A disease classification method, device and program product based on anti-nephrotic protein autoantibodies

Abstract

The present application relates to the field of intelligent medical treatment, and specifically to a disease classification method, device and program product based on anti-nephrotic protein autoantibodies. It includes S1: obtaining a patient serum sample, the patient is a patient with glomerular microlesions and primary focal segmental glomerulosclerosis; S2: performing antibody detection on the serum sample to obtain anti-nephrotic protein autoantibodies, the autoantibodies are IgM autoantibodies; S3: classifying the disease based on the anti-nephrotic protein autoantibodies to obtain a classification result, the classification result is whether it is refractory. The present application can classify diseases, provide medication guidance and predict prognosis by detecting antibodies, and has great clinical value.

Description

Technical Field

[0001] The present application relates to the field of intelligent medical treatment, and specifically to a disease classification method, device, program product and computer-readable storage medium based on anti-nephrotic protein autoantibodies. Background Art

[0002] Minimal Change Disease (MCD) and primary focal segmental glomerulosclerosis (FSGS), also known as podocytopathy, are important and common pathological diagnoses in adults and children with nephrotic syndrome. Clinically, MCD is characterized by massive proteinuria and hypoproteinemia. Pathologically, there are no obvious changes under light microscopy, immunofluorescence is usually negative, and electron microscopy often shows extensive foot process fusion. FSGS, on the other hand, shows focal segmental glomerulosclerosis under light microscopy. Similar to MCD, FSGS shows diffuse podocyte foot process effacement under electron microscopy. Compared with MCD, it usually leads to more severe proteinuria and worse renal outcomes, and may progress to end-stage renal disease (ESKD). The cell-cell junction between the foot processes is called the slit diaphragm, a zipper-like structure that serves as one of the filtration barriers in the glomerulus. Functional and structural changes in podocytes and slit diaphragms are at the core of the proteinuria observed in patients with MCD and primary FSGS. MCD and primary FSGS share some important similarities in their clinicopathological presentation and thus represent the same entity in some circumstances.

[0003] The incidence of MCD is 70%-90% in children and 10%-15% in adults. One characteristic of MCD is that it has a high recurrence rate. 56%-76% of patients relapse 1-4 times. Predicting the recurrence of patients can prevent it in advance. However, there are some difficulties in detecting the recurrence of MCD, mainly due to the heterogeneity and unpredictability of the disease. Some patients may experience frequent recurrences, while others may not relapse for a long time. In adult patients, some patients with hormone-sensitive primary FSGS also relapse, and their sustained remission rate is less than 50%. About 40-60% of patients eventually develop ESKD 10-20 years after onset.

[0004] In addition, relapse may be related to multiple factors such as treatment compliance, individual immune status, environmental factors, etc., which increases the complexity of monitoring and prevention. Therefore, the monitoring strategy needs to be personalized and formulated by professional doctors according to the specific situation of the patient. Summary of the invention

[0005] Nephrin is a transmembrane receptor molecule and a component of the slit diaphragm of glomerular podocytes. It plays a vital role in maintaining the integrity of the glomerular filtration barrier and is involved in the signaling pathway that regulates podocyte function. In view of the above problems, the present invention proposes a disease classification method for anti-nephrin autoantibodies, which specifically includes:

[0006] S1 obtains a serum sample from a patient, wherein the patient is a patient with glomerular minimal change disease and primary focal segmental glomerulosclerosis;

[0007] S2 performs antibody detection on the serum sample to obtain anti-nephrotic protein autoantibodies, wherein the autoantibodies are IgM autoantibodies;

[0008] S3 classifies the disease based on the anti-nephrin autoantibodies to obtain a classification result, wherein the classification result is whether the disease is refractory.

[0009] Furthermore, the S2 is replaced by performing antibody detection on the serum sample to obtain anti-nephrin autoantibodies, and the autoantibodies are IgG and IgM autoantibodies.

[0010] Furthermore, the refractory type is a disease with a relapse frequency greater than L times within half a year, where L is a natural number greater than or equal to 2;

[0011] Optionally, a classification result is obtained by performing disease classification based on the anti-nephrotic protein autoantibodies, specifically: when the value of the anti-nephrotic protein autoantibodies is greater than a preset threshold, the patient is a refractory patient;

[0012] Optionally, the antibody detection method is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic particle chemiluminescence, and immunofluorescence technology.

[0013] Inputting the anti-nephrin autoantibodies into a classifier for disease classification to obtain a classification result;

[0014] Optionally, the classifier is one or more of the following: support vector machine, random forest, convolutional neural network, XGBoost, AdaBoost.

[0015] The S3 is replaced by S32, obtaining clinical indicators of the patient, and inputting the clinical indicators and anti-nephrotic protein autoantibodies into a classifier for classification to obtain a classification result;

[0016] Optionally, the clinical indicators include one or more of the following: urine protein, eGFR.

[0017] The method further includes medication guidance, and administering medication based on the classification results;

[0018] Optionally, when the classification result is refractory, the patient is treated with cyclophosphamide, calcineurin inhibitors, leflunomide, and rituximab.

[0019] The method also includes predicting the frequency of follow-up visits for prognosis, and predicting the frequency of follow-up visits for the patient's prognosis based on the classification results.

[0020] The object of the present invention is to provide a computer device having a memory and a processor and a computer program / instruction stored in the memory, comprising:

[0021] The processor executes the computer program / instructions to implement the above-mentioned disease classification method based on anti-nephrin autoantibodies.

[0022] The object of the present invention is to provide a computer program product having a computer program / instruction thereon, wherein the computer program / instruction is executed by a processor to implement the above-mentioned disease classification method based on anti-nephrin autoantibodies.

[0023] The object of the present invention is to provide a computer-readable storage medium having a computer program / instruction stored thereon, wherein the computer program / instruction is executed by a processor to implement the above-mentioned disease classification method for anti-nephrin autoantibodies.

[0024] The purpose of the present invention is to provide a product for detecting anti-nephrotic protein autoantibodies for use in the preparation of a preparation for diagnosing whether the patient has refractory glomerular microlesions or primary focal segmental glomerulosclerosis; optionally, the method used for detecting anti-nephrotic protein autoantibodies is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic particle chemiluminescence, immunofluorescence technology;

[0025] Optionally, the autoantibodies are IgM and / or IgG type autoantibodies.

[0026] The object of the present invention is to provide a detection kit for detecting anti-nephrotic protein autoantibodies, the kit comprising: a substrate of nephrotic protein, an antibody of anti-human IgG and / or IgM antibody;

[0027] Optionally, the substrate is an extracellular region fragment of nephrin;

[0028] Optionally, the antibody is enzyme-labeled or fluorescently labeled;

[0029] Optionally, the species of the antibody include any one of the following: chicken, donkey, goat, mouse, rabbit, rat, sheep;

[0030] Optionally, the types of antibody fluorescence mainly include fluorescein isothiocyanate FITC, tetramethylrhodamine isothiocyanate TRITC, RRX, Texas Red, cyanine dyes, phycoerythrin or phycobilichrome protein, AMCA;

[0031] Optionally, the kit includes coating buffer, Tween20 blocking slow-release solution, HRP-labeled mouse anti-human IgG or IgM, FITC-labeled mouse anti-human IgG or IgM, PBST washing buffer, TMB substrate, and stop solution;

[0032] Optionally, the kit is used to diagnose whether it is refractory glomerular minimal change disease or primary focal segmental glomerulosclerosis.

[0033] Advantages of the present invention:

[0034] 1. The present invention classifies MCD patients and FSGS patients by IgM and / or IgG autoantibodies, and can effectively distinguish refractory patients from non-refractory patients by detecting IgM and / or IgG autoantibodies. Based on the classification type, corresponding medication guidance and prognosis prediction are given to patients, providing a more accurate disease diagnosis method, improving the disease treatment effect, and having important clinical practical value.

[0035] 2. Comprehensive evaluation of anti-Nephrin antibodies in a large cohort provides reliable data on their prevalence and impact in MCD or FSGS. Further support is provided for the autoimmune etiology. Anti-Nephrin antibodies can not only serve as a biomarker for monitoring disease activity, but also help clinicians more effectively predict disease progression and adjust treatment to improve the prognosis of patients with MCD or FSGS. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 A schematic flow chart of a disease classification method based on anti-nephrotic protein autoantibodies provided in an embodiment of the present invention;

[0038] Figure 2 A schematic diagram of a disease classification system based on anti-nephrin autoantibodies provided in an embodiment of the present invention;

[0039] Figure 3 A schematic diagram of a disease classification device based on anti-nephrotic protein autoantibodies provided in an embodiment of the present invention;

[0040] Figure 4 Anti-Nephrin antibodies (IgG and IgM) of MCD, primary FSGS, MN patients and healthy controls provided in the embodiments of the present invention. (A) Scatter plot of OD values ​​of anti-Nephrin IgG and IgM antibodies. The cutoff values ​​of anti-Nephrin IgG antibodies and anti-Nephrin IgM antibodies were 0.10 and 0.19, respectively. 71 / 313 (22.7%) MCD patients and 40 / 118 (33.9%) primary FSGS patients were positive for anti-Nephrin IgG antibodies (left side), and 124 / 313 (39.6%) MCD patients and 35 / 118 (29.7%) primary FSGS patients were positive for anti-Nephrin IgM antibodies (right side). All normal controls and PLA2R-related MN patients were negative for anti-Nephrin IgG and IgM antibodies. (B) The positive rates of anti-nephrin IgG and IgM autoantibodies in all MCD patients, MCD patients with nephrotic range proteinuria, MCD patients with nephrotic range proteinuria not receiving immunosuppressive therapy, all primary FSGS patients, primary FSGS patients with nephrotic range proteinuria, primary FSGS patients with nephrotic range proteinuria not receiving immunosuppressive therapy, and the control group. Nephrotic range proteinuria was defined as 24-hour urine protein quantitative >3.5 g / 24 hours;

[0041] Figure 5 Anti-Nephrin IgG and IgM antibodies in MCD and primary FSGS patients with active and remission nephrotic syndrome provided in the embodiments of the present invention. (A) Changes in anti-Nephrin IgG antibodies in MCD and primary FSGS patients. (B) Changes in anti-Nephrin IgM antibodies in MCD and primary FSGS patients;

[0042] Figure 6 The long-term clinical course of 4 MCD patients and 4 primary FSGS patients with positive anti-Nephrin antibodies provided in the embodiments of the present invention. (A) Changes in anti-Nephrin IgG antibodies and proteinuria in 2 MCD patients (1 MCD patient, 2 MCD patients) and 2 primary FSGS patients (1 FSGS patient, 2 FSGS patients) during follow-up. (B) Changes in anti-Nephrin IgM antibodies and proteinuria in 2 MCD patients (3 MCD patients, 4 MCD patients) and 2 primary FSGS patients (3 FSGS patients, 4 FSGS patients) during follow-up. The changes in anti-Nephrin antibodies are closely related to the quantification of urine protein;

[0043] Figure 7Patients with positive anti-Nephrin IgG or IgM antibodies provided in the embodiments of the present invention were subjected to immunofluorescence staining of IgG or IgM and Nephrin. (A) Immunofluorescence staining of IgG (green) and Nephrin (red) in normal renal tissue, anti-Nephrin-related MCD patients, anti-Nephrin-related primary FSGS patients and MN patients. Co-localization of IgG and Nephrin was observed in patients with MCD and primary FSGS who were IgG-positive in conventional renal pathology and anti-Nephrin IgG antibodies. No obvious co-localization was observed in normal kidneys and MN patients. (B) Immunofluorescence staining of IgM (green) and Nephrin (red) in renal tissue of normal renal tissue, anti-Nephrin-related MCD patients, anti-Nephrin-related primary FSGS patients and MN patients. Co-localization of IgM and Nephrin was observed in patients with MCD and primary FSGS who were IgM-positive in renal tissue pathology and anti-Nephrin IgM antibodies. No obvious co-localization was observed in normal kidneys and MN patients. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0045] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The sequence numbers of the operations, such as S101, S102, etc., are only used to distinguish between different operations, and the sequence numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0046] Figure 1 The schematic diagram of the disease classification method based on anti-nephrotic protein autoantibodies provided in the embodiment of the present invention specifically includes:

[0047] S1: Obtain serum samples from patients with minimal glomerular changes and primary focal segmental glomerulosclerosis;

[0048] In a specific embodiment, the present invention includes 313 patients with minimal glomerular change (MCD) diagnosed by renal biopsy at Peking University First Hospital from January 2013 to December 2023. Patients with known secondary causes, such as hepatitis B / C virus or HIV infection, malignant tumors, drugs, mercury poisoning and other untreated infections are excluded. In addition, according to the Columbia classification criteria, 118 patients with primary focal segmental glomerulosclerosis (FSGS) confirmed by renal biopsy are included. Familial, hereditary FSGS, as well as patients with clear secondary FSGS due to viral infection, drugs, obesity, etc. are excluded.

[0049] In a specific embodiment, the present invention includes 30 patients with anti-human phospholipase A2 receptor (hPLA2R) antibody-associated membranous nephropathy MN as disease controls, and 30 healthy individuals are evaluated as healthy controls. All blood specimens were collected on the day of renal biopsy. The samples were frozen at -80°C to avoid repeated freezing and thawing. In addition, clinical data were collected from medical records at the time of biopsy and during follow-up.

[0050] In a specific embodiment, complete remission is defined as urine protein quantification less than 0.3g / 24h, and blood creatinine is stable. Partial remission is defined as a decrease in urine protein excretion less than 3.5g / 24h, but greater than 0.3g / 24h and a decrease of more than 50% from baseline, and stable blood creatinine. Patients who do not achieve remission are considered to have no remission. Relapse is defined as urine protein quantification greater than 3.5g / 24h after remission. Frequent relapse is defined as 2 or more relapses within 6 months after remission, or 4 or more relapses within 12 months after remission. For the assessment of renal outcomes, the primary endpoint is ESKD, defined as dependence on renal replacement therapy; the secondary endpoint is renal insufficiency, defined as a decrease in eGFR of more than 30% from baseline (at the time of renal biopsy). The follow-up time is the time point when the patient reaches the endpoint. If the patient does not reach the endpoint during the entire follow-up period, it is the last visit. All eGFRs are calculated using the formula of the Chronic Kidney Disease Epidemiology Collaboration.

[0051] In one embodiment, patients with nephrotic syndrome, defined as urine protein ≥ 3.5 g / 24 h, serum albumin < 30 g / L, are treated with corticosteroids combined with immunosuppressants including cyclophosphamide and cyclosporin A. Oral prednisone is started at a rate of 1 mg / kg per day for up to 12-16 weeks, then tapered, oral cyclophosphamide is 1.5-2 mg / kg per day for 3 months, or cyclosporin A, 2-3 mg / kg per day, trough concentration is 100-150 mg / mL, for 6-12 months. All patients receive angiotensin converting enzyme inhibitors and / or angiotensin receptor blockers.

[0052] In a specific embodiment, a renal biopsy is performed at the time of diagnosis, and the renal biopsy specimen is evaluated using direct immunofluorescence, optical and electron microscopy. For direct immunofluorescence, IgG, IgM, IgA, C3c, C1q, fibrinogen, albumin and IgG subclasses are detected on frozen tissue by fluorescein isothiocyanate-coupled antibodies (Dako, Copenhagen, Den mark). Fluorescence intensity is measured using a semi-quantitative scale of 0 to 4+. For optical microscopy, paraffin sections are stained with hematoxylin and eosin, periodic acid-Schiff, hexamine silver and Masson's trichrome. For electron microscopy, the tissue is fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, then dehydrated in gradient acetone, and embedded in Epon812. Ultrathin sections are cut with a thickness of 80nm and placed on nickel grids. Then, ultrathin sections are stained with uranyl acetate and examined with a transmission electron microscope JEM-1230. The renal biopsy results of primary FSGS are classified according to the Columbia FSGS classification system.

[0053] In a specific embodiment, disodium EDTA is used as an anticoagulant to collect patient plasma samples on the day of renal biopsy. Plasma samples from 30 healthy blood donors matched in age and gender are collected as normal controls. 2000g of plasma is collected immediately after centrifugation at 4°C for 15 minutes. The plasma is then aliquoted and stored at -80°C for use. Repeated freeze-thaw cycles are avoided. At the same time, plasma samples from 60 MCD patients and 23 FSGS patients during follow-up are collected and stored until use.

[0054] S2 performs antibody detection on the serum sample to obtain anti-nephrotic protein autoantibodies, wherein the autoantibodies are IgM autoantibodies;

[0055] In one embodiment, the S2 is replaced by performing antibody detection on the serum sample to obtain anti-nephrin autoantibodies, and the autoantibodies are IgG and IgM autoantibodies.

[0056] In one embodiment, the antibody detection method is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic microparticle chemiluminescence, and immunofluorescence technology.

[0057] In one embodiment, the anti-nephrin antibody is expressed as an anti-Nephrin antibody. In a specific embodiment, the present invention measures circulating anti-Nephrin antibodies by enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay uses a Nunc MaxiSorpTM ELISA plate (Thermo Fisher), each well is coated with 100 ng of recombinant human Nephrin extracellular domain (R&D Systems) commercially diluted coating buffer (Bio Legend), and incubated O / N at 4°C. Uncoated control wells are used to determine the nonspecific binding of each patient sample (in the absence of antigen), which allows for background subtraction. Block with 300 μl of blocking buffer at room temperature for 1 hour, and then incubate with 100 μl of 1:100 or 1:20 diluted patient samples in a blocking buffer (Super T) containing 0.1% Tween20 at 4°C O / N.

[0058] The cells were washed five times with 300 μl of PBST, and 100 μl of 1:2000 diluted HRP-labeled mouse anti-human IgG Fc (Abcam) or 1:1000 diluted HRP-labeled mouse anti-human IgM (Abcam) were added, incubated in Super T, and shaken at 500 rpm for 1 hour at room temperature.

[0059] After washing five times with PBST, 100 μl of TMB substrate (BioLegend) was added and incubated at room temperature for 10 minutes. Then, 100 μl of stop solution (BioLegend) was added and the absorbance was measured at 450 nm.

[0060] Background subtraction was performed by subtracting the mean OD value of duplicate uncoated wells from the mean OD value of duplicate antigen-coated wells for each patient sample. The cutoff value was set to the mean + 2 standard deviations (2SD) of 30 healthy blood donors. The cutoff value for anti-Nephrin antibodies (IgG) was 0.10; the cutoff value for anti-Nephrin antibodies (IgM) was 0.19.

[0061] In a specific embodiment, the antibody is subjected to immunofluorescence staining:

[0062] For the colocalization of nephrin and IgG, frozen sections (5 μm) of human renal biopsy tissue were fixed in acetone for 15 minutes, then blocked in 3% BSA for 1 hour at room temperature and incubated with 1 μg / ml polyclonal goat anti-human nephrin antibody (AF4269, R&D Systems) and monoclonal mouse anti-human IgG antibody (ab200699, Abcam) at a dilution of 1:750 at 4°C overnight. Subsequently, after washing three times with PBS containing 0.05% Tween 20 (PBST), the sections were incubated with Alexa Fluor™ 568-conjugated donkey anti-goat IgG antibody (A21099, Invitrogen, Carlsbad, CA, USA) at a dilution of 1:500 and donkey anti-mouse IgG antibody (A21202, Invitrogen) at a dilution of 1:500 at 37°C for 30 minutes and examined under a fluorescence microscope.

[0063] For the colocalization of nephrin and IgM, frozen sections (5 μm) of human renal biopsy tissue were incubated with 1 μg / ml polyclonal goat anti-human nephrin antibody (AF4269, R&D Systems) at 4°C overnight. After washing three times with PBS containing 0.05% Tween 20 (Tween 20, PBST), the sections were incubated with 1:500 dilution of secondary antibody Alexa FluorTM 568-conjugated donkey anti-goat IgG antibody (A21099, Invitrogen) and 1:100 dilution of FITC-labeled rabbit anti-human IgM antibody (F0203, Dako, Glostrup, Denmark) at 37°C for 30 min, and finally observed under a fluorescence microscope.

[0064] In a specific embodiment, SPSS26.0 is used for statistical analysis. Categorical variables are expressed as frequency or percentage (%). Continuous variables are described as non-normally distributed variables using median and interquartile range (IQR), and normally distributed variables using mean ± standard deviation (SD). Normally distributed variables use Student's t test or one-way analysis of variance, and non-normally distributed variables use Kruskal-Wallis test or Mann-Whitney U test. The differences in categorical variables are evaluated by chi-square test or Fisher's exact test. All analyses were performed using two-sided tests, and P<0.05 was considered statistically significant.

[0065] In a specific embodiment, demographic and clinical characteristics: The present invention analyzed 313 patients with MCD confirmed by renal biopsy and 118 patients with primary FSGS confirmed by renal biopsy, and classified them according to the treatment received before renal biopsy. A detailed summary of demographic and clinical data is shown in Table 1. The average age of MCD patients was 44.4±18.9 years old, and the male to female ratio was 175 / 138. The median urine protein quantification was 6.3 (IQR: 3.5-10.0) g / 24h, and the mean serum albumin level was 25.4±9.2 g / L. The median serum creatinine level was 80.8 (IQR: 64.3-108.2) μmol / L, and the mean eGFR was 86.8±35.1 ml / min / 1.73m 2 For patients with primary FSGS, the mean age was 44.3 years ± 18.0 years, with a male to female ratio of 73 / 45. The median urine protein quantification was 7.3 (IQR: 4.2-12.0) g / 24h, and the serum albumin level was 23.2 ± 6.8 g / L. The proportion of patients with nephrotic syndrome was 78.8%. The median serum creatinine level was 99.3 (IQR: 80.0-148.0) μmol / L, and the mean eGFR was 80.1 ± 35.2 ml / min / 1.73 m 2 A total of 194 patients with MCD and 69 patients with FSGS were untreated at the time of renal biopsy.

[0066] Table 1 Characteristics of patients with MCD and primary FSGS.

[0067]

[0068]

[0069] ACEI: angiotensin-converting enzyme inhibitor; ARB: angiotensin receptor blocker; ESKD: end-stage renal disease.

[0070] In a specific embodiment, the present invention sets the cutoff value based on the average OD value of 30 healthy blood donors + two times the standard deviation (2SD). The cutoff values ​​of anti-Nephrin IgG antibody and anti-Nephrin IgM antibody are 0.10 and 0.19 respectively.

[0071] In a specific embodiment, in MCD patients, the positive rate of anti-Nephrin IgG antibodies was 22.7% (71 / 313), and the positive rate of anti-Nephrin IgM antibodies was 39.9% (124 / 313). At the same time, 33.9% (40 / 118) of primary FSGS patients were positive for anti-Nephrin IgG antibodies, and 29.7% (35 / 118) of primary FSGS patients were positive for anti-Nephrin IgM antibodies. Neither MN patients with positive anti-PLA2R antibodies nor healthy controls were detected with positive anti-Nephrin IgG and IgM antibodies ( Figure 4 A).

[0072] In a specific embodiment, the present invention also analyzes the difference in anti-Nephrin antibody positive rates between different subgroups, including patients with nephrotic range proteinuria (defined as 24-hour urine protein quantification> 3.5g / 24h) and patients with non-nephrotic range proteinuria, and patients who were receiving immunosuppressive therapy or not at the time of sample collection ( Figure 4 B). Among 313 MCD patients, 229 had nephrotic-range proteinuria. Among the 229 patients, 60 (26.2%) were positive for anti-Nephrin IgG antibodies and 98 (42.8%) were positive for anti-Nephrin IgM antibodies. Among the 229 MCD patients with nephrotic-range proteinuria, 160 patients were not receiving immunosuppressive therapy at the time of sample collection. Among the 160 patients, 52 (32.5%) were positive for anti-Nephrin IgG antibodies and 71 (44.4%) were positive for anti-Nephrin IgM antibodies. Among the 118 primary FSGS patients, 101 showed nephrotic-range proteinuria. Among the 101 patients, 37 (36.6%) were positive for anti-Nephrin IgG antibodies and 34 (33.7%) were positive for anti-Nephrin IgM antibodies. Similarly, among 101 patients with primary FSGS and nephrotic-range proteinuria, 59 patients were not receiving immunosuppressive therapy at the time of sample collection. Among the 59 patients, 26 (44.1%) were positive for anti-Nephrin IgG antibodies, and 20 (33.9%) were positive for anti-Nephrin IgM antibodies.

[0073] In a specific embodiment, anti-Nephrin antibodies and clinical outcomes: The anti-Nephrin antibody (IgG) status of MCD patients was compared.

[0074] In a specific embodiment, among 331 MCD patients, 194 patients were not receiving immunosuppressive therapy at the time of renal biopsy (when specimens were collected). Among the 194 untreated MCD patients, 54 were positive for anti-Nephrin IgG antibodies. Compared with anti-Nephrin IgG negative patients, anti-Nephrin IgG positive patients had significantly increased urine protein quantification (9.9 (IQR: 6.3-14.0)) g / 24h vs. 5.5 (IQR: 3.6-8.9) g / 24h (p<0.001), and eGFR (73.9±33.9 ml / min / 1.73 m 2 vs.85.5±33.7ml / min / 1.73m 2 , p=0.033) was significantly reduced (p<0.001). The proportion of nephrotic syndrome in anti-Nephrin IgG-positive patients was significantly higher than that in anti-Nephrin IgG-negative patients (96.3% vs. 72.9%, p<0.001). Interestingly, the relapse rate (61.5% vs. 14.2%, p<0.001) and the frequency of relapse (13.5% vs. 1.5%, p=0.002) in anti-Nephrin IgG-positive MCD patients were significantly higher than those in anti-Nephrin IgG-negative MCD patients, but there was no significant difference in the remission rate and remission duration (as shown in Table 2). Among the 118 primary FSGS patients, 69 patients did not receive immunosuppressive therapy at the time of renal biopsy (when the specimen was collected). Among the 69 untreated primary FSGS patients, 28 were positive for anti-Nephrin IgG antibodies. The urine protein level of anti-Nephrin IgG-positive patients was higher (9.0 (IQR: 4.3-15.6)) g / 24 h than that of anti-Nephrin IgG-negative patients (5.6 (IQR: 3.7-8.2) g / 24 h (p = 0.016), the serum creatinine level was higher (120.6 (IQR: 93.5-179.5)) μmol / L than that of anti-Nephrin IgG-negative patients (91.7 (IQR: 76.6-142.5) μmol / L (p = 0.010), and the eGFR was higher (68.9 ± 32.4 ml / min / 1.73 m 2 vs.88.6±33.9ml / min / 1.73m 2 ,p=0.018) was lower than that in anti-Nephrin IgG negative patients.

[0075] Table 2 Comparison of clinicopathological features between anti-Nephrin IgG antibody-positive and anti-Nephrin IgG antibody-negative patients with treatment-naive MCD.

[0076]

[0077]

[0078] Similar to MCD patients, the relapse rate (70.4% vs. 31.4%, p = 0.002) and relapse frequency ratio (55.6% vs. 5.7%, p < 0.001) of primary FSGS patients who were positive for anti-Nephrin IgG were significantly increased (see Table 3).

[0079] Table 3 Comparison of clinicopathological features of anti-Nephrin IgG antibody positive and anti-Nephrin IgG antibody negative patients with primary FSGS

[0080]

[0081]

[0082] In a specific embodiment, anti-Nephrin antibodies and clinical outcomes: The anti-Nephrin antibody (IgM) status of MCD patients was compared.

[0083] Among 194 untreated MCD patients, 82 were positive for anti-Nephrin IgM antibodies. Compared with anti-Nephrin IgM negative patients, anti-Nephrin IgM positive patients had significantly higher serum creatinine levels (86.4 (IQR: 67.0-129.9)) μmol / L vs. 70.5 (IQR: 63.9-98.8) μmol / L (p = 0.043), and eGFR (77.6 ± 34.3 ml / min / 1.73 m 2 vs.88.7±32.9ml / min / 1.73m 2 , p=0.025) was significantly reduced. The present invention also found that the serum IgM level (1.5 (IQR: 1.0-2.5)) g / L vs. 0.9 (IQR: 0.6-1.5) g / L, p=0.001) and the proportion of patients with IgM deposition in renal biopsy tissue (52.4% vs. 33.0%, p=0.007) were significantly higher in anti-Nephrin IgM positive patients than in anti-Nephrin IgM negative patients. The relapse rate (50.6% vs. 10.3%, p<0.001) and the frequency relapse ratio (11.4% vs. 0%, p=0.001) of MCD patients with anti-Nephrin IgM positive were significantly increased (as shown in Table 4).

[0084] Table 4 Comparison of clinicopathological features between anti-Nephrin IgM antibody-positive and anti-Nephrin IgM antibody-negative untreated MCD patients.

[0085]

[0086]

[0087] In a specific embodiment, 20 of 69 untreated primary FSGS patients were positive for anti-Nephrin IgM antibodies. The urine protein quantification of anti-Nephrin IgM positive patients was significantly higher than that of anti-Nephrin IgM negative patients ((8.1 (IQR: 4.6-16.6)) g / 24h vs. 5.8 (IQR: 3.6-8.9) g / 24h, p = 0.024). The serum IgM level (1.4 (IQR: 0.7-2.0)) g / L vs. 0.9 (IQR: 0.7-1.0) g / L, p = 0.037) and the proportion of patients with IgM deposition in renal biopsy tissue (70.0% vs. 34.7%, p = 0.007) were significantly higher in anti-Nephrin IgM positive patients than in anti-Nephrin IgM negative patients. The recurrence rate (90.0% vs. 28.6%, p<0.001) and recurrence frequency ratio (60.0% vs. 11.9%, p<0.001) of primary FSGS patients with positive anti-Nephrin IgM were significantly increased (Table 5).

[0088] Table 5 Comparison of clinicopathological features between untreated primary FSGS patients with anti-Nephrin IgM antibody positive and anti-Nephrin IgM antibody negative.

[0089]

[0090]

[0091] In a specific embodiment, anti-Nephrin antibodies and clinical outcomes: anti-Nephrin antibody (IgG and / or IgM) status of MCD patients are compared.

[0092] Among 194 untreated MCD patients, 90 patients were positive for anti-Nephrin IgG antibodies or anti-Nephrin IgM antibodies, or both. The urine protein quantification of patients with positive anti-Nephrin antibodies was significantly higher than that of patients with negative anti-Nephrin antibodies ((7.7 (IQR: 4.9-11.5)) g / 24h vs. 5.4 (IQR: 3.6-9.0) g / 24h, p = 0.001). The relapse rate (48.0% vs. 3.5%, p < 0.001) and the frequency relapse ratio (9.0% vs. 0%, p = 0.004) of patients with positive anti-Nephrin antibodies were significantly increased (as shown in Table 6). It is worth noting that no MCD patients with negative anti-Nephrin antibodies had a relapse.

[0093] Table 6 Comparison of clinicopathological characteristics of untreated MCD patients with positive anti-Nephrin IgG and / or IgM antibodies and negative anti-Nephrin antibodies.

[0094]

[0095]

[0096] Among 69 untreated primary FSGS patients, 32 patients were positive for anti-Nephrin IgG antibodies or anti-Nephrin IgM antibodies, or both. The urine protein quantification of patients with positive anti-Nephrin antibodies was significantly increased (7.0 (IQR: 4.3-13.2)) g / 24h vs. 5.6 (IQR: 3.6-8.5) g / 24h, p = 0.041). The relapse rate (74.2% vs. 22.6%, p < 0.001) and the frequency relapse ratio (51.6% vs. 3.2%, p < 0.001) of patients with positive anti-Nephrin antibodies were significantly increased (as shown in Table 7). Only one primary FSGS patient with negative anti-Nephrin antibodies had frequent relapse.

[0097] Table 7 Comparison of clinicopathological features of untreated primary FSGS patients with positive anti-Nephrin IgG and / or IgM antibodies and negative anti-Nephrin antibodies.

[0098]

[0099]

[0100] In a specific embodiment, a comparison of MCD and primary FSGS patients who are positive for both anti-Nephrin IgG and IgM antibodies:

[0101] Among 194 untreated MCD patients, 32 patients were positive for both anti-Nephrin IgG and IgM antibodies. The urine protein quantity ((10.5 (IQR: 6.8-15.3)) g / 24h) and the proportion of nephrotic syndrome (96.9%) in these 32 patients were higher than those in patients with only positive anti-Nephrin antibodies (urine protein quantity: 6.7 (IQR: 4.5-9.9)) g / 24h, p = 0.001; proportion of nephrotic syndrome: 79.2%, p = 0.044) and patients with negative anti-Nephrin antibodies (urine protein quantity: 5.4 (IQR: 3.6-9.0)) g / 24h, p < 0.001; proportion of nephrotic syndrome: 73.3%, p = 0.005).

[0102] The relapse rate after remission in patients with double antibody positive (77.4%) was significantly higher than that in patients with single antibody positive (34.8%, p<0.001) and patients with double antibody negative (3.5%, p<0.001). In addition, the relapse rate in the double antibody positive group (22.6%) was significantly higher than that in patients with single antibody positive (2.9%, P=0.005) and patients with double antibody negative (0%, P<0.001) (as shown in Table 8).

[0103] Table 8 Comparison of clinicopathological characteristics of untreated MCD patients who were positive for both anti-Nephrin IgG and IgM antibodies, positive for either anti-Nephrin IgG or IgM antibodies, or negative for both anti-Nephrin IgG and IgM antibodies.

[0104]

[0105]

[0106]

[0107] Among 69 untreated primary FSGS patients, 16 patients were positive for both anti-Nephrin IgG and IgM antibodies. The urine protein quantitative of these 16 patients ((11.6 (IQR: 4.7-19.7)) g / 24h) was significantly higher than that of patients with negative antibodies ((5.6 (IQR: 3.6-8.5)) g / 24h, p = 0.001) and patients with single antibody positive ((5.8 (IQR: 3.8-11.1)) g / 24h, p = 0.018). There was no statistically significant difference in serum creatinine and eGFR. The serum IgM level of patients with both positive anti-Nephrin IgG and IgM antibodies ((1.4 (IQR: 0.9-2.2)) g / L) was significantly higher than that of antibody-negative patients ((0.7 (IQR: 0.5-0.9)) g / L, p = 0.003) and single antibody-positive patients ((0.9 (IQR: 0.8-1.0)) g / L, p = 0.004). The relapse rate and frequency relapse rate of the group with both positive antibodies were significantly higher than those of the patients with both negative antibodies (relapse rate: 87.5% vs. 22.6%, p < 0.001; frequency relapse rate: 68.8% vs. 3.2%, p < 0.001). In addition, the frequency relapse rate of the double antibody-positive group was also slightly higher than that of the single antibody-positive patients (68.8% vs. 33.3%, p = 0.049). (See Table 9).

[0108] Table 9 Comparison of clinicopathological features of untreated primary FSGS patients who were positive for both anti-Nephrin IgG and IgM antibodies, positive for anti-Nephrin IgG or IgM monoclonal antibodies, or negative for both anti-Nephrin IgG and IgM antibodies.

[0109]

[0110]

[0111] In a specific embodiment, anti-Nephrin antibodies and disease activity: For patients with positive anti-Nephrin IgG antibodies, the present invention collected plasma samples from 26 MCD patients and 18 primary FSGS patients during the follow-up period, all of whom were in complete remission. The anti-Nephrin IgG antibody OD values ​​of MCD patients and primary FSGS patients in remission (MCD: 0.32±0.21 vs. 0.07±0.04, p<0.0001; primary FSGS: 0.62±0.38 vs. 0.08±0.07, p<0.0001) were significantly lower than those at the time of renal biopsy. 23 / 26 MCD patients in remission and 13 / 18 primary FSGS patients were negative for anti-Nephrin. The antibody OD values ​​of 3 MCD patients and 5 primary FSGS patients who were positive for anti-Nephrin IgG antibodies in remission were slightly higher than the critical value ( Figure 5 (as shown in A).

[0112] For patients with positive anti-Nephrin IgM antibodies, plasma samples were collected from 38 MCD patients and 15 primary FSGS patients in remission. The anti-Nephrin IgM antibody OD values ​​of MCD patients and primary FSGS patients in remission (MCD: 0.77±0.29 vs. 0.12±0.08, p<0.0001; primary FSGS: 0.61±0.32 vs. 0.12±0.09, p<0.0001) were significantly lower than those at the time of renal biopsy. In the remission period, 34 of the 38 MCD patients were negative for anti-Nephrin IgM antibodies, and 12 of the 15 primary FSGS patients were negative for anti-IgM antibodies. The antibody OD values ​​of the 4 MCD patients and 3 primary FSGS patients who were positive for anti-Nephrin IgM in remission were slightly higher than the critical value, ( Figure 5 In general, the levels of plasma anti-Nephrin IgG or IgM antibodies in patients with MCD and primary FSGS during remission were significantly lower than those in patients with active nephrotic syndrome.

[0113] In one embodiment, longitudinal clinical course and antibody changes: In order to better observe the relationship between anti-Nephrin antibodies and proteinuria, the present invention collects sequential plasma samples from multiple patients.

[0114] In 4 patients with positive anti-Nephrin IgG antibodies, including 2 MCD and 2 primary FSGS patients, the present invention detected a series of anti-Nephrin IgG antibodies during follow-up. The results showed that the anti-Nephrin IgG autoantibody levels of the 4 patients were closely correlated with urine protein quantification. ( Figure 6 (A). During the active phase of nephrotic syndrome, the OD value of anti-Nephrin IgG antibodies was relatively high. The OD value of the antibody decreased during remission, and increased again and exceeded the critical value during relapse. Interestingly, MCD patient 2 had complete remission of nephrotic syndrome on October 23, 2016 and November 30, 2016, but the OD value of anti-Nephrin IgG antibodies increased and turned positive on November 30, 2016. Nearly 1 month later, the urine protein quantification increased from 0.02g / 24h to 3.52g / 24h, and the OD value of anti-Nephrin IgG antibodies further increased. This suggests that increased anti-Nephrin IgG antibodies can predict the recurrence of proteinuria. A similar pattern was observed in primary FSGS patient 1 (indicated by a red arrow).

[0115] Similarly, in another 4 patients with positive anti-Nephrin IgM antibodies, including 2 MCD and 2 primary FSGS patients, the present invention also observed that the level of anti-Nephrin IgM autoantibodies was closely correlated with urine protein quantification ( Figure 6 (B). MCD patient 3 had a significantly increased anti-Nephrin IgM antibody level at the third follow-up, but proteinuria was relieved. Nearly 2 months later, urine protein quantification increased sharply from 0.27g / 24h to 4.06g / 24h, and finally peaked at 12.64g / 24h, and the antibody level increased further. Subsequently, as proteinuria decreased after treatment, the antibody level also decreased. In November 2016, urine protein quantification and anti-Nephrin IgM antibodies (indicated by red arrows) also showed similar changes. This shows that anti-Nephrin IgM antibodies are closely related to urine protein quantification and can predict the recurrence of proteinuria. A similar pattern was also observed in primary FSGS patient 3 (indicated by arrows).

[0116] In a specific embodiment, immunofluorescence validation: The present invention performed immunofluorescence staining to observe the co-localization of punctate IgG and nephrin or IgM and nephrin in renal biopsy tissue. Co-localization of IgG (green) and nephrin (red) was observed in MCD and primary FSGS patients with positive IgG in routine renal histopathology and positive circulating anti-nephrin IgG antibodies. No obvious co-localization was observed in normal kidneys and MN patients ( Figure 7Similarly, immunofluorescence staining showed co-localization of IgM (green) and nephrin (red) in patients with MCD and primary FSGS who were IgM-positive in routine renal histopathology and positive for circulating anti-nephrin IgM antibodies, but not in normal kidneys and MN patients ( Figure 7 This co-localization validates the specificity of anti-Nephrin antibody in some patients with MCD and primary FSGS.

[0117] These observations underscore the prognostic significance of anti-nephrin antibodies and highlight their utility as biomarkers for monitoring disease activity and treatment response in MCD. The dynamic association of these antibodies with clinical disease status suggests their potential role in guiding treatment strategies, reflecting their ability to effectively indicate changes in disease status and predict outcome.

[0118] In a specific embodiment, cross-comparison of IgG or IgM deposition with anti-Nephrin IgG or IgM antibodies: In the study of the present invention, immunofluorescence staining of IgG and IgM in patients diagnosed with MCD and primary FSGS from April 2023 to April 2024 was also reviewed, including 123 cases of MCD and 42 cases of primary FSGS. At the same time, the present invention also detected anti-Nephrin antibodies in these patients during renal biopsy and conducted a cross-comparison. Among the 123 MCD patients, punctate IgG deposition was observed in the renal biopsy tissue of 72 patients (58.5%), and IgM deposition was observed in the renal biopsy tissue of 77 patients (62.6%). In terms of anti-Nephrin antibodies, 33 (26.8%) MCD patients were positive for anti-Nephrin IgG antibodies, and 51 (41.5%) MCD patients were positive for anti-Nephrin IgM antibodies. Among 42 patients with primary FSGS, 17 (40.5%) had IgG punctate deposits, and 34 (81.0%) had positive IgM immunofluorescence staining. Among 42 patients with primary FSGS, 16 (38.1%) had positive anti-Nephrin IgG antibodies, and 14 (33.3%) had positive anti-Nephrin IgM antibodies. It is worth noting that in the immunofluorescence staining of renal biopsy tissue, patients with positive anti-Nephrin antibodies (IgG or IgM) were included in the subgroup of patients with positive renal tissue IgG or IgM.

[0119] In one specific embodiment, this study provides important insights into the role of anti-Nephrin antibodies in MCD, a common cause of nephrotic syndrome in children and adults. The findings of the present invention highlight the strong correlation between the presence of these antibodies and major clinical outcomes, particularly proteinuria and response to treatment. In addition, the present study not only confirmed the presence of anti-Nephrin antibodies in a subset of MCD patients, but also demonstrated their correlation with adverse clinical outcomes. The correlation of anti-Nephrin antibodies with disease severity highlights the potential of these antibodies as new targets for therapeutic intervention. Targeting these antibodies may provide a new avenue for treating patients who are resistant to standard treatments or who relapse frequently.

[0120] In a specific embodiment, the present invention first systematically studied the prevalence of anti-Nephrin IgG antibodies in Chinese adult MCD and primary FSGS patients. It was found that 22.7% of adult MCD patients and 33.9% of primary FSGS patients were positive for anti-Nephrin IgG antibodies. In patients with active MCD or primary FSGS who were not receiving immunosuppressive therapy, the positive rates were 32.5% and 44.1%, respectively. Anti-Nephrin IgG antibodies were not detected in patients with PLA2R-related MN and healthy controls. It is worth noting that compared with patients with negative anti-Nephrin IgG antibodies, patients with positive anti-Nephrin IgG antibodies showed higher proteinuria, lower eGFR, higher relapse rate and frequency relapse rate. At the same time, the present invention also found that anti-Nephrin IgG antibodies were significantly reduced when nephrotic syndrome was relieved, and the antibodies were closely related to urine protein quantification, which may indicate the recurrence of proteinuria in the longitudinal clinical course.

[0121] In the cohort of the present invention, the positive rate of anti-Nephrin IgG antibodies in all MCD patients or active MCD patients who did not receive immunosuppressive therapy was lower than that in the study of Hengel FE et al. The present invention speculates that the prevalence of anti-Nephrin IgG antibodies in MCD patients in the present study population may be underestimated for three reasons: first, the present study used a modified ELISA method to detect antibodies. As mentioned above, the biotin-avidin amplification system was not used, and the OD values ​​of some patients may be lower than the critical value; second, the present invention set a strict cutoff value based on the average OD value + 2SD of 30 healthy blood donors to avoid false positives; third, 38.0% of MCD patients received treatment before plasma sampling, which may lead to false negatives in some patients. At the same time, the present invention also found that the positive rate of anti-Nephrin IgG autoantibodies in primary FSGS was 33.9%, which may indicate that more primary FSGS in the Chinese population is an immune-mediated disease, which needs to be confirmed by global multicenter cohort studies.

[0122] In a specific embodiment, the present invention first found that 39.6% of adult MCD patients and 29.7% of adult primary FSGS patients were positive for anti-Nephrin IgM antibodies. Similar to anti-Nephrin IgG antibodies, anti-Nephrin IgM antibodies are associated with disease severity, especially relapse rate and frequent relapse rate. These findings suggest that IgM autoantibodies, like IgG autoantibodies, may play a pathogenic role in the pathophysiological process of some MCD and primary FSGS patients.

[0123] The longitudinal analysis of the present invention showed a close correlation between circulating anti-Nephrin autoantibody levels and urine protein quantification, reinforcing the potential of these antibodies as biomarkers of disease activity. Patients with elevated anti-Nephrin antibody levels but remission of proteinuria at this time are prone to recurrence of proteinuria the next time, suggesting that monitoring anti-Nephrin autoantibody levels during follow-up can guide treatment strategies and improve patient management.

[0124] In a specific embodiment, one of the main advantages of this study is the comprehensive evaluation of anti-Nephrin antibodies in a large cohort, providing reliable data for their significance in MCD / FSGS. The presence of anti-Nephrin autoantibodies was confirmed in the MCD / FSGS cohort of the present invention, and the findings of the present invention demonstrated that these antibodies are closely related to clinical manifestations and prognosis, and antibody-positive patients experience more severe disease and higher relapse rates. The results of the present invention suggest that anti-Nephrin autoantibodies may be involved in the pathogenesis of MCD / FSGS, providing further support for autoimmune etiology. Anti-Nephrin antibodies can not only serve as biomarkers for monitoring disease activity, but also help clinicians more effectively predict disease progression and adjust treatment to improve the prognosis of MCD / FSGS patients.

[0125] S3: Classifying the disease based on the anti-nephrin autoantibodies to obtain a classification result, wherein the classification result is whether the disease is refractory.

[0126] In one embodiment, the refractory type is a patient with more than L relapses within half a year, where L is a natural number greater than or equal to 2;

[0127] Optionally, the disease is classified based on the anti-nephrin autoantibodies to obtain a classification result, specifically: when the value of the anti-nephrin autoantibodies is greater than a preset threshold, the patient is a refractory patient.

[0128] In one embodiment, the preset threshold is a cutoff value of anti-nephrin autoantibodies.

[0129] In one embodiment, the anti-nephrin autoantibody is input into a classifier for disease classification to obtain a classification result;

[0130] Optionally, the classifier is one or more of the following: support vector machine, random forest, convolutional neural network, XGBoost, AdaBoost.

[0131] In one embodiment, S3 is replaced by S32, clinical indicators of the patient are obtained, and the clinical indicators and anti-nephrin autoantibodies are input into a classifier for classification to obtain classification results.

[0132] In one embodiment, the clinical indicators include one or more of the following: urine protein, eGFR.

[0133] In one embodiment, the method further comprises providing medication guidance, and administering medication based on the classification results;

[0134] Optionally, when the classification result is refractory, the patient is given treatment with cyclophosphamide, calcineurin inhibitors, leflunomide, rituximab, etc.

[0135] In one embodiment, the refractory type is treated with some other immunosuppressants or rituximab. Among them, for patients with IgM antibodies and refractory disease diagnosed with a combination of IgG and IgM (actually mainly prone to relapse and frequent relapse), the present invention may be beneficial to the treatment based on CD20 monoclonal antibodies. At the same time, these patients are more likely to relapse, so it is more cautious to reduce or stop the drug.

[0136] In one embodiment, the method further comprises predicting the frequency of follow-up visits for prognosis, and predicting the frequency of follow-up visits for the patient's prognosis based on the classification result.

[0137] In one embodiment, antibody-positive patients are initially judged to be more prone to relapse and relapse more frequently.

[0138] The embodiments of the present disclosure also provide a computer program product or system, including a computer program, which, when executed by a processor, implements the steps of the above-mentioned disease classification method based on anti-nephrin autoantibodies.

[0139] Figure 2 The schematic diagram of the disease classification system based on anti-nephrotic protein autoantibodies provided in the embodiment of the present invention specifically includes:

[0140] Acquisition unit: acquiring serum samples from patients with minimal glomerular changes and primary focal segmental glomerulosclerosis;

[0141] Detection unit: performing antibody detection on the serum sample to obtain anti-nephrotic protein autoantibodies, wherein the autoantibodies are IgM autoantibodies;

[0142] Classification unit: A classification result is obtained by classifying the disease based on the anti-nephrin autoantibodies, and the classification result is whether the disease is refractory.

[0143] Figure 3 The embodiment of the present invention provides a disease classification device based on anti-nephrotic protein autoantibodies, which specifically includes:

[0144] A memory and a processor; the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, any one of the above-mentioned disease classification methods based on anti-nephrotic protein autoantibodies is executed.

[0145] An embodiment of the present invention further provides a computer-readable storage medium storing a computer program, wherein the computer program, when executed by a processor, is any one of the above-mentioned disease classification methods based on anti-nephrin autoantibodies.

[0146] The embodiment of the present invention also provides a use of a product for detecting anti-nephrotic protein autoantibodies in the preparation of a preparation for diagnosing whether the patient has refractory glomerular microlesions or primary focal segmental glomerulosclerosis;

[0147] In one embodiment, the method used to detect anti-nephrin autoantibodies is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic microparticle chemiluminescence, immunofluorescence technology;

[0148] Optionally, the autoantibodies are IgM and / or IgG type autoantibodies.

[0149] The embodiment of the present invention also provides a detection kit for detecting anti-nephrotic protein autoantibodies, the kit comprising: a substrate of nephrotic protein, an antibody of anti-human IgG and / or IgM antibody;

[0150] Optionally, the substrate is an extracellular region fragment of nephrin;

[0151] Optionally, the antibody is enzyme-labeled or fluorescently labeled;

[0152] Optionally, the species of the antibody include any one of the following: chicken, donkey, goat, mouse, rabbit, rat, sheep;

[0153] Optionally, the types of antibody fluorescence mainly include fluorescein isothiocyanate FITC, tetramethylrhodamine isothiocyanate TRITC, RRX, Texas Red, cyanine dyes, phycoerythrin or phycobilichrome protein,

[0154] AMCA;

[0155] Optionally, the kit includes coating buffer, Tween20 blocking slow-release solution, HRP-labeled mouse anti-human IgG or IgM, FITC-labeled mouse anti-human IgG or IgM, PBST washing buffer, TMB substrate, and stop solution;

[0156] In one embodiment, the kit is used to diagnose refractory glomerular minimal change disease or primary focal segmental glomerulosclerosis.

[0157] The verification results of this verification embodiment show that assigning inherent weights to indications can improve the performance of the method relative to the default setting. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here. In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic, for example, the division of the units is only a logical function division, and there may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the coupling or direct coupling or communication connection between each other shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms. The unit described as a separate component may or may not be physically separated, and the component displayed as a unit may or may not be a physical unit, that is, it may be located in one place, or it may be distributed on multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the scheme of this embodiment. In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units. A person of ordinary skill in the art may understand that all or part of the steps in the various methods of the above-mentioned embodiments may be completed by instructing the relevant hardware through a program, and the program may be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0158] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be implemented by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned medium storage can be a read-only memory, a disk or an optical disk, etc.

[0159] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A disease classification method based on anti-nephrin autoantibodies, characterized in that: include: S1 obtains a serum sample from a patient, wherein the patient is a patient with glomerular minimal change disease and primary focal segmental glomerulosclerosis; S2 performs antibody detection on the serum sample to obtain anti-nephrin autoantibodies, wherein the autoantibodies are IgM autoantibodies; the autoantibodies are anti-Nephrin antibodies; S3 classifies the disease based on the anti-nephrotic protein autoantibody to obtain a classification result, wherein the classification result is whether the disease is refractory; The refractory type refers to a relapse rate greater than L times within half a year, where L is a natural number greater than or equal to 2; The disease classification is performed based on the anti-nephrin autoantibodies to obtain a classification result, specifically: when the value of the anti-nephrin autoantibodies is greater than a preset threshold, the patient is a refractory patient.

2. The disease classification method based on anti-nephrin autoantibodies according to claim 1, characterized in that: The S2 is replaced by performing antibody detection on the serum sample to obtain anti-nephrin autoantibodies, wherein the autoantibodies are IgG and IgM autoantibodies.

3. The disease classification method of anti-nephrin autoantibodies according to claim 1, characterized in that: The antibody detection method is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic particle chemiluminescence, and immunofluorescence technology.

4. The disease classification method based on anti-nephrin autoantibodies according to claim 1, characterized in that: The anti-nephrin autoantibodies are input into a classifier for disease classification to obtain a classification result.

5. The disease classification method based on anti-nephrin autoantibodies according to claim 4, characterized in that: The classifier is one or more of the following: support vector machine, random forest, convolutional neural network, XGBoost, AdaBoost.

6. The disease classification method based on anti-nephrin autoantibodies according to claim 1 or 2, characterized in that: The method also includes predicting the frequency of follow-up visits for prognosis, and predicting the frequency of follow-up visits for the patient's prognosis based on the classification results.

7. A computer device having a memory and a processor and a computer program / instruction stored in the memory, characterized in that: include: The processor executes the computer program / instructions to implement the disease classification method based on anti-nephrin autoantibodies as described in any one of claims 1-6.

8. A computer program product having a computer program / instructions thereon, characterized in that: The computer program / instructions are executed by a processor to implement the disease classification method based on anti-nephrin autoantibodies as described in any one of claims 1-6.

9. A computer-readable storage medium having a computer program / instruction stored thereon, characterized in that: The computer program / instructions are executed by a processor to implement the disease classification method for anti-nephrin autoantibodies according to any one of claims 1 to 6.

10. Use of a product for detecting anti-nephrin autoantibodies in the preparation of a preparation for diagnosing refractory glomerular microlesions or primary focal segmental glomerulosclerosis, wherein the autoantibody is an anti-Nephrin antibody.

11. Use of the product for detecting anti-nephrotic protein autoantibodies according to claim 10 in the preparation of a preparation for diagnosing whether the patient has refractory glomerular microlesions or primary focal segmental glomerulosclerosis, wherein the method used for detecting anti-nephrotic protein autoantibodies is one or more of the following: enzyme-linked immunosorbent assay, immunoprecipitation, magnetic particle chemiluminescence, and immunofluorescence technology.

12. Use of the product for detecting anti-nephrin autoantibodies according to claim 11 in the preparation of a preparation for diagnosing whether the disease is refractory glomerular minimal change disease or primary focal segmental glomerulosclerosis, wherein the autoantibodies are IgM and / or IgG autoantibodies.

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