Method and device for detecting urinary kidney injury molecule-1 protein by immunofluorescence

Abstract

The application provides a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection method and device, is applied to the biomedical detection technical field, through pretreatment to the sample, obtains the dilution urine sample, and then adjusts the pH range of the dilution urine sample, and then utilizes the specific recognition of the antibody to the antigen to form a complex, uses the capture antibody fixed in the detection area in advance to capture the immune complex, makes the immune complex enrich in the detection area, improves the detection signal. Drop the fluorescence substrate to the detection area, finally use the portable fluorescence detector to detect the fluorescence intensity of the detection area, obtain the concentration of the urine kidney injury molecule-1 protein in the urine sample, realize quantitative detection. Therefore, the application realizes the rapid, simple and quantitative detection of the urine kidney injury molecule-1 protein in the urine sample.

Description

Technical Field

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

[0002] In large-scale epidemiological screening programs for kidney disease, on-site urine sample collection is the primary step. To achieve efficient screening, it is usually necessary to rapidly collect a large number of samples at community health centers or mobile sites. Urinary kidney injury molecule-1 protein (NGAL), as a highly sensitive biomarker for early kidney injury, is crucial for early diagnosis and timely intervention when its concentration in urine is detected. Immunofluorescence assay, with its excellent sensitivity, has become an effective technique for detecting low concentrations of NGAL protein. However, in practice, on-site collected urine samples 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 shown that NGAL protein is highly susceptible to degradation during urine sample transportation, especially in environments lacking cold chain conditions. Protein degradation directly leads to signal attenuation, severely impacting the accuracy and reliability of test results. This issue is particularly prominent for screening early kidney injury, as patients with early kidney damage already have relatively low NGAL protein concentrations in their urine. Any degree of protein degradation can result in false negatives, thus 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 reduction in the 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] To address the aforementioned issues, existing technologies urgently need improvement. Summary of the Invention

[0006] In view of the shortcomings of the prior art, this application provides a detection method and device for urinary kidney injury molecule-1 protein immunofluorescence detection reagent, which has the advantages of rapid, simple and accurate detection of urinary kidney injury molecule-1 protein in urine samples under large-scale pathogen screening.

[0007] Firstly, a method for detecting urinary kidney injury molecule-1 protein using an immunofluorescence assay reagent, the method comprising the following steps: S1: Mix the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: Add the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjust the pH of the diluted urine sample to a preset target range; S3: Add chromatography solvent to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex. S4: Use a capture antibody to capture the immune complex and enrich the immune complex in the detection area; S5: Add a fluorescent substrate to the immune complex, and generate a fluorescent signal by horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity in 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] This application proposes an immunofluorescence assay method for detecting urinary kidney injury molecule-1 (UKIL-1) protein, used for the quantitative detection of UKIL-1 protein concentration in urine samples. The method first involves sample pretreatment. Step S1 aims to dilute the urine sample, reduce interference from the urine matrix in subsequent detection, and bring the UKIL-1 protein concentration within the detection range. Step S2 provides a suitable environment for the subsequent immune reaction. Step S3 utilizes the specific recognition of the antigen by the antibody to form a complex. Step S4 uses a capture antibody pre-immobilized in the detection area to capture the immune complex formed in Step S3, enriching the immune complex in the detection area and improving the detection signal. Step S5 adds a fluorescent substrate to the detection area; horseradish peroxidase catalyzes the fluorescent substrate to generate a fluorescence signal, converting the immune reaction into a detectable fluorescence signal. Step S6 uses a portable fluorescence detector to detect the fluorescence intensity in the detection area. The fluorescence intensity is correlated with the amount of UKIL-1 protein in the urine sample. By detecting the fluorescence intensity and based on a preset conversion relationship, the concentration of UKIL-1 protein in the urine sample can be determined, achieving quantitative detection. In summary, this method establishes a rapid, simple, and quantitative method for detecting urinary kidney injury molecule-1 protein in urine samples through a series of steps utilizing immunochromatography, enzyme-catalyzed fluorescence reaction, and portable detection instruments.

[0009] Furthermore, step S2 includes: S21: A compound sample stabilizer containing multiple protease inhibitors and pH buffers; S22: The composite sample stabilizer is pre-fixed in the sample area of ​​the immunochromatographic reagent strip in a lyophilized form; S23: Add the diluted urine sample to the sample area containing the composite sample stabilizer, and adjust the pH of the diluted urine sample to the preset target range.

[0010] This application proposes a detection method for urinary kidney injury molecule-1 protein immunofluorescence assay. By pre-fixing a composite sample stabilizer containing protease inhibitors and pH buffers in the sample area, the technical problem of easy degradation of urinary kidney injury molecule-1 protein during sample transportation is solved, thereby improving the reliability of the detection results.

[0011] Furthermore, step S21 includes: S211: Obtain the protease type in the diluted urine sample, and select serine protease inhibitor, cysteine ​​protease inhibitor, and metalloproteinase inhibitor as the protease inhibitor according to the protease type. S212: Obtain the optimal pH range for protease inhibition and the pH fluctuation range of urine samples, and prepare the protease inhibitors with a concentration of 1 mM for amino acid protease inhibitor, a concentration of 10 μM for cysteine ​​protease inhibitor, and a concentration of 10 μM for metalloproteinase inhibitor. S213: Based on the pH fluctuation range of the urine sample and the preset target range, prepare the pH buffer with a molar ratio of 2:1 between the phosphate buffer system and the Tris buffer system; S214: Mix the protease inhibitor with the pH buffer to obtain the composite sample stabilizer.

[0012] This application proposes a method for detecting urinary kidney injury molecule-1 protein using immunofluorescence assay. This method can configure more targeted and efficient composite sample stabilizers 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: Bovine serum albumin solution is added to the chromatographic membrane of the immunochromatographic reagent strip to block the non-specific binding sites on the chromatographic membrane; S32: Add dextran to the chromatography solvent to maintain the flow rate of the chromatography solvent on the immunochromatographic reagent strip with blocked non-specific binding sites at 0.5-1 cm / min; S33: Add a pH buffer to the chromatography solvent to adjust the pH of the chromatography solvent to 7.0, so as to ensure that the urinary kidney injury molecule-1 protein binds to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex.

[0014] This application proposes a method for detecting urinary kidney injury molecule-1 protein using immunofluorescence assay. Step S31 utilizes bovine serum albumin to block non-specific binding sites on the chromatographic membrane, effectively reducing the non-specific adsorption of other components in the urine sample to the membrane, lowering background signal, and improving detection specificity. Step S32 effectively adjusts the flow rate of the chromatography buffer on the immunochromatographic reagent strip, controlling the flow rate within the range of 0.5-1 cm / min, ensuring sufficient time for the immune reaction and improving reaction efficiency and detection sensitivity. Step S33 adjusts the pH of the chromatography buffer to 7.0, close to physiological pH, providing a suitable pH environment for the specific binding of urinary kidney injury molecule-1 protein to the antibody, ensuring the effective formation of immune complexes.

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

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

[0017] Furthermore, step S4 includes: S41: The capture antibody is covalently coupled to the surface of the detection area. 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 fully binds to the capture antibody and is enriched in the detection area.

[0018] Furthermore, in step S41, a nitrocellulose membrane is disposed on the surface of the detection area, 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.

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

[0020] Secondly, a urine kidney injury molecule-1 protein immunofluorescence detection reagent detection device, applied in the steps of any of the above-described methods, the device comprising: Sample acquisition module: Mix the urine sample to be tested with diluent to obtain a diluted urine sample; Sample adjustment module: Adds the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjusts the pH of the diluted urine sample to a preset target range; Chromatography binding module: Add chromatography solvent to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; Enrichment module: uses capture antibodies to capture the immune complexes and enriches the immune complexes in the detection area; Fluorescence addition module: Fluorescent substrate is added to the immune complex, and a fluorescent signal is generated by horseradish peroxidase catalysis; Concentration detection module: A portable fluorescence detector is used to detect the fluorescence intensity in the detection area, and the concentration of urinary kidney injury molecule-1 protein in the urine sample is determined based on the fluorescence intensity value.

[0021] Beneficial Effects: The immunofluorescence assay method and apparatus for urinary kidney injury molecule-1 protein proposed in this application pre-treats the sample to obtain a diluted urine sample, reducing interference from the urine matrix on subsequent detection and ensuring that the concentration of urinary kidney injury molecule-1 protein in the urine sample falls within the detection range. The diluted urine sample is added to 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 immunoreaction. Chromatography buffer is added, driving the sample to move on the reagent strip. Simultaneously, the urinary kidney injury molecule-1 protein in the urine sample binds to a pre-added anti-urinary kidney injury molecule-1 protein antibody conjugated with horseradish peroxidase, forming an immune complex. A capture antibody pre-immobilized in the detection area is used to capture the immune complex, enriching it in the detection area and improving the detection signal. A fluorescent substrate is added to the detection area; horseradish peroxidase catalyzes the fluorescent substrate to generate a fluorescent signal, converting the immunoreaction into a detectable fluorescent signal. A portable fluorescence detector is used to detect the fluorescence intensity in the detection area. The fluorescence intensity is correlated with the amount of urinary kidney injury molecule-1 protein in the urine sample. By detecting the fluorescence intensity and according to a preset conversion relationship, the concentration of urinary kidney injury molecule-1 protein in the urine sample can be determined, achieving quantitative detection. Therefore, this application utilizes immunochromatography, enzyme-catalyzed fluorescence reaction, and a portable detection instrument to establish a rapid, simple, and quantitative method for detecting urinary kidney injury molecule-1 protein in urine samples, improving the accuracy of large-scale pathogen screening. Attached Figure Description

[0022] Figure 1 This is a flowchart of the detection method for the urinary kidney injury molecule-1 protein immunofluorescence assay 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 Implementation

[0024] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. The components of the embodiments of this application described and marked in the accompanying drawings can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

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

[0026] Firstly, a method for detecting urinary kidney injury molecule-1 protein using an immunofluorescence assay reagent, the method comprising the following steps: S1: Mix the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: Add the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjust the pH of the diluted urine sample to the preset target range; S3: Add chromatography buffer to dilute the urine sample and allow the urine kidney injury molecule-1 protein in the diluted urine sample to bind with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex. S4: Use capture antibodies to capture immune complexes and enrich them in the detection area; S5: Add fluorescent substrate to the immune complex, and generate a fluorescent signal through horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity in 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 the diluent to reduce the matrix effect of the urine sample and to adjust the concentration of urinary kidney injury molecule-1 protein in the urine sample to a suitable range for detection.

[0028] In step S2, the diluted urine sample is added to the sample area of ​​the immunochromatographic reagent strip. Through the preset treatment of the sample area, the pH of the diluted urine sample is adjusted to the preset target range to provide a stable pH environment for the subsequent immune reaction.

[0029] In step S3, the chromatography solvent 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 with horseradish peroxidase to form an immune complex. The antibody-antigen specific binding reaction is utilized.

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

[0031] In step S5, the fluorescent substrate is dropped into the detection area, and horseradish peroxidase catalyzes the fluorescent substrate to generate a fluorescent signal, which is then converted into a detectable fluorescent signal.

[0032] In step S6, a portable fluorescence detector is used to detect the fluorescence intensity in 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 urinary kidney injury molecule-1 protein concentration 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 is pretreated by mixing with a diluent to reduce the impact of the complex urine matrix on the test results and to adjust the concentration of urinary kidney injury molecule-1 protein to a range suitable for immunoassay. Then, the pretreated urine sample is added to the sample area of ​​the immunochromatographic strip. This sample area may be pre-prepared with a pH buffer to quickly adjust the pH of the urine sample to a preset suitable range, ensuring that the subsequent immunoassay proceeds under optimal pH conditions. The addition of the chromatography solvent initiates the immunochromatographic process, driving the sample to move on the strip. Simultaneously, the horseradish peroxidase-conjugated anti-urinary kidney injury molecule-1 protein antibody carried in the chromatography solvent specifically binds to the urinary kidney injury molecule-1 protein in the urine sample, forming an immune complex.

[0034] When the immune complexes migrate to the detection area, the capture antibody pre-fixed in this area specifically captures the immune complexes, causing them to aggregate in the detection area and thus increasing the detection signal intensity. After the addition of the fluorescent substrate, horseradish peroxidase enriched in the detection area catalyzes an enzymatic reaction, generating a detectable fluorescent signal. Finally, a portable fluorescence detector is used to detect the fluorescence intensity in the detection area. The detector has a pre-set conversion formula that converts the fluorescence intensity value into the concentration value of urinary kidney injury molecule-1 protein, enabling quantitative detection of urinary kidney injury molecule-1 protein in urine samples. Through the above steps, this method achieves rapid, simple, and quantitative detection of urinary kidney injury molecule-1 protein.

[0035] Furthermore, step S2 includes: S21: A compound sample stabilizer containing multiple protease inhibitors and pH buffers; S22: The composite sample stabilizer is pre-fixed in the sample area of ​​the immunochromatographic reagent strip in a lyophilized form; S23: Add the diluted urine sample to the sample area containing the composite sample stabilizer, and adjust the pH of the diluted urine sample to the preset target range.

[0036] This scheme provides an improved method for pre-fixing a composite sample stabilizer into the sample area of ​​an immunochromatographic reagent strip. The composite sample stabilizer contains multiple 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 protease types in urine are serine proteases, cysteine ​​proteases, and metalloproteinases, so the corresponding protease inhibitors should be selected during detection. Furthermore, the concentration of the protease inhibitor should be configured with reference to the optimal pH range for protease inhibition and the pH fluctuation range of the urine sample to ensure that the protease inhibitor remains effective under pH fluctuations in the urine sample.

[0037] The prepared lyophilized composite sample stabilizer was then pre-fixed in lyophilized form onto the sample area of ​​the immunochromatographic reagent strip. The lyophilization method was chosen to ensure the long-term stability and activity of the composite sample stabilizer at room temperature, which is beneficial for the large-scale transport of samples.

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

[0039] pH buffers are used to adjust and stabilize the pH of urine samples, ensuring that subsequent testing steps are performed in 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 urinary kidney injury molecule-1 protein is effectively guaranteed.

[0040] 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 sample and the preset target range, with the aim of stabilizing the pH of urine sample within the preset target range.

[0041] Furthermore, step S21 includes: S211: Obtain the protease type 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 for amino acid protease inhibitors, a concentration of 10 μM for cysteine ​​protease inhibitors, and a concentration of 10 μM for metalloproteinase inhibitors. S213: Based on the pH fluctuation range of the urine sample and the preset target range, prepare a pH buffer with a molar ratio of 2:1 between the phosphate buffer system and the Tris buffer system. S214: A composite sample stabilizer is obtained by mixing a protease inhibitor with a pH buffer.

[0042] In step S211, the protease inhibitors selected are first analyzed based on the protease types in the diluted urine sample, making the selection of protease inhibitors more targeted and effectively inhibiting 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 was determined based on the optimal pH range for protease inhibition and the pH fluctuation range of the urine sample. This ensures that the protease inhibitor can effectively function under the pH conditions of the diluted urine sample, thereby achieving a better inhibitory effect.

[0044] In step S213, the pH fluctuation range of the urine sample and the preset target range are used as a reference. A pH buffer with a molar ratio of 2:1 between the phosphate buffer system and the Tris buffer system is prepared. This compound buffer system improves the buffering capacity, and can more effectively cope with the pH fluctuation of the urine sample. It is possible to stabilize the pH of the urine sample within the preset target range, thereby ensuring the pH environment stability of the subsequent immune response.

[0045] In step S214, the optimized protease inhibitor and pH buffer are mixed, and the preparation of the composite sample stabilizer is completed.

[0046] In existing technologies, for large-scale community screening, urine samples collected on-site often need to be transported to a central laboratory for testing, inevitably causing time delays. Studies have shown that NGAL proteins are highly susceptible to degradation during transport, especially under non-cold chain conditions. Protein degradation directly weakens the detection signal, severely affecting the accuracy and reliability of test results.

[0047] To address this issue, this application proposes a more targeted and efficient composite sample stabilizer based on the characteristics of highly diluted urine samples (the main protease types in urine are serine proteases, cysteine ​​proteases, and metalloproteinases). This stabilizer effectively inhibits protease activity, stabilizes the pH of urine samples, and ultimately improves the accuracy of NGAL protein immunofluorescence detection results.

[0048] Furthermore, step S3 includes: S31: Add 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: Add dextran to the chromatography buffer to maintain the flow rate of the chromatography buffer on the immunochromatographic reagent strip with blocked non-specific binding sites at 0.5-1 cm / min; S33: Add a pH buffer to the chromatography buffer to adjust the pH of the chromatography buffer to 7.0, so as to ensure that the urinary kidney injury molecule-1 protein binds to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex.

[0049] Regarding step S31, by applying bovine serum albumin solution to the chromatographic membrane, the non-specific binding sites on the membrane surface are effectively blocked. Specifically, bovine serum albumin, as an inert protein, can occupy sites on the chromatographic membrane that may non-specifically adsorb with other components in the urine sample, thereby reducing background signal and improving the specificity of the detection results.

[0050] For step S32, by incorporating dextran into the chromatography solvent, precise control of the solvent flow rate was achieved. The addition of dextran increases the viscosity of the chromatography solvent, thereby reducing the flow rate of the solvent on the immunochromatographic reagent strip. The flow rate was controlled within the range of 0.5-1 cm / min, ensuring sufficient time for the immunoreaction, thus improving the efficiency of the immunoreaction and the detection sensitivity.

[0051] In step S33, a pH buffer was added to the chromatography solvent, enabling precise adjustment of the solvent's pH. The pH of the chromatography solvent was adjusted to 7.0, which is close to physiological pH, providing a suitable environment for the specific binding of urinary kidney injury molecule-1 protein to the antibody and ensuring the effective formation of immune complexes.

[0052] In practical applications, to improve detection efficiency, the preparation of the chromatography solution and the coating of bovine serum albumin solution onto the chromatographic 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 buffer solution and adjust the pH to 7.2-7.4. S312: Apply the prepared bovine serum albumin solution evenly onto the chromatographic membrane of the immunochromatographic reagent strip at a rate of 5-10 μL / mm². S323: Place the immunochromatographic reagent strip coated with bovine serum albumin solution in a 37°C constant temperature drying oven and dry for 1-2 hours, controlling the humidity to be below 30%, so that bovine serum albumin is fully adsorbed on the surface of the chromatography membrane.

[0054] In step S311, a bovine serum albumin (BSA) solution with a concentration of 2%-5% is prepared in advance. This concentration range ensures effective blocking; too low a concentration may result in insufficient blocking, while too high a concentration may lead to waste. A phosphate buffer solution is used to prepare the BSA solution, and the pH is adjusted to 7.2-7.4. The selection of the phosphate buffer solution and pH range aims to simulate the physiological environment, maintain the stability and activity of BSA, and ensure effective adsorption of BSA onto the chromatographic membrane surface, achieving optimal blocking.

[0055] In step S312, the prepared bovine serum albumin solution is uniformly coated onto the chromatographic membrane of the immunochromatographic reagent strip at a rate of 5-10 μL / mm². This controlled coating amount ensures that the membrane surface is adequately covered, achieving complete sealing. Uniform coating avoids inadequate local sealing or blocking solution accumulation, ensuring the uniformity of the sealing effect.

[0056] In step S323, the immunochromatographic reagent strip coated with bovine serum albumin solution is placed in a 37°C constant temperature drying oven for 1-2 hours, with humidity controlled below 30% to ensure sufficient adsorption of bovine serum albumin onto the chromatographic membrane surface. Maintaining a temperature of 37°C, mimicking body temperature, facilitates the adsorption of bovine serum albumin onto the chromatographic membrane surface and avoids protein denaturation and inactivation due to excessively high temperatures. The limited drying time ensures sufficient adsorption of bovine serum albumin, forming a stable blocking layer. Controlling humidity below 30% accelerates moisture evaporation, promotes the drying process, and prevents excessive humidity from affecting the blocking effect.

[0057] Pre-preparing the immunochromatographic reagent strips with the sealed chromatographic membrane before testing can improve testing efficiency.

[0058] Furthermore, step S32 includes: S321: Add 0.5%-1% dextran to the chromatography solvent and filter the prepared chromatography solvent using a filter membrane with a pore size of 0.22μm; S322: Pump the filtered chromatography solution into the immunochromatographic reagent strip, monitor the flow rate of the chromatography solution in the immunochromatographic reagent strip, and control the pumping flow rate of the chromatography solution according to the monitoring results, so that the flow rate of the chromatography solution on the immunochromatographic reagent strip is maintained at 0.5-1 cm / min.

[0059] In step S321, dextran is added to the chromatography solvent to increase its viscosity, thereby reducing the flow rate of the chromatography solvent on the immunochromatographic reagent strip and keeping the flow rate within a reasonable range. The filtration process uses a 0.22 μm pore size filter membrane, through which the prepared chromatography solvent is filtered, effectively removing particulate impurities and preventing membrane clogging. This ensures the uniformity and stability of the chromatography solvent flow. In step S322, the filtered chromatography solvent is pumped into the immunochromatographic reagent strip. The flow rate monitoring step monitors the flow rate of the chromatography solvent in the immunochromatographic reagent strip, allowing for real-time monitoring of the flow rate. The pumping flow rate control step controls the pumping flow rate of the chromatography solvent based on the monitoring results, thus adjusting the actual flow rate and precisely maintaining it at 0.5-1 cm / min.

[0060] In practical applications, pre-preparing and filtering the chromatography solution makes it easier to add the solution directly during the subsequent detection process, thus improving detection efficiency.

[0061] Furthermore, step S4 includes: S41: The capture antibody is covalently coupled to the surface of the detection area. 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 complexes fully bind to the capture antibodies and are enriched in the detection area.

[0062] In step S41, the capture antibody is covalently coupled and immobilized. This results in a stronger binding of the capture antibody to the surface of the detection zone compared to physical adsorption, reducing the risk of antibody detachment during detection and ensuring capture efficiency and detection stability. The capture antibody is a monoclonal antibody targeting different epitopes of the urinary kidney injury molecule-1 protein, ensuring capture specificity. The use of monoclonal antibodies improves antibody homogeneity and batch stability, thereby ensuring the accuracy of the detection results. The fixed concentration of the capture antibody is set at 1 mg / mL. This concentration aims to ensure sufficient capture antibody in the detection zone, thereby effectively improving 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 at 30 minutes. These conditions are set to ensure that the immune complex and the capture antibody bind fully and are effectively enriched in the detection area. The suitable temperature and humidity conditions and incubation time provide a guarantee for the full progress of the immune reaction, thereby improving the detection sensitivity.

[0064] Furthermore, in step S41, a nitrocellulose membrane is disposed 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] Nitrocellulose membrane was chosen as the surface material for the detection zone because of its excellent protein adsorption properties. This allows captured antibodies to be effectively immobilized in the detection zone, ensuring their stable presence and facilitating subsequent capture of immune complexes. The average pore size of the nitrocellulose membrane is controlled within the range of 5-10 μm. This pore size range ensures smooth permeation of liquid samples while effectively trapping larger immune complexes, thereby promoting the enrichment of immune complexes in the detection zone. Surface modification of the nitrocellulose membrane with polyethylene glycol reduces the adsorption of non-specific components from biological samples in the detection zone, thereby lowering background signal and improving the signal-to-noise ratio and sensitivity of the detection.

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

[0067] In step S61, a portable fluorescence detector is used to detect the intensity of the fluorescence signal generated in the detection area. The detector has a pre-set conversion formula that converts fluorescence intensity values ​​to urinary nephropathy molecule-1 (NLM-1) protein concentration values. Specifically, the conversion formula can be a pre-established standard curve obtained by immunofluorescence detection of a known concentration of NLM-1 protein standard, reflecting the quantitative relationship between fluorescence intensity and urinary NLM-1 protein concentration. During actual detection, the detector reads the fluorescence intensity value of the detection area, then calls the built-in standard curve, substituting the read fluorescence intensity value into the standard curve to calculate the corresponding urinary NLM-1 protein concentration value.

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

[0069] Secondly, a detection device for urinary kidney injury molecule-1 protein immunofluorescence assay, applied in the steps of any of the above methods, the device comprising: Sample acquisition module 201: Mix the urine sample to be tested with diluent to obtain a diluted urine sample; Sample adjustment module 202: Adds the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjusts the pH of the diluted urine sample to the preset target range; Chromatography binding module 203: Add chromatography buffer to dilute urine sample and bind urinary kidney injury molecule-1 protein to horseradish peroxidase-coupled anti-urinary kidney injury molecule-1 protein antibody to form immune complex. Enrichment module 204: Uses capture antibodies to capture immune complexes and enriches the immune complexes in the detection area; Fluorescence addition module 205: Adds fluorescent substrate to immune complexes, and generates fluorescent signals through horseradish peroxidase catalysis; Concentration detection module 206: Uses a portable fluorescence detector to detect the fluorescence intensity in the detection area, and determines the concentration of urinary kidney injury molecule-1 protein in the urine sample based on the fluorescence intensity value.

[0070] The sample acquisition module 201 is configured to perform a mixing operation of urine sample and diluent to obtain a diluted urine sample. The sample acquisition module 201 may include an automatic pipetting device or a quantitative dispensing mechanism to accurately control the volume of urine sample and diluent, ensuring the accuracy of the dilution ratio.

[0071] The sample adjustment module 202 is designed to add diluted urine samples to the sample area of ​​the immunochromatographic reagent strip and adjust the pH. The sample adjustment module 202 can integrate a micro-pH adjustment solution addition mechanism and a mixing mechanism to achieve automatic pH adjustment of the sample.

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

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

[0074] The fluorescence addition module 205 is configured to drop fluorescent substrates onto immune complexes and generate fluorescent signals through horseradish peroxidase catalysis. The fluorescence addition module 205 can use a micro-injection pump or a spray device to uniformly add fluorescent substrates.

[0075] The concentration detection module 206 uses a portable fluorescence detector to detect the fluorescence intensity in the detection area and converts the fluorescence intensity value into the concentration value of urinary kidney injury molecule-1 protein 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, obtaining a diluted urine sample to reduce its viscosity and optimize subsequent testing procedures. Then, the diluted urine sample is added to the sample area of ​​the immunochromatographic reagent strip by the sample adjustment module 202, while simultaneously adjusting the pH to a preset target range to ensure the immune reaction proceeds under suitable pH conditions. Next, the chromatography binding module 203 adds chromatography solvent, which drives the diluted urine sample to move on the immunochromatographic reagent strip. During this process, the urinary kidney injury molecule-1 protein in the urine sample binds to a pre-added anti-urinary kidney injury molecule-1 protein antibody conjugated with horseradish peroxidase, forming an immune complex. Driven by the chromatography solvent, the immune complex continues to move to the detection area, where it is captured and enriched by the capture antibody immobilized in the detection area by the enrichment module.

[0077] After the immune complexes accumulate in the detection area, the fluorescence addition module 204 adds a fluorescent substrate to the detection area. Horseradish peroxidase catalyzes the fluorescent substrate to generate a fluorescence signal, and the intensity of the fluorescence signal is positively correlated with the concentration of urinary kidney injury molecule-1 protein. Finally, the concentration detection module 205 uses a portable fluorescence detector to detect the fluorescence intensity in the detection area. The detector converts the fluorescence intensity value into a urinary kidney injury molecule-1 protein concentration value, thereby achieving quantitative detection of urinary kidney injury molecule-1 protein concentration in urine samples. The entire detection process, through the coordinated work of various modules, automates and accelerates the immunofluorescence detection of urinary kidney injury molecule-1 protein, effectively solving the problems of cumbersome manual operation steps and low detection efficiency, and meeting the needs of large-scale rapid screening. The application of the portable fluorescence detector makes rapid on-site detection possible, solving the problem of protein degradation caused by untimely sample transportation, and ensuring the accuracy of the detection results.

[0078] In this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, without necessarily requiring or implying any such actual relationship or order between these entities or operations.

[0079] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A urine kidney injury molecule-1 protein immunofluorescence detection device, characterized in that, The device includes: Sample acquisition module: Mix the urine sample to be tested with diluent to obtain a diluted urine sample; Sample adjustment module: Adds the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjusts the pH of the diluted urine sample to a preset target range; Chromatography binding module: Add chromatography solvent to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex; Enrichment module: uses capture antibodies to capture the immune complexes and enriches the immune complexes in the detection area; Fluorescence addition module: Fluorescent substrate is added to the immune complex, and a fluorescent signal is generated by horseradish peroxidase catalysis; Concentration detection module: A portable fluorescence detector is used to detect the fluorescence intensity in the detection area, and the concentration of urinary kidney injury molecule-1 protein in the urine sample is determined based on the fluorescence intensity value; The device is used to implement an immunoassay method for urinary kidney injury molecule-1 protein, the method comprising the following steps: S1: Mix the urine sample to be tested with the diluent to obtain a diluted urine sample; S2: Add the diluted urine sample to the sample area of ​​the immunochromatographic reagent strip and adjust the pH of the diluted urine sample to a preset target range; Step S2 includes: S21: A compound sample stabilizer containing multiple protease inhibitors and pH buffers; S22: The composite sample stabilizer is pre-fixed in the sample area of ​​the immunochromatographic reagent strip in a lyophilized form; S23: Add the diluted urine sample to the sample area containing the composite sample stabilizer, and adjust the pH of the diluted urine sample to the preset target range; S3: Add chromatography solvent to allow the urinary kidney injury molecule-1 protein in the diluted urine sample to bind with the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex. S4: Use a capture antibody to capture the immune complex and enrich the immune complex in the detection area; S5: Add a fluorescent substrate to the immune complex, and generate a fluorescent signal by horseradish peroxidase catalysis; S6: Use a portable fluorescence detector to detect the fluorescence intensity in 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 urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 1, characterized in that, Step S21 includes: S211: Obtain the protease type in the diluted urine sample, and select serine protease inhibitor, cysteine ​​protease inhibitor, and metalloproteinase inhibitor as the protease inhibitor according to the protease type. S212: Obtain the optimal pH range for protease inhibition and the pH fluctuation range of urine samples, and prepare the protease inhibitors with a concentration of 1 mM for serine protease inhibitor, 10 μM for cysteine ​​protease inhibitor, and 10 μM for metalloproteinase inhibitor. S213: Based on the pH fluctuation range of the urine sample and the preset target range, prepare the pH buffer with a molar ratio of 2:1 between the phosphate buffer system and the Tris buffer system; S214: Mix the protease inhibitor with the pH buffer to obtain the composite sample stabilizer.

3. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 1, characterized in that, Step S3 includes: S31: Bovine serum albumin solution is added to the chromatographic membrane of the immunochromatographic reagent strip to block the non-specific binding sites on the chromatographic membrane; S32: Add dextran to the chromatography solvent to maintain the flow rate of the chromatography solvent on the immunochromatographic reagent strip with blocked non-specific binding sites at 0.5-1 cm / min; S33: Add a pH buffer to the chromatography solvent to adjust the pH of the chromatography solvent to 7.0, so as to ensure that the urinary kidney injury molecule-1 protein binds to the anti-urinary kidney injury molecule-1 protein antibody coupled with horseradish peroxidase to form an immune complex.

4. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 3, characterized in that, Step S31 includes: S311: Prepare a 2%-5% bovine serum albumin solution, wherein the bovine serum albumin solution is prepared using a phosphate buffer solution and the pH is adjusted to 7.2-7.4; S312: Apply the prepared bovine serum albumin solution evenly onto the chromatographic membrane of the immunochromatographic reagent strip at a rate of 5-10 μL / mm². S313: Place the immunochromatographic reagent strip coated with bovine serum albumin solution in a 37°C constant temperature drying oven and dry for 1-2 hours, controlling the humidity to be below 30%, so that bovine serum albumin is fully adsorbed on the surface of the chromatography membrane.

5. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 3, characterized in that, Step S32 includes: S321: Add 0.5%-1% dextran to the chromatography solvent and filter the prepared chromatography solvent using a filter membrane with a pore size of 0.22μm; S322: Pump the filtered chromatography solution into the immunochromatographic reagent strip, monitor the flow rate of the chromatography solution in the immunochromatographic reagent strip, and control the pumping flow rate of the chromatography solution according to the monitoring results, so that the flow rate of the chromatography solution on the immunochromatographic reagent strip is maintained at 0.5-1 cm / min.

6. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 1, characterized in that, Step S4 includes: S41: The capture antibody is covalently coupled to the surface of the detection area. 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 fully binds to the capture antibody and is enriched in the detection area.

7. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 6, characterized in that, In step S41, a nitrocellulose membrane is disposed on the surface of the detection area, 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.

8. The urinary kidney injury molecule-1 protein immunofluorescence detection device according to claim 1, characterized in that, Step S6 includes: S61: A portable fluorescence detector is used to detect the fluorescence intensity in the detection area. The detector converts the fluorescence intensity value into the concentration value of urinary kidney injury molecule-1 protein according to the built-in conversion formula. S62: The concentration of urinary kidney injury molecule-1 protein in a urine sample is obtained based on the concentration value of urinary kidney injury molecule-1 protein.

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