3 and SFRP-1 test strips provided by the present application are used to detect blood samples. By adding the sample once, the detection results of two indicators can be reported simultaneously within 5 minutes.

[0026] 3. During the detection process of the test strip of the present application, when Galectin-3 and SFRP-1 antigens are present in the sample, the antigens in the sample react with Galectin-3 antibody 1 and SFRP-1 antibody 1 labeled with quantum dot microspheres at the lower end of the nitrocellulose membrane, that is, the quantum dot conjugate, to form a labeled antigen-antibody complex. The complex chromatographs upward by capillary action and is captured by the corresponding test lines (T1 line coated with Galectin-3 antibody 2 and T2 line coated with SFRP-1 antibody 2) coated on the nitrocellulose membrane, and a fluorescent band appears. The complex continues to chromatograph upward and is captured by the antibody of the quality control line (goat anti-mouse IgG) coated on the nitrocellulose membrane, and a fluorescent band appears. If Galectin-3 and SFRP-1 antigens are not present in the sample, only the quality control line shows a fluorescent band.

[0027] 4. In the preparation process of the quantum dot conjugate of the present application, tris(hydroxymethyl)methyl amino acid + BSA is used as the blocking solution. Compared with the traditional blocking agent, the minimum detectable limit of the product is optimized. Description of the Drawings

[0028] Figure 1 is a schematic plan view of the test strip of the embodiment of the present application.

[0029] Figure 2 is a comparison chart of the test results of the test strip of Example 1 of the present application and the R&D ELISA kit for Galectin-3 patient serum samples.

[0030] Figure 3 is a comparison chart of the test results of the test strip of Example 1 of the present application and the R&D ELISA kit for SFRP-1 patient serum samples.

[0031] Description of the reference numerals: 1. Sample pad; 2. Blank pad; 3. Nitrocellulose membrane; 31. Labeling area; 32. Test line T1; 33. Test line T2; 34. Quality control line; 4. Absorbent pad. Detailed Embodiments

[0032] The following further describes the present application in detail with reference to the drawings and embodiments. Example

[0033] This example provides an antigen detection test strip. As Figure 1 shown, the test strip includes a bottom plate, and a sample pad 1, a blank pad 2, a nitrocellulose membrane 3, and an absorbent pad 4 that are sequentially overlapped on the bottom plate in the chromatography direction; a detection line and a quality control line 34 are provided on the nitrocellulose membrane 3. Among them, there are two detection lines in this example, which are respectively denoted as detection line T1 32 and detection line T2 33; the detection antibody is coated on the detection line, and the quality control antigen or antibody is coated on the quality control line 34.

[0034] A marking area 31 is also provided on the nitrocellulose membrane 3. The marking area 31, the detection line, and the quality control line 34 are sequentially located on the nitrocellulose membrane 3 in the chromatography direction. The chromatography direction of the sample is sample pad 1 - blank pad 2 - nitrocellulose membrane 3 (marking area 31) - nitrocellulose membrane 3 (detection line and quality control line 34).

[0035] The NC membrane treatment component and the quantum dot conjugate are sequentially fixed on the marking area 31, and the quantum dot conjugate is superimposed on the NC membrane treatment component; the NC membrane treatment component includes a macromolecular polymer, a protein, sucrose, and a surfactant, and the quantum dot conjugate is obtained by conjugating quantum dot microspheres with a labeled antibody.

[0036] In addition, it should be noted that the term "superimposed" in this application is because during the operation process, after scribing the NC membrane treatment solution on the nitrocellulose membrane and drying it, and then scribing the quantum dot conjugate working solution and drying it, the quantum dot conjugate is attached to the NC membrane treatment component. Since the operation process uses liquid, if part of the quantum dot conjugate "seeps down" between the NC membrane treatment components, it is a normal phenomenon. "Superimposed" does not limit the quantum dot conjugate to be completely above the NC membrane treatment component in the sense of mechanical structure.

[0037] The relevant solutions used in this example are prepared as follows: Coupling buffer: Weigh 1.066 g of MES (4-morpholineethanesulfonic acid) into a beaker, add 1 L of purified water, stir evenly, and store at 4°C for later use.

[0038] Coating solution preparation: Weigh 2.4 g of Tris (pH 8.0) and 20 g of trehalose into a beaker, add 1 L of purified water, stir and mix evenly, adjust the pH to 8 with HCL (hydrochloric acid) solution, and store at 4°C for later use.

[0039] Blocking solution preparation: Weigh 0.358 g of tris (hydroxymethyl) methyl amino acid and 1 g of BSA into a beaker, add 100 mL of purified water, stir evenly, and store at 4°C for later use.

[0040] Preservative solution preparation: Weigh 0.23 g / L of disodium hydrogen phosphate, 0.0456 g / L of sodium dihydrogen phosphate, 10 g of BSA, and 100 g of sucrose into a beaker, add 1 L of purified water, stir and mix well, then adjust the pH to 8 with HCL solution, and store at 4 °C for later use.

[0041] Sample pad treatment solution: Weigh 2.3 g / L of disodium hydrogen phosphate, 0.456 g / L of sodium dihydrogen phosphate, 5 g of Casein, 10 g of surfactant S21, 5 g of PVP10, and 0.2 g of RBC (red blood cells), add 1 L of purified water, stir and mix well, then store at 4 °C for later use.

[0042] In the NC membrane treatment component, the macromolecular polymers in this embodiment are preferably polyethylene glycol and polyvinylpyrrolidone; polyethylene glycol includes but is not limited to PEG20000, PEG6000, PEG4000, PEG2000; polyvinylpyrrolidone includes but is not limited to PVPK30, PVP40, PVP10.

[0043] In addition, it should be noted that: in the process of preparing the nitrocellulose membrane, the fixing order of the quality control line, the detection line, and the quantum dot conjugate can be adjusted according to the actual working conditions, and this application does not limit the fixing order.

[0044] Among them, Galectin-3 antibody 1 and SFRP-1 antibody 1 are labeled antibodies that can react with antigens, namely Galectin-3 monoclonal antibody 1 and SFRP-1 monoclonal antibody 1, which can be purchased from Jiangsu Shenji Biotechnology Co., Ltd.; Galectin-3 antibody 2 and SFRP-1 antibody 2 are detection antibodies that can capture the labeled antigen-antibody complex, which can be purchased from Jiangsu Shenji Biotechnology Co., Ltd.

[0045] The following is explained through specific examples. Example 1

[0046] This example provides a method for preparing a test strip, including the following steps: (1) Nitrocellulose membrane preparation: (1.1) Fixing of the quality control line and the detection line: Dilute Galectin-3 antibody 2 with the coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it on the fixed position of the nitrocellulose membrane at a parameter of 1 uL / cm to obtain the first detection line, denoted as detection line T1; Dilute SFRP-1 antibody 2 with the coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it on the fixed position of the nitrocellulose membrane at a parameter of 1 uL / cm to obtain the second detection line, denoted as detection line T2; Dilute goat anti-mouse IgG to 1 mg / mL with the coating solution, and use a membrane scribing instrument to coat it on the specified position of the nitrocellulose membrane at a parameter of 1 μL / cm to obtain the quality control line.

[0047] The interval between adjacent lines is 3.5 mm. Place the coated nitrocellulose membrane in an oven at 45 °C and dry it for 2 hours to obtain the nitrocellulose membrane with the quality control line and the detection line fixed.

[0048] (1.2)Preparation of the labeling area: Preparation of the quantum dot conjugate: Preparation of the blocking solution: Weigh 0.358 g of tris (hydroxymethyl) methyl amino acid and 1 g of BSA into a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use.

[0049] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, add 1250 μg of EDC, 100 μg of Galectin-3 antibody 1, and 100 μg of SFRP-1 antibody 1, and react at room temperature in the dark for 2 min. Centrifuge (temperature 4 °C, rotation speed 15000 rpm, 15 min), discard the supernatant, add 1 mL of blocking solution to block for 1 h, centrifuge (temperature 4 °C, rotation speed 15000 rpm, 15 min), and discard the supernatant to obtain the quantum dot conjugate.

[0050] Preparation of the quantum dot conjugate working solution: Add the quantum dot conjugate to 0.1 mL of the preservation solution for reconstitution to obtain the quantum dot conjugate working solution. Calculated by the mass of the quantum dot microspheres, the final concentration of the working solution is 2.5 mg / mL.

[0051] Preparation of the NC membrane treatment solution: Weigh 3 g of BSA, 3 g of PVPK30, 3 g of sucrose, and 0.05 g of Tween-20 into a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use; Fixation of the NC membrane treatment components: At 3 mm above the lower edge (near the sample loading end) of the nitrocellulose membrane obtained in step (1.1) (i.e., the labeling area), scribe the NC membrane treatment solution according to the parameter of 1.5 μL / cm, and dry it at 37 °C for 10 min to fix the NC membrane treatment components.

[0052] Fixation of the quantum dot conjugate: At the position where the NC membrane treatment components are fixed, scribe the quantum dot conjugate working solution according to the parameter of 0.2 μL / cm, and dry it at 37 °C for 0.5 h to obtain the nitrocellulose membrane.

[0053] (2)Treatment of the sample pad: Soak the blank spacer in the sample pad treatment solution, take it out and dry it at 37 °C.

[0054] (3)Assembly of test strip: The sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad are successively lapped and pasted on the PVC bottom plate in the chromatography direction, and then cut into test strips with a width of 3.5 mm. Example 2

[0055] This example provides a method for preparing a test strip, including the following steps: (1)Preparation of nitrocellulose membrane: (1.1)Fixation of quality control line and detection line: Dilute Galectin-3 antibody 2 with coating solution to 0.8 mg / mL, and coat it on the fixed position of the nitrocellulose membrane with a membrane scribing instrument at a parameter of 1 uL / cm to obtain the first detection line, denoted as detection line T1; Dilute SFRP-1 antibody 2 with coating solution to 0.8 mg / mL, and coat it on the fixed position of the nitrocellulose membrane with a membrane scribing instrument at a parameter of 1 uL / cm to obtain the second detection line, denoted as detection line T2; Dilute goat anti-mouse IgG with coating solution to 1 mg / mL, and coat it on the specified position of the nitrocellulose membrane with a membrane scribing instrument at a parameter of 1 uL / cm to obtain the quality control line.

[0056] The adjacent lines are spaced 4 mm apart. The coated nitrocellulose membrane is placed in an oven at 45 °C and dried for 2 hours to obtain a nitrocellulose membrane with a quality control line and a detection line fixed on it.

[0057] (1.2)Preparation of the labeling area: Preparation of quantum dot conjugate: Preparation of blocking solution: Weigh 0.896 g of tris (hydroxymethyl) methyl amino acid and 0.5 g of BSA into a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use.

[0058] Take 250 μg of 100 nm quantum dot microspheres and add them to 1 mL of coupling buffer, add 1250 μg of EDC, 80 μg of Galectin-3 antibody 1, and 80 μg of SFRP-1 antibody 1, and react at room temperature in the dark for 2 min. Centrifuge (at a temperature of 4 °C, a rotation speed of 15000 rpm, for 15 min), discard the supernatant, add 1 mL of blocking solution to block for 0.5 h, centrifuge (at a temperature of 4 °C, a rotation speed of 15000 rpm, for 15 min), and discard the supernatant to obtain the quantum dot conjugate.

[0059] Preparation of quantum dot conjugate working solution: Add the quantum dot conjugate to 0.1 mL of preservation solution for reconstitution to obtain the quantum dot conjugate working solution. Calculated by the mass of the quantum dot microspheres, the final concentration of the working solution is 2.5 mg / mL.

[0060] Preparation of NC membrane treatment solution: Weigh 3 g of BSA, 1.5 g of PVP10, 3 g of sucrose, and 0.05 g of Triton-100 into a beaker, add 100 mL of purified water, stir evenly, and store at 4°C for later use.

[0061] Fixation of NC membrane treatment components: At 3 mm above the lower edge (near the sample application end) of the nitrocellulose membrane obtained in step (1.1) (i.e., the marking area), draw the NC membrane treatment solution at a parameter of 1.2 μL / cm, dry it at 37°C for 10 min to fix the NC membrane treatment components.

[0062] Fixation of quantum dot conjugate: At the position where the NC membrane treatment components are fixed, draw the working solution of the quantum dot conjugate at a parameter of 0.3 μL / cm, dry it at 37°C for 0.5 h to obtain the nitrocellulose membrane.

[0063] (2) Treatment of sample pad: Soak the blank spacer in the sample pad treatment solution, take it out and dry it at 37°C.

[0064] (3) Assembly of test strip: Lap and paste the sample pad, blank glass fiber, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in the chromatography direction in sequence, and cut it into test strips with a width of 3.5 mm. Example 3

[0065] This example provides a method for preparing a test strip, including the following steps: (1) Preparation of nitrocellulose membrane: (1.1) Fixation of quality control line and detection line: Dilute Galectin-3 antibody 2 with the coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it at a parameter of 1 μL / cm at the fixed position of the nitrocellulose membrane to obtain the first detection line, denoted as detection line T1; Dilute SFRP-1 antibody 2 with the coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it at a parameter of 1 μL / cm at the fixed position of the nitrocellulose membrane to obtain the second detection line, denoted as detection line T2; Dilute goat anti-mouse IgG with the coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it at a parameter of 1 μL / cm at the specified position of the nitrocellulose membrane to obtain the quality control line.

[0066] The adjacent lines are spaced 3.5 mm apart.

[0067] The coated nitrocellulose membrane is placed in an oven at 45°C and dried for 2 hours to obtain the nitrocellulose membrane.

[0068] (1.2) Preparation of marking area: Preparation of quantum dot conjugate: Preparation of blocking solution: Weigh 0.358 g of tris (hydroxymethyl) methyl amino acid and 1 g of BSA in a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use.

[0069] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, add 1250 μg of EDC, 80 μg of Galectin-3 antibody 1, and 100 μg of SFRP-1 antibody 1, and react at room temperature in the dark for 2 min. Centrifuge (at 4 °C, 15000 rpm, for 15 min), discard the supernatant, add 1 mL of blocking solution to block for 1 h, centrifuge (at 4 °C, 15000 rpm, for 15 min), and discard the supernatant to obtain the quantum dot conjugate.

[0070] Preparation of working solution of quantum dot conjugate: Dissolve the quantum dot conjugate in 0.1 mL of preservation solution to obtain the working solution of the quantum dot conjugate. Calculated based on the mass of the quantum dot microspheres, the final concentration of the working solution is 2.5 mg / mL.

[0071] Preparation of NC membrane treatment solution: Weigh 60 mL of 5% (50 g / L) Casein solution, 1.5 g of PVPK30, 1 g of sucrose, and 0.1 g of Tween-80 in a beaker, add 40 mL of purified water, stir evenly, and store at 4 °C for later use.

[0072] Fixation of NC membrane treatment components: At 3 mm above the lower edge (near the sample application end) of the nitrocellulose membrane obtained in step (1.1) (i.e., the marking area), draw the NC membrane treatment solution according to the parameter of 1.2 μL / cm, and dry at 37 °C for 20 min to fix the NC membrane treatment components.

[0073] Fixation of quantum dot conjugate: At the position where the NC membrane treatment components are fixed, draw the working solution of the quantum dot conjugate according to the parameter of 0.2 μL / cm, and dry at 37 °C for 0.5 h to obtain the nitrocellulose membrane.

[0074] (2) Treatment of sample pad: Soak the blank spacer in the sample pad treatment solution, take it out and dry at 37 °C.

[0075] (3) Assembly of test strip: Lap and paste the sample pad, blank glass fiber, nitrocellulose membrane, and absorbent pad on the PVC bottom plate in the chromatography direction in sequence, and cut into test strips with a width of 3.5 mm. Example 4

[0076] This example provides a method for preparing a test strip, including the following steps: (1)Preparation of nitrocellulose membrane: (1.1)Fixation of quality control line and detection line: Dilute Galectin-3 antibody 2 with coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it on the fixed position of the nitrocellulose membrane at a parameter of 1 μL / cm to obtain the first detection line, denoted as detection line T1; Dilute SFRP-1 antibody 2 with coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it on the fixed position of the nitrocellulose membrane at a parameter of 1 μL / cm to obtain the second detection line, denoted as detection line T2; Dilute goat anti-mouse IgG with coating solution to 1 mg / mL, and use a membrane scribing instrument to coat it on the specified position of the nitrocellulose membrane to obtain the quality control line.

[0077] The adjacent lines are spaced 4 mm apart.

[0078] Place the coated nitrocellulose membrane in an oven at 45 °C and dry it for 2 hours to obtain the nitrocellulose membrane.

[0079] (1.2)Preparation of the labeling area: Preparation of quantum dot conjugate: Preparation of blocking solution: Weigh 0.358 g of tris (hydroxymethyl) methyl amino acid and 1 g of BSA in a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use.

[0080] Take 250 μg of 100 nm quantum dot microspheres and add them to 1 mL of coupling buffer. Add 1250 μg of EDC, 100 μg of Galectin-3 antibody 1, and 80 μg of SFRP-1 antibody 1, and react at room temperature in the dark for 2 min. Centrifuge (temperature 4 °C, rotation speed 15000 rpm, 15 min), discard the supernatant, add 1 mL of blocking solution to block for 0.5 h, centrifuge (temperature 4 °C, rotation speed 15000 rpm, 15 min), and discard the supernatant to obtain the quantum dot conjugate.

[0081] Preparation of quantum dot conjugate working solution: Add the quantum dot conjugate to 0.1 mL of preservation solution for reconstitution to obtain the quantum dot conjugate working solution. Calculated based on the mass of the quantum dot microspheres, the final concentration of the working solution is 2.5 mg / mL.

[0082] Configuration of NC membrane treatment solution: Weigh 20 mL of 5% (50 g / L) Casein solution, 3 g of PEG20000, 3 g of sucrose, and 0.05 g of Triton-114 in a beaker, add 100 mL of purified water, stir evenly, and store at 4 °C for later use.

[0083] Fixation of NC membrane treatment components: At 3 mm above the lower edge (near the sample application end) of the nitrocellulose membrane obtained in step (1.1) (i.e., the marking area), draw the NC membrane treatment solution at a parameter of 1.5 μL / cm, dry it at 37 °C for 30 min to fix the NC membrane treatment components.

[0084] Quantum dot conjugate fixation: At the position where the NC membrane treatment components are fixed, draw the quantum dot conjugate working solution at a parameter of 0.3 μL / cm, dry it at 37 °C for 1 h to obtain the nitrocellulose membrane.

[0085] (2) Treatment of the sample pad: Soak the blank spacer in the sample pad treatment solution, take it out and dry it at 37 °C.

[0086] (3) Assembly of the test strip: Lap and paste the sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad in sequence in the chromatography direction on the PVC bottom plate, and cut it into test strips with a width of 3 mm.

[0087] Comparative example The difference between Comparative Example 1 and Example 1 is that when preparing the quantum dot conjugate, the blocking solution is prepared as follows: Weigh 0.5 g of BSA in a beaker, add 100 mL of purified water, stir evenly, and store it at 4 °C for later use.

[0088] The difference between Comparative Example 2 and Example 1 is that: The quantum dot conjugate working solution is sprayed on a conventional conjugate pad (glass fiber) to obtain the nitrocellulose membrane.

[0089] The difference between Comparative Example 3 and Example 1 is that: The preparation of the NC membrane treatment solution and the fixation of the NC membrane treatment components are not carried out.

[0090] The difference between Comparative Example 4 and Example 1 is that: The NC membrane treatment solution is prepared as follows: Weigh 3 g of PVPK30, 3 g of sucrose, 0.05 g of Tween-20 in a beaker, add 100 mL of purified water, stir evenly, and store it at 4 °C for later use.

[0091] The difference between Comparative Example 5 and Example 1 is that: The NC membrane treatment solution is prepared as follows: Weigh 3 g of BSA, 3 g of sucrose, 0.05 g of Tween-20 in a beaker, add 100 mL of purified water, stir evenly, and store it at 4 °C for later use.

[0092] Performance detection 1. Interpretation time test Three Galectin-3 positive samples confirmed by the R&D ELISA kit (weak positive: 0.15 ng / mL, medium positive: 1.38 ng / mL, strong positive: 4.12 ng / mL), and three SFRP-1 positive samples confirmed by the R&D ELISA kit (weak positive: 0.18 ng / mL, medium positive: 1.45 ng / mL, strong positive: 4.19 ng / mL) were taken. The weak positive samples of Galectin-3 and SFRP-1 were mixed in equal proportions, the medium positive samples were mixed in equal proportions, and the strong positive samples were mixed in equal proportions, and then were recorded as Sample 1, Sample 2, and Sample 3 in sequence. The test strips prepared in the examples and comparative examples were used to detect them. The samples were serum samples, and the test results are shown in Table 1.

[0093] Table 1 Interpretation time Test results

[0094] As can be seen from Table 1, for Examples 1-4 and Comparative Example 1, the results can be interpreted in 3-5 minutes, and the readings at 5 minutes, 8 minutes, 15 minutes, and 30 minutes are consistent, without time evolution; for Comparative Example 2, the results can be interpreted in 15 minutes, and the value still increases by 5% at 30 minutes, with a large time evolution. Table 1 shows that the method of fixing the quantum dot conjugate on the nitrocellulose membrane in this application can give results quickly. Subsequently, for Examples 1-4 and Comparative Example 1, the results were interpreted at 5 minutes, and for Comparative Example 2, the results were interpreted at 15 minutes.

[0095] 2. Repeatability test A serum sample with a concentration of 1.43 ng / mL was obtained by mixing Galectin-3 positive samples confirmed by the R&D ELISA kit, and a serum sample with a concentration of 3.439 ng / mL was obtained by mixing SFRP-1 positive samples confirmed by the R&D ELISA kit. The repeatability was detected after mixing the Galectin-3 mixed sample and the SFRP-1 mixed sample in equal proportions. The test results are shown in Table 2-1 and Table 2-2. Among them, AV is the average value, SD is the standard deviation, and CV is the coefficient of variation.

[0096] Table 2-1 Repeatability test results of examples

[0097] Table 2-2 Repeatability test results of comparative examples

[0098] As can be seen from Table 2-1 and Table 2-2, the repeatability of Examples 1-4 and Comparative Example 1 is significantly better than that of Comparative Example 2, indicating that the method of fixing the quantum dot conjugate on the nitrocellulose membrane in this application has better product repeatability; The repeatability of Example 1 is significantly better than that of Comparative Example 3, indicating that the NC membrane treatment solution provided by the present application can promote the uniform and effective release of the quantum dot conjugate; The repeatability of Example 1 is better than that of Comparative Example 4 and Comparative Example 5, indicating that in the NC membrane treatment components provided by the present application, the macromolecular polymer and the protein can promote the better release of the quantum dot conjugate.

[0099] 3. Sample testing Take the Galectin-3 positive serum samples confirmed by the R&D ELISA kit and dilute them with negative serum to 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 1.6 ng / mL, 3.2 ng / mL, 6.4 ng / mL, and test the samples. The fluorescence value test results are shown in Table 3.

[0100] Take the SFRP-1 positive serum samples confirmed by the R&D ELISA kit and dilute them with negative serum to 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 1.6 ng / mL, 3.2 ng / mL, 6.4 ng / mL, and test the samples. The signal value (fluorescence value) test results are shown in Table 4.

[0101] Table 3 Detection results of Galectin-3 samples

[0102] Table 4 Detection results of SFRP-1 samples

[0103]

[0104] The greater the difference between the signal value at the 0.05 ng / mL concentration point and the signal value of the negative sample, the lower the minimum detection limit and the better the product sensitivity; the closer the ratio of the signal value at the 6.4 ng / mL concentration point to the signal value at the 0.05 ng / mL concentration point is to 128 (the multiple difference between the concentrations of 6.4 ng / mL and 0.05 ng / mL), the better. The greater the multiple difference, the more accurate the conversion result; As can be seen from Table 3 and Table 4, in Examples 1-4 and Comparative Example 2, the difference between the signal value at the 0.05 ng / mL concentration point and the signal value of the negative sample is larger than that in Comparative Example 1, indicating that its minimum detection limit is lower than that in Comparative Example 1, indicating that the blocking solution used in the present application can improve the product sensitivity; As can be seen from Table 3 and Table 4, in Examples 1-4, the ratio of the signal value at the 6.4 ng / mL concentration point to the signal value at the 0.05 ng / mL concentration point is larger than that in Comparative Example 2, indicating that its linear gradient is better than that in Comparative Example 2, indicating that the method of fixing the quantum dot conjugate on the nitrocellulose membrane in the present application can obtain a better linear gradient; As can be seen from Table 3 and Table 4, the ratio of the signal value at the 6.4 ng / mL concentration point to the signal value at the 0.05 ng / mL concentration point in Example 1 is larger than that in Comparative Example 3 and Comparative Example 4, indicating that its linear gradient is better than that in Comparative Example 3 and Comparative Example 4, which shows that the NC membrane treatment component in this application can better promote the release of the quantum dot conjugate and obtain a better linear gradient.

[0105] Examples 1-4 can establish a polynomial mathematical model of signal value-concentration value with the T value signal value as the abscissa and the concentration value as the ordinate to back-calculate the sample concentration value according to the detected signal value.

[0106] 4. Sample type test Take peripheral blood and venous whole blood from Galectin-3 positive patients, and separate serum and plasma from the venous whole blood; take peripheral blood and venous whole blood from SFRP-1 positive patients, and separate serum and plasma from the venous whole blood. Use Examples 1-4 to establish a polynomial mathematical model of signal value-concentration value with the T value signal value as the abscissa and the concentration value as the ordinate, and use the test strip of Example 1 to detect the above sample types respectively. The test results are shown in Table 5 below.

[0107] Table 5 Test results of sample types

[0108] As can be seen from Table 5, in Example 1, the test results of Galectin-3 samples and SFRP-1 samples for peripheral blood, venous whole blood, serum, and plasma are consistent. The kit for rapid detection of Galectin-3 and SFRP-1 in blood prepared in this application can be used for the detection of peripheral blood / venous whole blood / serum / plasma.

[0109] 5. Accuracy Take 50 serum samples from Galectin-3 patients and 50 serum samples from SFRP-1 patients, compare the test results of the test strip prepared in Example 1 and the test results of the R&D ELISA kit. The test results are shown in Figure 2 and Figure 3 . From Figure 2 and Figure 3 it can be seen that the test results of the test strip prepared in Example 1 are highly consistent with the test results of the R&D ELISA kit. The test strip prepared in this application can obtain test results quickly and has high accuracy.

[0110] Generally speaking, the test strip prepared in the examples of this application can not only quickly measure the results within five minutes, but also has high accuracy and excellent repeatability. A small amount of peripheral blood / venous whole blood / serum / plasma samples can be detected, and it has the advantages of convenient use, simple operation, and easy promotion.

[0111] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. An antigen detection test strip, characterized in that, It includes a bottom plate, a sample pad, a blank pad, a nitrocellulose membrane, and an absorbent pad that are sequentially lapped on the bottom plate in the chromatographic direction; a marking area, a detection line, and a quality control line are provided on the nitrocellulose membrane; the marking area, the detection line, and the quality control line are sequentially located on the nitrocellulose membrane along the chromatographic direction. The detection line is coated with a detection antibody, and the quality control line is coated with a quality control antigen or antibody; an NC membrane treatment component and a quantum dot conjugate are sequentially fixed on the marking area, and the quantum dot conjugate is superimposed on the NC membrane treatment component; the NC membrane treatment component includes a macromolecular polymer, a protein, sucrose, and a surfactant, and the quantum dot conjugate is obtained by conjugating a quantum dot microsphere with a labeled antibody.

2. The antigen detection test strip according to claim 1, characterized in that, In the NC membrane treatment component, the mass ratio of the macromolecular polymer, the protein, sucrose, and the surfactant is (30 - 60):(20 - 60):(20 - 60):(1 - 2).

3. The antigen detection test strip according to claim 1, wherein, The macromolecular polymer includes at least one of polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol.

4. An antigen detection test strip according to claim 1, characterized in that, The protein includes at least one of BSA and Casein.

5. An antigen detection test strip according to claim 1, characterized in that, The surfactant includes at least one of Tween and Triton.

6. The antigen detection test strip according to claim 1, wherein The labeled antibody is Galectin-3 antibody 1 and SFRP-1 antibody 1; the detection antibody is Galectin-3 antibody 2 and SFRP-1 antibody 2, and Galectin-3 antibody 2 and SFRP-1 antibody 2 are coated on different detection lines.

7. A method for preparing an antigen detection test strip according to any one of claims 1-6, characterized in that, It includes: Lap and paste the sample pad, the blank pad, the nitrocellulose membrane, and the absorbent pad on the bottom plate in sequence along the chromatographic direction. Among them, the preparation method of the nitrocellulose membrane includes: Coat the detection antibody on the nitrocellulose membrane to form a detection line on the nitrocellulose membrane. Coat the quality control antigen or antibody on the nitrocellulose membrane to form a quality control line on the nitrocellulose membrane; use the dissolution and drying method to sequentially fix the NC membrane treatment component and the quantum dot conjugate on the marking area to obtain the prepared marking area.

8. The preparation method of an antigen detection test strip according to claim 7, wherein The quantum dot conjugate is obtained by conjugating a quantum dot microsphere with a labeled antibody, the labeled antibody is Galectin-3 antibody 1 and SFRP-1 antibody 1; the detection antibody is Galectin-3 antibody 2 and SFRP-1 antibody 2; The quality control antibody is goat anti-mouse IgG. The preparation method of the marking area includes: Add the NC membrane treatment component to purified water to obtain an NC membrane treatment solution, demarcate the NC membrane treatment solution with a parameter of 1.2 - 1.5 uL / cm on the marking area, and dry it to obtain a marking area containing the NC membrane treatment component; dissolve and reconstitute the quantum dot conjugate in a preservation solution to obtain a quantum dot conjugate working solution, demarcate the quantum dot conjugate working solution with a parameter of 0.2 - 0.3 uL / cm on the marking area, and dry it to obtain the prepared marking area.

9. The preparation method of an antigen detection test strip according to claim 8, characterized in that, In the quantum dot conjugate working solution, calculated by quantum dot microspheres, the concentration of the quantum dot conjugate working solution is 2.0 - 3.0 mg / ml; in the NC membrane treatment solution, the concentration of the macromolecular polymer is 15 - 30 g / L, the protein concentration is 10 - 30 g / L, the sucrose concentration is 10 - 30 g / L, and the surfactant concentration is 0.5 - 1 g / L.

10. The preparation method of an antigen detection test strip according to claim 8, characterized in that, The preparation method of the quantum dot conjugate includes the following steps: Add quantum dot microspheres into the coupling buffer solution, add the activator, then add Galectin-3 antibody 1 and SFRP-1 antibody 1, react in the dark, centrifuge to discard the supernatant, add the blocking solution to redissolve and block, centrifuge to discard the supernatant to obtain the quantum dot conjugate; the composition of the blocking solution is: 3.58 - 8.96 g / L tris(hydroxymethyl)methyl amino acid, 5 - 10 g / L BSA.

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