Nano-chelate-labeled KIM-1 time-resolved immunofluorescence detection reagent and nano-chelate-labeled KIM-1 time-resolved immunofluorescence detection method

Abstract

The invention relates to the technical field of biological detection, in particular to a nano chelate labeled KIM-1 time-resolved immunofluorescence detection reagent and a nano chelate labeled KIM-1 time-resolved immunofluorescence detection method, the detection reagent comprises a solid-phase antibody, a EuNPs (at) PAA (at) SiO2-OH nano chelate coupled KIM-1 antibody, 0.05-20ng / mL series KIM-1 standard substance solution and an auxiliary reagent; the detection method is based on a time-resolved immunofluorescence technology, a KIM-1 antigen is captured through a double-antibody sandwich reaction, the fluorescence value is detected and the concentration is calculated after washing and fluorescence enhancement, the nano chelate bears more Eu < 3 + > through a high specific surface area and amplifies a signal in cooperation with a fluorescence enhancement liquid, so that the detection sensitivity is improved by 5 times compared with that of traditional TRFIA, KIM-1 as low as 0.01 ng / mL can be accurately detected, and the detection method is suitable for industrial production. The kit can rapidly quantify the concentration of KIM-1 in serum or urine, is suitable for early diagnosis and disease evaluation of chronic kidney diseases, diabetic nephropathy, lupus nephritis and the like, and has good result consistency and remarkable clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a time-resolved immunofluorescence detection reagent and method for KIM-1 labeled with nano-chelates. Background Technology

[0002] Kidney injury molecule 1 (KIM-1) is a type I transmembrane glycoprotein that is almost not expressed in normal kidney tissue, but its expression is significantly upregulated in proximal tubular cells during kidney injury. KIM-1 functions as both a phosphatidylserine receptor and an oxidized lipoprotein receptor, and can participate in the recognition and clearance of apoptotic cells. Its expression level is closely related to renal interstitial fibrosis and macrophage infiltration, and is positively correlated with serum creatinine, but negatively correlated with creatinine clearance and estimated glomerular filtration rate (eGFR). Existing clinical studies have confirmed that KIM-1 is a specific biomarker for renal tubular injury: the "Expert Consensus on Diagnosis, Prognostic Assessment and Application of Biomarkers in Diabetic Kidney Disease" and the 2022 edition of the "Expert Consensus on Diabetic Nephropathy" both list it as a biomarker for renal tubular injury, which can predict the occurrence of diabetic nephropathy (DKD) and the progression of the disease; in patients with chronic kidney disease (CKD), except for minimal change disease, KIM-1 is expressed to varying degrees, and the level is positively correlated with the degree of kidney injury; in patients with lupus nephritis, the concentration of KIM-1 is significantly higher than that in healthy individuals (p<0.001).

[0003] Current methods for detecting KIM-1 have significant limitations. While traditional time-resolved immunofluorescence assays (TRFIA) possess a certain level of sensitivity, they are limited by the signal carrying capacity of the labeling vector, making it difficult to meet the demand for accurate detection of low concentrations of KIM-1 in early kidney injury. Furthermore, inconsistencies in sensitivity can easily lead to inconsistent test results, failing to provide reliable technical support for the diagnosis of early kidney injury. Therefore, in light of these limitations, there is an urgent need to develop a nano-chelate-labeled KIM-1 time-resolved immunofluorescence assay reagent and method to overcome the shortcomings in current practical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a time-resolved immunofluorescence detection reagent and method for KIM-1 labeled with nano-chelates, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A time-resolved immunofluorescence assay reagent for KIM-1 labeled with nanochelates comprises: a solid-phase antibody, a KIM-1 antibody conjugated with EuNPs@PAA@SiO2-OH nanochelates, a KIM-1 standard solution, and auxiliary reagents; The solid-phase antibody is a carrier coated with KIM-1 monoclonal antibody, and the EuNPs@PAA@SiO2-OH nanochelate can improve the detection sensitivity of KIM-1.

[0006] As a further aspect of the present invention: the carrier of the solid-phase antibody is a 96-well plate, and the KIM-1 monoclonal antibody is diluted with coating buffer and fixed on the surface of the carrier, and then blocked by blocking solution for unbound sites and dried for storage.

[0007] As a further aspect of the present invention, the preparation of the EuNPs@PAA@SiO2-OH nanochelate coupled with KIM-1 antibody includes the following steps: Solvent replacement, carboxyl activation, removal of excess activator, antibody conjugation, end-capping, and purification.

[0008] As a further aspect of the present invention: the solvent replacement is performed by replacing the aqueous dispersion of EuNPs@PAA@SiO2-OH particles with a gradient solvent of acetone and DMF; The carboxyl activation was performed by activating the carboxyl groups on the surface of the nanoparticles using DSC.

[0009] As a further aspect of the present invention: the auxiliary reagents include coating buffer, blocking solution, PBS buffer containing BSA, fluorescence enhancement solution, magnetic bead preservation solution and washing solution; The pH of the BSA-containing PBS buffer is 7.4.

[0010] As a further aspect of the present invention: the fluorescence enhancement liquid can make Eu 3+ After dissociation from the nano-chelate, it chelates with the enhancer to form colloidal molecular clusters, thereby enhancing the fluorescence signal.

[0011] As a further aspect of the present invention: the KIM-1 standard solution is a series of KIM-1 antigen solutions with concentration gradients ranging from 0.05 ng / mL to 20 ng / mL, prepared using analytical buffer and stored at 2–8°C.

[0012] A time-resolved immunofluorescence detection method for KIM-1 labeled with nanochelates, using the detection reagents described above, based on time-resolved immunofluorescence technology and a double-antibody sandwich reaction, includes the following steps: The sample to be tested and the labeled antibody were added to the solid-phase antibody-coated carrier and reacted. After washing, fluorescence enhancement solution was added, and the fluorescence value was measured by a time-resolved fluorescence immunoassay analyzer. The concentration of KIM-1 in the sample to be tested was calculated based on the standard curve.

[0013] As a further aspect of the present invention: the sample to be tested is serum or urine, used for comparative detection of KIM-1 concentration in healthy individuals and patients with kidney damage-related diseases; The EuNPs@PAA@SiO2-OH nano-chelates in the detection reagent work synergistically with the fluorescence enhancement solution to increase the detection sensitivity by 5 times.

[0014] As a further aspect of the present invention: the reaction temperature is 37°C, the reaction time is 1 hour; the washing is performed no less than 3 times to remove unbound substances.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Significantly enhanced sensitivity, enabling early diagnosis: EuNPs@PAA@SiO2-OH nanochelates can carry more Eu through their high specific surface area. 3+ The triple effect of surface modification to enhance coupling stability and nano-quantum effect to amplify the signal with fluorescence enhancement solution improves the detection sensitivity of KIM-1 by 5 times compared with the traditional TRFIA method. It can accurately detect KIM-1 as low as 0.01 ng / mL, effectively identify early kidney damage (KIM-1 concentration 0.15-0.25 ng / mL), and solve the problem that traditional methods cannot make early diagnosis. 2. High specificity and stability: The large Stokes shift (maximum 290nm) of TRFIA technology avoids interference from background fluorescence and environmental factors, and the double-antibody sandwich mode further enhances specificity; the stable structure of EuNPs@PAA@SiO2-OH nanoparticles improves antibody conjugation efficiency by 20%, and the non-radioactive Eu... 3+ Labeling enhances reagent stability; it remains stable for more than 6 months when stored at 4°C in the dark, and the test results show good consistency (CV<5%). 3. Simple operation and wide clinical applicability: The entire testing process takes about 2 hours, and samples can be processed in batches, making it suitable for routine clinical testing; it can detect serum or urine samples, and can achieve early diagnosis of chronic kidney disease, diabetic nephropathy and lupus nephritis through its high sensitivity, and can also accurately assess disease progression through quantitative assessment, making it suitable for a wide range of clinical applications. Attached Figure Description

[0016] Figure 1 This is a comparison chart of KIM-1 concentrations between healthy individuals and patients with lupus nephritis in an embodiment of the present invention; The horizontal axis represents the type of test subject (healthy individuals and patients with lupus nephritis), and the vertical axis represents the KIM-1 concentration (ng / mL). The figure shows that the average KIM-1 concentration in patients with lupus nephritis (0.3446 ng / mL) was significantly higher than that in healthy individuals (0.0997 ng / mL), and the difference between the two groups met the p<0.001 standard, indicating that the test reagent has good clinical discrimination. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0019] Please see Figure 1 The present invention provides a time-resolved immunofluorescence detection reagent and method for KIM-1 labeled with nano-chelates, the specific details of which are as follows: Example 1: Preparation of detection reagents (1) Preparation of solid-phase antibodies: Dilute the KIM-1 monoclonal antibody (KIM-1 MAb) to 1 μg / mL with coating buffer, add 100 μL to each well of a 96-well plate, and incubate overnight at 4°C to allow the antibody to fully adsorb onto the well walls. Discard the coating solution in the well and wash once with washing solution to remove unadsorbed free antibodies. Add 150 μL of blocking solution to each well and shake at room temperature for 2 hours to block the blank sites on the plate that have not bound antibodies, thereby reducing non-specific binding in subsequent detection. After discarding the sealing liquid, the 96-well plate was dried in a vacuum dryer, sealed, and then stored in a -20°C refrigerator for later use.

[0020] This preparation method optimizes the coating concentration, coating temperature, and blocking conditions to ensure stable antibody immobilization in 96-well plates, laying the foundation for subsequent high-sensitivity detection.

[0021] (2) Preparation of KIM-1 antibody conjugated with EuNPs@PAA@SiO2-OH nanochelates (EuP nanoparticles@polyacrylic acid@silica nanochelates with hydroxyl groups on the surface): Solvent replacement: Take an aqueous dispersion containing 10 mg EuNPs@PAA@SiO2-OH particles, centrifuge at 12000 rpm for 15 min, and discard the supernatant; Subsequently, 500 μL of 50% acetone, 75% acetone, 100% acetone (twice), 50% DMF (N,N-dimethylformamide) / 50% acetone, 75% DMF / 25% acetone, and 100% DMF (twice) were added sequentially for gradient solvent replacement. After each replacement, the mixture was ultrasonically dispersed in an ice bath for 5-10 seconds, then centrifuged at 12000 rpm for 10 min and the supernatant was discarded. Finally, a 200 μL suspension with a concentration of 50 mg / mL was prepared using 100% DMF and placed in a desiccator for later use.

[0022] Gradient solvent replacement effectively ensured the good dispersion of nanoparticles in the subsequent reaction system.

[0023] DSC (1,3-dicyclohexylcarbodiimide) activation of carboxyl groups: 10 μL of 0.2 M DSC stock solution (prepared by dissolving 5.1 mg DSC in 100 μL of anhydrous DMF) was added to the suspension prepared above to make the final concentration of DSC in the system 0.02 M. After sealing with sealing film, the system was shaken at room temperature in the dark for 2 hours to activate the carboxyl groups on the surface of the nanoparticles.

[0024] Removal of excess DSC: After the reaction is complete, centrifuge at 12,000 rpm for 10 min to collect the activated nanoparticles and remove the unreacted excess DSC in the system to avoid interference with the subsequent antibody conjugation reaction.

[0025] Antibody conjugation: The collected activated particles were rapidly resuspended in 800 μL of pre-cooled (4°C) 0.1 M sodium phosphate buffer (pH 7.2), and 200 μL of KIM-1 antibody solution was added. The mixture was then placed on a reverse mixer and reacted in the dark for 4 hours to ensure that the activated nanoparticles were fully conjugated with the KIM-1 antibody.

[0026] EuNPs@PAA@SiO2-OH nanoparticles possess a high specific surface area, increasing it by 3-4 times compared to conventional microsphere carriers used in TRFIA, thus enabling them to carry more Eu. 3+ Meanwhile, surface hydroxyl modification can enhance the coupling stability between particles and antibodies, reduce antibody shedding, and ensure the continuity of detection signals.

[0027] Capping, purification and storage: Add 100mM ethanolamine (pH 7.2-7.4) to the above reaction solution and react at room temperature for 20 min (this reaction time is within the optimized range of 15-30 min) to block the activation sites of the nanoparticles that are not bound to antibodies. Then, centrifuge at 12000 rpm for 15 min and discard the supernatant. Resuspend the particles in 1 mL of PBS (phosphate buffer) containing 1% BSA (bovine serum albumin) (pH 7.4), centrifuge and wash 4 times (meeting the requirement of 3-4 washes) to remove unconjugated antibodies and impurities. Finally, the purified particles were dispersed in magnetic bead preservation solution and stored at 4°C in the dark.

[0028] Upon testing, the Eu of the labeled antibody was found to be... 3+ The loading capacity is 4.8 times higher than that of traditional TRFIA-labeled antibodies. Combined with the enhancement effect of nanomaterials on fluorescence signals, this lays a solid foundation for improving detection sensitivity.

[0029] (3) Preparation of KIM-1 standard solution: Take a high-concentration KIM-1 antigen solution and dilute it with analytical buffer to a series of concentrations of 0.05 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, and 20 ng / mL. Aliquot 1 mL into each vial, seal, and store at 2–8°C.

[0030] This series of concentration gradients covers the KIM-1 concentration range for healthy individuals (average 0.0997 ng / mL), patients with early-stage kidney injury (KIM-1 concentration 0.15-0.25 ng / mL), and patients with intermediate-to-late-stage kidney injury (e.g., lupus nephritis patients, average 0.3446 ng / mL). Combined with the high sensitivity of the nano-chelates, it can ensure accurate quantification of low concentrations of KIM-1 in early-stage kidney injury, meeting the needs of early clinical diagnosis.

[0031] (4) Preparation of auxiliary reagents: The auxiliary reagents include coating buffer, blocking solution, PBS buffer containing 1% BSA (pH 7.4), fluorescence enhancement solution, magnetic bead preservation solution, and washing solution.

[0032] Among them, the fluorescence enhancement solution can make Eu 3+ After dissociation from the chelate, it chelates with the enhancer to form colloidal molecular clusters, which enhances the original fluorescence signal by one million times. This, combined with the sensitivity-enhancing effect of EuNPs@PAA@SiO2-OH nano-chelates, further consolidates the 5-fold sensitivity advantage. The washing solution effectively removes unbound substances, reduces background interference, and ensures the specificity of high-sensitivity detection. PBS buffer (pH 7.4) containing 1% BSA is used for antibody resuspension and washing, and the magnetic bead preservation solution maintains the stability of the labeled antibody.

[0033] Example 2: Implementation of the KIM-1 detection method (1) Sample reaction: Add 50 μL of the sample to be tested (serum or urine) and 50 μL of KIM-1 antibody solution conjugated with EuNPs@PAA@SiO2-OH nano-chelate to the solid-phase antibody-coated 96-well plate prepared above, and place it in a 37°C environment with shaking reaction for 1 hour.

[0034] These reaction conditions ensure that low-concentration KIM-1 antigens bind sufficiently, avoiding insufficient binding due to excessively low concentrations, thus guaranteeing accurate subsequent detection.

[0035] (2) Washing: After the reaction, the 96-well plate is washed 6 times with washing solution to thoroughly remove unbound labeled antibodies and impurities in the sample, avoid background interference from affecting the high-sensitivity detection results, and ensure the specificity of the detection signal.

[0036] (3) Fluorescence detection: Add fluorescence enhancement solution to the washed 96-well plate to make Eu 3+ The fluorescent molecular clusters were formed by dissociation from the nano-chelate and chelation with the enhancer, and the fluorescence value of each well was measured using a time-resolved fluorescence immunoassay analyzer.

[0037] Thanks to the EuNPs@PAA@SiO2-OH nanochelates carrying more Eu 3+ It also exhibits a fluorescence synergistic enhancement effect, increasing the detection signal intensity by 5 times compared to the traditional TRFIA method, and can accurately identify low concentrations of KIM-1.

[0038] (4) Concentration calculation: Based on the KIM-1 series standard solutions prepared above, the corresponding fluorescence values ​​were obtained by detection, and a concentration-fluorescence value standard curve was established; By substituting the fluorescence value of the sample into the standard curve, the concentration of KIM-1 in the sample can be calculated, enabling accurate quantification of KIM-1 and providing reliable data support for the identification of early kidney injury.

[0039] Among them, based on the high sensitivity of EuNPs@PAA@SiO2-OH nanochelates, KIM-1 can be accurately detected as low as 0.01 ng / mL.

[0040] Example 3: Verification of Detection Results (1) Sensitivity comparison experiment: Experimental grouping: The detection reagent group of this invention (nano-chelate TRFIA group) and the traditional TRFIA detection reagent group (ordinary microsphere labeling group) were set up, and each group was tested in parallel 3 times to ensure the reliability of the experimental results.

[0041] Sample preparation: Low-concentration standard samples of KIM-1 with concentrations of 0.05 ng / mL, 0.10 ng / mL, 0.15 ng / mL, and 0.20 ng / mL were prepared to simulate the KIM-1 levels in patients with early kidney injury.

[0042] Detection procedure: The nano-chelate TRFIA group was performed according to the steps in "Implementation of KIM-1 Detection Method" above, and the conventional TRFIA group was performed according to the conventional double antibody sandwich TRFIA procedure.

[0043] Results Recording: The fluorescence values ​​and detection concentrations of the two groups were measured, and the relative error (RE) and coefficient of variation (CV) were calculated. The specific results are shown in the table below: Theoretical concentration of KIM-1 (ng / mL) Fluorescence values ​​of the nano-chelate TRFIA group fluorescence values ​​of the traditional TRFIA group Concentration of the nano-chelate TRFIA group (ng / mL) Traditional TRFIA group detection concentration (ng / mL) RE (%) of the nano-chelate TRFIA group CV (%) in the traditional TRFIA group 0.05 12865 2542 0.051 0.054 2.0 8.0 0.10 25730 5210 0.102 0.107 2.0 7.0 0.15 38595 7823 0.148 0.160 1.3 6.7 0.20 51460 10435 0.203 0.212 1.5 6.0 Results analysis: The data in the table above show that in the detection of low concentration KIM-1, the fluorescence value of the nano-chelate TRFIA group is 4.9-5.1 times higher than that of the traditional TRFIA group, the relative error of detection (RE<3%) is significantly lower than that of the traditional TRFIA group (RE<8%), and the coefficient of variation (CV<4%) is lower than that of the traditional TRFIA group (CV<7%).

[0044] The results confirm that the sensitivity of the detection reagents and methods of the present invention is 5 times higher than that of the traditional TRFIA method, and the accuracy and repeatability are better, which can meet the needs of accurate detection of low concentrations of KIM-1 in early kidney injury.

[0045] (2) Clinical sample testing experiments: Sample collection: Twenty serum samples were collected from suspected early-stage lupus nephritis patients (with mild clinical symptoms and normal SCr and BUN levels), and 20 serum samples were collected from healthy individuals as controls.

[0046] Detection procedure: The KIM-1 concentration of all samples was detected using the detection reagents and methods of this invention.

[0047] Results verification: Renal biopsy was performed on suspected early lupus nephritis patients, and 12 cases were diagnosed with early lupus nephritis (mild tubulointerstitial damage).

[0048] Results analysis: The test data showed that the KIM-1 concentration in the 12 confirmed early lupus nephritis patients was 0.16-0.24 ng / mL, all of which were accurately identified by the test reagent of this invention; The KIM-1 concentration in 8 patients without lupus nephritis ranged from 0.07 to 0.12 ng / mL, which was not significantly different from that in healthy individuals (0.00 to 0.11 ng / mL).

[0049] Calculations show that the detection reagent of this invention has a sensitivity of 100% (12 / 12) and a specificity of 90% (18 / 20) in diagnosing early lupus nephritis. This confirms that it can achieve accurate diagnosis of early kidney damage through its high sensitivity advantage, and is suitable for the auxiliary diagnosis and disease assessment of kidney damage-related diseases such as chronic kidney disease (CKD), diabetic nephropathy (DKD), and lupus nephritis.

[0050] In summary, this invention achieves high sensitivity, high specificity, and high stability detection of KIM-1 through specific reagent composition and preparation process, combined with optimized detection steps. Specifically, the large Stokes shift (maximum 290 nm) of TRFIA technology avoids interference from background fluorescence and environmental factors, while the double-antibody sandwich mode further enhances specificity; the stable structure of EuNPs@PAA@SiO2-OH nanoparticles improves antibody conjugation efficiency by 20%, and the non-radioactive Eu... 3+ The labeling enhances reagent stability, allowing it to remain stable for over 6 months when stored at 4°C in the dark, resulting in good consistency of test results (CV < 5%). The entire testing process takes approximately 2 hours, enabling batch processing of samples. It is suitable for routine clinical testing and has significant clinical application value.

[0051] It should be noted that, in this invention, although the specification describes the embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A time-resolved immunofluorescence assay reagent for KIM-1 labeled with nanochelates, characterized in that, Includes: solid-phase antibody, KIM-1 antibody conjugated with EuNPs@PAA@SiO2-OH nano-chelate, KIM-1 standard solution, and auxiliary reagents; The solid-phase antibody is a carrier coated with KIM-1 monoclonal antibody, and the EuNPs@PAA@SiO2-OH nanochelate can improve the detection sensitivity of KIM-1.

2. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nanochelates according to claim 1, characterized in that, The solid-phase antibody is carried by a 96-well plate. The KIM-1 monoclonal antibody is diluted with coating buffer and fixed on the surface of the carrier. After blocking unbound sites with blocking solution, it is dried and stored.

3. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nano-chelates according to claim 1, characterized in that, The preparation of the EuNPs@PAA@SiO2-OH nanochelate conjugated with KIM-1 antibody includes the following steps: Solvent replacement, carboxyl activation, removal of excess activator, antibody conjugation, end-capping, and purification.

4. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nano-chelates according to claim 3, characterized in that, The solvent replacement is performed by replacing the aqueous dispersion of EuNPs@PAA@SiO2-OH particles with a gradient solvent of acetone and DMF. The carboxyl activation was performed by activating the carboxyl groups on the surface of the nanoparticles using DSC.

5. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nano-chelates according to claim 1, characterized in that, The auxiliary reagents include coating buffer, blocking solution, BSA-containing PBS buffer, fluorescence enhancement solution, magnetic bead preservation solution, and washing solution; The pH of the BSA-containing PBS buffer is 7.

4.

6. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nano-chelates according to claim 5, characterized in that, The fluorescence enhancement liquid can make Eu 3+ After dissociation from the nano-chelate, it chelates with the enhancer to form colloidal molecular clusters, thereby enhancing the fluorescence signal.

7. The KIM-1 time-resolved immunofluorescence assay reagent labeled with nano-chelates according to claim 1, characterized in that, The KIM-1 standard solution is a series of KIM-1 antigen solutions with concentration gradients ranging from 0.05 ng / mL to 20 ng / mL. It is prepared using analytical buffer and stored at 2–8°C.

8. A time-resolved immunofluorescence detection method for KIM-1 labeled with nanochelates, characterized in that, Using the detection reagent according to any one of claims 1-7, based on time-resolved immunofluorescence technology and double-antibody sandwich reaction, the following steps are included: The sample to be tested and the labeled antibody were added to the solid-phase antibody-coated carrier and reacted. After washing, fluorescence enhancement solution was added, and the fluorescence value was measured by a time-resolved fluorescence immunoassay analyzer. The concentration of KIM-1 in the sample to be tested was calculated based on the standard curve.

9. The time-resolved immunofluorescence detection method for KIM-1 labeled with nanochelates according to claim 8, characterized in that, The test sample is serum or urine, used to compare the KIM-1 concentration in healthy individuals and patients with kidney damage-related diseases. The EuNPs@PAA@SiO2-OH nano-chelates in the detection reagent work synergistically with the fluorescence enhancement solution to increase the detection sensitivity by 5 times.

10. The time-resolved immunofluorescence detection method for KIM-1 labeled with nanochelates according to claim 8, characterized in that, The reaction temperature is 37°C, and the reaction time is 1 hour; the washing is performed no less than 3 times to remove unbound substances.

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