Urine microalbumin (MALB) detection kit and preparation method thereof
A urine microalbumin detection kit was prepared using quantum dot-labeled mouse anti-albumin monoclonal antibody and a competitive immunoassay method. This method solves the problem of insufficient sensitivity in existing technologies and achieves high sensitivity and specificity in the detection of urine microalbumin, meeting the precision requirements of clinical applications.
Patent Information
- Application Number
- CN202511847674.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-02-27
AI Technical Summary
Existing fluorescent immunochromatographic assay strips have insufficient sensitivity and poor specificity when detecting urinary microalbumin, making it difficult to meet the precise clinical needs for dynamic monitoring of renal function.
A urine microalbumin detection kit was prepared using quantum dot-labeled mouse anti-albumin monoclonal antibody combined with a competitive immunoassay. The kit includes a urine microalbumin detection kit card and sample diluent. The kit is sealed and stored using a desiccant and aluminum foil bags to ensure its stability and sensitivity.
It achieves high sensitivity and specificity in the detection of urinary microalbumin, with high accuracy in routine quantitative detection, a wide linear range, an intra-assay coefficient of variation of less than 15%, and a correlation coefficient of not less than 0.990, meeting the needs of clinical applications.
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Figure CN121577880A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of albumin detection kits, in particular to a urine microalbumin (MALB) detection kit and a preparation method thereof. BACKGROUND
[0002] Albumin is an important plasma protein in the human body. Under normal circumstances, it cannot penetrate the glomerular basement membrane due to its large molecular weight. Healthy people's urine contains only a very low concentration of albumin (no more than 20 mg per liter), which is called "urine microalbumin (MALB)". MALB detection is a core diagnostic indicator for early kidney disease and kidney damage. Pathological increase is often seen in diseases such as diabetic nephropathy, hypertensive nephropathy, and pre-eclampsia in pregnancy, and can occur in the reversible stage of kidney damage, providing key evidence for early intervention, termination, or reversal of kidney disease progression. In addition, MALB can also be used as an indicator of early kidney damage caused by urinary tract infection and other inflammation, a predictor of complications of acute pancreatitis, and is suitable for kidney function monitoring for those taking nephrotoxic drugs. It has irreplaceable clinical value for kidney disease localization diagnosis and disease condition assessment, and there is an urgent need to develop efficient and accurate MALB detection technology. Existing methods for detecting urine microalbumin mainly include colloidal gold immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunoturbidimetry, and fluorescent immunochromatography. Among them, fluorescent immunochromatography has become the mainstream development direction due to its high sensitivity, good stability, and low natural fluorescence interference. Commonly used markers include fluorescein, quantum dots, and upconversion nanoparticles. Quantum dot markers have the outstanding characteristics of strong light stability, excellent anti-bleaching ability, and long fluorescence lifetime, and have significant potential in improving detection performance, and have been gradually applied to the development of related detection reagents. However, the existing technology still has the following technical problems: (1) The mainstream fluorescent immunochromatographic test strips on the market are fast in detection, but generally have problems such as insufficient sensitivity, narrow linear range, and poor specificity, making it difficult to accurately quantify the changes in MALB content, and unable to meet the precise needs of clinical kidney function dynamic monitoring. (2) In other detection methods, colloidal gold immunochromatography has low sensitivity, enzyme-linked immunosorbent assay is cumbersome and time-consuming, and chemiluminescence and immunoturbidimetry require high-performance instruments and are difficult to popularize at the grassroots level. These problems restrict the clinical application effect of MALB detection, therefore, there is an urgent need for a urine microalbumin fluorescent immunochromatographic test kit with high specificity, strong sensitivity, and wide linear range, and a preparation method thereof, to solve the technical problems of insufficient sensitivity in detecting MALB using existing fluorescent immunochromatographic test strips. SUMMARY
[0003] The present application aims to provide a urine microalbumin (MALB) detection kit and a preparation method thereof to solve the technical problem of insufficient sensitivity when detecting MALB using existing fluorescent immunochromatographic test strips.
[0004] To achieve the above objectives, the present invention adopts the following technical solution: a urine microalbumin (MALB) detection kit, comprising a urine microalbumin detection reagent card and a urine microalbumin sample diluent: the urine microalbumin detection reagent card comprises a card shell and a reagent strip; the reagent strip comprises a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially pasted on the PVC base plate; the urine microalbumin sample diluent is phosphate buffer; The conjugation pad contains a quantum dot-labeled mouse anti-albumin monoclonal antibody; the nitrocellulose membrane has a detection T line and a control C line arranged sequentially, with the detection T line near the sample pad coated with urinary microalbumin antigen, and the control C line near the absorbent paper coated with goat anti-mouse IgG.
[0005] Preferably, as an improvement, the test kit also includes a desiccant, an aluminum foil bag, an instruction manual, and an SD card.
[0006] Beneficial Effects: This solution, employing the above-mentioned method, facilitates assembly into a commercially viable product. The desiccant absorbs moisture within the aluminum foil bag, maintaining a dry environment for the reagent kit (especially the reagent cards), preventing humidity-induced inactivation of fluorescently labeled antibodies and deformation of the NC membrane due to moisture absorption, thus ensuring the stability of the reagent kit within its shelf life. The aluminum foil bag is used to seal and store the reagent cards and desiccant, isolating them from external air, moisture, and light, preventing photobleaching of fluorescent labels (especially since quantum dots are sensitive to strong light) and oxidative inactivation of antibodies; it also prevents microbial contamination, extending the shelf life of the reagent kit.
[0007] Preferably, as an improvement, this solution also provides a method for preparing a urine microalbumin detection kit, comprising the following steps: Step 1, NC membrane coating: First, prepare the C-line working solution and dilute the quality control antibody with the printing buffer; second, prepare the T-line working solution and dilute the urinary microalbumin antigen with the printing buffer; finally, attach the NC membrane to the PVC substrate, coat the C-line working solution and T-line working solution onto the NC membrane respectively, and dry at 57°C before storage. Step 2: Preparation of the conjugate pad: First, immerse the glass fiber membrane in the pre-prepared conjugate pad optimization solution for 30-60 minutes, then lay it flat to dry until the humidity is below 30% before storage; next, biotin-label the mouse anti-albumin monoclonal antibody to obtain a biotin-labeled mouse anti-albumin monoclonal antibody solution; then, add the biotin-labeled mouse anti-albumin monoclonal antibody and quantum dots to the quantum dot diluent in a certain proportion to obtain a quantum dot-labeled mouse anti-albumin monoclonal antibody solution; finally, spray the quantum dot-labeled mouse anti-albumin monoclonal antibody solution onto the glass fiber membrane, dry it at 37°C, and then store it to obtain the conjugate pad; Step 3: Preparation of sample pads: Prepare a sample pad optimization solution, immerse the glass fiber membrane in it for 30-60 minutes, spread it out and dry it until the humidity is below 30% before storing it to obtain the sample pads; Step 4: Assembly: Assemble the prepared sample pad, conjugate pad, absorbent paper, and base plate with the coated NC membrane by pasting and pasting. Then cut it into reagent strips, seal and dry them to obtain the test reagent card.
[0008] Preferably, as an improvement, in step one, the C-line working solution is obtained by diluting goat anti-mouse IgG with printing buffer to 0.8~1.5 mg / mL; the T-line working solution is obtained by diluting MALB antigen with printing buffer to 0.5~1.0 mg / mL; and the spraying volume of the C-line working solution and the T-line working solution is 1.0~2.0 μL / cm.
[0009] Preferably, as an improvement, in step one, the printing buffer comprises PBS buffer with a concentration of 0.01~0.03 mol / L and pH 7.0-7.4, 0.8~1.2 wt% trehalose, EDTA-12 mmol / L, 0.08~0.12 wt% proclin_300, and purified water.
[0010] Preferably, as an improvement, in step two, the binding pad optimization buffer comprises 50-55 mmol / L of tris(hydroxymethyl)aminomethane (tris) at pH 8.2-8.4, 1-1.2 wt% of bovine serum albumin (BSA), 0.5-1.0 wt% of Triton X-100, 2-2.5 wt% of sucrose, 5-10 wt% of trehalose, 0.1-0.2 wt% of proclin_300, and purified water.
[0011] Preferably, as an improvement, in step two, the amount of the quantum dot-labeled mouse anti-albumin monoclonal antibody solution sprayed onto the conjugate pad is 2.0~3.0 μL / cm; the quantum dot-labeled mouse anti-albumin monoclonal antibody solution is prepared by the following steps: S1. Biotin-labeled antibody: Biotin and mouse anti-albumin monoclonal antibody were dissolved separately in PBS solution to obtain biotin diluent and mouse anti-albumin monoclonal antibody diluent. The biotin mass was 1-5% of the mass of mouse anti-human urinary microalbumin monoclonal antibody. The biotin diluent and mouse anti-albumin monoclonal antibody diluent were mixed and reacted in the dark by rotating for 1-2 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed 2-3 times with PBS solution. 5-15% of the recovered amount of BSA was added, mixed well, aliquoted and stored at -18℃ to obtain biotin-labeled mouse anti-albumin monoclonal antibody solution. S2. Quantum dot labeling: Dilute the quantum dots with quantum dot diluent and mix them with a biotin-labeled mouse anti-albumin monoclonal antibody solution and react for 0.5-7 hours to obtain a quantum dot-labeled mouse anti-albumin monoclonal antibody solution.
[0012] Preferably, as an improvement, in S1, the concentration of the PBS solution is 0.2M; the concentration of biotin in the biotin diluent is 5.56~5.60 mg / mL; and the concentration of mouse anti-albumin monoclonal antibody in the mouse anti-albumin monoclonal antibody diluent is 1~1.05 mg / mL.
[0013] Preferably, as an improvement, in S2, the quantum dot diluent comprises 90-110 mmol / L of tris(hydroxymethyl)aminomethane, 0.8-1.2 wt% of bovine serum albumin, 0.4-0.6 wt% of Tween 20, 1.8-2.2 wt% of sucrose, 0.08-0.12 wt% of proclin_300, and purified water.
[0014] Preferably, as an improvement, in S2, the dilution volume ratio of the quantum dots and the quantum dot diluent is 1:5~15, and the mixing volume ratio of the diluted quantum dot solution to the biotin-labeled mouse anti-albumin monoclonal antibody solution is 1:0.05~0.5.
[0015] Preferably, as an improvement, in step two, the conjugation pad optimization treatment solution comprises tris(hydroxymethyl)aminomethane (tris) at a concentration of 50 mmol / L and pH 8.2, 1 wt% bovine serum albumin (BSA), 0.5 wt% Triton X-100, 2 wt% sucrose, 5 wt% trehalose, 0.1 wt% proclin_300, and purified water.
[0016] Preferably, as an improvement, in step three, the sample pad optimization solution comprises tris(hydroxymethyl)aminomethane at a concentration of 50-55 mmol / L and pH 8.2-8.4, 0.5-0.8 wt% polyvinylpyrrolidone, 0.5-1.0 wt% Tween 20, 0.2-0.5 wt% sodium casein, 0.10-0.2 wt% proclin_300, and purified water.
[0017] The principles and advantages of this scheme are: 1. The detection method of this invention is based on competitive immunoassay. Fluorescent immunochromatography is a microanalysis method with advantages such as simple operation, high sensitivity, and low cost. The quantum dots used in this method have advantages such as long fluorescence lifetime, large Stokes shift, excellent optical properties, good photostability and biocompatibility, and a broad excitation spectrum and narrow emission spectrum. These advantages help to overcome the interference of scattered light caused by excitation light on the measurement, further reduce fluorescence background, and achieve high resolution, ultimately improving the sensitivity and specificity of the reagent, thereby improving the performance of the product.
[0018] 2. Compared with the insufficient sensitivity of existing conventional fluorescent immunochromatographic assay strips for detecting MALB, the urine microalbumin assay kit of this protocol is simple to use, highly accurate and stable, and enables routine quantitative detection of urine microalbumin (MALB). The recovery rate is in the range of 90%-110%, the linear range is 5~300mg / L, the correlation coefficient (r) is not less than 0.990, and the intra-assay coefficient of variation (CV) is less than 15%. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of the structure of the urine microalbumin detection reagent strip in Example 1 of the present invention.
[0020] Figure 2 This is a schematic diagram of the structure of the urine microalbumin detection reagent strip in Comparative Example 1 of the present invention.
[0021] Figure 3 This is a schematic diagram of the structure of the urine microalbumin detection reagent strip in Comparative Example 2 of the present invention.
[0022] Figure 4 This is a schematic diagram of the structure of the urine microalbumin detection reagent strip in Comparative Example 3 of the present invention. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.
[0024] Example 1 This solution provides a urine microalbumin (MALB) detection kit, including a urine microalbumin test kit card, urine microalbumin sample diluent, desiccant, aluminum foil bag, instruction manual, and SD card. The urine microalbumin test kit card includes a card shell and a test strip. The test strip includes a PVC base plate and, sequentially, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper (e.g., ...) pasted onto the PVC base plate. Figure 1(As shown); the binding pad contains quantum dot-labeled mouse anti-albumin monoclonal antibody; the nitrocellulose membrane has a detection T line and a control C line set sequentially, with the detection T line close to the sample pad and coated with urinary microalbumin antigen, and the control C line close to the absorbent paper and coated with goat anti-mouse IgG.
[0025] As a reference, the diluent for urine microalbumin samples is phosphate-buffered saline (PBS).
[0026] This protocol also provides a method for preparing a urine microalbumin (MALB) detection kit, comprising the following steps: Step 1, NC membrane coating: First, prepare the C-line working solution and dilute the quality control antibody with the printing buffer; second, prepare the T-line working solution and dilute the urinary microalbumin antigen with the printing buffer; finally, attach the NC membrane to the PVC substrate, coat the C-line working solution and T-line working solution onto the nitrocellulose membrane, and dry it at 57°C before storage. As a reference, the C-line working solution was obtained by diluting goat anti-mouse IgG with printing buffer to 1.0 mg / mL; the T-line working solution was obtained by diluting MALB antigen with printing buffer to 0.8 mg / mL; the spraying volume of both the C-line and T-line working solutions was 1.2 μL / cm. The printing buffer consisted of 0.02 mol / L PBS buffer at pH 7.2, 1.0 wt% trehalose, 10 mmol / L EDTA, 0.1 wt% proclin_300, and purified water.
[0027] Step 2: Preparation of the binding pad: First, the glass fiber membrane was immersed in the pre-prepared conjugate pad optimization solution for 45 minutes, then laid flat to dry until the humidity was below 30% before storage. As a reference, the conjugate pad optimization solution included 50 mmol / L tris(hydroxymethyl)aminomethane (tris), pH 8.2, 1 wt% bovine serum albumin (BSA), 0.5 wt% Triton X-100, 2 wt% sucrose, 5 wt% trehalose, 0.1 wt% proclin_300, and purified water.
[0028] A quantum dot-labeled mouse anti-albumin monoclonal antibody solution was then sprayed onto the conjugate pad at a rate of 2.0 μL / cm. The pad was then dried at 37°C and stored to obtain the conjugate pad. The quantum dot-labeled mouse anti-albumin monoclonal antibody solution was prepared by the following steps: S1. Biotin-labeled antibody: Biotin and mouse anti-albumin monoclonal antibody were dissolved separately in 0.2M PBS solution to obtain a 5.56 mg / mL biotin dilution and a 1 mg / mL mouse anti-albumin monoclonal antibody dilution. The biotin dilution and mouse anti-albumin monoclonal antibody dilution were mixed and reacted in the dark by rotating for 1 hour. After the reaction, the reaction solution was transferred to a dialysis bag and dialyzed 2-3 times with PBS solution. 5-15% of the recovered amount of BSA was added, mixed well, aliquoted, and stored at -18℃ to obtain a biotin-labeled mouse anti-albumin monoclonal antibody solution. S2, Quantum dot labeling: Dilute quantum dots 10 times with quantum dot diluent, mix the resulting quantum dot diluent with biotin-labeled mouse anti-albumin monoclonal antibody solution at a volume ratio of 1:0.3 and react for 0.5 h to obtain quantum dot-labeled mouse anti-albumin monoclonal antibody solution; As a reference, the quantum dot diluent consists of 95 mmol / L tris(hydroxymethyl)aminomethane, 1.0 wt% bovine serum albumin, 0.5 wt% Tween 20, 2.0 wt% sucrose, 0.1 wt% proclin 300, and purified water.
[0029] Step 3: Preparation of sample pads: Prepare sample pad optimization solution, immerse the glass fiber membrane in it for 45 minutes, spread it out and dry it until the humidity is below 30% before storing it to obtain the sample pads; As a reference, the sample pad optimization solution consisted of 50 mmol / L tris(hydroxymethyl)aminomethane (tris), 0.5 wt% polyvinylpyrrolidone (PVP), 0.5 wt% Tween 20, 0.2 wt% sodium casein, 0.10 wt% proclin 300, and purified water.
[0030] Step 4: Assembly: Assemble the prepared sample pad, conjugate pad, absorbent paper, and base plate with the coated NC membrane by pasting and pasting. Then cut it into reagent strips, seal and dry them to obtain the test reagent card.
[0031] Finally, the test kit is placed inside the casing, and then packaged with the desiccant, instruction manual, and SD card in an aluminum foil bag to obtain the urinary microalbumin (MALB) test kit.
[0032] Comparative Example 1 In this example, the control antibody coated on the NC membrane control C line of the urinary microalbumin (MALB) test strip uses a secondary antibody with the same function, such as rabbit anti-mouse IgG (e.g., Figure 2 As shown in the figure, the other structures and preparation methods are the same as in Example 1, and will not be repeated here.
[0033] Comparative Example 2 In this example, the conjugation pad of the urinary microalbumin (MALB) test strip contains quantum dot mouse anti-albumin monoclonal antibody and QD-MIgG (such as...). Figure 3 (as shown) The method for preparing the conjugate pad is as follows: First, the glass fiber membrane was immersed in the pre-prepared conjugate pad optimization solution for 30 minutes, then laid flat to dry until the humidity was below 30% before storage. As a reference, the conjugate pad optimization solution included 50 mmol / L tris(hydroxymethyl)aminomethane (tris), pH 8.2, 1 wt% bovine serum albumin (BSA), 0.5 wt% Triton X-100, 2 wt% sucrose, 5 wt% trehalose, 0.1 wt% proclin_300, and purified water.
[0034] Next, a mixture of quantum dot-labeled mouse anti-albumin monoclonal antibody and QD-MIgG was sprayed onto the conjugate pad at a concentration of 2.0 μL / cm. The mixture of quantum dot-labeled mouse anti-albumin monoclonal antibody and QD-MIgG was prepared by the following steps: S1. Biotin-labeled antibody: Biotin and mouse anti-albumin monoclonal antibody were dissolved separately in 0.2M PBS solution to obtain a 5.56 mg / mL biotin dilution and a 1 mg / mL mouse anti-albumin monoclonal antibody dilution. The biotin dilution and mouse anti-albumin monoclonal antibody dilution were mixed and reacted in the dark by rotating for 1 hour. After the reaction, the reaction solution was transferred to a dialysis bag and dialyzed 2-3 times with PBS solution. 5-15% of the recovered amount of BSA was added, mixed well, aliquoted, and stored at -18℃ to obtain a biotin-labeled mouse anti-albumin monoclonal antibody solution. S2, Quantum dot labeling: Dilute quantum dots 10 times with quantum dot diluent, mix the resulting quantum dot diluent with biotin-labeled mouse anti-albumin monoclonal antibody solution at a volume ratio of 1:0.3, then add 1 / 50 of the volume of quantum dot diluent QD-MIgG and react for 0.5 h to obtain a mixed solution of quantum dot-labeled mouse anti-albumin monoclonal antibody + QD-MIgG. As a reference, the quantum dot diluent consists of 95 mmol / L tris(hydroxymethyl)aminomethane, 1.0 wt% bovine serum albumin, 0.5 wt% Tween 20, 2.0 wt% sucrose, 0.1 wt% proclin 300, and purified water.
[0035] The other structures and preparation methods are the same as in Example 1, and will not be repeated here.
[0036] Comparative Example 3 This solution provides a urine microalbumin (MALB) detection kit, including a urine microalbumin test kit card, urine microalbumin sample diluent, desiccant, aluminum foil bag, instruction manual, and SD card. The urine microalbumin test kit card includes a card shell and a test strip. The test strip includes a PVC base plate and, sequentially, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper (e.g., ...) pasted onto the PVC base plate. Figure 4 (As shown); the mat contains quantum dot-labeled mouse anti-albumin monoclonal antibody and quantum dot-labeled chicken IgY; the nitrocellulose membrane has a detection T line and a control C line set in sequence, the detection T line is close to the sample mat and is coated with urinary microalbumin antigen, and the control C line is close to the absorbent paper and is coated with goat anti-chicken IgY.
[0037] As a reference, the diluent for urine microalbumin samples is phosphate-buffered saline (PBS).
[0038] This protocol also provides a method for preparing a urine microalbumin (MALB) detection kit, comprising the following steps: Step 1, NC membrane coating: First, prepare the C-line working solution and dilute the quality control antibody with the printing buffer; second, prepare the T-line working solution and dilute the urinary microalbumin antigen with the printing buffer; finally, attach the NC membrane to the PVC substrate, coat the C and T-line working solutions onto the nitrocellulose membrane, and dry it at 57°C before storage. As a reference, the C-line working solution was prepared by diluting goat anti-chicken IgY with printing buffer to a concentration of 1.0 mg / mL; the T-line working solution was prepared by diluting MALB antigen with printing buffer to a concentration of 0.8 mg / mL; the spraying volume of both C and T-line working solutions was 1.2 μL / cm. The printing buffer consisted of 0.02 mol / L PBS buffer at pH 7.2, 1.0 wt% trehalose, 10 mmol / L EDTA, 0.1 wt% proclin 300, and purified water.
[0039] Step 2: Preparation of the binding pad: First, the glass fiber membrane is immersed in the pre-prepared conjugate pad optimization solution for 30-60 minutes, then laid flat to dry until the humidity is below 30% before storage. As a reference, the conjugate pad optimization solution includes 50 mmol / L tris(hydroxymethyl)aminomethane (tris), pH 8.2, 1 wt% bovine serum albumin (BSA), 0.5 wt% Triton X-100, 2 wt% sucrose, 5 wt% trehalose, 0.1 wt% proclin_300, and purified water.
[0040] Next, a mixture of quantum dot-labeled mouse anti-albumin monoclonal antibody and quantum dot-labeled chicken IgY solution was sprayed onto the conjugate pad at a spraying volume of 2.0 μL / cm; and the quantum dot-labeled mouse anti-albumin monoclonal antibody solution was prepared by the following steps: S1. Biotin-labeled antibodies: Biotin, mouse anti-albumin monoclonal antibody, and chicken IgY were dissolved in 0.2M PBS solution to obtain 5.56 mg / mL biotin dilution, 1 mg / mL mouse anti-albumin monoclonal antibody dilution, and 1 mg / mL chicken IgY dilution. The biotin was added to the mouse anti-albumin monoclonal antibody dilution and the chicken IgY dilution at 3% of the mass of the mouse anti-human urinary microalbumin monoclonal antibody and chicken IgY, respectively, and the mixture was rotated in the dark for 1 hour. After the reaction, the reaction solution was transferred to a dialysis bag and dialyzed 2-3 times with PBS solution. 5-15% of the recovered BSA was added, mixed well, aliquoted, and stored at -18℃ to obtain biotin-labeled mouse anti-albumin monoclonal antibody solution and biotin-labeled chicken IgY solution. S2, Quantum Dot Labeling: After diluting the quantum dots 10 times with quantum dot diluent, the resulting quantum dot diluent was mixed with biotin-labeled mouse anti-albumin monoclonal antibody and biotin-labeled chicken IgY at volume ratios of 1:0.3 and 1:0.08, respectively. Then, the quantum dot-labeled mouse anti-albumin monoclonal antibody solution and the quantum dot-labeled chicken IgY solution were mixed and reacted for 0.5 h to obtain a mixed solution of quantum dot-labeled mouse anti-albumin monoclonal antibody and quantum dot-labeled chicken IgY. As a reference, the quantum dot diluent consists of 95 mmol / L tris(hydroxymethyl)aminomethane, 1.0 wt% bovine serum albumin, 0.5 wt% Tween 20, 2.0 wt% sucrose, 0.1 wt% proclin 300, and purified water.
[0041] Step 3: Prepare sample pad: Prepare sample pad optimization solution, immerse the glass fiber membrane in it for 30-60 minutes, spread it out and dry it until the humidity is below 30% before storing it; As a reference, the sample pad optimization solution consisted of 50 mmol / L tris(hydroxymethyl)aminomethane (tris), 0.5 wt% polyvinylpyrrolidone (PVP), 0.5 wt% Tween 20, 0.2 wt% sodium casein, 0.10 wt% proclin 300, and purified water.
[0042] Step 4, Assembly: Packaging: Assemble the prepared sample pad, conjugate pad, absorbent paper, and base plate with coated NC membrane by pasting and pasting. Then cut into reagent strips, seal and store under dry conditions to obtain test reagent cards.
[0043] Finally, the test kit is placed inside the casing, and then packaged with the desiccant, instruction manual, and SD card in an aluminum foil bag to obtain the urinary microalbumin (MALB) test kit.
[0044] Results and Analysis: Performance Evaluation Comparison of the Urinary Microalbumin Detection Kits Obtained in Example 1 and Comparative Examples 1-3 1. Precision Thirty test strips were randomly selected and tested for the company's reference standard for urinary microalbumin (MALB) at concentrations of 10 mg / L, 50 mg / L, and 200 mg / L. 80 μL of the sample was added, and after reacting for 10 min, the sample was detected using a dry fluorescence immunoassay analyzer. Each of the three concentration levels was tested 10 times. The test results are detailed in Table 1.
[0045] Table 1 Intra-batch precision
[0046] As shown in the table above, the intra-batch precision of Example 1 and Comparative Examples 1 to 3 all meet the requirement of precision within 15%. Among them, Comparative Example 2 has the highest precision at 11.41%, while Example 1 has the best precision (3.03% to 4.06%) compared to the previous example.
[0047] 2. Accuracy (1) Recovery experiment The reagent kit prepared above was subjected to a recovery experiment. 80 μL of the sample was added, and after reacting for 10 min, it was detected using a dry fluorescence immunoassay analyzer. The detection results are shown in Tables 2 to 5.
[0048] Table 2 Recovery Experiment of Example 1
[0049] Table 3 Comparative Example 1 Recovery Experiment
[0050] Table 4 Comparative Example 2 Recovery Experiment
[0051] Table 5. Recovery experiment of Comparative Example 3
[0052] As shown in Tables 2-5 above, the average recovery rate of Example 1 was 102% (98%~105%), the average recovery rate of Comparative Example 1 was 96% (93%~99%), which was the best result. The average recovery rate of Comparative Example 2 was 91% (88%~95%), which was relatively low overall. The average recovery rate of Comparative Example 3 was 110% (100%~119%), which was relatively high overall. However, the average recovery rates of the four examples all met the requirement of a recovery rate of 90%~110%.
[0053] (2) Accuracy of enterprise reference materials Nine test strips were randomly selected to test the enterprise reference standards for urinary microalbumin (MALB) concentrations of 10 mg / L, 50 mg / L, and 200 mg / L, respectively. 80 μL of the sample was added, and after reacting for 10 min, the results were detected using a dry fluorescence immunoassay analyzer. The results are shown in Table 6.
[0054] Table 6. Accuracy of Enterprise Reference Materials
[0055] As shown in the table above, the maximum relative deviation of the enterprise reference in Example 1 is 5.20% (-6.04%~5.20%), which meets the requirement of a relative deviation of ±15%. Comparative Examples 1 and 3 are generally too low, with the deviation of the reference concentration point being below -15%, which is unqualified. Comparative Example 2 is generally too high, with some deviations exceeding 15%, which is also unqualified.
[0056] 3. Linear range Urinary microalbumin (MALB) antigen was diluted to 2.50 mg / L, 5.00 mg / L, 20.00 mg / L, 50.00 mg / L, 100.00 mg / L, 200.00 mg / L, 300.00 mg / L, and 600.00 mg / L using a urinary microalbumin (MALB) antigen diluent. 80 μL of each solution was added and spotted. After reacting for 10 min, the antigen was detected using a dry fluorescence immunoassay analyzer. The results are shown in Table 7.
[0057] Table 7 Linear Range
[0058] As shown in the table above, within the linear range of 5~300mg / L, the linear correlation coefficient of Example 1 is r=0.9995, which meets the research and development target of r≥0.990. However, the linear correlation coefficients of Comparative Examples 1~3 all fail to meet the requirement of r≥0.99 and are therefore unqualified.
[0059] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. A urine microalbumin (MALB) detection kit, characterized in that: The test includes a urine microalbumin test kit and a urine microalbumin sample diluent. The urine microalbumin test kit includes a card shell and a reagent strip. The reagent strip includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially pasted onto the PVC base plate. The urine microalbumin sample diluent is phosphate buffer. The conjugation pad contains a quantum dot-labeled mouse anti-albumin monoclonal antibody; the nitrocellulose membrane has a detection T line and a control C line arranged sequentially, with the detection T line near the sample pad coated with urinary microalbumin antigen, and the control C line near the absorbent paper coated with goat anti-mouse IgG.
2. The urine microalbumin (MALB) detection kit according to claim 1, characterized in that: The test kit also includes a desiccant, an aluminum foil bag, an instruction manual, and an SD card.
3. A method for preparing a urine microalbumin (MALB) detection kit according to any one of claims 1 to 2, characterized in that: Includes the following steps: Step 1, NC membrane coating: First, prepare the C-line working solution and dilute the quality control antibody with the printing buffer; second, prepare the T-line working solution and dilute the urinary microalbumin antigen with the printing buffer; finally, attach the NC membrane to the PVC substrate, coat the C-line working solution and T-line working solution onto the NC membrane respectively, and dry at 57°C before storage. Step 2: Preparation of the conjugate pad: First, immerse the glass fiber membrane in the pre-prepared conjugate pad optimization solution for 30-60 minutes, then lay it flat to dry until the humidity is below 30% before storage; next, biotin-label the mouse anti-albumin monoclonal antibody to obtain a biotin-labeled mouse anti-albumin monoclonal antibody solution; then, add the biotin-labeled mouse anti-albumin monoclonal antibody and quantum dots to the quantum dot diluent in a certain proportion to obtain a quantum dot-labeled mouse anti-albumin monoclonal antibody solution; finally, spray the quantum dot-labeled mouse anti-albumin monoclonal antibody solution onto the glass fiber membrane, dry it at 37°C, and then store it to obtain the conjugate pad; Step 3: Preparation of sample pads: Prepare a sample pad optimization solution, immerse the glass fiber membrane in it for 30-60 minutes, spread it out and dry it until the humidity is below 30% before storing it to obtain the sample pads; Step 4: Assembly: Assemble the prepared sample pad, conjugate pad, absorbent paper, and base plate with the coated NC membrane by pasting and pasting. Then cut it into reagent strips, seal and dry them to obtain the test reagent card.
4. The method for preparing a urine microalbumin (MALB) detection kit according to claim 3, characterized in that: In step one, the C-line working solution is obtained by diluting goat anti-mouse IgG with printing buffer to 0.8-1.5 mg / mL; the T-line working solution is obtained by diluting MALB antigen with printing buffer to 0.5-1.0 mg / mL; the spraying volume of the C-line and T-line working solutions is 1.0-2.0 μL / cm; the printing buffer includes PBS buffer with a concentration of 0.01-0.03 mol / L and pH 7.0-7.4, 0.8-1.2 wt% trehalose, 8-12 mmol / L ethylenediaminetetraacetic acid, 0.08-0.12 wt% proclin_300, and purified water.
5. The method for preparing a urine microalbumin (MALB) detection kit according to claim 3, characterized in that: In step two, the binding pad optimization buffer comprises 50-55 mmol / L of tris(hydroxymethyl)aminomethane (tris) at pH 8.2-8.4, 1-1.2 wt% of bovine serum albumin (BSA), 0.5-1.0 wt% of Triton X-100, 2-2.5 wt% of sucrose, 5-10 wt% of trehalose, 0.1-0.2 wt% of proclin_300, and purified water.
6. The method for preparing a urine microalbumin (MALB) detection kit according to claim 5, characterized in that: In step two, the amount of the quantum dot-labeled mouse anti-albumin monoclonal antibody solution sprayed onto the conjugate pad is 2.0~3.0 μL / cm; the quantum dot-labeled mouse anti-albumin monoclonal antibody solution is prepared by the following steps: S1. Biotin-labeled antibody: Biotin and mouse anti-albumin monoclonal antibody were dissolved separately in PBS solution to obtain biotin diluent and mouse anti-albumin monoclonal antibody diluent. The biotin mass was 1-5% of the mass of mouse anti-human urinary microalbumin monoclonal antibody. The biotin diluent and mouse anti-albumin monoclonal antibody diluent were mixed and reacted in the dark by rotating for 1-2 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed 2-3 times with PBS solution. 5-15% of the recovered amount of BSA was added, mixed well, aliquoted and stored at -18℃ to obtain biotin-labeled mouse anti-albumin monoclonal antibody solution. S2. Quantum dot labeling: Dilute the quantum dots with quantum dot diluent and mix them with a biotin-labeled mouse anti-albumin monoclonal antibody solution and react for 0.5-7 hours to obtain a quantum dot-labeled mouse anti-albumin monoclonal antibody solution.
7. The method for preparing a urine microalbumin (MALB) detection kit according to claim 6, characterized in that: In S1, the concentration of the PBS solution is 0.2M; the concentration of biotin in the biotin diluent is 5.56~5.60 mg / mL; and the concentration of mouse anti-albumin monoclonal antibody in the mouse anti-albumin monoclonal antibody diluent is 1~1.05 mg / mL.
8. The method for preparing a urine microalbumin (MALB) detection kit according to claim 6, characterized in that: In S2, the quantum dot diluent comprises 90-110 mmol / L of tris(hydroxymethyl)aminomethane, 0.8-1.2 wt% of bovine serum albumin, 0.4-0.6 wt% of Tween 20, 1.8-2.2 wt% of sucrose, 0.08-0.12 wt% of proclin_300, and purified water.
9. The method for preparing a urine microalbumin (MALB) detection kit according to claim 8, characterized in that: In S2, the dilution volume ratio of the quantum dots and the quantum dot diluent is 1:5~15, and the mixing volume ratio of the diluted quantum dot solution to the biotin-labeled mouse anti-albumin monoclonal antibody solution is 1:0.05~0.
5.
10. A method for preparing a urine microalbumin (MALB) detection kit according to any one of claims 4 to 9, characterized in that: In step two, the conjugation pad optimization solution comprises 50 mmol / L of tris(hydroxymethyl)aminomethane (tris) at pH 8.2, 1 wt% bovine serum albumin (BSA), 0.5 wt% Triton X-100, 2 wt% sucrose, 5 wt% trehalose, 0.1 wt% proclin_300, and purified water. In step three, the sample pad optimization solution comprises 50-55 mmol / L of tris(hydroxymethyl)aminomethane at pH 8.2-8.4, 0.5-0.8 wt% polyvinylpyrrolidone, 0.5-1.0 wt% Tween 20, 0.2-0.5 wt% sodium casein, 0.10-0.2 wt% proclin_300, and purified water.