NCC antibody solution and application thereof

Abstract

The invention discloses an NCC antibody solution and application thereof. An NCC antibody in the NCC antibody solution is an antibody generated by a polypeptide immune animal with a sequence shown as SEQ ID NO: 1. According to the invention, the self-designed NCC polypeptide antigen is used as the immunogen to prepare the NCC antibody solution, and the obtained antibody has good specificity and can be applied to different NCC content detection methods. The NCC antibody solution disclosed by the invention can be used for detecting different species, has a good linear relationship, is particularly suitable for detecting the NCC content of the urine exosome of a hypertensive patient, can be used as an objective basis for clinically selecting antihypertensive drugs as a detection result, can also be used for detecting kidney samples and urine exosome samples of rats and mice, and has a wide application prospect. The method is used for fundamental research of pathogenesis. Compared with the existing NCC antibody produced by using fusion protein as immunogen, the method disclosed by the invention is more time-saving and money-saving, and the produced NCC antibody is higher in sensitivity when the content of NCC is detected.

Description

Technical Field

[0001] The invention belongs to the technical field of antibodies, and in particular relates to an NCC antibody solution and application thereof. Background Art

[0002] Hypertension is currently the most common chronic disease. It is also a major risk factor for stroke, ischemic heart disease, other cardiovascular diseases and chronic kidney disease, causing more than 10 million deaths worldwide each year. The pathogenesis of hypertension is complex and diverse. Due to the lack of objective basis for selecting drugs based on target protein levels, its drug treatment is blind, inefficient and has superimposed side effects, which increases the burden on society and families.

[0003] First-line antihypertensive diuretics include three major categories, represented by furosemide, hydrochlorothiazide and amiloride, and the corresponding target proteins are sodium-potassium-chloride cotransporter (NKCC2, SLC12A1), sodium-chloride cotransporter (NCC, SLC12A3) and epithelial cell sodium channels (ENaCs). NCC is expressed in the distal convoluted tubules of the renal tubules and excreted in the urine with exosomes. The level of this protein in urinary exosomes of hypertensive animal models reflects the kidney level and is consistent with the antihypertensive effect of hydrochlorothiazide. Although animal experiments have shown that the level of these proteins in the kidney is positively correlated with the antihypertensive effect of the corresponding diuretics, such as hypertensive rats with increased renal NCC have a better antihypertensive effect on its inhibitor hydrochlorothiazide. However, there is a lack of practical sampling / detection methods in the clinic, resulting in no basis for drug selection. We can only turn our attention to non-invasive urine exosome samples. Detection of NCC content in urinary exosomes of hypertensive patients can serve as one of the objective bases for clinical selection of antihypertensive drugs according to target protein levels, achieve personalized molecular diagnosis, effectively lower blood pressure, and thus protect important organs such as the heart, brain, and kidneys of hypertensive patients.

[0004] However, the traditional enzyme-linked immunosorbent assay (ELISA) kit has limited use and is not suitable for the study of tissue target proteins or the detection of exosome biomarkers. These two types of samples are mostly tested by Western hybridization (or immunohybridization) and other immunological methods. In recent years, multi-factor high-throughput antibody chip technology has also attracted more and more attention. In any case, the core principle of these immunoassay technologies is the specific reaction of antigens and antibodies, which depends on the preparation of high-quality antibodies. Therefore, antibody design has become the central link of these immunoassay technologies. However, the NCC antibodies currently on the market are all imported from foreign countries for consignment sales. They are not only expensive and in small quantities, but also cannot be guaranteed in quality. Therefore, the development and production of high-quality NCC antibodies is of great significance for the molecular diagnosis and subclassification of clinical hypertension patients by promoting the detection of urinary exosome NCC. Summary of the invention

[0005] The purpose of the invention is to provide a high-quality NCC antibody solution and apply the solution to the detection and analysis of hypertension and acute kidney injury.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides an NCC antibody solution, wherein the NCC antibody in the NCC antibody solution is an antibody produced by immunizing an animal with a polypeptide having a sequence as shown in SEQ ID NO:1.

[0008] The present invention designs an antigenic polypeptide with high antigenicity and specificity and taking into account the homology between humans and mice according to the primary structure and antigenic polypeptide sequence of human NCC protein, and its amino acid sequence is PGEPRKVRPTLADLHSFLKQEG (SEQ ID NO: 1). Compared with the primary structure and antigenic polypeptide sequence of human NCC protein: NCC, Gene ID: 6559 (https: / / www.ncbi.nlm.nih.gov / gene / 6559) Gene bank Official Symbol: SLC12A3, with a total length of 1030 amino acid residues. Although the antigenic polypeptide designed by the present invention is only 23 amino acid residues in length, it has high antigenicity and specificity, and the antigen prepared by using the immunized animal is of high quality and can be applied to immunoblotting detection of human samples.

[0009] In an embodiment of the present invention, the type of the antibody is IgG or IgY.

[0010] According to some specific embodiments of the present invention, the antibody is rabbit IgG or chicken IgY.

[0011] More preferably, the NCC antibody is a rabbit IgG produced by immunizing a rabbit with a polypeptide having a sequence as shown in SEQ ID NO:1.

[0012] In an embodiment of the present invention, the solvent of the NCC antibody solution is a PBS buffer solution with a pH value between 7.2 and 7.5.

[0013] Furthermore, the concentration of the PBS buffer is between 0.1 mol / L and 0.2 mol / L.

[0014] In an embodiment of the present invention, the preparation method of the NCC antibody solution is as follows:

[0015] (1) using the artificially synthesized polypeptide to immunize an animal to obtain antiserum or highly immune egg yolk fluid;

[0016] (2) The antiserum or high-immune egg yolk solution is purified by Protein A / G affinity chromatography, concentrated by ultrafiltration centrifuge tube, and resuspended in PBS buffer to obtain the NCC antibody solution.

[0017] According to some specific embodiments of the present invention, the filler used in the Protein A / G affinity chromatography is PurKine TM Antibody purification SulfoLink resin.

[0018] The second aspect of the present invention provides the use of the above-mentioned NCC antibody solution in the preparation of a reagent or a kit for detecting the content of NCC in a sample to be tested.

[0019] In the embodiment of the present invention, the sample to be tested is urinary exosomes, and the urinary exosome NCC detection can be promoted to the molecular diagnosis and subclassification of clinical hypertension patients.

[0020] The NCC antibody solution of the present invention can be used to detect the NCC content in urinary exosomes of hypertensive patients, as one of the objective bases for clinical selection of antihypertensive drugs according to the target protein level, to achieve individualized molecular diagnosis, effectively lower blood pressure, and thus protect important organs such as the heart, brain, and kidneys of hypertensive patients.

[0021] The NCC antibody solution of the present invention can also be used for the detection of kidney tissue samples and urine exosome samples of rats and mice, and for basic research such as pathogenesis.

[0022] The third aspect of the present invention provides the use of the above-mentioned NCC antibody solution in the preparation of a hypertension detection reagent or kit.

[0023] Studies have shown that there are large individual differences in the expression of NCC in urinary exosomes, and there is an increasing trend in the urinary exosomes of patients with primary hypertension. The NCC antibody solution of the present invention can be used to detect changes in the level of NCC in urinary exosomes of patients with primary hypertension, which is conducive to the realization of individualized molecular diagnosis in the clinical diagnosis of hypertension. The test results can be used as an objective basis for the clinical selection of antihypertensive drugs.

[0024] The NCC antibody solution of the present invention can also be used for immunoblotting detection of kidneys of rats with essential hypertension.

[0025] The fourth aspect of the present invention provides the use of the above-mentioned NCC antibody solution in the preparation of an acute kidney injury detection reagent or kit.

[0026] Studies have shown that in dopamine receptor 4 knockout mice, the expression of NCC in the kidneys and distal convoluted tubule cells is increased. The NCC antibody solution of the present invention can be used to detect the increase of NCC in the kidneys and distal convoluted tubule cells mentioned above, and can also be used for double immunofluorescence staining of mouse kidneys, and the luminal membrane of the thick ascending limb of the loop of Meuer in the outer medulla of the mouse kidney is well stained.

[0027] The NCC antibody solution of the present invention can also be used for immunoblotting detection of mouse kidney membrane components to detect the expression of 17k (plasma membrane) and 200k (intracellular particles) NCC in mouse kidney.

[0028] The NCC antibody solution of the present invention can also be used for immunofluorescence staining of distal convoluted tubule cells to locate the distal convoluted tubule cell membrane and lysosome.

[0029] The NCC antibody solution of the present invention can also be used in immunohistochemistry / co-immunoprecipitation (CO-IP) experiments.

[0030] In an embodiment of the present invention, the reagent or kit includes a fluorescent staining detection reagent or kit, an immunoblotting detection reagent or kit, an immunoprecipitation detection reagent or kit, and an antibody chip reagent or kit.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The present invention uses a self-designed NCC polypeptide antigen with good antigenicity and the ability to completely block antibodies as an immunogen to prepare an NCC antibody solution. The obtained antibody has good specificity and can be applied to different NCC content detection methods. The NCC antibody solution of the present invention can be used for detection of different species, has a good linear relationship, and is particularly suitable for detecting the NCC content of urine exosomes in patients with hypertension. The test results can be used as one of the objective bases for clinical selection of antihypertensive drugs based on the target protein level, and realize individualized molecular diagnosis. The NCC antibody solution of the present invention can also be used for the detection of kidney samples and urine exosome samples of rats and mice, and for basic research such as pathogenesis. The present invention uses polypeptide antigens as immunogens to produce NCC antibodies. Compared with the existing NCC antibodies produced using fusion proteins as immunogens, the present invention saves more time and money. When the produced NCC antibodies are used to detect the NCC content, they have higher sensitivity and effectively improve the positive detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The NCC antibody solutions of Example 1 and Comparative Example 1 are applied to human samples for immunoblotting detection results;

[0034] Figure 2 This is a graph showing the results of immunofluorescence detection of the distal convoluted tubules of the kidney using the NCC antibody solution of Example 1;

[0035] Figure 3 This is a diagram showing the results of immunohistochemical detection of the distal convoluted tubules of the kidney using the NCC antibody solution of Example 1;

[0036] Figure 4 This is a graph showing the results of the NCC antibody solution of Example 1 applied to the urine exosome NCC detection of patients with essential hypertension (Western blotting method);

[0037] Figure 5 This is a graph showing the results of NCC antibody solution of Example 1 applied to mouse kidney NCC detection (Western blotting);

[0038] Figure 6 This is a graph showing the results of NCC detection in mouse kidney using the NCC antibody solution of Example 1 (immunofluorescence method);

[0039] Figure 7 This is a diagram showing the results of immunofluorescence staining of mouse distal tubule cells using the NCC antibody solution of Example 1;

[0040] Figure 8 This is a graph showing the results of the application of the NCC antibody solution of Example 1 in the detection of NCC in the kidneys of essential hypertensive rats (immunoblotting);

[0041] Fig. 9 This is a graph showing the results of immunohistochemical detection of the distal convoluted tubules of mouse kidney using the NCC antibody solution of Example 1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0045] Unless otherwise specified, the present invention will be implemented using conventional polypeptide artificial synthesis methods, polypeptide antigen animal immunization methods, antibody purification methods, biotechnology detection methods, etc. Experimental methods without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0046] Example 1

[0047] In this example, a polypeptide antigen is used to prepare an NCC antibody solution, as follows:

[0048] 1. Artificially synthesize NCC polypeptide antigen.

[0049] Peptide antigen design: The primary structure and antigen peptide sequence of human NCC protein were selected: NCC Gene ID: 6559 (https: / / www.ncbi.nlm.nih.gov / gene / 6559) Gene bank Official Symbol: SLC12A3, with a total length of 1030 amino acid residues. The peptide segment consisting of the 76th to 97th amino acid sequence was selected as the peptide antigen. The amino acid sequence was: PGEPRKVRPTLADLHSFLKQEG (SEQ ID NO: 1). The peptide was 23 amino acid residues long, highly antigenic and specific, and had homology with the corresponding mouse protein. The NCC peptide antigen was artificially synthesized according to the amino acid sequence shown in SEQ ID NO: 1.

[0050] 2. Prepare NCC antibody solution.

[0051] 2.1 Animal immunization

[0052] The synthetic NCC polypeptide antigen was used to immunize New Zealand rabbits weighing 2-2.5 kg. The NCC polypeptide antigen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, and the New Zealand rabbits were immunized intravenously. The second immunization was performed 15 days after the first immunization, and then booster immunization was performed every 15 days, and a total of five injections were injected, with an injection volume of 0.5 mL / time. One week after the fifth immunization, the New Zealand rabbits were blooded by carotid artery bleeding, and the whole blood was placed in a 37°C constant temperature box for 1 hour, and then placed in a 4°C refrigerator for 3 hours. After the blood coagulated and the blood clot contracted, the serum was drawn with a capillary dropper, centrifuged at 3000 rpm for 15 minutes, and the supernatant was the antiserum, which was placed in a 4°C refrigerator for standby after being packaged. The serum antibody titer of the present embodiment was 1:64000 or more when the ELISA method was used to detect the titer of the serum antibody of the present embodiment.

[0053] 2.2 Antibody purification

[0054] Protein A / G affinity chromatography column was used to purify the antibody, and the column packing was PurKine TM Antibody purification SulfoLink resin.

[0055] (1) Sample pretreatment: The antiserum was centrifuged at 4°C and 12,000 r / min for 15 min. The supernatant was diluted with loading buffer and filtered through a 0.45 μm filter membrane.

[0056] (2) Equilibration: Equilibrate the column with 5-10 column volumes of loading buffer at a flow rate of 1 mL / min until the pH of the effluent reaches 7.4.

[0057] (3) Loading: Load the pretreated antiserum onto the column, maintain a flow rate of 1 mL / min, and continue to wash with loading buffer for 5-10 times the volume of the column until the baseline is stable.

[0058] (4) Elution: The antibody bound to the column was eluted with an eluent and real-time monitoring was performed using a fast protein liquid chromatography (FPLC) system. When the baseline began to rise, i.e., an elution peak appeared, the effluent was collected until the elution peak returned to the baseline and the pH of the collected effluent from each tube was immediately adjusted to 7.0 with 1.0 mol / L pH 9.0 Tris-HCl buffer.

[0059] (5) Regeneration: Maintain a flow rate of 1 mL / min and elute the column with 3-5 times the volume of the column regeneration solution.

[0060] (6) Equilibration: Continue to re-equilibrate the column with 5-10 times the volume of the column loading buffer until the pH of the effluent is neutral, then re-equilibrate with 10 times the volume of the column triple distilled water. It can be reused.

[0061] (7) Concentration: Combine the eluates adjusted to neutrality in each tube, concentrate using an ultrafiltration centrifuge tube, detect the absorbance at a wavelength of 280 using a Thermo Fisher nano protein analyzer, estimate the protein concentration, identify the antibody purity using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis, and quantify the antibody using the bicinchoninic acid assay (BCA) method. Finally, adjust the antibody concentration to 1 mg / ml using a phosphate buffered saline (PBS) solution (pH between 7.2 and 7.5, such as pH 7.4) with a concentration between 0.1 mol / L and 0.2 mol / L (e.g., 0.15 mol / L), which is the desired NCC antibody solution, and store it in aliquots for later use. The PBS buffer solution mainly contains Na2HPO4, KH2PO4, NaCl, and KCl.

[0062] Comparative Example 1

[0063] In this comparative example, an existing NCC polypeptide having an amino acid sequence of DGRPGHELTDGLVEDETGANSEK (SEQ ID NO: 2) was used as an NCC polypeptide antigen, and an NCC antibody solution was prepared according to the method of Example 1.

[0064] Functional verification of the NCC antibody solution prepared in Example 1 and Comparative Example 1

[0065] 1. Western Blot Detection

[0066] (1) Human renal cortical tissue samples (Department of Pathology, BenQ Hospital) and urine exosome samples (Health Check Center, BenQ Hospital) were used as test samples. Human renal cortical tissue was loaded at 20 μg, and urine exosomes were loaded according to creatinine.

[0067] (2) Glue preparation: Prepare separation gel and concentrated gel according to the instructions of the Novozymes One-Step Rapid Western Blot Gel Kit. First, add the separation gel slowly with a pipette, and then add water to seal. Add distilled water to seal quickly and evenly to prevent the liquid surface from being uneven after the separation gel solidifies. After the separation gel solidifies, add the concentrated gel. Be careful not to have bubbles. Insert a comb and let the concentrated gel stand for use after it is completely solidified;

[0068] (3) Sample loading: Remove the prepared gel and place it on the electrophoresis tank. Add electrophoresis solution to the tank, gently pull out the comb, and load the protein and protein maker samples.

[0069] (4) Electrophoresis: Fill the tank with electrophoresis solution, adjust the voltage to 80 V, and start electrophoresis. When the protein maker bands are clearly separated, adjust the voltage to 100 V and continue electrophoresis until the bromophenol blue in the protein sample runs to the bottom of the gel. Then stop electrophoresis.

[0070] (5) Transfer: Use wet transfer. Remove the gel after electrophoresis, cut off the excess part, soak it in the prepared electrotransfer solution, measure the size of the gel with a ruler, cut a nitrocellulose membrane (NC) slightly larger than the gel, and then put it into the electrotransfer solution. Cut a thick filter paper of similar size to the gel and soak it in the electrotransfer solution. Use the sandwich method to place a black foam board, thick filter paper, NC membrane, gel, thick filter paper, and black foam board on the white transfer plate in sequence and clamp them. Insert the obtained "sandwich" into the electrotransfer tank, fill the electrotransfer tank with ice cubes to prevent the temperature from being too high during the electrotransfer process, and transfer the membrane at a constant voltage of 100V for 100 minutes.

[0071] (6) Blocking: Prepare 5% skimmed milk powder in advance and block the NC membrane on a shaker at room temperature for 1 h;

[0072] (7) Incubation with primary antibody: dilute the NCC antibody solution with 5% skim milk (volume ratio 1:1000) according to the ratio. After blocking, place the NC membrane in the diluted primary antibody and incubate at 4°C on a shaker overnight.

[0073] (8) Washing the membrane: Recover the primary antibody and wash with TBST (TBS with Tween-20, 10X) for 15 min, three times;

[0074] (9) Incubation with secondary antibody: dilute goat anti-rabbit IgG antibody at a volume ratio of 1:5000 with 5% skim milk, and incubate the polyvinylidene fluoride (PVDF) membrane on a shaker at room temperature for 1 h;

[0075] (10) Washing the membrane: Recover the secondary antibody and wash with TBST for 15 min, three times;

[0076] (11) Color development: Prepare ultrasensitive enhanced chemiluminescence (ECL) chemiluminescence solution, soak the NC membrane in the luminescence solution for 2 min, expose and photograph the film using a Tanon colorimeter, and detect the signal band intensity. Figure 1 .

[0077] Figure 1 It was shown that the target protein NCC can be detected in human renal cortical tissue and urine exosomes using the NCC antibody solution of Example 1 (see Figure 1 The target protein NCC could not be detected in human renal cortical tissue and urine exosomes using the NCC antibody solution of Comparative Example 1 (see Figure 1 (middle right picture). The NCC antibody solution of Example 1 can be applied to human sample immunoblotting detection, and the bands are clear.

[0078] 2. Immunofluorescence detection

[0079] (1) Conventional dewaxing and dehydration: The paraffin-embedded mouse kidney was cut into 2 μm thick slices and baked in an oven at 60°C for 2 hours.

[0080] (2) Dewaxing: sections were immersed in xylene I (10 min) → xylene II (10 min);

[0081] (3) Dehydration of sections with gradient alcohol: wash twice with 100% ethanol / 5 min → wash once with 95%, 75%, and 50% ethanol / 5 min → wash three times with PBS buffer / 5 min;

[0082] (4) Antigen repair: Add 600 ml of sodium citrate repair solution to the pressure cooker. After the repair solution boils, place the slices in the pot and continue boiling for 2.5 minutes. Then open the lid and cool to room temperature.

[0083] (5) Washing: Place the slices cooled to room temperature in PBS buffer for washing;

[0084] (6) Blocking: Block with 5% bovine serum albumin (BSA) for 1 h at room temperature;

[0085] (7) Washing: Wash the slices in PBS buffer;

[0086] (8) Primary antibody: dilute the prepared NCC antibody solution with PBS buffer at a volume ratio of 1:200 and incubate at 4°C overnight;

[0087] (9) Washing: Wash the slices in PBS buffer;

[0088] The following steps need to be protected from light

[0089] (10) Secondary antibody: incubate with goat anti-rabbit IgG fluorescent secondary antibody diluted 1:400 in PBS buffer at room temperature for 1 h;

[0090] (11) Washing: Wash the slices in PBS buffer;

[0091] (12) Nuclear staining: Add 4',6-diamidino-2-phenylindole (DAPI) dropwise onto the slice and stain the nucleus for 5-10 min;

[0092] (13) Washing: Wash the sections in PBS buffer;

[0093] (14) Sealing and photographing: After sealing, analyze and photograph the positive results.

[0094] The antibody produced by the NCC polypeptide antigen stains well on the luminal membrane of the distal convoluted tubules of mouse kidney (green) (see Figure 2 Left middle image), and also significant staining in the luminal membrane of the distal convoluted tubules of human kidney (see Figure 2 center right).

[0095] 3. Immunohistochemistry

[0096] (1) Paraffin sectioning: Paraffin-embedded mouse kidneys were cut into 2 μm thick sections and baked in an oven at 60°C for 2 h.

[0097] (2) Dewaxing: sections were immersed in xylene I (10 min) → xylene II (10 min);

[0098] (3) Dehydration of sections with gradient alcohol: wash twice with 100% ethanol / 5 min → wash once with 95%, 75%, and 50% ethanol / 5 min → wash three times with PBS buffer / 5 min;

[0099] (4) Antigen repair: Add 600 ml of sodium citrate repair solution to the pressure cooker. After the repair solution boils, place the slices in the pot and continue boiling for 2.5 minutes. Then open the lid and cool to room temperature.

[0100] (5) Washing: Wash tissue sections three times with PBS buffer, 5 min each time;

[0101] (6) Elimination of peroxidase activity: incubate tissue sections with 3% H2O2 at room temperature for 20 min;

[0102] (7) Washing: Wash tissue sections three times with PBS buffer, 5 min each time;

[0103] (8) Cell membrane disruption: Dissolve 0.1% triton X-100 in a water bath and add dropwise to tissue sections for incubation for 10 min.

[0104] (9) Washing: Wash tissue sections three times with PBS buffer, 5 min each time;

[0105] (10) Primary antibody: dilute the prepared NCC antibody solution with PBS buffer at a volume ratio of 1:200 and incubate at 4°C overnight;

[0106] (11) Washing: The next day, wash the tissue sections three times with PBS buffer, 5 min each time;

[0107] (12) Secondary antibody: incubate tissue sections with goat anti-rabbit IgG diluted 1:400 in PBS buffer at room temperature for 30 min;

[0108] (13) Washing: Wash tissue sections three times with PBS buffer, 5 min each time;

[0109] (14) Add peroxidase (Strept Avidin-Biotin Complex, SABC): Add SABC to the tissue sections and incubate at room temperature for 30 min;

[0110] (15) Washing: Wash tissue sections three times with PBS buffer, 5 min each time;

[0111] (16) Color development: DAB color developing solution was added to the sliced ​​tissue and observed under an optical microscope until the tissue turned brown. The slice was placed in distilled water to stop color development.

[0112] (17) Rinse: Rinse tissue sections under running tap water for 20 min;

[0113] (18) Counterstaining: Add hematoxylin solution to the tissue sections and incubate for 10 min;

[0114] (19) Return to blue: Rinse continuously under running tap water for 30 minutes;

[0115] (20) Dehydration: Tissue sections were immersed in 50%, 75%, 95% ethanol, 100% ethanol I, and 100% ethanol II, respectively, for 5 min each time;

[0116] (21) Transparency: Tissue sections were placed in xylene I and xylene II for 5 min each;

[0117] (22) Sealing: Add neutral gum to the slices and seal with a coverslip;

[0118] (23) Observation and photography: Observe positive results under a fluorescence microscope and take photos.

[0119] The antibody produced by the NCC polypeptide antigen clearly stains the luminal membrane of the distal convoluted tubules of the mouse kidney (brown) (see Figure 3 ).

[0120] Application Example 1

[0121] The NCC antibody solution of Example 1 was used to detect the NCC level in urine exosomes of patients with essential hypertension according to the above-mentioned immunoblotting detection method. The results showed that the level of NCC in patients with essential hypertension had an increasing trend (see Figure 4 ).

[0122] Application Example 2

[0123] The NCC antibody solution of Example 1 was used to detect the dopamine receptor 4 non-knockout mice (Drd4 + / + ) and dopamine receptor 4 knockout mice (Drd4 - / - ) in the kidney NCC, the results showed that Drd4 - / - Increased NCC protein in mouse kidneys (see Figure 5 ).

[0124] Application Example 3

[0125] The NCC antibody solution of Example 1 was used to detect multiple Drd4 + / + Mice and Drd4 - / - Expression of NCC in mouse kidneys. Results of double immunofluorescence staining of mouse kidneys are shown in Figure 6 , NCC antibody stains the luminal membrane of the thick ascending limb of the loop of Henle in the outer medulla of mouse kidney (green) clearly. D4R (red): dopamine receptor 4, DAPI (blue): nuclear staining.

[0126] Application Example 4

[0127] The NCC antibody solution of Example 1 was used to locate the distal tubule cell membrane and lysosome of the mouse according to the above-mentioned immunofluorescence detection method. The results of immunofluorescence staining of the distal tubule cells of the mouse are shown in Figure 7 , NCC antibody stains clearly in distal tubule cells. D4R: dopamine receptor 4, LAMP1: lysosomal marker protein, DAPI: nuclear staining.

[0128] Application Example 5

[0129] The NCC antibody solution of Example 1 was used to detect the expression of NCC in the kidney of essential hypertension rats according to the above-mentioned immunoblotting detection method. The results showed that the NCC in the kidney homogenate of essential hypertension rats increased significantly (see Figure 8 ).

[0130] Application Example 6

[0131] Using the NCC antibody solution of Example 1, immunoprecipitation and immunoblotting were performed to detect the effects of silencing and stimulating dopamine receptor 4 (D4R) in distal convoluted tubule cells on NCC ubiquitination (Ub-NCC). The results showed that D4R can promote the formation of Ub-NCC (see Fig. 9 ).

[0132] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the protection scope of the present invention. The present invention is not limited to the above embodiments, and any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A NCC antibody solution, characterized in that The NCC antibody in the NCC antibody solution is an antibody produced by immunizing an animal with a polypeptide whose sequence is shown in SEQ ID NO:

1.

2. The NCC antibody solution according to claim 1, characterized in that The type of the antibody is IgG or IgY.

3. The NCC antibody solution according to claim 2, characterized in that The antibody is rabbit IgG or chicken IgY.

4. The NCC antibody solution according to claim 1, characterized in that The NCC antibody is a rabbit IgG produced by immunizing a rabbit with a polypeptide having a sequence as shown in SEQ ID NO:

1.

5. The NCC antibody solution according to claim 1, characterized in that The solvent of the NCC antibody solution is a PBS buffer solution with a pH value between 7.2 and 7.

5.

6. The NCC antibody solution according to claim 1, characterized in that The preparation method of the NCC antibody solution is as follows: (1) using the artificially synthesized polypeptide to immunize an animal to obtain antiserum or highly immune egg yolk fluid; (2) The antiserum or high-immune egg yolk solution is purified by Protein A / G affinity chromatography, concentrated by ultrafiltration centrifuge tube, and resuspended in PBS buffer to obtain the NCC antibody solution.

7. Use of the NCC antibody solution according to any one of claims 1 to 6 in preparing a reagent or a kit for detecting the content of NCC in a sample to be tested.

8. The use according to claim 7, characterized in that: The sample to be tested is urine exosomes or kidney tissue.

9. Use of the NCC antibody solution according to any one of claims 1 to 6 in the preparation of a hypertension detection reagent or kit.

10. Use of the NCC antibody solution according to any one of claims 1 to 6 in the preparation of an acute kidney injury detection reagent or kit.

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