Application of ATP6V1D as marker for IgA nephropathy diagnosis and / or disease risk assessment

Abstract

The invention provides application of ATP6V1D as an IgA nephropathy diagnosis and / or disease risk assessment marker, and belongs to the technical field of in-vitro diagnosis. The invention provides an application of ATP6V1D as a marker for IgA nephropathy diagnosis and / or disease risk assessment. The research finds that the ATP6V1D protein has different expressions in IgA nephropathy and healthy people, membranous nephropathy and slight lesion nephropathy, and can be used as a marker for IgA nephropathy diagnosis and / or disease risk assessment. The invention further provides a kit for detecting the ATP6V1D, and compared with an existing KM55 kit, the kit provided by the invention is low in price and high in detection sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to the use of ATP6V1D as a marker for diagnosing IgA nephropathy and / or assessing the risk of the disease. Background Art

[0002] IgA nephropathy is the most common primary glomerular disease, with proteinuria being its earliest manifestation. The higher the degree of proteinuria, the greater the risk of kidney disease progression. While there are currently relatively precise and specific treatment options for IgA nephropathy, there are no specific therapeutic agents for other primary glomerular diseases. Therefore, early differentiation of primary glomerular diseases from IgA nephropathy is of great clinical value. Currently, the etiology of proteinuria relies on renal biopsy, and effective, non-invasive methods for diagnosing IgA nephropathy are lacking. Few non-invasive diagnostic markers exist for IgA nephropathy. Hypogalactosylated IgA1 (Gd-IgA1), a recognized etiology, has been shown to be useful in differentiating IgA nephropathy from other glomerular diseases. However, as a glycosylated protein, Gd-IgA1 presents significant challenges in accurate detection. The most widely used KM55 assay targets a specific epitope in the hinge region of Gd-IgA1 that is not properly glycosylated. It is the newest and most stable reagent for detecting Gd-IgA1. However, studies have shown that the Gd-IgA1 antibody KM55, which deposits in renal tissue, cannot effectively distinguish between primary and secondary IgA nephropathy. Furthermore, the classical lectin assay for Gd-IgA1 is unstable and poorly able to differentiate between IgA nephropathy and healthy individuals.

[0003] ATP6V1D is a key subunit of the vacuolar H+-ATPase (V-ATPase). Mutations in ATP6V1D can impair the assembly or stability of the V-ATPase complex. Reduced expression of ATP6V1D can lead to elevated lysosomal pH and decreased lysosomal degradation capacity. Currently, there are no reports detailing the specific role of ATP6V1D in IgA nephropathy. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of ATP6V1D as a marker for the diagnosis and / or risk assessment of IgA nephropathy, thereby providing a new target for the identification and diagnosis of IgA nephropathy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Use of ATP6V1D as a marker for the diagnosis and / or risk assessment of IgA nephropathy.

[0007] Another object of the present invention is to provide a kit for detecting ATP6V1D, the kit comprising: an ELISA plate, an ATP6V1D protein standard, a negative control, an ATP6V1D antibody, a biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxide, and an o-phenylenediamine staining substrate solution.

[0008] Preferably, the negative control is artificial urine containing creatinine.

[0009] Preferably, the o-phenylenediamine dyeing substrate solution is 0.4 mg / mL o-phenylenediamine hydrochloride + 0.4 mg / mL urea hydrogen peroxide + 0.05 M phosphate citrate, pH 4.9-5.1.

[0010] Preferably, the kit further comprises a diluent, a blocking solution, a washing solution and a stop solution.

[0011] Preferably, the diluent is 0.05M bicarbonate buffer; the blocking solution is 2% bovine serum albumin solution; the washing solution is phosphate buffer containing 0.1% Tween-20; and the stopping solution is 8M H2SO4 solution.

[0012] Another object of the present invention is to provide the use of the kit in any one or more of the following (1) to (4);

[0013] (1) Differential diagnosis of IgA nephropathy and other renal diseases;

[0014] (2) Assessment of the risk of IgA nephropathy;

[0015] (3) Evaluation of the therapeutic effects of drugs for IgA nephropathy;

[0016] (4) Diagnosis of IgA nephropathy.

[0017] Another object of the present invention is to provide a method for detecting ATP6V1D for non-diagnostic and non-therapeutic purposes, wherein the detection is performed using the kit, comprising the following steps:

[0018] The sample to be tested was coated on the ELISA plate and blocked with blocking solution. After washing, ATP6V1D antibody was added and incubated. After washing, biotinylated goat anti-rabbit IgG was added and incubated. After incubation, streptavidin-horseradish peroxide was added and incubated again. After incubation, o-phenylenediamine staining substrate solution was added, and then stop solution was added to stop the reaction. The optical density value was measured at a wavelength of 490 nm.

[0019] Preferably, the sample to be tested includes urine.

[0020] Preferably, the plate washing step comprises washing the ELISA plate 5 times using a washing solution.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides the use of ATP6V1D as a marker for the diagnosis and / or risk assessment of IgA nephropathy. The present invention's research found that the expression of ATP6V1D protein is different in IgA nephropathy and healthy people, membranous nephropathy, and minimal change nephropathy, and can be used as a marker for the diagnosis and / or risk assessment of IgA nephropathy.

[0023] The present invention also provides a kit for detecting ATP6V1D. Compared with the current KM55 kit, the present invention is low in price and has high detection sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the slope drift rate in the thermal stability experiment;

[0025] Figure 2 The results of competitive inhibition assays of ATP6V1D and ATP6V1A;

[0026] Figure 3 The test results of ATP6V1D in the patient's urine; A is the ROC curve of ATP6V1D concentration; B is the ROC curve of ATP6V1D / urinary protein quantitative ratio; C is the correlation analysis result of ATP6V1D concentration and urine protein;

[0027] Figure 4 : is the ROC curve of the detection results of the kit of the present invention, wherein the red line represents the detection concentration of ATP6V1D, and the blue line represents the ratio of ATP6V1D concentration divided by urine protein concentration;

[0028] Figure 5 Figure 3 is the ROC curve of the KM55 test results, where the red line represents the detection concentration of Gd-IgA1 and the blue line represents the ratio of Gd-IgA1 concentration to urine protein concentration. DETAILED DESCRIPTION

[0029] The present invention provides the use of ATP6V1D as a marker for the diagnosis and / or risk assessment of IgA nephropathy. The present invention has found that ATP6V1D is differentially expressed in IgA nephropathy and healthy individuals, membranous nephropathy, and minimal change disease. This marker can be used as a marker for the diagnosis and / or risk assessment of IgA nephropathy, providing a new target for the identification and diagnosis of IgA nephropathy.

[0030] The present invention also provides a kit for detecting ATP6V1D, which includes: an ELISA plate, an ATP6V1D protein standard, a negative control, an ATP6V1D antibody, biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxide, and an o-phenylenediamine staining substrate solution.

[0031] In the kit provided by the present invention, the preferred negative control is artificial urine containing creatinine. The preferred concentration range of the ATP6V1D protein standard is 0 to 250 ng / mL, preferably including 5 to 7 standard concentrations, and the more preferred concentrations of the ATP6V1D protein standard are 0, 15.625, 17.86, 31.25, 50, 62.5 and 125 ng / mL, respectively. The o-phenylenediamine dyeing substrate solution of the present invention is preferably prepared and used immediately, and the prepared and used immediately is preferably used within 0 to 20 minutes after preparation. The o-phenylenediamine dyeing substrate solution is 0.4 mg / mL o-phenylenediamine hydrochloride (OPD) + 0.4 mg / mL urea hydrogen peroxide + 0.05 M phosphate citrate, pH 4.9 to 5.0, and the concentration of each reagent is its final concentration in the o-phenylenediamine dyeing substrate solution.

[0032] The kit of the present invention preferably further comprises a diluent, a blocking solution, a washing solution, and a stop solution. The diluent is preferably a 0.05M bicarbonate buffer solution; the blocking solution is preferably a 2% (w / v) bovine serum albumin solution; the washing solution is preferably a phosphate buffer solution containing 0.1% (v / v) Tween-20, the concentration of the phosphate buffer solution preferably being 0.01M; and the stop solution is preferably an 8M H2SO4 solution.

[0033] The present invention also provides application of the kit in differential diagnosis of IgA nephropathy and other kidney diseases.

[0034] The present invention also provides the use of the kit in the assessment of the risk of IgA nephropathy.

[0035] The present invention also provides the use of the kit in evaluating the therapeutic effect of IgA nephropathy drugs.

[0036] The present invention also provides application of the kit in the diagnosis of IgA nephropathy.

[0037] The present invention also provides a method for detecting ATP6V1D for non-diagnostic and non-therapeutic purposes, which is detected using the kit, comprising the following steps: coating a sample to be tested on an enzyme labeling plate, adding a blocking solution for blocking, washing the plate, adding an ATP6V1D antibody for incubation, washing the plate, adding biotinylated goat anti-rabbit IgG for incubation, adding streptavidin-horseradish peroxide for incubation again, adding an o-phenylenediamine staining substrate solution after incubation, adding a stop solution to stop the reaction, and measuring the optical density value at a wavelength of 490 nm.

[0038] In the method for detecting ATP6V1D of the present invention, the test sample preferably includes urine. The urine is preferably collected in the morning and then centrifuged to obtain the supernatant. The centrifugation speed is preferably 2500 to 3500 rpm, more preferably 3000 rpm, and the centrifugation time is preferably 8 to 12 minutes, more preferably 10 minutes. Before testing the test sample, the test sample is preferably diluted with a diluent before testing. The plate washing method preferably involves washing the ELISA plate five times with a washing solution.

[0039] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] A kit for detecting ATP6V1D, comprising:

[0042] ELISA plate;

[0043] ATP6V1D protein (Proteintech, Ag6742) standard: concentrations were 0, 15.625, 17.86, 31.25, 50, 62.5, and 125 ng / mL;

[0044] Interferor ATP6V1A protein (Proteintech, Ag10820)

[0045] Negative control: artificial urine containing creatinine (Leye-Bio, pH 5.0);

[0046] Blank control: 0.05 M bicarbonate buffer, pH 9.6;

[0047] ATP6V1D antibody (proteintech, 14920-1-AP, 500 μg / mL): diluted to 1:8000 using primary antibody diluent (Servicebio, G2025-100ML);

[0048] Biotinylated goat anti-rabbit IgG (Proteintech, SA0004-2): Dilute to 1:4000 in phosphate buffered saline (PBS) containing 0.1% (v / v) Tween-20 (PBS concentration is 0.01 M).

[0049] Streptavidin-horseradish peroxide (Proteintech, SA00001-0): Dilute to 1:1000 using phosphate buffered saline (PBS) containing 0.1% (v / v) Tween-20, where the concentration of phosphate buffered saline is 0.01 M.

[0050] O-phenylenediamine dyeing substrate solution (prepared and used within 0-20 min after preparation): 0.4 mg / mL o-phenylenediamine hydrochloride (OPD) + 0.4 mg / mL urea hydrogen peroxide + 0.05 M phosphate citrate, pH 5.0. OPD was purchased from SIGMAFAST TM ;

[0051] Diluent: 0.05 M bicarbonate buffer, pH 9.6;

[0052] Blocking solution: 2% (w / v) bovine serum albumin solution;

[0053] Washing solution: phosphate buffer containing 0.1% (v / v) Tween-20, the concentration of phosphate buffer is 0.01M;

[0054] Stop solution: 8M H2SO4 solution.

[0055] Example 2

[0056] A method for detecting ATP6V1D for non-diagnostic and non-therapeutic purposes, using the kit provided in Example 1 for detection, comprises the following steps:

[0057] Collect morning urine, centrifuge at 3000 rpm and 4°C for 10 minutes, and extract the urine supernatant as the test sample.

[0058] Dilute the sample to be tested and the negative control with diluent at a volume ratio of 1:7 and set aside.

[0059] Coat the diluted test sample, diluted negative control, blank control, and ATP6V1D protein standard onto an ELISA plate, loading 100 μL per well and incubating overnight at 4°C.

[0060] Wash the plate 5 times with washing solution, pat dry, then add 100 μL of blocking solution to each well and incubate at 37°C for 2 h. Wash the plate 5 times with washing solution and pat dry.

[0061] Then, add 100 μL of ATP6V1D antibody to each well and incubate overnight at 4°C. Wash the plate five times with washing buffer and pat dry.

[0062] 100 μL of biotinylated goat anti-rabbit IgG was added to each well and incubated at 37°C for 1 h. Streptavidin-horseradish peroxide was added and incubated at 37°C for 1 h.

[0063] Add 100 μL of o-phenylenediamine dyeing substrate solution to each well and incubate at 37°C for 30 minutes.

[0064] Add 25 μL of 8 M H2SO4 solution to each well to stop the reaction. Measure the optical density at a wavelength of 490 nm.

[0065] A standard curve was constructed using the difference between the optical density of the ATP6V1D protein standard and the corresponding concentration. The difference between the optical density of each sample and the blank control was substituted into the standard curve to calculate the ATP6V1D concentration in the sample.

[0066] Example 3

[0067] Assay performance of the kit for detecting ATP6V1D.

[0068] The kit in Example 1 and the method for detecting ATP6V1D provided in Example 2 were used.

[0069] Sensitivity: The negative control was tested three times with five replicates each time. The difference between the average optical density value of the negative control + 3SD minus the average value of the blank control was substituted into the corresponding standard curve. The detection concentration was calculated as the detection sensitivity. The average of the three results was calculated, and the detection sensitivity of the kit of the present invention was 1.78 ng / mL.

[0070] Precision:

[0071] Intra-group precision: ATP6V1D protein was prepared at concentrations of 55 ng / mL, 25 ng / mL, and 10 ng / mL. Samples of each concentration were tested 12 times. The difference between the optical density of the test sample and the optical density of the blank control was substituted into the standard curve to determine the test concentration. The arithmetic mean and standard deviation of the test concentration were calculated. The coefficient of variation (CV) = standard deviation / mean*100%.

[0072] The results showed that the CV value was 0.67% at a concentration of 55 ng / mL, 1.50% at a concentration of 25 ng / mL, and 2.95% at a concentration of 10 ng / mL.

[0073] Interassay precision: ATP6V1D protein was prepared at concentrations of 55 ng / mL, 25 ng / mL, and 10 ng / mL. Twelve replicates of each concentration were tested on three different days by different researchers. The arithmetic mean and standard deviation of all test results were calculated. The coefficient of variation (CV) = standard deviation / mean * 100%. The CV of the three replicates for each concentration was calculated. The CV was 1.91% at 55 ng / mL, 5.03% at 25 ng / mL, and 5.63% at 10 ng / mL.

[0074] Recovery rate:

[0075] 10 ng of ATP6V1D protein standard was added to a sample with an ATP6V1D protein concentration of 25 ng / mL. The obtained samples were tested three times on different days and by different experimenters, with four replicates each time. The measured values ​​were substituted into the standard curve. The recovery rate of each test was calculated separately: recovery rate = (spike sample measured value - sample background value) ÷ spike amount × 100%. The average of the three recovery rates was used as the final recovery rate. The calculated recovery rate was 93.82%;

[0076] 20 ng of ATP6V1D protein standard was added to a sample with an ATP6V1D protein concentration of 55 ng / mL, and the recovery was calculated as described above. The calculated recovery was 101.21%.

[0077] 10 ng of ATP6V1D protein standard was added to a sample with an ATP6V1D protein concentration of 75 ng / mL, and the recovery was calculated as described above. The calculated recovery was 100.31%.

[0078] Example 4

[0079] Stability test.

[0080] The test was performed using the kit in Example 1 and the method for detecting ATP6V1D provided in Example 2.

[0081] Remove each component reagent (ATP6V1D antibody, biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxide) from the kit, divide them into sterile containers, and divide them into centrifuge tubes by volume (200 μL per tube to avoid repeated freezing and thawing). Mark the temperature and time point. Set the temperature gradient to 4°C, 25°C, and 37°C, and seal the tubes with parafilm. Place the centrifuge tubes in a water bath or incubator at the preset temperature, ensuring complete immersion or uniform heating to prevent water evaporation. Treat at 4°C for 1 hour, 4 hours, 24 hours, and 6 months; at 25°C for 4 hours, 24 hours; and at 37°C for 4 hours, 24 hours, and 14 days.

[0082] The reagents at the corresponding time points were removed and returned to room temperature (15-30°C). Standards stored at -80°C (concentrations of 0, 15.625, 17.86, 31.25, 50, 62.5, and 125 ng / mL) were used to construct a standard curve for calculation. The assay was repeated three times in parallel, and the OD values ​​were recorded.

[0083] Activity retention rate = (OD value of standard after treatment / initial OD value) × 100%.

[0084] Recovery rate = (test value of treatment group / test value of control group) × 100%.

[0085] Artificial urine samples containing ATP6V1D standard at concentrations of 10 ng / mL and 115 ng / mL were tested using the kit after being treated at different temperatures and time periods.

[0086] The OD value recorded from the reagent test standard curve in the group treated at 4°C for 1 hour was used as the initial OD value, and the OD values ​​recorded from the reagent test standard curve in the other treatment groups were used as the post-treatment standard OD value. The values ​​measured by the reagent test in the group treated at 4°C for 1 hour were used as the control value, and the values ​​measured by the reagent test in the other treatment groups were used as the treatment group value. Activity retention and recovery rates were calculated.

[0087] Artificial urine without a standard served as a negative control; artificial urine containing 60 ng / mL of the ATP6V1D standard served as a positive control; and an ATP6V1A standard (Proteintech, Ag10820) was added to the positive control to achieve a concentration of 600 ng / mL as an interference control. Assays were performed using the kit after treatment at different temperatures and time periods. Six replicate wells were tested each time. The relative rate of change in OD values ​​was calculated to assess the stability of the reagent specificity at different temperatures and storage times. The experiment was repeated three times. The three results were averaged.

[0088] Relative change rate (%) = [(OD value of interference group - OD value of negative control) / (OD value of positive control - OD value of negative control)] × 100%.

[0089] Table 1 Thermal stability

[0090]

[0091] The results in Table 1 indicate that the reagent provided by the present invention can be stably stored at 4°C for 6 months and at 37°C for 14 days. The isotype interferor ATP6V1A exhibited minimal impact on the test samples at high concentrations (relative change rates were less than 10% under various temperature and time conditions), indicating that the kit exhibited good specificity and thermal stability.

[0092] 2. Use reagents treated at different temperatures to detect standards stored at -80°C (0, 15.625, 17.86, 31.25, 50, 62.5, and 125 ng / mL). Construct a standard curve and calculate the drift rate of the slope.

[0093] Drift rate = | slope of the curve after treatment - initial slope | / initial slope × 100%. The results are as follows Figure 1 Based on the results of three experiments, compared with the slope of the standard curve after treatment at 4°C for 24 hours, the drift rate after storage at 37°C for 24 hours was 4.07%, and the drift rate after storage at 37°C for 14 days was 13.56%.

[0094] Example 5

[0095] Specific detection.

[0096] The test was performed using the kit in Example 1 and the method for detecting ATP6V1D provided in Example 2.

[0097] ATP6V1A, a homologous protein of ATP6V1D, was used as an antigen control. The following experimental groups were set up:

[0098] Blank control: Contains only diluent (0.05 M bicarbonate buffer, pH 9.6); no sample or antibody is added. This control is used to eliminate background signal from the reagent itself. In subsequent experiments, all OD values ​​used for calculations will be subtracted from the average of the blank control from the same batch.

[0099] ATP6V1D inhibition: ATP6V1D antibody (200 ng / mL) was mixed with various concentrations of ATP6V1D standard (1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, and 1.0 ng / mL) at a 1:1 volume ratio and preincubated for 30 minutes (4°C). The mixture was then added to the coated wells. The coating antigen concentration was 100 ng / mL.

[0100] ATP6V1A inhibition: ATP6V1D antibody (200 ng / mL) was mixed with various concentrations of ATP6V1A standard (1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, and 1.0 ng / mL) at a 1:1 volume ratio and preincubated for 30 minutes (4°C). The mixture was then added to the coated wells. The coating antigen concentration was 100 ng / mL.

[0101] The inhibition curve was drawn using four-parameter logistic curve fitting to calculate the IC of ATP6V1D and ATP6V1A competitive inhibition assay. 50 (like Figure 2 ). The average value of the three test results was calculated to obtain the IC of ATP6V1D. 50 The IC50 of ATP6V1A was 13.53 ng / mL, the IC50 of ATP6V1A was 462.29 ng / mL, and the cross-reaction rate was 2.93%, indicating high specificity.

[0102] Combined with the results of the relative change rate of ATP6V1A in Example 4, it can be shown that the kit has good specificity under normal storage conditions.

[0103] Example 6

[0104] The expression of ATP6V1D in urine was detected in 27 patients with IgA nephropathy and 30 patients with other glomerular diseases in the Second Affiliated Hospital of South China University. Figure 3 As shown. It was found that ATP6V1D expression was positively correlated with urine protein concentration ( Figure 3 C in ). Urinary ATP6V1D concentration ( Figure 3 A) and ATP6V1D concentration corrected for 24-hour urine protein ( Figure 3 B) in both cases have differential diagnostic significance.

[0105] Patients from the Second Affiliated Hospital of the University of South China were reselected, including 31 patients in the IgA nephropathy group, 20 patients in the membranous nephropathy group, and 10 patients with minimal change nephropathy. Urine protein concentration was directly detected after the patient was admitted to the hospital. ATP6V1D was detected in urine samples from the above patients using the kit in Example 1 and the method for detecting ATP6V1D provided in Example 2. At the same time, the KM55 kit (purchased from Shenzhen Kerunda Biotechnology) was used to detect urine samples from the above patients.

[0106] According to the test results, the cutoff value was obtained by calculating the sensitivity and specificity using the ROC curve.

[0107] The test results of the kit of the present invention are as follows Figure 4 As shown, the red line represents the ROC curve for the detection concentration of ATP6V1D, with an area under the curve of 0.976, a cutoff value of 311.0225 ng / mL, a sensitivity of 96.8%, and a specificity of 90%. The blue line represents the ROC curve for the ratio of ATP6V1D to urine protein concentration, with an area under the curve of 0.886.

[0108] The KM55 test kit test results are as follows Figure 5 As shown, the blue line represents the ratio of Gd-IgA1 concentration to urine protein concentration, with an area under the curve of 0.942. The red line represents the detected Gd-IgA1 concentration, with an area under the curve of 0.956, a cutoff value of 3.53 μg / dL, a sensitivity of 80.6%, and a specificity of 100%.

[0109] Example 7

[0110] This example compares the accuracy of the KM55 kit and the kit of the present invention in diagnosing IgA nephropathy.

[0111] The present invention tested a urine sample from a patient with a urine protein concentration of 10,366.7 mg / 24 hours (diagnosed with membranous nephropathy by renal biopsy). The KM55 test kit (purchased from Shenzhen Kerunda Biotechnology Co., Ltd.) revealed a Gd-IgA1 concentration of 4.47 μg / dL. Using the kit described in Example 1 and the method for detecting ATP6V1D provided in Example 2, the sample was tested for ATP6V1D concentration of 757.9 ng / mL.

[0112] According to Example 6, the KM55 test kit has a cutoff value of 3.53 μg / dL, so the sample would be classified as IgA nephropathy based on the KM55 test results. However, the ATP6V1D cutoff value of the present invention kit is 311.0225 ng / mL, so the sample would not be classified as IgA nephropathy based on the present invention kit's test results. These examples suggest that in samples with high proteinuria concentrations, the KM55 test may interfere with the judgment due to the absolute increase in Gd-IgA1 concentration.

[0113] Gd-IgA1 is present at elevated levels in the urine of patients with IgA nephropathy, but it has a strong correlation with the patient's protein concentration (R = 0.42). In membranous nephropathy or minimal change nephropathy, elevated levels of Gd-IgA1 may be detected when the urine protein level is high, thus interfering with the diagnosis. However, the detection indicator ATP6V1D of the present invention is highly expressed in healthy individuals and in patients with other kidney diseases, but is low in IgA nephropathy. Therefore, among patients with high urine protein concentrations, ATP6V1D expression is higher in the urine of patients with membranous nephropathy and minimal change nephropathy. ATP6V1D concentration is low in the urine of patients with IgA nephropathy. The relative difference between the two is not significantly affected by urine protein concentration. Furthermore, the urine ATP6V1D concentration alone has high sensitivity and specificity as a marker, eliminating the need for further urine protein level testing for correction, making detection more convenient. This provides a safe and convenient early differential diagnosis method for patients with kidney disease who have high proteinuria and a high risk of progression.

[0114] Therefore, in patients with high proteinuria, ATP6V1D has better performance in differentiating IgA nephropathy from membranous nephropathy and minimal change nephropathy. The present invention can accurately identify IgA nephropathy, especially IgA nephropathy with high proteinuria levels, which is beneficial for disease-specific treatment and early control.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of ATP6V1D as a biomarker for the diagnosis and / or risk assessment of IgA nephropathy.

2. A kit for detecting ATP6V1D, characterized in that: The kit comprises: an ELISA plate, an ATP6V1D protein standard, a negative control, an ATP6V1D antibody, a biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxide and an o-phenylenediamine staining substrate solution.

3. The kit according to claim 2, wherein The negative control was artificial urine containing creatinine.

4. The kit according to claim 2, wherein The o-phenylenediamine dyeing substrate solution is 0.4 mg / mL o-phenylenediamine hydrochloride+0.4 mg / mL urea hydrogen peroxide+0.05 M phosphate citrate, with a pH of 4.9-5.

1.

5. The kit according to claim 2, wherein The kit further comprises a diluent, a blocking solution, a washing solution and a stop solution.

6. The kit according to claim 5, characterized in that The diluent is 0.05M bicarbonate buffer; the blocking solution is 2% bovine serum albumin solution; the washing solution is phosphate buffer containing 0.1% Tween-20; and the stopping solution is 8M H2SO4 solution.

7. Use of the kit according to any one of claims 2 to 6 in any one or more of the following (1) to (4); (1) Differential diagnosis of IgA nephropathy and other renal diseases; (2) Assessment of the risk of IgA nephropathy; (3) Evaluation of the therapeutic effects of drugs for IgA nephropathy; (4) Diagnosis of IgA nephropathy.

8. A method for detecting ATP6V1D for non-diagnostic and non-therapeutic purposes, characterized in that: The detection is performed using the kit according to any one of claims 2 to 6, comprising the following steps: The sample to be tested was coated on the ELISA plate and blocked with blocking solution. After washing, ATP6V1D antibody was added and incubated. After washing, biotinylated goat anti-rabbit IgG was added and incubated. After incubation, streptavidin-horseradish peroxide was added and incubated again. After incubation, o-phenylenediamine staining substrate solution was added, and then stop solution was added to stop the reaction. The optical density value was measured at a wavelength of 490 nm.

9. The method for detecting ATP6V1D according to claim 8, wherein The sample to be tested includes urine.

10. The method for detecting ATP6V1D according to claim 8, characterized in that The plate washing step is to wash the ELISA plate 5 times using a washing solution.

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