Urine kidney injury molecule-1 protein immunofluorescence detection reagent detection method and device

Abstract

The invention provides a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection method and a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device, which are applied to the technical field of biomedical detection, a sample is pretreated to obtain a diluted urine sample, then the pH range of the diluted urine sample is adjusted, and then a compound is formed by using specific recognition of an antibody to an antigen. The immune complex is captured by using a capture antibody fixed in a detection area in advance, so that the immune complex is enriched in the detection area, and a detection signal is improved. Dropwise adding a fluorescent substrate into the detection area, and finally detecting the fluorescence intensity of the detection area by using a portable fluorescence detector to obtain the concentration of the urine kidney injury molecule-1 protein in the urine sample so as to realize quantitative detection. Therefore, rapid, simple and quantitative detection of the urine kidney injury molecule-1 protein in the urine sample is realized.

Description

Technical Field

[0001] The present application relates to the field of biomedical detection technology, and in particular to a method and device for detecting urinary kidney injury molecule-1 protein immunofluorescence detection reagent. Background Art

[0002] In large-scale epidemiological screening programs for kidney disease, on-site collection of urine samples is the primary step. To achieve efficient screening, large numbers of samples must be collected quickly at community health centers or mobile sites. Urinary kidney injury molecule-1 protein (NGAL) is a highly sensitive marker for early kidney injury, and its concentration detection in urine is crucial for early diagnosis and timely intervention of the disease. Immunofluorescence detection technology, with its excellent sensitivity, has become an effective technical means to detect low concentrations of NGAL protein. However, in actual operations, urine samples collected on-site often need to be transported to a central laboratory for testing, and the sample transportation process inevitably causes time delays.

[0003] Multiple studies have clearly demonstrated that NGAL protein is susceptible to degradation during the transportation of urine samples, particularly in environments lacking cold chain conditions. Protein degradation directly leads to attenuation of the test signal, severely impacting the accuracy and reliability of test results. This issue is particularly acute for screening for early-stage renal injury, as urine NGAL protein concentrations in patients with early-stage renal injury are already relatively low. Any degree of protein degradation can lead to false-negative results, delaying diagnosis and treatment.

[0004] Existing technologies still have significant shortcomings and limitations in addressing the challenges of NGAL protein degradation caused by sample transportation delays in large-scale community screening scenarios, and the resulting reduced accuracy of test results. Specifically, existing technologies lack effective technical means to achieve rapid, simple, and accurate detection of urinary kidney injury molecule-1 protein.

[0005] In view of the above problems, the existing technology is in urgent need of improvement. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the existing technology, the present application provides a method and device for detecting urine kidney injury molecule-1 protein immunofluorescence detection reagent, which has the advantages of realizing rapid, simple and accurate detection of urine kidney injury molecule-1 protein in urine samples in large-scale pathogen screening.

[0007] In a first aspect, a method for detecting urinary kidney injury molecule-1 protein with an immunofluorescence detection reagent is provided, the method comprising the steps of: S1: mixing the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: adding the diluted urine sample dropwise to the sample area of the immunochromatographic reagent strip, and adjusting the pH of the diluted urine sample to within a preset target range; S3: adding a chromatography solution to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; S4: capturing the immune complex using a capture antibody and enriching the immune complex in a detection zone; S5: adding a fluorescent substrate dropwise to the immune complex to generate a fluorescent signal through horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determine the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

[0008] The present application proposes a method for detecting a urine kidney injury molecule-1 protein immunofluorescence detection reagent for quantitatively detecting the concentration of urine kidney injury molecule-1 protein in a urine sample. The method first performs sample pretreatment. The purpose of step S1 is to dilute the urine sample, reduce the interference of the urine matrix on subsequent detection, and bring the concentration of urine kidney injury molecule-1 protein in the urine sample into the detection range. Step S2 provides a suitable environment for the subsequent immune reaction. Step S3 uses the specific recognition of the antibody to the antigen to form a complex. Step S4 uses a capture antibody pre-immobilized in the detection zone to capture the immune complex formed in step S3, so that the immune complex is enriched in the detection zone and the detection signal is increased. Step S5 adds a fluorescent substrate to the detection zone, and horseradish peroxidase catalyzes the fluorescent substrate to produce a fluorescent signal, converting the immune reaction into a detectable fluorescent signal. Step S6 uses a portable fluorescence detector to detect the fluorescence intensity of the detection zone. The fluorescence intensity is related to the amount of urine kidney injury molecule-1 protein in the urine sample. By detecting the fluorescence intensity and based on the preset conversion relationship, the concentration of urine kidney injury molecule-1 protein in the urine sample can be determined to achieve quantitative detection. In summary, this method, through a series of steps, uses immunochromatographic technology, enzyme-catalyzed fluorescence reaction and portable detection instruments to establish a method for rapid, simple and quantitative detection of urinary kidney injury molecule-1 protein in urine samples.

[0009] Furthermore, step S2 includes: S21: Prepare a composite sample stabilizer containing multiple protease inhibitors and pH buffers; S22: pre-fixing the composite sample stabilizer in a freeze-dried form on the sample area of the immunochromatographic reagent strip; S23: adding the diluted urine sample to the sample area containing the composite sample stabilizer, and adjusting the pH of the diluted urine sample to within the preset target range.

[0010] The present application proposes a method for detecting a urine kidney injury molecule-1 protein immunofluorescence detection reagent, which solves the technical problem of easy degradation of urine kidney injury molecule-1 protein during sample transportation by pre-fixing a composite sample stabilizer containing a protease inhibitor and a pH buffer in the sample area, thereby improving the reliability of the detection results.

[0011] Furthermore, step S21 includes: S211: Obtaining the type of protease in the diluted urine sample, and selecting a serine protease inhibitor, a cysteine protease inhibitor, or a metalloprotease inhibitor as the protease inhibitor according to the protease type; S212: Obtaining the optimal pH range for protease inhibition and the pH fluctuation range of the urine sample, and preparing the protease inhibitors with a concentration of 1 mM for amino acid protease inhibitors, 10 μM for cysteine protease inhibitors, and 10 μM for metalloproteinase inhibitors; S213: preparing the pH buffer with a molar ratio of phosphate buffer system to Tris buffer system of 2:1 according to the pH fluctuation range of the urine sample and the preset target range; S214: mixing the protease inhibitor and the pH buffer to obtain the composite sample stabilizer.

[0012] This application proposes a method for detecting a urinary kidney injury molecule-1 protein immunofluorescence detection reagent. This scheme can configure a more targeted and efficient composite sample stabilizer according to the characteristics of different urine samples, thereby more effectively inhibiting protease activity, stabilizing the pH of urine samples, and ultimately improving the accuracy of fluorescence detection results.

[0013] Furthermore, step S3 includes: S31: adding bovine serum albumin solution to the chromatographic membrane of the immunochromatographic reagent strip to block nonspecific binding sites on the chromatographic membrane; S32: adding dextran to the chromatographic fluid, so that the flow rate of the chromatographic fluid on the immunochromatographic reagent strip that blocks nonspecific binding sites is maintained at 0.5-1 cm / min; S33: Adding a pH buffer to the chromatographic fluid to adjust the pH of the chromatographic fluid to 7.0 to ensure that the URI-1 protein binds to the anti-URI-1 antibody coupled to horseradish peroxidase to form an immune complex.

[0014] The present application proposes a method for detecting a urine kidney injury molecule-1 protein immunofluorescence detection reagent. In step S31, bovine serum albumin is used to block the nonspecific binding sites on the chromatographic membrane, thereby effectively reducing the nonspecific adsorption of other components in the urine sample to the chromatographic membrane, reducing the background signal, and improving the detection specificity. Step S32 effectively adjusts the flow rate of the chromatographic fluid on the immunochromatographic reagent strip so that the flow rate is controlled within the range of 0.5-1 cm / min, ensuring that the immune reaction has sufficient time to proceed, thereby improving the efficiency of the immune reaction and the detection sensitivity. Step S33 adjusts the pH of the chromatographic fluid to 7.0, which is close to the physiological pH, providing a suitable pH environment for the specific binding of the urine kidney injury molecule-1 protein and the antibody, and ensuring the effective formation of the immune complex.

[0015] Furthermore, step S31 includes: S311: preparing a bovine serum albumin solution with a concentration of 2%-5%, wherein the bovine serum albumin solution is prepared using a phosphate buffered saline solution and the pH is adjusted to 7.2-7.4; S312: Evenly apply the prepared bovine serum albumin solution at a volume of 5-10 μL / mm² onto the chromatographic membrane of the immunochromatographic reagent strip; S323: Place the immunochromatographic reagent strip coated with the bovine serum albumin solution in a 37°C constant temperature drying oven and dry for 1-2 hours. Control the humidity below 30% to allow the bovine serum albumin to be fully adsorbed on the surface of the chromatography membrane.

[0016] Furthermore, step S32 includes: S321: adding dextran at a concentration of 0.5% to 1% to the chromatographic solution, and filtering the prepared chromatographic solution using a filter membrane with a pore size of 0.22 μm; S322: Pump the filtered chromatographic fluid into the immunochromatographic reagent strip, monitor the flow rate of the chromatographic fluid in the immunochromatographic reagent strip, and control the pumping flow rate of the chromatographic fluid based on the monitoring result, so that the flow rate of the chromatographic fluid on the immunochromatographic reagent strip is maintained at 0.5-1 cm / min.

[0017] Furthermore, step S4 includes: S41: immobilizing a capture antibody on the surface of the detection area by covalent coupling, wherein the capture antibody is a monoclonal antibody targeting different epitopes of the urinary kidney injury molecule-1 protein, and the fixed concentration is 1 mg / mL; S42: Place the immunochromatographic reagent strip in an environment with a temperature of 25° C. and a humidity of 50% for 30 minutes to ensure that the immune complex is fully combined with the capture antibody and is enriched in the detection area.

[0018] Furthermore, in step S41, a nitrocellulose membrane is provided on the surface of the detection area, the capture antibody is immobilized on the nitrocellulose membrane, the average pore size of the nitrocellulose membrane is 5-10 μm, and the surface is modified with polyethylene glycol.

[0019] Furthermore, step S6 includes: S61: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area. The detector converts the fluorescence intensity value into a urine kidney injury molecule-1 protein concentration value according to a built-in conversion formula; S62: The urine kidney injury molecule-1 protein concentration in the urine sample is obtained according to the urine kidney injury molecule-1 protein concentration value.

[0020] In a second aspect, a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device is used in the steps of any of the above methods, the device comprising: Sample acquisition module: mixes the urine sample to be tested with the diluent to obtain a diluted urine sample; Sample adjustment module: adding the diluted urine sample to the sample area of the immunochromatographic reagent strip, and adjusting the pH of the diluted urine sample to a preset target range; Chromatography binding module: adding chromatography fluid to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; Enrichment module: using a capture antibody to capture the immune complex and enrich the immune complex in the detection area; Fluorescence addition module: adding fluorescent substrate to the immune complex and generating fluorescent signal through horseradish peroxidase catalysis; Concentration detection module: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determine the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

[0021] Beneficial effects: The urine kidney injury molecule-1 protein immunofluorescence detection reagent detection method and device proposed in the present application obtains a diluted urine sample by performing sample pretreatment, reduces the interference of the urine matrix on subsequent detection, and makes the urine kidney injury molecule-1 protein concentration in the urine sample enter the detection range. The diluted urine sample is dripped into the sample area of the immunochromatographic reagent strip, and the pH is adjusted to the preset target range to provide a suitable environment for the subsequent immune reaction. The chromatography fluid is added, and the chromatography fluid drives the sample to move on the reagent strip. At the same time, the urine kidney injury molecule-1 protein in the urine sample is combined with the anti-urine kidney injury molecule-1 protein antibody coupled to horseradish peroxidase added in advance to form an immune complex. The capture antibody pre-fixed in the detection area is used to capture the immune complex, so that the immune complex is enriched in the detection area, thereby increasing the detection signal. A fluorescent substrate is dripped into the detection area, and horseradish peroxidase catalyzes the fluorescent substrate to produce a fluorescent signal, thereby converting the immune reaction into a detectable fluorescent signal. A portable fluorescence detector is used to detect the fluorescence intensity of the detection area. The fluorescence intensity is related to the amount of urinary kidney injury molecule-1 protein in the urine sample. By detecting the fluorescence intensity and based on the preset conversion relationship, the concentration of urinary kidney injury molecule-1 protein in the urine sample can be determined to achieve quantitative detection. Therefore, this application uses immunochromatographic technology, enzyme-catalyzed fluorescence reaction and portable detection instrumentation to establish a method for rapid, simple and quantitative detection of urinary kidney injury molecule-1 protein in urine samples, thereby improving the accuracy of large-scale pathogen screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the detection method of the urinary kidney injury molecule-1 protein immunofluorescence detection reagent proposed in this application.

[0023] Figure 2 This is a structural diagram of the detection device for the urinary kidney injury molecule-1 protein immunofluorescence detection reagent proposed in this application. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. The components of the embodiments of the present application generally described and marked in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work fall within the scope of protection of the present application.

[0025] It should be noted that similar reference numerals and letters represent similar items in the following figures. Therefore, once an item is defined in one figure, it does not need to be further defined or explained in subsequent figures. At the same time, in the description of this application, the terms "first", "second", etc. are only used to distinguish the description and should not be understood as indicating or implying relative importance.

[0026] In a first aspect, a method for detecting urinary kidney injury molecule-1 protein with an immunofluorescence detection reagent is provided, the method comprising the steps of: S1: mixing the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: adding the diluted urine sample to the sample area of the immunochromatographic reagent strip, and adjusting the pH of the diluted urine sample to within a preset target range; S3: adding chromatography buffer to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; S4: Capture the immune complex using a capture antibody and enrich the immune complex in the detection zone; S5: Add fluorescent substrate to the immune complex and generate fluorescent signal through horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determine the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

[0027] In step S1 , the urine sample to be tested is mixed with a diluent to reduce the matrix effect of the urine sample and adjust the concentration of the urinary kidney injury molecule-1 protein in the urine sample to an appropriate detection range.

[0028] In step S2, the diluted urine sample is dripped into the sample area of the immunochromatographic reagent strip. Through the preset processing of the sample area, the pH of the diluted urine sample is adjusted to the preset target range, providing a stable pH environment for subsequent immune reactions.

[0029] In step S3, a chromatography fluid is added, which drives the sample to move on the reagent strip. At the same time, the urinary kidney injury molecule-1 protein in the urine sample binds to the anti-urinary kidney injury molecule-1 protein antibody coupled to horseradish peroxidase to form an immune complex, and the specific binding reaction between the antibody and the antigen is utilized.

[0030] In step S4, the capture antibody pre-immobilized in the detection zone is used to capture the immune complex formed in step S3. The immune complex is enriched in the detection zone, thereby enhancing the detection signal.

[0031] In step S5, the fluorescent substrate is added dropwise to the detection area, horseradish peroxidase catalyzes the fluorescent substrate to generate a fluorescent signal, and the immune reaction signal is converted into a detectable fluorescent signal.

[0032] In step S6, a portable fluorescence detector is used to detect the fluorescence intensity of the detection area. The fluorescence intensity value is correlated with the content of urinary kidney injury molecule-1 protein in the urine sample, thereby achieving quantitative detection of the concentration of urinary kidney injury molecule-1 protein in the urine sample.

[0033] Specifically, this method aims to quantitatively detect the concentration of urinary kidney injury molecule-1 protein in urine samples. The detection principle is as follows: First, the urine sample to be tested is pretreated by mixing it with a diluent to reduce the impact of the complex matrix of urine on the test results and adjust the concentration of urine kidney injury molecule-1 protein to a range suitable for immunoassay. Subsequently, the pretreated urine sample is dripped into the sample area of the immunochromatographic reagent strip. The sample area may be pre-set with a pH buffer substance to quickly adjust the pH value of the urine sample to a preset appropriate range, ensuring that subsequent immune reactions are carried out under optimal pH conditions. The addition of the chromatography fluid starts the immunochromatography process, driving the sample to move on the reagent strip. At the same time, the anti-urinary kidney injury molecule-1 protein antibody coupled to horseradish peroxidase carried in the chromatography fluid specifically binds to the urine kidney injury molecule-1 protein in the urine sample to form an immune complex.

[0034] When the immune complex moves to the detection area, the capture antibody pre-fixed in this area can specifically capture the immune complex, causing the complex to aggregate in the detection area, thereby increasing the detection signal intensity. After the addition of the fluorescent substrate, the horseradish peroxidase enriched in the detection area catalyzes the enzymatic reaction of the fluorescent substrate to produce a detectable fluorescent signal. Finally, a portable fluorescence detector is used to detect the fluorescence intensity of the detection area. The detector has a preset conversion formula that can convert the fluorescence intensity value into the concentration value of the urine kidney injury molecule-1 protein, thereby realizing the quantitative detection of the urine kidney injury molecule-1 protein in the urine sample. Through the above steps, this method realizes the rapid, simple and quantitative detection of urine urine kidney injury molecule-1 protein.

[0035] Furthermore, step S2 includes: S21: Prepare a composite sample stabilizer containing multiple protease inhibitors and pH buffers; S22: pre-fixing the composite sample stabilizer in a freeze-dried form on the sample area of the immunochromatographic reagent strip; S23: Adding the diluted urine sample to the sample area containing the composite sample stabilizer, and adjusting the pH of the diluted urine sample to within a preset target range.

[0036] Among them, this scheme provides an improved method in which a composite sample stabilizer is pre-fixed on the sample area of the immunochromatographic reagent strip. The composite sample stabilizer contains a variety of protease inhibitors and pH buffers. The selection of protease inhibitors is based on the type of protease in the urine sample to be tested. For example, in practical applications, the main types of proteases in urine are serine proteases, cysteine proteases, and metalloproteases, so the corresponding protease inhibitors should be selected when conducting the test. And the concentration configuration of the protease inhibitor needs to refer to the optimal inhibition pH range of the protease and the pH fluctuation range of the urine sample to ensure that the protease inhibitor can still work effectively when the pH of the urine sample fluctuates.

[0037] The prepared lyophilized composite sample stabilizer is then pre-fixed in the sample area of the immunochromatographic reagent strip in a lyophilized form. The lyophilized form is chosen to ensure the long-term stability and activity of the composite sample stabilizer at room temperature, facilitating the transportation of large-scale samples.

[0038] The pre-fixed freeze-dried composite sample stabilizer allows it to quickly dissolve and take effect when the sample is added. When the diluted urine sample to be tested is added to the sample area pre-fixed with the composite sample stabilizer, the composite sample stabilizer quickly dissolves, and the protease inhibitors within it immediately inhibit the activity of proteases in the urine sample, thereby reducing the degradation of the urinary kidney injury molecule-1 protein during the initial stage of sample addition.

[0039] The pH buffer is used to adjust and stabilize the pH of the urine sample, ensuring that subsequent testing steps are performed under an optimized pH environment. Ultimately, through the action of the composite sample stabilizer, the pH of the urine sample is adjusted to the preset target range, while the stability of the urinary KIM-1 protein is effectively guaranteed.

[0040] Among them, the selection and ratio of pH buffers, such as the molar ratio of phosphate buffer system to Tris buffer system of 2:1, are determined based on the pH fluctuation range of urine samples and the preset target range, aiming to stabilize the pH of urine samples within the preset target range.

[0041] Furthermore, step S21 includes: S211: Obtain the type of protease in the diluted urine sample, and select serine protease inhibitors, cysteine protease inhibitors, and metalloproteinase inhibitors as protease inhibitors according to the protease type; S212: Obtain the optimal pH range for protease inhibition and the pH fluctuation range of urine samples, and prepare protease inhibitors with a concentration of 1 mM amino acid protease inhibitor, 10 μM cysteine protease inhibitor, and 10 μM metalloproteinase inhibitor; S213: preparing a pH buffer having a molar ratio of phosphate buffer system to Tris buffer system of 2:1 according to the pH fluctuation range of the urine sample and a preset target range; S214: Mixing a protease inhibitor and a pH buffer to obtain a composite sample stabilizer.

[0042] In step S211, the selected protease inhibitor is first analyzed based on the protease types in the diluted urine sample, so that the selection of the protease inhibitor is more targeted and can effectively inhibit the main protease types in the diluted urine sample, thereby improving the protease inhibition effect.

[0043] In step S212, the concentration ratio of the protease inhibitor is determined based on the optimal pH range for protease inhibition and the pH fluctuation range of the urine sample, so that the protease inhibitor can effectively function under the pH conditions of the diluted urine sample, thereby achieving a better inhibition effect.

[0044] In step S213, the pH fluctuation range of the urine sample and the preset target range are used as a reference, and a pH buffer with a molar ratio of 2:1 between the phosphate buffer system and the Tris buffer system is configured. The buffering capacity of this composite buffer system is improved, and the fluctuation of the pH of the urine sample can be more effectively dealt with. It becomes possible to stabilize the pH of the urine sample within the preset target range, thereby ensuring the stability of the pH environment of the subsequent immune reaction.

[0045] In step S214 , the optimally selected protease inhibitor and the pH buffer are mixed, and the composite sample stabilizer is prepared.

[0046] In existing technologies, in the practical application of large-scale community screening, urine samples collected on-site often need to be transported to a centralized laboratory for testing, which inevitably leads to time delays during transportation. Studies have shown that NGAL protein is highly susceptible to degradation during the transportation of urine samples, especially under non-cold chain conditions. Protein degradation directly leads to a weakened detection signal, seriously affecting the accuracy and reliability of test results.

[0047] In order to solve this problem, the present application is able to configure a more targeted and efficient composite sample stabilizer based on the characteristics of a large number of diluted urine samples (the main types of proteases in urine are serine proteases, cysteine proteases and metalloproteases), thereby more effectively inhibiting protease activity, stabilizing the pH of urine samples, and ultimately improving the accuracy of NGAL protein immunofluorescence detection results.

[0048] Furthermore, step S3 includes: S31: Adding bovine serum albumin solution to the chromatographic membrane of the immunochromatographic reagent strip to block the non-specific binding sites on the chromatographic membrane; S32: Adding dextran to the chromatography fluid to maintain a flow rate of 0.5-1 cm / min on the immunochromatographic reagent strip that blocks nonspecific binding sites; S33: Add a pH buffer to the chromatographic fluid to adjust the pH of the chromatographic fluid to 7.0 to ensure that the urinary kidney injury molecule-1 protein and the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase are combined to form an immune complex.

[0049] In step S31, the application of a bovine serum albumin solution to the chromatographic membrane effectively blocks nonspecific binding sites on the membrane surface. Specifically, bovine serum albumin, as an inert protein, can occupy sites on the membrane that might otherwise nonspecifically adsorb to other components in the urine sample, thereby reducing background signal and improving the specificity of the test results.

[0050] In step S32, the flow rate of the chromatographic fluid is precisely controlled by adding dextran to the fluid. The addition of dextran increases the viscosity of the chromatographic fluid, thereby reducing its flow rate through the immunochromatographic reagent strip. The flow rate is controlled within the range of 0.5-1 cm / min, ensuring ample time for the immune reaction to proceed, thereby improving both its efficiency and detection sensitivity.

[0051] In step S33, the pH of the chromatographic fluid is precisely adjusted by adding a pH buffer. The pH of the chromatographic fluid is adjusted to 7.0, which is close to physiological pH. This provides a suitable environment for the specific binding of the urinary kidney injury molecule-1 protein and the antibody, ensuring the effective formation of the immune complex.

[0052] In actual applications, in order to improve detection efficiency, the preparation of the chromatography fluid and the coating of the bovine serum albumin solution on the chromatography membrane of the immunochromatographic reagent strip are prepared in advance.

[0053] Furthermore, step S31 includes: S311: Prepare a 2%-5% bovine serum albumin solution using phosphate buffered saline and adjust the pH to 7.2-7.4; S312: Evenly apply the prepared bovine serum albumin solution at a volume of 5-10 μL / mm² onto the chromatographic membrane of the immunochromatographic reagent strip; S323: Place the immunochromatographic reagent strip coated with the bovine serum albumin solution in a 37°C constant temperature drying oven and dry for 1-2 hours. Control the humidity below 30% to allow the bovine serum albumin to be fully adsorbed on the surface of the chromatography membrane.

[0054] In step S311, a 2%-5% bovine serum albumin solution is prepared in advance. This concentration range ensures a blocking effect; too low a concentration may not adequately block the membrane, while too high a concentration may result in waste. A phosphate-buffered saline solution is used to prepare the bovine serum albumin solution, and the pH is adjusted to 7.2-7.4. The selection of the phosphate-buffered saline solution and pH range is intended to simulate a physiological environment, maintain the stability and activity of bovine serum albumin, and ensure effective adsorption of bovine serum albumin to the chromatographic membrane surface for optimal blocking.

[0055] In step S312, the prepared bovine serum albumin solution is evenly applied to the chromatographic membrane of the immunochromatographic reagent strip at a concentration of 5-10 μL / mm². The limited coating ensures sufficient coverage of the membrane surface, achieving complete blocking. This uniform coating prevents partial blocking or accumulation of blocking solution, ensuring uniform blocking.

[0056] In step S323, the immunochromatographic reagent strip coated with the bovine serum albumin solution is placed in a 37°C constant temperature drying oven and dried for 1-2 hours. The humidity is controlled below 30% to allow the bovine serum albumin to fully adsorb on the surface of the chromatography membrane. The temperature is controlled at 37°C to simulate body temperature, which is conducive to the adsorption of bovine serum albumin on the surface of the chromatography membrane and avoids protein denaturation and inactivation due to excessive temperature. The limited drying time ensures that the bovine serum albumin is fully adsorbed to form a stable sealing layer. Controlling the humidity below 30% accelerates water evaporation and promotes the drying process, while preventing excessive humidity from affecting the sealing effect.

[0057] Preparing the immunochromatographic reagent strip with the closed chromatographic membrane before testing is beneficial to improving the efficiency of testing.

[0058] Furthermore, step S32 includes: S321: adding dextran at a concentration of 0.5% to 1% to the chromatography fluid, and filtering the prepared chromatography fluid using a filter membrane with a pore size of 0.22 μm; S322: Pump the filtered chromatographic fluid into the immunochromatographic reagent strip, monitor the flow rate of the chromatographic fluid in the immunochromatographic reagent strip, and control the pumping flow rate of the chromatographic fluid based on the monitoring results to maintain the flow rate of the chromatographic fluid on the immunochromatographic reagent strip at 0.5-1 cm / min.

[0059] Among them, in step S321, dextran is added to the chromatography fluid to increase the viscosity of the chromatography fluid, thereby reducing the flow rate of the chromatography fluid on the immunochromatographic reagent strip, so that the flow rate is controlled within a reasonable range; the filtration process uses a filter membrane with a pore size of 0.22 μm, and the configured chromatography fluid is filtered through the filter membrane, so that the particulate impurities in the chromatography fluid are effectively removed, and the situation of impurities clogging the chromatography membrane is prevented, thereby ensuring the uniformity and stability of the chromatography fluid flow; in step S322, the filtered chromatography fluid is pumped into the immunochromatographic reagent strip, and the flow rate monitoring step monitors the flow rate of the chromatography fluid in the immunochromatographic reagent strip, so that the flow rate situation is thus grasped in real time; the pumping flow rate control step controls the pumping flow rate of the chromatography fluid according to the monitoring results, so that the actual flow rate situation is adjusted, and the flow rate is accurately maintained at 0.5-1 cm / min.

[0060] In actual applications, the chromatography fluid is prepared in advance and filtered so that the chromatography fluid can be directly added during the subsequent detection process to improve detection efficiency.

[0061] Furthermore, step S4 includes: S41: immobilizing the capture antibody on the surface of the detection area by covalent coupling. The capture antibody is a monoclonal antibody targeting different epitopes of the urinary kidney injury molecule-1 protein at a fixed concentration of 1 mg / mL; S42: Place the immunochromatographic reagent strip in an environment with a temperature of 25°C and a humidity of 50% for 30 minutes to ensure that the immune complex is fully bound to the capture antibody and enriched in the detection area.

[0062] Among them, in step S41, the capture antibody is fixed by covalent coupling. Therefore, compared with physical adsorption, the binding of the capture antibody to the surface of the detection area is stronger, which reduces the risk of antibody shedding during the detection process and ensures the capture efficiency and detection stability. The capture antibody uses monoclonal antibodies targeting different epitopes of the urinary kidney injury molecule-1 protein to ensure the specificity of capture. The use of monoclonal antibodies improves the uniformity and batch stability of the antibodies, thereby ensuring the accuracy of the test results. The fixed concentration of the capture antibody is set to 1 mg / mL. This concentration is intended to ensure that the detection area has sufficient capture antibodies, thereby effectively improving the capture efficiency and detection signal intensity.

[0063] In step S42, the environmental conditions for the binding of the immune complex and the capture antibody are set, the temperature is controlled at 25°C, the humidity is controlled at 50%, and the incubation time is set to 30 minutes. These conditions are set to ensure that the immune complex and the capture antibody are fully bound and effectively enriched in the detection area. Appropriate temperature and humidity conditions and incubation time provide guarantees for the full progress of the immune reaction, thereby improving the detection sensitivity.

[0064] Furthermore, in step S41, a nitrocellulose membrane is provided on the surface of the detection area, and the capture antibody is fixed on the nitrocellulose membrane. The average pore size of the nitrocellulose membrane is 5-10 μm, and the surface is modified with polyethylene glycol.

[0065] Among them, nitrocellulose membrane is selected as the surface material of the detection area, considering that nitrocellulose membrane has good adsorption properties for proteins, so that the capture antibody can be effectively fixed in the detection area, ensuring the stable presence of the capture antibody in the detection area, which is conducive to the subsequent capture of the immune complex. The average pore size of the nitrocellulose membrane is controlled at 5-10μm. This pore size range setting not only ensures that the liquid sample can penetrate smoothly, but also can effectively intercept larger immune complexes, thereby promoting the enrichment of immune complexes in the detection area. Surface modification of the nitrocellulose membrane by polyethylene glycol can reduce the adsorption of non-specific components in the biological sample in the detection area, thereby reducing the background signal and improving the signal-to-noise ratio and sensitivity of the detection.

[0066] Furthermore, step S6 includes: S61: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area. The detector converts the fluorescence intensity value into a urine kidney injury molecule-1 protein concentration value according to a built-in conversion formula; S62: The urine kidney injury molecule-1 protein concentration in the urine sample is obtained according to the urine kidney injury molecule-1 protein concentration value.

[0067] Among them, in step S61, a portable fluorescence detector is used to detect the fluorescence signal intensity generated in the detection area. A conversion formula is preset inside the detector, and the conversion formula can realize the conversion of the fluorescence intensity value to the urine kidney injury molecule-1 protein concentration value. Specifically, the conversion formula can be a pre-established standard curve, which is obtained by immunofluorescence detection of a known concentration of urine kidney injury molecule-1 protein standard, reflecting the quantitative relationship between fluorescence intensity and urine kidney injury molecule-1 protein concentration. In the actual detection process, the detector reads the fluorescence intensity value of the detection area, and then calls the built-in standard curve, and substitutes the read fluorescence intensity value into the standard curve to calculate the corresponding urine kidney injury molecule-1 protein concentration value.

[0068] In step S62, the concentration level of the urinary kidney injury molecule-1 protein in the urine sample is finally determined based on the urinary kidney injury molecule-1 protein concentration value converted in step S61.

[0069] In a second aspect, a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device is used in the steps of any of the above methods, the device comprising: Sample acquisition module 201: mixing the urine sample to be tested with the diluent to obtain a diluted urine sample; Sample adjustment module 202: adding the diluted urine sample to the sample area of the immunochromatographic reagent strip and adjusting the pH of the diluted urine sample to a preset target range; Chromatography binding module 203: adding chromatography fluid to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; Enrichment module 204: using a capture antibody to capture the immune complex and enrich the immune complex in the detection area; Fluorescence adding module 205: adding fluorescent substrate to the immune complex and generating fluorescent signal through horseradish peroxidase catalysis; Concentration detection module 206: uses a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determines the concentration of urinary kidney injury molecule-1 protein in the urine sample according to the fluorescence intensity value.

[0070] The sample acquisition module 201 is configured to perform a mixing operation of the urine sample and the diluent to obtain a diluted urine sample. The sample acquisition module 201 may include an automatic pipetting device or a quantitative liquid addition mechanism for precisely controlling the volume of the urine sample and the diluent to ensure the accuracy of the dilution ratio.

[0071] The sample adjustment module 202 is designed to drop the diluted urine sample into the sample area of the immunochromatographic reagent strip and adjust the pH. The sample adjustment module 202 can integrate a micro-pH adjustment liquid adding mechanism and a mixing mechanism to achieve automatic adjustment of the sample pH.

[0072] The chromatography binding module 203 is configured to add chromatography fluid to promote the binding of the urinary kidney injury molecule-1 protein in the urine sample with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex. The chromatography binding module 203 can include a fluid delivery device such as a peristaltic pump or a syringe pump to accurately control the amount and flow rate of the chromatography fluid added.

[0073] The enrichment module 204 uses the capture antibodies pre-immobilized in the detection area to capture the immune complex and achieve enrichment in the detection area. The performance of the enrichment module 204 depends on the design of the immunochromatographic reagent strip and the effectiveness of the capture antibodies.

[0074] The fluorescence adding module 205 is configured to drop a fluorescent substrate into the immune complex to generate a fluorescent signal through horseradish peroxidase catalysis. The fluorescence adding module 205 can use a microinjection pump or a spray device to evenly add the fluorescent substrate.

[0075] The concentration detection module 206 uses a portable fluorescence detector to detect the fluorescence intensity of the detection area, and converts the fluorescence intensity value into a urinary kidney injury molecule-1 protein concentration value according to a preset conversion formula. The core component of the concentration detection module 206 is the portable fluorescence detector, which has a built-in signal processing and data analysis unit.

[0076] Specifically, the working principle of the urine kidney injury molecule-1 protein immunofluorescence detection device is as follows: First, the sample acquisition module 201 dilutes the urine sample to be tested to obtain a diluted urine sample, reduces the viscosity of the urine sample, and optimizes the subsequent detection process. Subsequently, the diluted urine sample is added dropwise to the sample area of the immunochromatographic reagent strip by the sample adjustment module 202, and the pH is simultaneously adjusted to the preset target range to ensure that the immune reaction is carried out under appropriate pH conditions. Afterwards, the chromatography binding module 203 adds a chromatography fluid, and the chromatography fluid drives the diluted urine sample to move on the immunochromatographic reagent strip. During this process, the urine sample contains the urine kidney injury molecule-1 protein and the pre-added horseradish peroxidase-coupled anti-urine kidney injury molecule-1 protein antibody to form an immune complex. Driven by the chromatography fluid, the immune complex continues to move to the detection area and is captured and enriched by the capture antibody fixed in the detection area by the enrichment module.

[0077] After the immune complex is enriched in the detection area, the fluorescence addition module 204 drips the fluorescent substrate into the detection area, and the horseradish peroxidase catalyzes the fluorescent substrate to produce a fluorescent signal. The intensity of the fluorescent signal is positively correlated with the concentration of the urinary kidney injury molecule-1 protein. Finally, the concentration detection module 205 uses a portable fluorescence detector to detect the fluorescence intensity of the detection area, and the detector converts the fluorescence intensity value into a urinary kidney injury molecule-1 protein concentration value, thereby realizing the quantitative detection of the urinary kidney injury molecule-1 protein concentration in the urine sample. The entire detection process is realized through the collaborative work of various modules, which realizes the automation and rapidity of the immunofluorescence detection of the urinary kidney injury molecule-1 protein, effectively solves the problems of cumbersome manual operation steps and low detection efficiency, and meets the needs of large-scale rapid screening. The application of portable fluorescence detectors makes on-site rapid detection possible, solves the problem of protein degradation caused by untimely sample transportation, and ensures the accuracy of the test results.

[0078] In this document, relational terms such as first and second, etc. are used merely to distinguish one entity or operation from another entity or operation, but do not necessarily require or imply any actual relationship or order between these entities or operations.

[0079] The foregoing is merely an embodiment of the present application and is not intended to limit the scope of protection of the present application. Persons skilled in the art will readily appreciate that the present application may be modified and altered in various ways. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for detecting urinary kidney injury molecule-1 protein immunofluorescence detection reagent, characterized in that: The method comprises the steps of: S1: mixing the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: adding the diluted urine sample dropwise to the sample area of the immunochromatographic reagent strip, and adjusting the pH of the diluted urine sample to within a preset target range; S3: adding a chromatography solution to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; S4: capturing the immune complex using a capture antibody and enriching the immune complex in a detection zone; S5: adding a fluorescent substrate dropwise to the immune complex to generate a fluorescent signal through horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determine the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

2. The method for detecting a urinary kidney injury molecule-1 protein immunofluorescence detection reagent according to claim 1, characterized in that: Step S2 includes: S21: Prepare a composite sample stabilizer containing multiple protease inhibitors and pH buffers; S22: pre-fixing the composite sample stabilizer in a freeze-dried form on the sample area of the immunochromatographic reagent strip; S23: adding the diluted urine sample to the sample area containing the composite sample stabilizer, and adjusting the pH of the diluted urine sample to within the preset target range.

3. The method for detecting a urinary kidney injury molecule-1 protein immunofluorescence detection reagent according to claim 2, characterized in that: Step S21 includes: S211: Obtaining the type of protease in the diluted urine sample, and selecting a serine protease inhibitor, a cysteine protease inhibitor, or a metalloprotease inhibitor as the protease inhibitor according to the protease type; S212: Obtaining the optimal pH range for protease inhibition and the pH fluctuation range of the urine sample, and preparing the protease inhibitors with a concentration of 1 mM for amino acid protease inhibitors, 10 μM for cysteine protease inhibitors, and 10 μM for metalloproteinase inhibitors; S213: preparing the pH buffer with a molar ratio of phosphate buffer system to Tris buffer system of 2:1 according to the pH fluctuation range of the urine sample and the preset target range; S214: mixing the protease inhibitor and the pH buffer to obtain the composite sample stabilizer.

4. The method for detecting a urinary kidney injury molecule-1 protein immunofluorescence detection reagent according to claim 1, characterized in that: Step S3 includes: S31: adding bovine serum albumin solution to the chromatographic membrane of the immunochromatographic reagent strip to block nonspecific binding sites on the chromatographic membrane; S32: adding dextran to the chromatographic fluid, so that the flow rate of the chromatographic fluid on the immunochromatographic reagent strip that blocks nonspecific binding sites is maintained at 0.5-1 cm / min; S33: Adding a pH buffer to the chromatographic fluid to adjust the pH of the chromatographic fluid to 7.0 to ensure that the URI-1 protein binds to the anti-URI-1 antibody coupled to horseradish peroxidase to form an immune complex.

5. The method for detecting urinary kidney injury molecule-1 protein by immunofluorescence detection reagent according to claim 4, characterized in that: Step S31 includes: S311: preparing a bovine serum albumin solution with a concentration of 2%-5%, wherein the bovine serum albumin solution is prepared using a phosphate buffered saline solution and the pH is adjusted to 7.2-7.4; S312: Evenly apply the prepared bovine serum albumin solution at a volume of 5-10 μL / mm² onto the chromatographic membrane of the immunochromatographic reagent strip; S323: Place the immunochromatographic reagent strip coated with the bovine serum albumin solution in a 37°C constant temperature drying oven and dry for 1-2 hours. Control the humidity below 30% to allow the bovine serum albumin to be fully adsorbed on the surface of the chromatography membrane.

6. The method for detecting urinary kidney injury molecule-1 protein by immunofluorescence detection reagent according to claim 4, characterized in that: Step S32 includes: S321: adding dextran at a concentration of 0.5% to 1% to the chromatographic solution, and filtering the prepared chromatographic solution using a filter membrane with a pore size of 0.22 μm; S322: Pump the filtered chromatographic fluid into the immunochromatographic reagent strip, monitor the flow rate of the chromatographic fluid in the immunochromatographic reagent strip, and control the pumping flow rate of the chromatographic fluid based on the monitoring result, so that the flow rate of the chromatographic fluid on the immunochromatographic reagent strip is maintained at 0.5-1 cm / min.

7. The method for detecting urinary kidney injury molecule-1 protein by immunofluorescence detection reagent according to claim 1, characterized in that: Step S4 includes: S41: immobilizing a capture antibody on the surface of the detection area by covalent coupling, wherein the capture antibody is a monoclonal antibody targeting different epitopes of the urinary kidney injury molecule-1 protein, and the fixed concentration is 1 mg / mL; S42: Place the immunochromatographic reagent strip in an environment with a temperature of 25° C. and a humidity of 50% for 30 minutes to ensure that the immune complex is fully combined with the capture antibody and is enriched in the detection area.

8. The method for detecting urinary kidney injury molecule-1 protein by immunofluorescence detection reagent according to claim 7, characterized in that: In step S41, a nitrocellulose membrane is provided on the surface of the detection area, the capture antibody is immobilized on the nitrocellulose membrane, the average pore size of the nitrocellulose membrane is 5-10 μm, and the surface is modified with polyethylene glycol.

9. The method for detecting urinary kidney injury molecule-1 protein by immunofluorescence detection reagent according to claim 1, characterized in that: Step S6 includes: S61: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area. The detector converts the fluorescence intensity value into a urine kidney injury molecule-1 protein concentration value according to a built-in conversion formula; S62: The urine kidney injury molecule-1 protein concentration in the urine sample is obtained according to the urine kidney injury molecule-1 protein concentration value.

10. A urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device, characterized in that: In the steps of the method according to any one of claims 1 to 9, the device comprises: Sample acquisition module: mixes the urine sample to be tested with the diluent to obtain a diluted urine sample; Sample adjustment module: adding the diluted urine sample to the sample area of the immunochromatographic reagent strip, and adjusting the pH of the diluted urine sample to a preset target range; Chromatography binding module: adding chromatography fluid to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; Enrichment module: using a capture antibody to capture the immune complex and enrich the immune complex in the detection area; Fluorescence addition module: adding fluorescent substrate to the immune complex and generating fluorescent signal through horseradish peroxidase catalysis; Concentration detection module: Use a portable fluorescence detector to detect the fluorescence intensity of the detection area, and determine the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

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