Chemiluminescence detection kit for creatinine in urine and its preparation and detection method

Abstract

This invention relates to a chemiluminescent detection kit for creatinine in urine and its preparation and detection method. The kit includes calibrators, quality controls, a creatinine antigen-1 reagent solution coated with magnetic microparticles, and an alkaline phosphatase-labeled polyclonal antibody reagent solution. The calibrators include multiple concentrations of creatinine antigen-2; the quality controls include one or more concentrations of creatinine antigen-2; the creatinine antigen-1 reagent solution coated with magnetic microparticles is obtained by reacting magnetic beads with creatinine antigen-1, followed by blocking and dilution; the alkaline phosphatase-labeled polyclonal antibody reagent solution is obtained by labeling desalted, activated, and desalted creatinine polyclonal antibody with desalted, activated, and desalted alkaline phosphatase, followed by dilution. This invention employs a one-step competitive reaction mode, requiring only one antibody, saving costs, improving detection sensitivity, and increasing the linear range; it has advantages such as high specificity, high sensitivity, wide applicability, and broad detection range.

Description

Technical Field

[0001] This invention belongs to the field of creatinine detection technology, specifically, it relates to a chemiluminescent detection kit for creatinine in urine and its preparation and detection method. Background Technology

[0002] Creatinine is a metabolic product of creatine, with 98% of it found in muscles. Creatine, under the action of phosphokinase, forms phosphocreatine with high-energy bonds, serving as an energy source and reserve for muscle contraction. Phosphocreatine releases energy and is then dehydrated to become creatinine, which is excreted by the kidneys. Urinary creatinine mainly originates from creatinine filtered from the blood through the glomeruli and excreted in urine. Urinary creatinine is minimally absorbed in the renal tubules, with only a small amount secreted into the urine. Blood creatinine levels reflect kidney function, especially glomerular filtration rate. When kidney function is impaired, creatinine cannot be effectively excreted, leading to elevated blood creatinine levels.

[0003] Common methods for creatinine testing: 1. Picric acid method: This is a traditional method. The principle is that creatinine reacts with picric acid under alkaline conditions to form a red complex. The concentration is calculated by measuring the color depth using a colorimeter. However, its specificity is poor and it is easily interfered with by other substances, such as ketone bodies, glucose, and proteins. These substances may react with picric acid, leading to false increases or decreases in the measurement results, especially when the creatinine concentration is low, the error is more obvious.

[0004] 2. Enzymatic method: A commonly used modern method, utilizing specific enzymes such as creatinine enzymes to catalyze a reaction, producing a measurable substance. Changes in absorbance are detected using a photometer. Absorbance at a specific wavelength (usually 546 nm or nearby) is directly proportional to creatinine concentration. Concentration is calculated by measuring color depth using a colorimeter. However, because the resolution of light absorption detection is not as sensitive as chemiluminescence, its linear range is narrow. High-concentration samples in clinical testing require dilution before detection, increasing costs, workload for laboratory physicians, and time to obtain results.

[0005] 3. Other methods: These include high-performance liquid chromatography (HPLC), used for research or special cases, which has extremely high precision but is complex to operate and expensive to use; and dry chemistry methods, which use test strips for rapid detection, suitable for emergency or home testing, but with relatively low accuracy. Summary of the Invention

[0006] To overcome the technical problems existing in the prior art, this invention provides a chemiluminescent detection kit for creatinine in urine and its preparation method. This not only improves detection sensitivity but also increases the linear range.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A chemiluminescent assay kit for creatinine in urine, the kit being used for the direct detection of creatinine in urine without sample dilution; the kit includes calibrators, quality controls, a creatinine antigen-1 reagent solution coated with magnetic microparticles, and an alkaline phosphatase-labeled polyclonal antibody reagent solution, the calibrators including multiple concentrations of creatinine antigen-2; the quality controls including one or more concentrations of creatinine antigen-2; the creatinine antigen-1 reagent solution coated with magnetic microparticles is obtained by reacting magnetic beads with creatinine antigen-1, followed by blocking and dilution; the alkaline phosphatase-labeled polyclonal antibody reagent solution is obtained by labeling desalted, activated, and desalted creatinine polyclonal antibody with desalted, activated, and desalted alkaline phosphatase, followed by dilution; creatinine antigen-1 is the creatinine antigen used for coating magnetic beads, and creatinine antigen-2 is the creatinine antigen used for calibrators / quality controls.

[0008] Preferably, the creatinine antigen-reagent solution coated with magnetic microparticles is prepared by the following steps: (a) Take magnetic beads into centrifuge tubes and place them on a magnetic rack to magnetically adsorb the supernatant; then add magnetic bead pretreatment solution to centrifuge tubes, vortex thoroughly to mix, and place them on a magnetic rack to magnetically adsorb the supernatant. (b) Add magnetic bead pretreatment solution, ammonium sulfate vortex magnetic beads to the magnetic beads treated above, add creatinine antigen and vortex the magnetic beads thoroughly, and react under constant temperature conditions; (c) After the reaction is complete, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add blocking solution to clean, add blocking solution again, and continue to mix and block under constant temperature conditions. (d) After sealing, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add magnetic bead preservation solution to vortex the magnetic beads, magnetically adsorb the supernatant, and then add magnetic bead preservation solution to dilute the magnetic bead concentration to obtain magnetic beads coated with creatinine antigen 1. (e) Dilute the magnetic beads coated with good creatinine antigen 1 with magnetic bead preservation solution to a working solution concentration of 0.1 to 0.4 mg / mL.

[0009] Preferably, the magnetic bead pretreatment solution is borate buffer, the blocking solution consists of phosphate buffer (PBS), bovine serum albumin (BSA), Tween 20, and Proclin 300, and the magnetic bead preservation solution consists of tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin, Tween 20, and Proclin 300.

[0010] Preferably, the alkaline phosphatase-labeled polyclonal antibody reagent solution is prepared by the following steps: (I) Take two desalting columns and centrifuge them to remove the liquid inside the tubes; (II) Add the polyclonal antibody to one of the desalting columns; add alkaline phosphatase to the other desalting column, and centrifuge both desalting columns simultaneously; collect the desalted liquid separately. (III) Activate the desalted antibody with a diluted 2-iminothione solution; (IV) Activation of desalted alkaline phosphatase with sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate; (V) Pass the activated antibody and alkaline phosphatase through a desalting column, centrifuge, and collect the desalted liquid separately; (VI) The antibody desalted by the desalting column and alkaline phosphatase were mixed in proportion and allowed to stand at 0-7℃ for more than 12 hours to obtain the antibody labeled with alkaline phosphatase. (VII) Dilute the alkaline phosphatase-labeled antibody to a working solution concentration of 0.1-3 ug / mL.

[0011] Preferably, in step (III), the components of the diluent used for dilution include triethanolamine and disodium ethylenediaminetetraacetate (EDTA·2Na), and in step (VII), the components of the diluent used for dilution include 2-morpholinoethanesulfonic acid (MES), bovine serum albumin (BSA), Tween 20, Proclin 300, zinc chloride, and magnesium chloride.

[0012] The present invention also provides a method for preparing the above-mentioned chemiluminescent detection kit for creatinine in urine, comprising the following steps: (1) Preparation of calibrators: creatinine antigen II was diluted with a diluent containing bovine serum albumin, tris(hydroxymethyl)aminomethane buffer and Proclin 300 preservative to prepare calibrators. The concentration of the calibrators was 0-25 mg / mL. (2) Preparation of quality control material: creatinine antigen II was diluted with a diluent containing bovine serum albumin, tris(hydroxymethyl)aminomethane buffer and Proclin 300 preservative to prepare quality control material. The concentration of quality control material was 0.5-20 mg / mL. (3) Preparation of creatinine antigen-1 reagent solution coated with magnetic microparticles: After the magnetic beads react with creatinine antigen-1, they are then blocked and diluted to obtain the solution. (4) Preparation of alkaline phosphatase-labeled polyclonal antibody reagent solution: After desalting, activating and desalting alkaline phosphatase is used to label desalted, activated and desalted creatinine polyclonal antibody, the solution is diluted.

[0013] As a preferred option, step (3) specifically involves: (a) Take the magnetic beads into the centrifuge tube and place it on the magnetic rack to magnetically remove the supernatant; then add borate buffer to the centrifuge tube, vortex to mix thoroughly, and place it on the magnetic rack to magnetically remove the supernatant. (b) Add borate buffer and ammonium sulfate vortex magnetic beads to the magnetic beads treated above, add creatinine antigen and vortex magnetic beads thoroughly, and react under constant temperature conditions; (c) After the reaction, place the centrifuge tubes on a magnetic rack to magnetically remove the supernatant, add blocking buffer to wash, then add more blocking buffer and continue mixing and blocking under constant temperature conditions; the blocking buffer consists of phosphate-buffered saline (PBS), bovine serum albumin (BSA), Tween 20, and Proclin 300. (d) After sealing, place the centrifuge tube on a magnetic rack to magnetically remove the supernatant, add magnetic bead preservation solution, vortex the magnetic beads, magnetically remove the supernatant, and then add magnetic bead preservation solution to dilute the magnetic bead concentration to obtain magnetic beads coated with creatinine antigen 1; the components of the magnetic bead preservation solution include tris(hydroxymethyl)aminomethane (Tris), bovine serum albumin, Tween 20 and Proclin 300; (e) Dilute the magnetic beads coated with good creatinine antigen to a working solution concentration of 0.1–0.4 mg / mL.

[0014] As a preferred option, step (4) specifically involves: (I) Take two desalting columns and centrifuge them to remove the liquid inside the tubes; (II) Add the polyclonal antibody to one of the desalting columns; add alkaline phosphatase to the other desalting column, and centrifuge both desalting columns simultaneously; collect the desalted liquid separately. (III) Activate the desalted antibody with a diluted 2-iminothione solution; the diluent used for dilution consists of triethanolamine and disodium ethylenediaminetetraacetate (EDTA·2Na). (IV) Activation of desalted alkaline phosphatase with sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate; (V) Pass the activated antibody and alkaline phosphatase through a desalting column, centrifuge, and collect the desalted liquid separately; (VI) The antibody desalted by the desalting column and alkaline phosphatase were mixed in proportion and allowed to stand at 0-7℃ for more than 12 hours to obtain the antibody labeled with alkaline phosphatase. (VII) Dilute the alkaline phosphatase-labeled antibody to a working solution concentration of 0.1-3 ug / mL; the diluent components include 2-morpholinoethanesulfonic acid (MES), bovine serum albumin (BSA), Tween 20, Proclin 300, zinc chloride, and magnesium chloride.

[0015] This invention also provides a chemiluminescent detection method for creatinine in urine for non-diagnostic purposes. Using the above-mentioned kit, the operation steps are as follows: The sample is incubated simultaneously with a creatinine antigen-reagent solution coated with magnetic microparticles and an alkaline phosphatase-labeled polyclonal antibody reagent solution in a constant temperature environment to form an enzyme-labeled antibody-magnetic bead antigen complex or an enzyme-labeled antibody-antigen complex. Under the action of an external magnetic field, the magnetic microparticles are adsorbed onto the reaction tube wall, and unbound substances are washed away by washing solution. A luminescent substrate solution is added, and luminescence is catalyzed by the complex. The luminescence intensity is inversely proportional to the creatinine content. The concentration of creatinine in the urine sample is obtained using a standard curve.

[0016] Preferably, the incubation temperature is 35–39°C, the incubation time is 12–18 min, and the luminescent substrate solution is AMPPD.

[0017] The beneficial effects of this invention are as follows: This invention's kit can be used for the direct detection of creatinine in urine without sample dilution, reducing usage costs and workload for laboratory physicians, and shortening result time. The invention employs a one-step competitive reaction mode, requiring only one antibody, saving costs. Magnetic bead coating is used, making the coating process simple and easy to operate. Through alkaline phosphatase-catalyzed AMPPD luminescence, compared to traditional and currently commonly used methods, it not only improves detection sensitivity but also increases the linear range, exhibiting advantages such as high specificity, high sensitivity, wide applicability, and broad detection range. Furthermore, this invention does not require other light sources, effectively preventing the influence of light scattering and achieving a higher signal-to-noise ratio. Attached Figure Description

[0018] Figure 1 A graph showing the correlation between the concentration points of the calibrator and the luminescence value; Figure 2 The graph shows the test results of the present invention and the comparison creatinine detection kit (enzymatic method); Figure 3 The percentage difference between the present invention and the comparison creatinine detection kit (enzymatic method) was calculated using the software MedCalc to obtain the result graph. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and the technical solutions of the present invention will be further described in detail. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0020] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0021] Creatinine antigen 1 (creatinine antigen for coated magnetic beads): purchased from Xiamen Huasheng Times Biotechnology Co., Ltd., product name: CRE-BSA, product code: HSJ03; Creatinine antigen 2 (creatinine antigen for calibrators / quality control products): purchased from Aladdin, product name: creatinine, product code: C108393; Creatinine polyclonal antibody: purchased from Xiamen Huasheng Times Biotechnology Co., Ltd.; AMPPD: purchased from Changsha Xinlizhihe Technology Co., Ltd.

[0022] Example 1 Reagent kit preparation Preparation of calibrators: creatinine antigen II was diluted with 50mM Tris buffer containing 1% BSA, pH 7.2 and 0.05% Proclin 300 preservative to prepare calibrators, and aliquoted into 0 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 1 mg / mL, 5 mg / mL and 20 mg / mL.

[0023] Preparation of quality control samples: The creatinine antigen II was diluted with 50mM Tris buffer containing 1% BSA and pH 7.2 and 0.05% Proclin 300 preservative to prepare quality control samples, which were then dispensed into 0.5mg / mL and 2.5mg / mL solutions.

[0024] Preparation of creatinine antigen-reagent solution (R1) coated with magnetic microparticles: (a) Take 10 mg of Tosyl magnetic beads into a 5 mL centrifuge tube and place it on a magnetic rack to magnetically adsorb the supernatant; then add 2 mL of magnetic bead pretreatment solution (0.1 M borate buffer, pH 9.5) to the centrifuge tube, vortex thoroughly to mix, and place it on a magnetic rack to magnetically adsorb the supernatant. (b) Add vortex magnetic beads containing 0.1M borate buffer (pH 9.5) and 3M ammonium sulfate (volume ratio of 0.1M borate buffer to 3M ammonium sulfate is 2:1) to the magnetic beads treated above, add creatinine antigen I and vortex the magnetic beads thoroughly (the ratio of creatinine antigen I to magnetic beads is 20 μg of creatinine antigen I per mg of magnetic beads), and continue to mix at a constant temperature of 37°C for more than 8 hours; (c) After the reaction is complete, place the centrifuge tube on a magnetic rack to magnetically remove the supernatant, add 2 mL of blocking solution (100 mM PBS (pH 7.2), 0.5% BSA, 0.05% Tween 20, 0.05% Proclin 300) to wash once, then add another 2 mL of blocking solution and continue to mix and block at 37°C for 4 hours. (d) After sealing, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add 1 mL of magnetic bead preservation solution (50 mM Tris, 1% BSA, 0.2% Tween 20, 0.05% Proclin 300, pH 7.2) to vortex the magnetic beads, magnetically adsorb the supernatant, and then add another 1 mL of magnetic bead preservation solution to dilute the magnetic bead concentration to 10 mg / mL for storage. (e) Preparation of reagent R1: Dilute the magnetic beads coated with creatinine antigen 1 to the working solution (magnetic bead concentration 0.1-0.4 mg / mL) and store at 4°C for later use.

[0025] Preparation of alkaline phosphatase-labeled polyclonal antibody reagent solution (R2): (I) Take two G25 desalting columns, centrifuge the G25 desalting columns to remove the liquid in the tubes, centrifuge at 5000 rpm for 1 min; (II) Add 0.5 mg of creatinine polyclonal antibody to one of the G25 desalting columns; add 0.6 mg of alkaline phosphatase to the other desalting column, and centrifuge both desalting columns at 5000 rpm for 1 min simultaneously; collect the desalted liquid separately. (III) Dissolve 2-iminothionane in diluent (0.1M triethanolamine, 0.1% EDTA.2Na) to a concentration of 10 mg / mL; activate the desalted antibody by adding 1% of the antibody volume for 5 minutes. (IV) Sulfosuccinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylate was dissolved in N,N-dimethylformamide to a concentration of 5 mg / mL; the desalted alkaline phosphatase was activated by adding 1% of the volume of alkaline phosphatase for 5 minutes. (V) Pass the activated antibody and alkaline phosphatase through a G25 desalting column, centrifuge at 5000 rpm for 1 min, and collect the desalted liquid separately; (VI) The antibody and alkaline phosphatase desalted by the desalting column were mixed at a mass ratio of antibody:alkaline phosphatase = 1:1 and left to stand at 4°C for more than 12 hours. (VII) Preparation of reagent R2: Dilute the alkaline phosphatase-labeled antibody with diluent (50mM MES, 1% BSA, 0.2% Tween 20, 0.05% Proclin 300, 0.1 mM ZnCl2, 0.1 mM MgCl2, pH 6.5) to the working solution (working concentration 0.1-3ug / mL) and store at 4℃ for later use.

[0026] The detection method of the above kit is based on a competitive method. Creatinine antigen-reagent R1 coated with magnetic microparticles, the creatinine to be tested in the sample, and alkaline phosphatase-labeled polyclonal antibody reagent R2 are incubated together at 37°C for 15 min to form an enzyme-labeled antibody-magnetic bead antigen complex or an enzyme-labeled antibody-antigen complex. Under the action of an external magnetic field, the magnetic microparticles are adsorbed onto the reaction tube wall, and unbound substances are washed away by washing buffer. The luminescent substrate solution AMPPD is added, and the alkaline phosphatase in the complex catalyzes the AMPPD to emit light. The luminescence intensity is inversely proportional to the creatinine content. Within the linear range, the concentration of creatinine in the urine sample can be calculated using a standard curve.

[0027] Example 2 Reagent kit performance testing: The reagent kit prepared in Example 1 was used for testing. Linearity: Detection was performed at 0 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 1 mg / mL, 5 mg / mL, and 20 mg / mL. The corresponding luminescence values ​​at each concentration point of the calibrator are shown in Table 1; the correlation curves are shown below. Figure 1 As shown, the linear correlation coefficient R of the calibrator curve can be obtained through a four-parameter logistic regression (4PL) model. 2 =0.998939, indicating a very good linear correlation.

[0028] Table 1 Accuracy: The certified reference material PHR1462 (sigma) for creatinine was prepared into two concentration points of 0.5 mg / mL and 2.5 mg / mL for testing. Each concentration point was tested 3 times, and the relative deviation between each test value and the theoretical value was calculated. The results are shown in Table 2. Accuracy = (Measured concentration - Certified reference material target value) / Certified reference material target value × 100% Table 2 As can be seen from Table 2, the relative deviation between each test value and the theoretical value is within 8%. When using certified reference materials for comparison, the smaller the deviation between the measurement results and the certified reference materials, the smaller the systematic error, and thus the higher the accuracy.

[0029] Sensitivity: The calibrator was tested at zero concentration for 20 times. The luminescence value (RLU) of the 20 measurements was obtained, and the mean (M) and standard deviation (SD) were calculated. The results are shown in Table 3.

[0030] Table 3 The corresponding concentration value of 0.004 mg / mL can be obtained through a four-parameter logistic regression model, which means that the sensitivity of the kit of this invention is 0.004 mg / mL.

[0031] Repeatability: Q1 and Q2 in the test kit were tested 10 times each, and the coefficient of variation (CV) was calculated. The results are shown in Table 4.

[0032] Table 4 As can be seen from Table 4, the test CV value is within 3%, and the smaller the CV value, the higher the precision.

[0033] Clinical urine sample test results: Table 5 shows the test results of 40 clinical urine samples tested using the present invention and the compared creatinine detection kit (enzymatic method) from a certain manufacturer. Figure 2 As shown, a correlation curve was established using the test results of a certain manufacturer's creatinine detection kit (enzymatic method) as the x-axis and the test results of this invention as the y-axis. The fitting equation is y = 0.9831x + 255.69, and the correlation coefficient R0 is 0.9831x + 255.69. 2 =0.9929. Data analysis shows that the results of this invention and the comparative creatinine detection kit (enzymatic method) are in good agreement. Figure 3 The percentage difference results were calculated using the MedCalc software: the x-axis represents the average measurement values ​​of the present invention and the control reagent, and the y-axis represents the ratio of the difference between the measured values ​​of the present invention and the control reagent to their average values. When calculating the conformity limits using the percentage difference, the 95% conformity limits were [-8.7%, 9.3%], within the ±15% acceptable standard. This verifies that the test results of the present invention are stable and reliable.

[0034] Table 5 The performance comparison results of the kit of the present invention and the comparative creatinine detection kit (enzymatic method) are shown in Table 6.

[0035] Table 6 As can be seen from Table 6, the sensitivity of the kit of the present invention is significantly higher than that of existing creatinine detection kits (enzymatic method); the linear range is wider than that of existing creatinine detection kits (enzymatic method); at the same time, urine samples do not need to be diluted, which reduces the cost of use and the workload of doctors in the laboratory department, and shortens the time to obtain results.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chemiluminescent detection kit for creatinine in urine, characterized in that, The kit is used for the direct detection of creatinine in urine without sample dilution. The kit includes calibrators, quality controls, a creatinine antigen-1 reagent solution coated with magnetic microparticles, and an alkaline phosphatase-labeled polyclonal antibody reagent solution. The calibrators include multiple concentrations of creatinine antigen-2. The quality controls include more than one concentration of creatinine antigen-2. The creatinine antigen-1 reagent solution coated with magnetic microparticles is obtained by reacting magnetic beads with creatinine antigen-1, followed by blocking and dilution. The alkaline phosphatase-labeled polyclonal antibody reagent solution is obtained by labeling desalted, activated, and desalted creatinine polyclonal antibodies with alkaline phosphatase, followed by dilution; creatinine antigen one is creatinine antigen for coating magnetic microbeads, and creatinine antigen two is creatinine antigen for calibrators / quality control products.

2. The chemiluminescent detection kit for creatinine in urine according to claim 1, characterized in that, The creatinine antigen-reagent solution coated with magnetic microparticles was prepared by the following steps: (a) Take magnetic beads into centrifuge tubes and place them on a magnetic rack to magnetically adsorb the supernatant; then add magnetic bead pretreatment solution to centrifuge tubes, vortex thoroughly to mix, and place them on a magnetic rack to magnetically adsorb the supernatant. (b) Add magnetic bead pretreatment solution, ammonium sulfate vortex magnetic beads to the magnetic beads treated above, add creatinine antigen and vortex the magnetic beads thoroughly, and react under constant temperature conditions; (c) After the reaction is complete, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add blocking solution to clean, add blocking solution again, and continue to mix and block under constant temperature conditions. (d) After sealing, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add magnetic bead preservation solution to vortex the magnetic beads, magnetically adsorb the supernatant, and then add magnetic bead preservation solution to dilute the magnetic bead concentration to obtain magnetic beads coated with creatinine antigen 1. (e) Dilute the magnetic beads coated with good creatinine antigen 1 with magnetic bead preservation solution to a working solution concentration of 0.1 to 0.4 mg / mL.

3. The chemiluminescent detection kit for creatinine in urine according to claim 2, characterized in that, The magnetic bead pretreatment solution is borate buffer, the blocking solution consists of phosphate buffer, bovine serum albumin, Tween 20 and Proclin 300, and the magnetic bead preservation solution consists of tris(hydroxymethyl)aminomethane, bovine serum albumin, Tween 20 and Proclin 300.

4. The chemiluminescent detection kit for creatinine in urine according to claim 1, characterized in that, The alkaline phosphatase-labeled polyclonal antibody reagent solution was prepared by the following steps: (I) Take two desalting columns and centrifuge them to remove the liquid inside the tubes; (II) Add the polyclonal antibody to one of the desalting columns; add alkaline phosphatase to the other desalting column, and centrifuge both desalting columns simultaneously; collect the desalted liquid separately. (III) Activate the desalted antibody with a diluted 2-iminothione solution; (IV) Activation of desalted alkaline phosphatase with sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate; (V) Pass the activated antibody and alkaline phosphatase through a desalting column, centrifuge, and collect the desalted liquid separately; (VI) The antibody desalted by the desalting column and alkaline phosphatase were mixed in proportion and allowed to stand at 0-7℃ for more than 12 hours to obtain the antibody labeled with alkaline phosphatase. (VII) Dilute the alkaline phosphatase-labeled antibody to a working solution concentration of 0.1-3 ug / mL.

5. The chemiluminescent detection kit for creatinine in urine according to claim 4, characterized in that, In step (III), the components of the diluent used for dilution include triethanolamine and disodium ethylenediaminetetraacetate. In step (VII), the components of the diluent used for dilution include 2-morpholine ethanesulfonic acid, bovine serum albumin, Tween 20, Proclin 300, zinc chloride, and magnesium chloride.

6. A method for preparing a chemiluminescent detection kit for creatinine in urine as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of calibrators: creatinine antigen II was diluted with a diluent containing bovine serum albumin, tris(hydroxymethyl)aminomethane buffer and Proclin 300 preservative to prepare calibrators. The concentration of the calibrators was 0-25 mg / mL. (2) Preparation of quality control material: creatinine antigen II was diluted with a diluent containing bovine serum albumin, tris(hydroxymethyl)aminomethane buffer and Proclin 300 preservative to prepare quality control material. The concentration of quality control material was 0.5-20 mg / mL. (3) Preparation of creatinine antigen-1 reagent solution coated with magnetic microparticles: After the magnetic beads react with creatinine antigen-1, they are then blocked and diluted to obtain the solution. (4) Preparation of alkaline phosphatase-labeled polyclonal antibody reagent solution: After desalting, activating and desalting alkaline phosphatase is used to label desalted, activated and desalted creatinine polyclonal antibody, the solution is diluted.

7. The method for preparing the chemiluminescent detection kit for creatinine in urine according to claim 6, characterized in that, Step (3) is as follows: (a) Take the magnetic beads into the centrifuge tube and place it on the magnetic rack to magnetically remove the supernatant; then add borate buffer to the centrifuge tube, vortex to mix thoroughly, and place it on the magnetic rack to magnetically remove the supernatant. (b) Add borate buffer and ammonium sulfate vortex magnetic beads to the magnetic beads treated above, add creatinine antigen and vortex magnetic beads thoroughly, and react under constant temperature conditions; (c) After the reaction is complete, place the centrifuge tube on a magnetic rack to magnetically remove the supernatant, add blocking solution to wash, add more blocking solution, and continue to mix and block under constant temperature conditions; the components of the blocking solution include phosphate buffer, bovine serum albumin, Tween 20 and Proclin 300. (d) After sealing, place the centrifuge tube on a magnetic rack to magnetically adsorb the supernatant, add magnetic bead preservation solution, vortex the magnetic beads, magnetically adsorb the supernatant, and then add magnetic bead preservation solution to dilute the magnetic bead concentration to obtain magnetic beads coated with creatinine antigen 1; the components of the magnetic bead preservation solution include tris(hydroxymethyl)aminomethane, bovine serum albumin, Tween 20 and Proclin 300; (e) Dilute the magnetic beads coated with good creatinine antigen to a working solution concentration of 0.1–0.4 mg / mL.

8. The method for preparing the chemiluminescent detection kit for creatinine in urine according to claim 6, characterized in that, Step (4) is as follows: (I) Take two desalting columns and centrifuge them to remove the liquid inside the tubes; (II) Add the polyclonal antibody to one of the desalting columns; add alkaline phosphatase to the other desalting column, and centrifuge both desalting columns simultaneously; collect the desalted liquid separately. (III) Activate the desalted antibody with a diluted 2-iminothione solution; the diluent used for dilution consists of triethanolamine and disodium ethylenediaminetetraacetate. (IV) Activation of desalted alkaline phosphatase with sulfosuccinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate; (V) Pass the activated antibody and alkaline phosphatase through a desalting column, centrifuge, and collect the desalted liquid separately; (VI) The antibody desalted by the desalting column and alkaline phosphatase were mixed in proportion and allowed to stand at 0-7°C for more than 12 hours to obtain the antibody labeled with alkaline phosphatase. (VII) Dilute the alkaline phosphatase-labeled antibody to a working solution concentration of 0.1–3 μg / mL; the diluent components include 2-morpholine ethanesulfonic acid, bovine serum albumin, Tween 20, Proclin 300, zinc chloride, and magnesium chloride.

9. A chemiluminescent detection method for creatinine in urine for non-diagnostic purposes, characterized in that, Using the kit described in claim 1, the operation steps are as follows: The sample is simultaneously incubated with a creatinine antigen-reagent solution coated with magnetic microparticles and an alkaline phosphatase-labeled polyclonal antibody reagent solution in a constant temperature environment to form an enzyme-labeled antibody-magnetic bead antigen complex or an enzyme-labeled antibody-antigen complex. Under the action of an external magnetic field, the magnetic microparticles are adsorbed onto the reaction tube wall, and the unbound substances are washed away by the washing solution. A luminescent substrate solution is added, and luminescence is catalyzed by the complex. The luminescence intensity is inversely proportional to the creatinine content. The creatinine concentration in the urine sample is obtained using a standard curve.

10. The detection method according to claim 9, characterized in that: The incubation temperature was 35–39℃, the incubation time was 12–18 min, and the luminescent substrate solution was AMPPD.

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