Antigen detection test strip and preparation method thereof

Abstract

The present application relates to the field of rapid diagnostic technology, and more specifically, discloses an antigen detection test strip and a preparation method thereof; the test strip comprises a base plate and a sample pad, a blank pad, a nitrocellulose membrane and a water-absorbing pad sequentially overlapped on the base 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; 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 present application can achieve rapid release of the labeled conjugate, and can be detected using a small amount of peripheral blood / venous whole blood / serum / plasma sample, and a report can be issued within 5 minutes. It has the advantages of being easy to use, simple to operate and easy to promote.

Description

Technical Field

[0001] The present application relates to the field of rapid diagnosis technology, and more specifically, to an antigen detection test strip and a preparation method thereof. Background Art

[0002] Diabetic nephropathy (DN) is a condition characterized by proteinuria and a progressive decrease in glomerular filtration rate (GFR) due to long-term diabetes. It is one of the most important complications in diabetic patients. Its incidence in my country is also on the rise, making it the second leading cause of end-stage renal disease, second only to various glomerulonephritides. Due to the complex metabolic disorders involved, DN, once it develops, is often more challenging to treat than other kidney diseases. Therefore, timely prevention and treatment are crucial for delaying the progression of diabetic nephropathy.

[0003] Galectin-3 (Gal-3) is a cytoplasmic protein and a member of the Gal family. It can mediate immune responses, participate in regulating renal cell apoptosis and renal tissue fibrosis, and is associated with various types of renal damage. Studies have found that Galectin-3 expression levels are significantly higher in patients with diabetic nephropathy than in diabetic patients without diabetic nephropathy. It can serve as a biomarker for the progression and prognosis of diabetic nephropathy.

[0004] Secreted frizzled-related protein-1 (SFRP-1), a member of the SFRP family, is a secreted glycoprotein that is associated with the development of renal interstitial fibrosis. As diabetic nephropathy progresses, most patients develop glomerular sclerosis and atrophy, as well as renal interstitial fibrosis.

[0005] Currently, the primary method for detecting DN, Galectin-3 and SFRP-1, on the market is enzyme-linked immunosorbent assay (ELISA). ELISA is complex and time-consuming, requiring patients to wait half a day or the next day for test results. Furthermore, ELISA requires supporting equipment, which takes up considerable desktop space, and can only detect a single indicator. ELISA can only test serum or plasma samples, and cannot be performed immediately after sample acquisition. Summary of the Invention

[0006] The present application provides an antigen detection test strip and a preparation method thereof. The present application fixes the quantum dot conjugate on the nitrocellulose membrane through the NC membrane processing component, which can achieve the rapid release of the quantum dot conjugate. A small amount of peripheral blood / venous whole blood / serum / plasma sample can be used for detection, and a report can be issued within 5 minutes. It has the advantages of easy use, simple operation, and easy promotion.

[0007] In a first aspect, the present application provides an antigen detection test strip, which adopts the following technical solution:

[0008] An antigen detection test strip comprises a base plate and a sample pad, a blank pad, a nitrocellulose membrane and a water-absorbing pad sequentially overlapped on the base plate in a 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;

[0009] The detection line is coated with a detection antibody, and the quality control line is coated with a quality control antigen or antibody; the NC membrane treatment component and the quantum dot conjugate are fixed in sequence on the labeling area, and the quantum dot conjugate is superimposed on the NC membrane treatment component; the NC membrane treatment component includes a macromolecular polymer, protein, sucrose and a surfactant, and the quantum dot conjugate is prepared by coupling quantum dot microspheres with labeled antibodies.

[0010] By adopting the above technical solution, the NC membrane treatment component is fixed to the nitrocellulose membrane, and then the quantum dot conjugate is fixed. The macromolecular polymer and protein in the NC membrane treatment component, combined with the small molecule sucrose, jointly block the internal pores of the nitrocellulose membrane, allowing the quantum dot conjugate to float on the membrane surface. The surfactant increases the hydrophilicity of the nitrocellulose membrane, promoting the rapid release of the NC membrane treatment component and the quantum dot conjugate when the sample flows through. The nitrocellulose membrane can then be used for rapid chromatography, thereby quickly and accurately obtaining test results. NC membrane is the abbreviation for nitrocellulose membrane.

[0011] Furthermore, in the NC membrane treatment components, the mass ratio of the macromolecular polymer, protein, sucrose and surfactant is (30-60): (20-60): (20-60): (1-2).

[0012] By adopting the above technical solution and adjusting the appropriate content of the NC membrane treatment components, the quantum dot conjugates can be released more quickly and stably.

[0013] Furthermore, the macromolecular polymer includes at least one of polyvinyl pyrrolidone, polyvinyl alcohol, and polyethylene glycol.

[0014] Furthermore, the protein includes at least one of BSA and Casein, wherein BSA is bovine serum albumin and Casein is casein.

[0015] Furthermore, the surfactant is a Tween or Triton surfactant, wherein Tween surfactants include but are not limited to Tween-80 and Tween-20; Triton surfactants include but are not limited to Triton-114 and Triton-100.

[0016] Furthermore, 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.

[0017] In a second aspect, the present application provides a method for preparing an antigen detection test strip, which adopts the following technical solution:

[0018] A method for preparing an antigen detection test strip, comprising:

[0019] Paste the sample pad, blank pad, nitrocellulose membrane and absorbent pad on the bottom plate in sequence according to the chromatography direction;

[0020] The preparation method of the nitrocellulose membrane comprises:

[0021] The detection antibody is coated on the nitrocellulose membrane to form a detection line on the nitrocellulose membrane; the quality control antigen or antibody is coated on the nitrocellulose membrane to form a quality control line on the nitrocellulose membrane; the NC membrane treatment component and the quantum dot conjugate are fixed on the labeling area in sequence by using the dissolution and drying method to obtain a prepared labeling area.

[0022] Furthermore, the method for preparing the marking region includes:

[0023] The quantum dot conjugate is added to the preservation solution for redissolution to obtain a quantum dot conjugate working solution; the NC membrane treatment component is added to purified water to obtain an NC membrane treatment solution; the NC membrane treatment solution is drawn on the specified marking area of ​​the nitrocellulose membrane, dried, and the NC membrane treatment component is fixed on the nitrocellulose membrane; the quantum dot conjugate working solution is then drawn on the marking area where the NC membrane treatment component is fixed, dried, and the quantum dot conjugate is fixed on the nitrocellulose membrane to obtain a prepared marking area.

[0024] Furthermore, the blank pad comprises blank glass fiber or blank polyester film, preferably glass fiber. Furthermore, the sample pad treatment step comprises: soaking the blank pad in a sample pad treatment solution, taking it out and drying it at 37°C.

[0025] Furthermore, the quantum dot conjugate is prepared by coupling 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;

[0026] The method for preparing the labeling region comprises:

[0027] The NC membrane treatment component is added to purified water to obtain an NC membrane treatment solution, and the NC membrane treatment solution with a parameter of 1.2-1.5 uL / cm is delineated in the marking area, and dried to obtain a marking area containing the NC membrane treatment component; the quantum dot conjugate is added to the preservation solution for re-dissolution to obtain a quantum dot conjugate working solution, and the quantum dot conjugate working solution with a parameter of 0.2-0.3 uL / cm is delineated in the marking area, and dried to obtain a prepared marking area.

[0028] Furthermore, 3-4 mm above the lower edge of the nitrocellulose membrane (near the sample loading end), draw the NC membrane treatment solution at a parameter of 1.2-1.5 uL / 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 at a parameter of 0.2-0.3 uL / cm, and dry it at 37°C for 0.5-1 h.

[0029] Furthermore, the Galectin-3 antibody 2 was diluted with coating solution to a working concentration of 0.8-1 mg / mL, the SFRP-1 antibody 2 was diluted with coating solution to a working concentration of 0.8-1 mg / mL, and the goat anti-mouse IgG was diluted with coating solution to a working concentration of 0.8-1 mg / mL. The Galectin-3 antibody 2 and SFRP-1 antibody 2 were respectively coated on the corresponding positions of the nitrocellulose membrane and dried to form two detection lines on the nitrocellulose membrane.

[0030] Furthermore, in the quantum dot conjugate working solution, the concentration of the quantum dot conjugate working solution is 2.0-3.0 mg / ml, calculated as quantum dot microspheres; 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.

[0031] Furthermore, the preparation method of the quantum dot conjugate includes the following steps: adding quantum dot microspheres to a coupling buffer, adding an activator and then adding Galectin-3 antibody 1 and SFRP-1 antibody 1, reacting in the dark, centrifuging and discarding the supernatant, adding a blocking solution for re-dissolution and blocking, centrifuging and discarding the supernatant, to obtain a quantum dot conjugate; the blocking solution is composed of: 3.58-8.96g / L tris(hydroxymethyl)methylamino acid, 5-10g / L BSA.

[0032] By adopting the above technical solution, tris(hydroxymethyl)methylamino acid, a zwitterionic buffer, can maintain the stability of the reaction system. At the same time, its hydroxyl and amino groups can synergistically block sites that BSA fails to block. Activators include but are not limited to EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride).

[0033] In summary, this application has the following beneficial effects:

[0034] 1. This application processes the quantum dot conjugates on a nitrocellulose membrane and fixes the NC membrane processing components on the nitrocellulose membrane in advance. Compared with the traditional process of processing on a binding pad, the reaction time is greatly shortened and the product reproducibility is better.

[0035] 2. The Galectin-3 and SFRP-1 test strips provided in this application detect blood samples, add the sample once, and report the test results of both indicators simultaneously within 5 minutes.

[0036] 3. During the testing process of the test strips of this application, when Galectin-3 or SFRP-1 antigens are present in the sample, the antigens in the sample react with the quantum dot microspheres labeled with Galectin-3 antibody 1 and SFRP-1 antibody 1, i.e., the quantum dot conjugate, at the bottom of the nitrocellulose membrane, forming a labeled antigen-antibody complex. The complex flows upward through capillary action and is captured by the corresponding test lines coated on the nitrocellulose membrane (line T1 coated with Galectin-3 antibody 2, line T2 coated with SFRP-1 antibody 2), resulting in the development of fluorescent bands. The complex continues to flow upward and is captured by the control line (goat anti-mouse IgG) antibody coated on the nitrocellulose membrane, resulting in the development of fluorescent bands. If the sample does not contain Galectin-3 or SFRP-1 antigens, only the control line will develop fluorescent bands.

[0037] 4. In the preparation process of quantum dot conjugates, this application uses tris (hydroxymethyl) methyl amino acid + BSA as a blocking solution. Compared with traditional blocking agents, the minimum detection limit of the product is optimized. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a plan view of the test strip according to the embodiment of the present application.

[0039] Figure 2 This is a comparison chart of the test results of Galectin-3 patient serum samples using the test strips in Example 1 of the present application and the R&D Elisa kit.

[0040] Figure 3 This is a comparison chart of the test results of the test strip in Example 1 of the present application and the R&D Elisa kit for SFRP-1 patient serum samples.

[0041] Explanation of the accompanying symbols: 1. Sample pad; 2. Blank pad; 3. Nitrocellulose membrane; 31. Marking area; 32. Detection line T1; 33. Detection line T2; 34. Quality control line; 4. Water-absorbing pad. DETAILED DESCRIPTION

[0042] The present application is further described in detail below with reference to the accompanying drawings and examples. Example

[0043] This embodiment provides an antigen detection test strip, such as Figure 1 As shown, the test strip includes a base plate and a sample pad 1, a blank pad 2, a nitrocellulose membrane 3 and a water-absorbing pad 4 sequentially overlapped on the base plate in the chromatography direction; a detection line and a quality control line 34 are provided on the nitrocellulose membrane 3, wherein there are two detection lines in this embodiment, respectively denoted as detection line T1 32 and detection line T2 33; the detection line is coated with a detection antibody, and the quality control line 34 is coated with a quality control antigen or antibody.

[0044] The nitrocellulose membrane 3 is also provided with a marking area 31. The marking area 31, the detection line and the quality control line 34 are located on the nitrocellulose membrane 3 in sequence along 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).

[0045] The labeling area 31 is fixed with NC membrane treatment components and quantum dot conjugates in sequence, and the quantum dot conjugates are superimposed on the NC membrane treatment components; the NC membrane treatment components include macromolecular polymers, proteins, sucrose and surfactants, and the quantum dot conjugates are prepared by coupling quantum dot microspheres with labeled antibodies.

[0046] It should also be noted that the "superposition" mentioned in this application is because during the operation, the nitrocellulose membrane is smeared with NC membrane treatment liquid and then dried, and then the quantum dot conjugate working liquid is applied and dried, so that the quantum dot conjugate is attached to the NC membrane treatment component. Since the operation process uses liquid, if some quantum dot conjugates "seep" into the NC membrane treatment components, it is a normal phenomenon. "Superposition" does not limit the quantum dot conjugate to being completely above the NC membrane treatment component in the mechanical structure sense.

[0047] The relevant solutions used in this embodiment are configured as follows:

[0048] Coupling buffer: Weigh 1.066 g of MES (4-morpholineethanesulfonic acid) into a beaker, add 1 L of purified water, stir well, and store at 4°C until use.

[0049] Coating solution preparation: Weigh 2.4g Tris (pH 8.0) and 20g trehalose in a beaker, add 1L purified water, stir and mix, adjust the pH to 8 with HCl (hydrochloric acid) solution, and store at 4℃ until use.

[0050] Preparation of blocking solution: weigh 0.358 g tris(hydroxymethyl)methylamino acid and 1 g BSA into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0051] Preparation of storage solution: Weigh 0.23 g / L disodium hydrogen phosphate, 0.0456 g / L sodium dihydrogen phosphate, 10 g BSA, and 100 g sucrose into a beaker, add 1 L purified water, stir to mix, adjust the pH to 8 with HCl solution, and store at 4°C until use.

[0052] Sample pad treatment solution: Weigh 2.3 g / L disodium hydrogen phosphate, 0.456 g / L sodium dihydrogen phosphate, 5 g Casein, 10 g surfactant S21, 5 g PVP10, and 0.2 g RBC (red blood cells), add 1 L of purified water, stir to mix, and store at 4°C until used.

[0053] Among the NC membrane treatment components, the macromolecular polymers in this embodiment are preferably polyethylene glycol and polyvinyl pyrrolidone; polyethylene glycol includes but is not limited to PEG20000, PEG6000, PEG4000, PEG2000; polyvinyl pyrrolidone includes but is not limited to PVPK30, PVP40, PVP10.

[0054] It should also be noted that: in the process of preparing 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.

[0055] 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 labeled antigen-antibody complexes, which can be purchased from Jiangsu Shenji Biotechnology Co., Ltd.

[0056] The following is an explanation through specific examples. Example 1

[0057] This embodiment provides a method for preparing a test strip, comprising the following steps:

[0058] (1) Preparation of nitrocellulose membrane:

[0059] (1.1) Fixing of quality control line and test line:

[0060] Galectin-3 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain the first detection line, which was recorded as detection line T1;

[0061] SFRP-1 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain a second detection line, which was recorded as detection line T2;

[0062] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it on the specified position of nitrocellulose membrane with a film streaker at a parameter of 1 uL / cm to obtain a quality control line.

[0063] The interval between adjacent lines was 3.5 mm. The coated nitrocellulose membrane was placed in a 45°C oven and dried for 2 hours to obtain a nitrocellulose membrane with fixed quality control lines and test lines.

[0064] (1.2) Preparation of the labeling area:

[0065] Preparation of quantum dot conjugates:

[0066] Preparation of blocking solution: weigh 0.358 g tris(hydroxymethyl)methylamino acid and 1 g BSA into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0067] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, followed by 1250 μg of EDC, 100 μg of Galectin-3 antibody 1, and 100 μg of SFRP-1 antibody 1. Incubate at room temperature in the dark for 2 minutes. Centrifuge (4°C, 15,000 rpm, 15 minutes), discard the supernatant, add 1 mL of blocking buffer, block for 1 hour, and centrifuge (4°C, 15,000 rpm, 15 minutes). Discard the supernatant to obtain the quantum dot conjugate.

[0068] Preparation of quantum dot conjugate working solution:

[0069] The quantum dot conjugate was added to 0.1 mL of the preservation solution for re-dissolution to obtain the quantum dot conjugate working solution. The final concentration of the working solution was 2.5 mg / mL based on the mass of the quantum dot microspheres.

[0070] NC membrane treatment liquid configuration:

[0071] Weigh 3 g BSA, 3 g PVPK30, 3 g sucrose, and 0.05 g Tween-20 into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use;

[0072] NC membrane treatment component fixation:

[0073] 3 mm above the lower edge of the nitrocellulose membrane (near the sample loading end) obtained in step (1.1) (i.e., the marked area), draw the NC membrane treatment solution at a parameter of 1.5 uL / cm, and dry it at 37°C for 10 min to fix the NC membrane treatment components.

[0074] Quantum dot conjugate immobilization:

[0075] At the position where the NC membrane treatment component is fixed, the quantum dot conjugate working solution is applied at a parameter of 0.2 uL / cm, and dried at 37° C. for 0.5 h to obtain a nitrocellulose membrane.

[0076] (2) Sample pad processing:

[0077] Soak the blank pad in the sample pad treatment solution and take it out to dry at 37℃.

[0078] (3) Test strip assembly:

[0079] The sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3.5 mm. Example 2

[0080] This embodiment provides a method for preparing a test strip, comprising the following steps:

[0081] (1) Preparation of nitrocellulose membrane:

[0082] (1.1) Fixing of quality control line and test line:

[0083] Galectin-3 antibody 2 was diluted to 0.8 mg / mL with coating solution and coated on a fixed position of nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain the first detection line, which was recorded as detection line T1;

[0084] SFRP-1 antibody 2 was diluted to 0.8 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain a second detection line, which was recorded as detection line T2;

[0085] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it on the specified position of nitrocellulose membrane with a film streaker at a parameter of 1 uL / cm to obtain a quality control line.

[0086] The interval between adjacent lines is 4 mm. The coated nitrocellulose membrane is placed in a 45°C oven and dried for 2 hours to obtain a nitrocellulose membrane with fixed quality control lines and test lines.

[0087] (1.2) Preparation of the labeling area:

[0088] Preparation of quantum dot conjugates:

[0089] Preparation of blocking solution: weigh 0.896 g tris(hydroxymethyl)methylamino acid and 0.5 g BSA into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0090] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, followed by 1250 μg of EDC, 80 μg of Galectin-3 antibody 1, and 80 μg of SFRP-1 antibody 1. Incubate at room temperature in the dark for 2 minutes. Centrifuge (4°C, 15,000 rpm, 15 minutes), discard the supernatant, add 1 mL of blocking buffer, block for 0.5 hours, and centrifuge (4°C, 15,000 rpm, 15 minutes). Discard the supernatant to obtain the quantum dot conjugate.

[0091] Preparation of quantum dot conjugate working solution:

[0092] The quantum dot conjugate was added to 0.1 mL of the preservation solution for re-dissolution to obtain the quantum dot conjugate working solution. The final concentration of the working solution was 2.5 mg / mL based on the mass of the quantum dot microspheres.

[0093] NC membrane treatment liquid configuration:

[0094] Weigh 3 g BSA, 1.5 g PVP10, 3 g sucrose, and 0.05 g Triton-100 into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0095] NC membrane treatment component fixation:

[0096] 3 mm above the lower edge of the nitrocellulose membrane (near the sample loading end) obtained in step (1.1) (i.e., the marked area), draw the NC membrane treatment solution at a parameter of 1.2 uL / cm, and dry it at 37°C for 10 min to fix the NC membrane treatment components.

[0097] Quantum dot conjugate immobilization:

[0098] At the position where the NC membrane treatment component is fixed, the quantum dot conjugate working solution is applied at a parameter of 0.3 uL / cm, and dried at 37° C. for 0.5 h to obtain a nitrocellulose membrane.

[0099] (2) Sample pad processing:

[0100] Soak the blank pad in the sample pad treatment solution and take it out to dry at 37℃.

[0101] (3) Test strip assembly:

[0102] The sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3.5 mm. Example 3

[0103] This embodiment provides a method for preparing a test strip, comprising the following steps:

[0104] (1) Preparation of nitrocellulose membrane:

[0105] (1.1) Fixing of quality control line and test line:

[0106] Galectin-3 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain the first detection line, which was recorded as detection line T1;

[0107] SFRP-1 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain a second detection line, which was recorded as detection line T2;

[0108] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it on the specified position of nitrocellulose membrane with a film streaker at a parameter of 1 uL / cm to obtain a quality control line.

[0109] The interval between adjacent lines is 3.5mm.

[0110] The coated nitrocellulose membrane was placed in a 45°C oven and dried for 2 hours to obtain a nitrocellulose membrane.

[0111] (1.2) Preparation of the labeling area:

[0112] Preparation of quantum dot conjugates:

[0113] Preparation of blocking solution: weigh 0.358 g tris(hydroxymethyl)methylamino acid and 1 g BSA into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0114] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, followed by 1250 μg of EDC, 80 μg of Galectin-3 antibody 1, and 100 μg of SFRP-1 antibody 1. Incubate at room temperature in the dark for 2 minutes. Centrifuge (4°C, 15,000 rpm, 15 minutes), discard the supernatant, add 1 mL of blocking buffer, block for 1 hour, and centrifuge (4°C, 15,000 rpm, 15 minutes). Discard the supernatant to obtain the quantum dot conjugate.

[0115] Preparation of quantum dot conjugate working solution:

[0116] The quantum dot conjugate was added to 0.1 mL of the preservation solution for re-dissolution to obtain the quantum dot conjugate working solution. The final concentration of the working solution was 2.5 mg / mL based on the mass of the quantum dot microspheres.

[0117] NC membrane treatment liquid configuration:

[0118] 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 into a beaker, add 40 mL of purified water, stir well, and store at 4°C until use.

[0119] NC membrane treatment component fixation:

[0120] 3 mm above the lower edge of the nitrocellulose membrane (near the sample loading end) obtained in step (1.1) (i.e., the marked area), draw the NC membrane treatment solution at a parameter of 1.2 uL / cm, and dry it at 37°C for 20 min to fix the NC membrane treatment components.

[0121] Quantum dot conjugate immobilization:

[0122] At the position where the NC membrane treatment component is fixed, the quantum dot conjugate working solution is applied at a parameter of 0.2 uL / cm, and dried at 37° C. for 0.5 h to obtain a nitrocellulose membrane.

[0123] (2) Sample pad processing:

[0124] Soak the blank pad in the sample pad treatment solution and take it out to dry at 37℃.

[0125] (3) Test strip assembly:

[0126] The sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3.5 mm. Example 4

[0127] This embodiment provides a method for preparing a test strip, comprising the following steps:

[0128] (1) Preparation of nitrocellulose membrane:

[0129] (1.1) Fixing of quality control line and test line:

[0130] Galectin-3 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain the first detection line, which was recorded as detection line T1;

[0131] SFRP-1 antibody 2 was diluted to 1 mg / mL with coating solution and coated on a fixed position of the nitrocellulose membrane using a membrane streaker at a parameter of 1 uL / cm to obtain a second detection line, which was recorded as detection line T2;

[0132] Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat it on the specified position of nitrocellulose membrane with a film streaker at a parameter of 1 uL / cm to obtain a quality control line.

[0133] The interval between adjacent lines is 4mm.

[0134] The coated nitrocellulose membrane was placed in a 45°C oven and dried for 2 hours to obtain a nitrocellulose membrane.

[0135] (1.2) Preparation of the labeling area:

[0136] Preparation of quantum dot conjugates:

[0137] Preparation of blocking solution: weigh 0.358 g tris(hydroxymethyl)methylamino acid and 1 g BSA into a beaker, add 100 mL of purified water, stir well, and store at 4°C until use.

[0138] Add 250 μg of 100 nm quantum dot microspheres to 1 mL of coupling buffer, along with 1250 μg of EDC, 100 μg of Galectin-3 antibody 1, and 80 μg of SFRP-1 antibody 1. Incubate in the dark for 2 minutes at room temperature. Centrifuge (4°C, 15,000 rpm, 15 minutes), discard the supernatant, add 1 mL of blocking buffer, and block for 0.5 hours. Centrifuge (4°C, 15,000 rpm, 15 minutes), and discard the supernatant to obtain the quantum dot conjugate.

[0139] Preparation of quantum dot conjugate working solution:

[0140] The quantum dot conjugate was added to 0.1 mL of the preservation solution for re-dissolution to obtain the quantum dot conjugate working solution. The final concentration of the working solution was 2.5 mg / mL based on the mass of the quantum dot microspheres.

[0141] NC membrane treatment liquid configuration:

[0142] 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 into a beaker, add 100 mL of purified water, stir well, and store at 4°C until ready for use.

[0143] NC membrane treatment component fixation:

[0144] 3 mm above the lower edge of the nitrocellulose membrane (near the sample loading end) obtained in step (1.1) (i.e., the marked area), draw the NC membrane treatment solution at a parameter of 1.5 uL / cm, and dry it at 37°C for 30 min to fix the NC membrane treatment components.

[0145] Quantum dot conjugate immobilization:

[0146] At the position where the NC membrane treatment component is fixed, the quantum dot conjugate working solution is applied at a parameter of 0.3 uL / cm, and dried at 37° C. for 1 h to obtain a nitrocellulose membrane.

[0147] (2) Sample pad processing:

[0148] Soak the blank pad in the sample pad treatment solution and take it out to dry at 37℃.

[0149] (3) Test strip assembly:

[0150] The sample pad, blank glass fiber, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3 mm.

[0151] Comparative Example

[0152] 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 BSA in a beaker, add 100 mL of purified water, stir evenly, and store at 4° C. for later use.

[0153] 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 a nitrocellulose membrane.

[0154] The difference between Comparative Example 3 and Example 1 is that the NC membrane treatment solution is not prepared and the NC membrane treatment components are not fixed.

[0155] The difference between Comparative Example 4 and Example 1 is that the NC membrane treatment solution is prepared as follows: 3 g PVPK30, 3 g sucrose, and 0.05 g Tween-20 are weighed in a beaker, 100 mL of purified water is added, the mixture is stirred evenly, and the mixture is stored at 4° C. for later use.

[0156] The difference between Comparative Example 5 and Example 1 is that the NC membrane treatment solution is prepared as follows: 3 g BSA, 3 g sucrose, and 0.05 g Tween-20 are weighed in a beaker, 100 mL of purified water is added, the mixture is stirred evenly, and stored at 4° C. for later use.

[0157] Performance testing

[0158] 1. Reading time test

[0159] Three Galectin-3 positive samples confirmed by the R&D Elisa kit (weakly positive 0.15 ng / mL, moderately positive 1.38 ng / mL, strongly positive 4.12 ng / mL) and three SFRP-1 positive samples confirmed by the R&D Elisa kit (weakly positive 0.18 ng / mL, moderately positive 1.45 ng / mL, strongly positive 4.19 ng / mL) were taken. The Galectin-3 and SFRP-1 weakly positive samples were mixed in equal proportions, the moderately positive samples were mixed in equal proportions, and the strongly positive samples were mixed in equal proportions. The samples were recorded as sample 1, sample 2, and sample 3, respectively. The test strips prepared in the examples and comparative examples were used to test them. The samples were serum samples. The test results are shown in Table 1.

[0160] Table 1 Reading time test results

[0161]

[0162] As can be seen from Table 1, in Examples 1-4 and Comparative Example 1, the results can be read in 3-5 minutes, and the readings at 5 minutes, 8 minutes, 15 minutes, and 30 minutes are consistent, with no time variability; in Comparative Example 2, the results can be read in 15 minutes, and the value at 30 minutes still increases by 5%, with a large time variability. Table 1 shows that the method of fixing the quantum dot conjugate on the nitrocellulose membrane in this application can quickly produce results. In subsequent Examples 1-4, Comparative Example 1 reads the results according to 5 minutes, and Comparative Example 2 reads the results according to 15 minutes.

[0163] 2. Repeatability test

[0164] A serum sample containing 1.43 ng / mL was obtained by mixing a Galectin-3-positive sample confirmed by the R&D Elisa kit with a serum sample containing 3.439 ng / mL of SFRP-1-positive sample confirmed by the R&D Elisa kit. The Galectin-3 and SFRP-1 mixed samples were mixed in equal proportions and then tested for repeatability. The test results are shown in Tables 2-1 and 2-2. Where AV is the mean, SD is the standard deviation, and CV is the coefficient of variation.

[0165] Table 2-1 Repeatability test results of the embodiment

[0166]

[0167] Table 2-2 Repeatability test results of comparative examples

[0168]

[0169] 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 conjugates on the nitrocellulose membrane in the present application has better product repeatability;

[0170] 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 quantum dot conjugates;

[0171] The repeatability of Example 1 is better than that of Comparative Example 4 and Comparative Example 5, indicating that the macromolecular polymer and protein in the NC membrane treatment component provided by the present application can promote better release of quantum dot conjugates.

[0172] 3. Sample testing

[0173] Galectin-3 positive serum samples confirmed by the R&D Elisa kit were diluted 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, and 6.4 ng / mL. The samples were tested, and the fluorescence value test results are shown in Table 3.

[0174] SFRP-1 positive serum samples confirmed by the R&D Elisa kit were diluted 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, and 6.4 ng / mL, and the samples were tested. The signal value (fluorescence value) test results are shown in Table 4.

[0175] Table 3 Galectin-3 sample test results

[0176]

[0177] Table 4 SFRP-1 sample test results

[0178]

[0179] The greater the difference between the signal value at the 0.05 ng / mL concentration point and the negative sample signal value, 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 larger the multiple difference, the more accurate the conversion result.

[0180] As shown in Tables 3 and 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 negative sample signal value is greater than that in Comparative Example 1, indicating that the minimum detection limit is lower than that in Comparative Example 1, indicating that the blocking solution used in this application can improve the sensitivity of the product;

[0181] As shown in Tables 3 and 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 greater than that in Comparative Example 2, indicating that its linear gradient is better than that in Comparative Example 2, indicating that the method of immobilizing the quantum dot conjugate on the nitrocellulose membrane in the present application can obtain a better linear gradient;

[0182] It can be seen from Tables 3 and 4 that the ratio of the signal value at the concentration point of 6.4 ng / mL to the signal value at the concentration point of 0.05 ng / mL in Example 1 is larger than that in Comparative Examples 3 and 4, indicating that its linear gradient is better than that in Comparative Examples 3 and 4, indicating that the NC membrane treatment component in this application can better promote the release of quantum dot conjugates and obtain a better linear gradient.

[0183] In Examples 1-4, a polynomial mathematical model of signal value-concentration value can be established with the T value signal value as the horizontal coordinate and the concentration value as the vertical coordinate to calculate the sample concentration value based on the detection signal value.

[0184] 4. Sample type test

[0185] Peripheral blood and venous whole blood were collected from Galectin-3-positive patients, and serum and plasma were separated from the venous whole blood. Peripheral blood and venous whole blood were collected from SFRP-1-positive patients, and serum and plasma were separated from the venous whole blood. Using Examples 1-4, a polynomial mathematical model of signal value-concentration value was established, with T value signal value as the horizontal axis and concentration value as the vertical axis. The above sample types were tested using the test strips of Example 1. The test results are shown in Table 5 below.

[0186] Table 5 Sample type test results

[0187]

[0188] As can be seen from Table 5, in Example 1, the test results of Galectin-3 samples and SFRP-1 samples in 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.

[0189] 5. Accuracy

[0190] Serum samples from 50 patients with Galectin-3 and 50 patients with SFRP-1 were collected and the test results of the test strips prepared in Example 1 were compared with those of the R&D Elisa kit. Figure 2 and Figure 3 .Depend on 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 test kit. The test strip prepared in this application can quickly obtain test results while having high accuracy.

[0191] In summary, the test strips prepared in the embodiments of the present application can not only quickly test the results within five minutes, but also have high accuracy and excellent repeatability. A small amount of peripheral blood / venous whole blood / serum / plasma samples can be used for testing, and have the advantages of being easy to use, simple to operate, and easy to promote.

[0192] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. An antigen detection test strip, characterized in that: The method comprises a bottom plate and a sample pad, a blank pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped 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 NC membrane treatment component and the quantum dot conjugate are fixed in sequence on the labeling area, and the quantum dot conjugate is superimposed on the NC membrane treatment component; the NC membrane treatment component includes a macromolecular polymer, protein, sucrose and a surfactant, and the quantum dot conjugate is prepared by coupling quantum dot microspheres with labeled antibodies; 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. The macromolecular polymer includes at least one of polyvinyl pyrrolidone, polyvinyl alcohol, and polyethylene glycol. The NC membrane treatment components are delineated on the nitrocellulose membrane.

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

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

4. An antigen detection test strip according to claim 1, characterized in that: The surfactant includes at least one of Tweens and Tritons.

5. An antigen detection test strip according to claim 1, characterized in that: 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.

6. A method for preparing an antigen detection test strip according to any one of claims 1 to 5, characterized in that: include: Paste the sample pad, blank pad, nitrocellulose membrane and absorbent pad on the bottom plate in sequence according to the chromatography direction; The preparation method of the nitrocellulose membrane comprises: The detection antibody is coated on the nitrocellulose membrane to form a detection line on the nitrocellulose membrane; The quality control antigen or antibody is coated on the nitrocellulose membrane to form a quality control line on the nitrocellulose membrane; the NC membrane treatment component and the quantum dot conjugate are fixed on the labeling area in sequence by the dissolution and drying method to obtain the prepared labeling area.

7. The method for preparing an antigen detection test strip according to claim 6, characterized in that: The quantum dot conjugate is prepared by coupling quantum dot microspheres with labeled antibodies, wherein 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 method for preparing the labeling region comprises: The NC membrane treatment component is added to purified water to obtain an NC membrane treatment solution, and the NC membrane treatment solution with a parameter of 1.2-1.5uL / cm is delineated in the marking area, and dried to obtain a marking area containing the NC membrane treatment component; the quantum dot conjugate is added to the preservation solution for re-dissolution to obtain a quantum dot conjugate working solution, and the quantum dot conjugate working solution with a parameter of 0.2-0.3uL / cm is delineated in the marking area, and dried to obtain a prepared marking area.

8. The method for preparing an antigen detection test strip according to claim 7, characterized in that: In the quantum dot conjugate working solution, the concentration of the quantum dot conjugate working solution is 2.0-3.0 mg / ml, calculated as quantum dot microspheres; 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.

9. The method for preparing an antigen detection test strip according to claim 7, wherein: The method for preparing the quantum dot conjugate comprises the following steps: The quantum dot microspheres were added to a coupling buffer, and after adding an activator, Galectin-3 antibody 1 and SFRP-1 antibody 1 were added. The reaction was protected from light, and the supernatant was discarded by centrifugation. A blocking solution was added for re-dissolution and blocking, and the supernatant was discarded by centrifugation to obtain a quantum dot conjugate. The blocking solution was composed of: 3.58-8.96 g / L tris(hydroxymethyl)methylamino acid, 5-10 g / L BSA.

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