HP and KIM-1 joint detection kit and preparation method thereof

Abstract

The invention discloses an HP and KIM-1 combined detection kit, which is used for early diagnosis of diabetic nephropathy and comprises a reagent card, the reagent card comprises a PVC (polyvinyl chloride) rubber plate, a nitrocellulose membrane arranged on the PVC rubber plate, and a combination pad, a sample pad and absorbent paper which are sequentially arranged on the nitrocellulose membrane; the nitrocellulose membrane is coated with an HP antibody, a KIM-1 antibody and a quality control antibody; and an HP antibody and a KIM-1 antibody are marked as tracers on the combination pad through a coupling method. The invention also discloses a preparation method of the HP and KIM-1 joint detection kit. The kit can be used for combined detection of HP and KIM-1 in urine, and early diagnosis of diabetic nephropathy is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, and in particular to a HP and KIM-1 combined detection kit and a preparation method thereof. Background Art

[0002] Diabetic kidney disease (DKD) is one of the most important complications of diabetes and the primary cause of end-stage kidney disease (ESKD). Studies have reported that approximately 26.8% of patients with type 2 diabetes will develop DKD. In recent years, studies have shown that tubulointerstitial lesions can be used as an effective indicator to predict the progression of DKD. Therefore, research on juxtatubular cell markers is expected to become a potential treatment direction for DKD.

[0003] Haptoglobin (HP), also known as haptoglobin, is an acidic glycoprotein with a molecular weight of 85,000, which is widely present in the serum and body fluids of humans and various mammals. In recent years, with the application of proteomics technology, it has been found that HP is closely related to the occurrence and development of type 2 diabetic nephropathy, and its specificity and sensitivity are better than traditional indicators. HP is considered to be a new biomarker of kidney damage.

[0004] Kidney Injury Molecular 1 (KIM-1) is a transmembrane glycoprotein of renal proximal tubular epithelial cells and belongs to the immunoglobulin superfamily. The content is very low or undetectable in normal renal tissue, but the expression level of KIM-1 is upregulated in renal injury and renal disease, resulting in high levels of KIM-1 protein at the top of the cell membrane of dedifferentiated proximal tubular cells. Studies have shown that in patients with type 1 diabetes, KIM-1 in both blood and urine can predict the progression of diabetic nephropathy.

[0005] The combined detection of HP and KIM-1 can comprehensively predict and evaluate type 2 diabetic nephropathy and type 1 diabetic nephropathy, which will bring good news to the majority of diabetic patients. However, there is currently no HP and KIM-1 combined detection reagent for clinical use.

[0006] Therefore, it is urgent to provide a new HP and KIM-1 combined detection kit to solve the above problems. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a HP and KIM-1 combined detection kit and a preparation method thereof, which can perform combined detection of HP and KIM-1 in urine and realize early diagnosis of diabetic nephropathy.

[0008] In order to solve the above technical problems, a technical solution adopted by the present invention is: to provide a combined detection kit of HP and KIM-1 for early diagnosis of diabetic nephropathy, including a reagent card; The reagent card comprises a PVC rubber plate, a membrane carrier arranged on the PVC rubber plate, a binding pad, a sample pad and absorbent paper arranged in sequence on the membrane carrier; the membrane carrier is coated with HP antibody, KIM-1 antibody and quality control antibody; the HP antibody and KIM-1 antibody are marked with tracers on the binding pad by a coupling method.

[0009] In a preferred embodiment of the present invention, the T1 line on the membrane carrier is coated with HP antibody at a concentration of 0.1-0.5 mg / ml; the T2 line is coated with KIM-1 antibody at a concentration of 0.2-1.0 mg / ml; the C line is coated with quality control antibody at a concentration of 0.1-0.5 mg / ml; and the coating amount of T1, T2 and C lines is 1 ul / cm.

[0010] In order to solve the above technical problems, another technical solution adopted by the present invention is: to provide a method for preparing the HP and KIM-1 combined detection kit as described above, comprising the following steps: (1) Membrane carrier antibody coating: (1.1) Prepare coating solution; (1.2) Preparation of coating antibodies: Place HP antibody, KIM-1 antibody and quality control antibody in ultrafiltration tubes respectively, centrifuge and replace with phosphate buffer, collect the antibodies in the ultrafiltration tubes into new centrifuge tubes, add an equal volume of 2× coating solution to dilute and mix, and prepare HP coating antibody, KIM-1 coating antibody and quality control coating antibody; (1.3) Antibody coating: The T1 line on the membrane carrier is coated with HP antibody, the T2 line is coated with KIM-1 antibody, and the C line is coated with quality control antibody, and then dried to obtain a coated ruled PVC rubber plate; (2) Preparation of bonding pad: (2.1) Quantum dot activation and antibody labeling: add the quantum dot solution to 10 mM MES solution, add EDC and NHS, and react at room temperature; add HP labeled antibody and KIM-1 labeled antibody respectively, mix thoroughly, and react at room temperature with a vortex mixer; add 10% BSA solution, mix thoroughly, react at room temperature with a vortex mixer, centrifuge, discard the supernatant, and retain the precipitate to label the HP antibody and KIM-1 antibody respectively; or Preparation of colloidal gold and antibody labeling: Take the prepared colloidal gold solution, add K2CO3 solution and mix well, then add HP labeled antibody and KIM-1 labeled antibody respectively, mix well and react, add blocking solution A, mix well and react, then add blocking solution B, after mixing and reacting, HP antibody and KIM-1 antibody are labeled respectively; (2.2) Prepare the reconstitution solution; (2.3) Redissolving the labeled antibodies and spraying the membrane: Use the re-dissolving solution to re-dissolve the prepared HP labeled antibodies and KIM-1 labeled antibodies, mix the two labeled antibodies in a 1:1 ratio, spray them on the glass fiber membrane, and obtain the quantum dot binding pad after drying; (3) Sample pad preparation: preparing a sample pad treatment solution; soaking the glass fiber membrane in the sample pad treatment solution, and drying to obtain a sample pad; (4) Paste the conjugate pad, sample pad, and absorbent paper onto the membrane carrier PVC plate coated with the antibody in sequence, cut them into strips and put them into the cartridge, put them into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature.

[0011] In a preferred embodiment of the present invention, the coating solution includes 0.5% BSA, 0.05% S17, 0.2% trehalose, 0.1% casein sodium, and 0.02% PEG6000 in 0.01M phosphate buffer at pH 6.5-8.0.

[0012] In a preferred embodiment of the present invention, the reconstitution solution includes 1% BSA, 0.5% trehalose, 0.5% L-tyrosine, 0.2% gelatin, and 0.05% S17 in 0.01M phosphate buffer at pH 6.5-8.0.

[0013] In a preferred embodiment of the present invention, the sample pad treatment solution includes 0.01M, pH 6.5-8.0 phosphate buffer containing 0.5% trehalose, 0.1% polyethyleneimine, 0.02% sodium dodecyl sulfate, and 0.05% S17.

[0014] In a preferred embodiment of the present invention, the method further comprises preparing a composite standard and a composite quality control product preservation solution, wherein the composite standard and the composite quality control product preservation solution comprises 0.01M phosphate buffer, added with 0.1%-0.5% BSA, 0.5%-1.0% trehalose, 0.1%-2% mannitol, 0.1%-0.5% caseinate sodium, 0.2-0.9% sodium chloride, 0.01-0.05% β-cyclodextrin, and 0.05% Tween-20.

[0015] Furthermore, the pH of the phosphate buffer is 6.5-8.0.

[0016] The beneficial effects of the present invention are as follows: the present invention adopts the immunoassay technology of antigen-antibody reaction, HP antibody, KIM-1 antibody and quality control antibody are coated on nitrocellulose membrane or other carriers, another HP antibody and KIM-1 antibody are marked with tracers and sprayed on glass fiber membrane or other carriers, and HP and KIM-1 in urine can be jointly detected. Its sensitivity, repeatability and accuracy meet clinical requirements. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0018] Embodiments of the present invention include: A HP and KIM-1 combined detection kit for early diagnosis of diabetic nephropathy, comprising a reagent card.

[0019] The reagent card comprises a PVC board, a membrane carrier arranged on the PVC board, a binding pad, a sample pad, and a blotting paper arranged on the nitrocellulose membrane in sequence. Specifically, the binding pad, the sample pad, and the blotting paper are sequentially attached to the membrane carrier coated with the antibody in an overlapping manner.

[0020] Specifically, the membrane carrier may be a nitrocellulose membrane or other membrane materials. In this example, the membrane carrier is a nitrocellulose membrane. The binding pad may be a glass fiber membrane or other membrane materials.

[0021] The nitrocellulose membrane is coated with three antibodies, namely HP antibody, KIM-1 antibody and quality control antibody. In this example, the quality control antibody is rabbit anti-mouse IgG antibody. Specifically, the T1 line on the nitrocellulose membrane is coated with HP antibody at a concentration of 0.1-0.5 mg / ml; the T2 line is coated with KIM-1 antibody at a concentration of 0.2-1.0 mg / ml; the C line is coated with quality control antibody at a concentration of 0.1-0.5 mg / ml; the coating amount of T1, T2 and C lines is 1 ul / cm.

[0022] HP antibody and KIM-1 antibody are labeled with tracers on the conjugate pad by a coupling method. The tracers may be quantum dots, fluorescein isothiocyanate, phycoerythrin, time-resolved fluorescent substances, colloidal gold, etc. In this example, the tracers are quantum dots and colloidal gold.

[0023] The present invention also provides a method for preparing the kit, comprising the following steps: (1) Nitrocellulose membrane antibody coating: (1.1) Prepare coating solution: 0.01M phosphate buffer (pH 6.5-8.0) containing 0.5% BSA, 0.05% S17, 0.2% trehalose, 0.1% sodium caseinate, and 0.02% PEG6000.

[0024] (1.2) Preparation of coating antibody: (1.2.1) Take 0.1 mg of HP coated antibody and place it in an ultrafiltration tube. Centrifuge it with 0.01M-0.05M, pH 6.5-8.5 phosphate buffer. Centrifuge parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 0.5mg / ml; (1.2.2) Take 0.2 mg of KIM-1 coated antibody and place it in an ultrafiltration tube. Centrifuge it with 0.01M-0.05M, pH 6.5-8.5 phosphate buffer. Centrifuge parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 1.0mg / ml; (1.2.3) Take 0.1 mg of rabbit anti-mouse IgG antibody and place it in an ultrafiltration tube. Centrifuge it with the prepared 0.01M-0.05M, pH 6.5-8.5 phosphate buffer. The centrifugation parameters are: 2-8℃, 12000rpm, 5min. Collect the antibody in the ultrafiltration tube into a new centrifuge tube (the volume is generally 100ul), add an equal volume of 2× coating solution to dilute and mix, so that the coating antibody concentration is 0.5mg / ml.

[0025] (1.3) Coating antibody: The T1 line on the nitrocellulose membrane is coated with HP antibody at a concentration of 0.1-0.5 mg / ml; the T2 line is coated with KIM-1 antibody at a concentration of 0.2-1.0 mg / ml; the C line is coated with rabbit anti-mouse IgG antibody at a concentration of 0.1-0.5 mg / ml; the coating amount of T1, T2, and C lines is 1 ul / cm, and the lines are placed face down in parallel and dried at 37°C for 2 hours to obtain a coated ruled rubber plate.

[0026] (2) Preparation of bonding pad: (2.1) Quantum dot activation and antibody labeling: (2.1.1) Take 30-60ul of quantum dot solution and add it to 10mM MES solution (pH5.5), with a final volume of 200ul. Add 200-400ul of 20mg / ml EDC and 200-400ul of 30mg / ml NHS, and react at room temperature for 30min. Add 10ug HP labeled antibody and mix thoroughly, and react at room temperature for 2h using a vortex mixer. Add 20ul of 10% BSA solution and mix thoroughly, and react at room temperature for 1h using a vortex mixer. Centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate; (2.1.2) Take 30-60ul of quantum dot solution and add it to 10mM MES solution (pH5.5), with a final volume of 200ul. Add 200-400ul of 20mg / ml EDC and 200-400ul of 30mg / ml NHS, and react at room temperature for 30min. Add 10ug of KIM-1 labeled antibody and mix thoroughly, and react at room temperature for 2h using a vortex mixer. Add 20ul of 10% BSA solution and mix thoroughly, and react at room temperature for 1h using a vortex mixer. Centrifuge at 2-8℃, 12000rpm for 10min, discard the supernatant, and keep the precipitate; Or (2.1') ​​using colloidal gold preparation and antibody labeling: (2.1.1') Gold label rehydration solution: Weigh 1.000g sucrose, 1.000g trehalose, and 0.010g caseinate sodium, add 10ml 10mmol / L, pH7.4PB, mix and dissolve thoroughly, and filter through a 0.45μm microporous filter membrane to obtain the gold-labeled complex solution.

[0027] (2.1.2') Preparation of colloidal gold: (1) Preparation of reducing solution Weigh 0.400 g of trisodium citrate, dissolve it completely in 20 ml of purified water, and filter it through a 0.45 μm microporous filter membrane to obtain a reducing solution.

[0028] (2) Add 480ml of purified water to a 1L round-bottom flask, then add 5ml of 2% chloroauric acid solution, add a stirrer, place on a magnetic heating mantle for heating and stirring, cover the flask with a glass watch glass, and quickly add 5.5ml of reducing solution after the solution boils. Continue heating and stirring for 5min after the solution turns purple-red, turn off the heating mantle, wait for the colloidal gold solution to cool, and dilute the cooled colloidal gold solution to 500ml. Use a spectrophotometer to test the maximum absorption peak wavelength of colloidal gold and the corresponding OD value under a 1cm optical path.

[0029] (2.1.3') Gold label pad pretreatment: (1) Prepare gold label pad pretreatment solution 3.000 g polyvinyl pyrrolidone K30, 2.000 g Tween 20, 1.000 g Triton, 3.000 g bovine serum albumin and 1.000 g trehalose were added to 10 mM / L PB with a pH of 7.40, and the mixture was fully dissolved and mixed to obtain the gold label pad pretreatment solution.

[0030] (2) Take a glass cellulose membrane and soak it completely in the gold label pad pretreatment solution for 1 hour. Take it out and place it face down in parallel and dry it at 37℃.

[0031] (2.1.4') Preparation of gold standard pad: (1) Antibody labeling (1.1) Take HP antibody and KIM-1 antibody and dilute them to 0.1 mg / ml respectively with 10 mmol / L, pH 7.4 PB.

[0032] (1.2) Take 1 ml of colloidal gold solution, add 15 μl of 0.1 mol / L K2CO3 and mix well. Then add 85 μl of 0.1 mg / ml HP antibody and mix well for 30 minutes. Then add 50 μl of blocking solution A and mix well for 15 minutes. Then add 50 μl of blocking solution B and mix well for 15 minutes to obtain HP antibody marker.

[0033] Take 1 ml of colloidal gold solution, add 17 μl of 0.1 mol / L K2CO3 and mix well, then add 100 μl of 0.1 mg / ml KIM-1 antibody, mix well for 30 minutes, add 50 μl of blocking solution A, mix well for 15 minutes, then add 50 μl of blocking solution B, mix well for 15 minutes to obtain the KIM-1 antibody marker.

[0034] The blocking solution A is obtained by adding 0.1-0.5 g of bovine serum albumin to a 0.01 mol / L phosphate buffer at a pH of 7.4±0.05, and the blocking solution B is obtained by adding 0.1-0.25 g of polyethylene glycol 20000 to a 0.01 mol / L phosphate buffer at a pH of 7.4±0.05.

[0035] (1.2) Centrifuge the markers separately at 8000 rpm for 35 min, discard the supernatant, collect the precipitate, take the gold label solution to resuspend and redissolve the precipitate, mix the HP antibody complex and the KIM-1 antibody complex at a ratio of 1.0:1.5, and finally mix and redissolve to 100 μl.

[0036] (1.3) Gold spraying Set the gold spraying parameters to 3.0-8.0 μl / cm and spray it on the glass fiber membrane. Place it parallelly with the front side facing down and dry it at 37°C to obtain the binding pad.

[0037] (2.2) Prepare the reconstitution solution: 1% BSA, 0.5% trehalose, 0.5% L-tyrosine, 0.2% gelatin, and 0.05% S17 in 0.01M phosphate buffer at pH 6.5-8.0; (2.3) Redissolving the labeled antibodies and spraying: Use the reconstitution solution to reconstitute the prepared HP labeled antibody to a concentration of 0.5 mg / ml, and the KIM-1 labeled antibody to a concentration of 0.5 mg / ml. The two labeled antibodies are fully mixed in a 1:1 ratio, sprayed on the glass fiber membrane at a parameter of 5-15 ul / cm, placed face down in parallel, and dried at 37°C to obtain the binding pad.

[0038] (3) Sample pad preparation: Prepare the sample pad treatment solution: 0.01M pH 6.5-8.0 phosphate buffer containing 0.5% trehalose, 0.1% polyethyleneimine, 0.02% sodium dodecyl sulfate, and 0.05% S17; The glass fiber membrane is immersed in a sample pad treatment solution and dried to obtain a sample pad; (4) Paste the conjugate pad, sample pad, and absorbent paper onto the antibody-coated nitrocellulose membrane PVC plate in sequence, cut them into strips and put them into the cartridge, put them into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature.

[0039] Furthermore, the preparation method also includes preparing composite standard and composite quality control preservation solutions. In this example, two composite standard products C1 and C2 are prepared, and the coating antibody concentration of C1 is HP: 20.0ng / ml, KIM-1: 0.20ng / ml; the coating antibody concentration of C2 is HP: 80.0ng / ml, KIM-1: 1.00ng / ml. The preparation method of the composite quality control preservation solution is: 0.1%-0.5% BSA, 0.5%-1.0% trehalose, 0.1%-2% mannitol, 0.1%-0.5% casein sodium, 0.2-0.9% sodium chloride, 0.01-0.05% β-cyclodextrin, and 0.05% Tween-20 are added to 0.01M phosphate buffer (pH 6.5-8.0).

[0040] The detection method of the kit comprises the following steps: Step 1: Place the reagent card at room temperature for 30-60 minutes (the sealed aluminum foil bag cannot be opened); Step 2: Open the aluminum foil bag, take out the reagent card and place it parallel to the desktop, and take 15ul-40ul of the sample to be tested into the sample well of the test card; Step 3: After 5-10 minutes, place the reagent card in the instrument card slot for testing; Step 4: Using the calibration curve with known concentration, substitute the obtained fluorescence value into the standard curve equation to calculate the concentration of HP and KIM-1 in the measured sample.

[0041] The kit prepared by the present invention adopts the immunoassay technology of antigen-antibody reaction, and HP antibody, KIM-1 antibody and quality control antibody are coated on nitrocellulose membrane or other carriers, and another HP antibody and KIM-1 antibody are marked with tracers and sprayed on glass fiber membrane or other carriers, so as to jointly detect HP and KIM-1 in urine. Its sensitivity, repeatability and accuracy meet clinical requirements.

[0042] The performance of the kit was evaluated by the following experiments: 1. Linearity and blank limits Set 20 replicate wells for the zero concentration standard (S0), and set single wells for the other 5 concentration standards S1-S5. Draw a standard curve based on the theoretical concentration and fluorescence value of the standard to obtain the dose-response curve of the kit. Calculate the mean (Mean) and standard deviation (SD) of the 20 S0 fluorescence values, and calculate the concentration value of Mean+2SD based on the fitting equation, which is the blank limit of the kit.

[0043] Composite standard concentration: S0: 0ng / ml; S1: (HP: 10.00ng / ml, KIM-1: 0.05ng / ml) S2: (HP: 40.00ng / ml, KIM-1: 0.10ng / ml) S3: (HP: 100.00ng / ml, KIM-1: 0.50ng / ml) S4: (HP: 200.00ng / ml, KIM-1: 1.50ng / ml) S5: (HP: 400.00ng / ml, KIM-1: 7.5ng / ml) Dose-response curve R 2 The values ​​are: 0.9999 (HP); 0.9995 (KIM-1).

[0044] The blank limits of the kit are: 0.1651 (HP); 0.00129 (KIM-1).

[0045] Table 1 Blank limits of test kit

[0046] 2. Repeatability Take high and low concentration composite quality control products, and use three batches of kits to perform 10 repeated tests on each sample. The results are shown in Table 2-1 and Table 2-2: Intra-batch variation of the kit: HP<3.5%; KIM-1<6.0%; The batch difference of the kit was: HP<4.0%; KIM-1<5.5%.

[0047] Table 2-1 Kit repeatability test results (HP)

[0048] Table 2-2 Kit repeatability test results (KIM-1)

[0049] 3. Specificity Add trace albumin (m-ALB), creatinine (Cr), and neutrophil gelatinase-associated lipocalin (NGAL) to low-concentration samples (HP: 20.0 ng / ml, KIM-1: 0.20 ng / ml) to obtain the test samples. Each sample is tested using batch 1 reagents, and the mean of the measurement results should be within the range of the target concentration mean (M) ± 2 standard deviations (SD).

[0050] The test results are shown in Table 3: HP was between 19.33 ng / ml-20.30 ng / ml, and KIM-1 was between 0.18 ng / ml-0.21 ng / ml, both within the target concentration range.

[0051] Table 3 Results of low concentration sample specificity experiments

[0052] 4. Clinical sample testing Urine from 44 normal people and 45 diabetic patients were collected for 2-3 consecutive morning urine tests with UACR in the range of 30-300 mg / g (excluding interference from factors such as infection, exercise, and heart failure). HP and KIM-1 values ​​were jointly tested using the reagent developed by this patent. The differences in HP and KIM-1 test values ​​between normal human urine and diabetic urine were analyzed, and a significant P<0.05 indicated that the difference was significant.

[0053] Combined with Table 4, the results of the HP project T test: Through the SPSS26.0 analysis results, it can be seen that P < 0.05, there is a statistically significant difference in the HP content in the urine of normal people and diabetic patients.

[0054] Table 4 Statistics of HP detection cases and mean values ​​in normal urine and diabetic urine

[0055] Table 5 Independent sample test results of normal urine and diabetic urine HP test data

[0056] As shown in Table 5, the results of the Levene's variance equality test show that P is 5.357E-10, <0.05, and the variances of the two groups of data are not the same. Looking at the t value and P value in the second row, the results show that P is 1.7355E-9, <0.05, and there is a significant difference in the means of the two groups of data.

[0057] Combined with Table 6, the T test results of the KIM-1 project: Through the SPSS26.0 analysis results, it can be seen that P < 0.05, there is a statistically significant difference in the KIM-1 content in the urine of normal people and diabetic patients.

[0058] Table 6 Statistics of the number and mean values ​​of KIM-1 detection in normal urine and diabetic urine

[0059] Table 7 Independent sample test results of KIM-1 detection data of normal urine and diabetic urine

[0060] As shown in Table 7, the results of the Levene's variance equality test show that P is 1.0935E-10, <0.05, and the variances of the two groups of data are not the same. Looking at the t value and P value in the second row, the results show that P is 0.000069, <0.05, and there is a significant difference in the means of the two groups of data.

[0061] 5. Stability assessment of composite quality control products Take 4 sets of composite quality control products, place 1 set at 2-8℃ and 3 sets at 37℃. After 5 days at 37℃, take the composite quality control products at 2-8℃ and 37℃ for 5 days, and test them with batch 1 reagents at the same time, and record the results. Take the other two sets of composite quality control products placed at 37℃ for 9 days and 11 days at 37℃, and test them respectively, and record the results. Combined with Table 8, the stability data shows that the composite quality control products have good stability.

[0062] Table 8 Stability evaluation results of composite quality control products

[0063]

[0064] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0065] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A combined detection kit for HP and KIM-1, characterized in that: Used for early diagnosis of diabetic nephropathy, including reagent cards; The reagent card comprises a PVC rubber plate, a membrane carrier arranged on the PVC rubber plate, a binding pad, a sample pad and absorbent paper arranged in sequence on the membrane carrier; the membrane carrier is coated with HP antibody, KIM-1 antibody and quality control antibody; the HP antibody and KIM-1 antibody are marked with tracers on the binding pad by a coupling method.

2. The HP and KIM-1 combined detection kit according to claim 1, characterized in that: The T1 line on the membrane carrier is coated with HP antibody at a concentration of 0.1-0.5 mg / ml; the T2 line is coated with KIM-1 antibody at a concentration of 0.2-1.0 mg / ml; the C line is coated with quality control antibody at a concentration of 0.1-0.5 mg / ml; and the coating amount of T1, T2 and C lines is 1 ul / cm.

3. A method for preparing the HP and KIM-1 combined detection kit as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Membrane carrier antibody coating: (1.1) Prepare coating solution; (1.2) Preparation of coating antibodies: Place HP antibody, KIM-1 antibody and quality control antibody in ultrafiltration tubes respectively, centrifuge and replace with phosphate buffer, collect the antibodies in the ultrafiltration tubes into new centrifuge tubes, add an equal volume of 2× coating solution to dilute and mix, and prepare HP coating antibody, KIM-1 coating antibody and quality control coating antibody; (1.3) Antibody coating: The T1 line on the membrane carrier is coated with HP antibody, the T2 line is coated with KIM-1 antibody, and the C line is coated with quality control antibody, and then dried to obtain a coated ruled PVC rubber plate; (2) Preparation of bonding pad: (2.1) Quantum dot activation and antibody labeling: add the quantum dot solution to 10 mM MES solution, add EDC and NHS, and react at room temperature; add HP labeled antibody and KIM-1 labeled antibody respectively, mix thoroughly, and react at room temperature with a vortex mixer; add 10% BSA solution, mix thoroughly, react at room temperature with a vortex mixer, centrifuge, discard the supernatant, and retain the precipitate to label the HP antibody and KIM-1 antibody respectively; or Preparation of colloidal gold and antibody labeling: Take the prepared colloidal gold solution, add K2CO3 solution and mix well, then add HP labeled antibody and KIM-1 labeled antibody respectively, mix well and react, add blocking solution A, mix well and react, then add blocking solution B, after mixing and reacting, HP antibody and KIM-1 antibody are labeled respectively; (2.2) Prepare the reconstitution solution; (2.3) Redissolving the labeled antibodies and spraying the membrane: Use the re-dissolving solution to re-dissolve the prepared HP labeled antibodies and KIM-1 labeled antibodies, mix the two labeled antibodies in a 1:1 ratio, spray them on the glass fiber membrane, and obtain the quantum dot binding pad after drying; (3) Sample pad preparation: preparing a sample pad treatment solution; soaking the glass fiber membrane in the sample pad treatment solution, and drying to obtain a sample pad; (4) Paste the conjugate pad, sample pad, and absorbent paper onto the membrane carrier PVC plate coated with the antibody in sequence, cut them into strips and put them into the cartridge, put them into an aluminum foil bag together with the desiccant, seal the bag, and store at room temperature.

4. The method for preparing the HP and KIM-1 combined detection kit according to claim 3, characterized in that: The coating solution includes 0.5% BSA, 0.05% S17, 0.2% trehalose, 0.1% casein sodium, and 0.02% PEG6000 in 0.01M phosphate buffer with a pH of 6.5-8.

0.

5. The method for preparing the HP and KIM-1 combined detection kit according to claim 3, characterized in that: The reconstitution solution includes 1% BSA, 0.5% trehalose, 0.5% L-tyrosine, 0.2% gelatin, and 0.05% S17 in 0.01M phosphate buffer at pH 6.5-8.

0.

6. The method for preparing the HP and KIM-1 combined detection kit according to claim 3, characterized in that: The sample pad treatment solution includes 0.5% trehalose, 0.1% polyethyleneimine, 0.02% sodium dodecyl sulfate, and 0.05% S17 in 0.01M phosphate buffer at pH 6.5-8.

0.

7. The method for preparing the HP and KIM-1 combined detection kit according to claim 3, characterized in that: The method also includes preparing a composite standard and a composite quality control preservation solution, wherein the composite standard and the composite quality control preservation solution include 0.01M phosphate buffer, added with 0.1%-0.5% BSA, 0.5%-1.0% trehalose, 0.1%-2% mannitol, 0.1%-0.5% caseinate sodium, 0.2-0.9% sodium chloride, 0.01-0.05% β-cyclodextrin, and 0.05% Tween-20.

8. The method for preparing the HP and KIM-1 combined detection kit according to claim 7, characterized in that: The pH of the phosphate buffer is 6.5-8.0.

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