Application of urine protein as a diagnostic marker for primary biliary cholangitis

Abstract

The present invention relates to the use of urine protein as a diagnostic marker for primary biliary cholangitis (PBC). The urine protein includes one or a combination of osteopontin (OPN), receptor activity-modifying protein 3 (RAMP3), and calcium-binding protein (S100A8). This application uses urine as a non-invasive test sample, making it more readily accepted by patients than blood. It is of great significance for the early, non-invasive diagnosis of PBC patients.

Description

Technical Field

[0001] The present invention relates to the technical field of non-invasive diagnosis, and in particular to the application of urine protein as a diagnostic marker for primary biliary cholangitis. Background Art

[0002] Primary biliary cholangitis (PBC) is an immune-mediated, chronic cholestatic liver disease characterized histologically by non-suppurative destruction of the epithelial cells of small and medium-sized intrahepatic bile ducts, which can gradually progress to cirrhosis and even liver failure. PBC is a multifactorial disease, and its onset is related to environmental, genetic, and immune factors. Over the past few decades, PBC has been on the rise worldwide. However, due to the atypical early clinical manifestations of PBC patients and the limitations of existing clinical diagnostic criteria, many patients are diagnosed in the clinical terminal stage, leading to delayed treatment. Therefore, early diagnosis and treatment of PBC patients are very important.

[0003] When extrahepatic biliary obstruction is excluded by imaging examination, the current diagnostic criteria for PBC are mainly based on any two of the following: (1) biochemical indicators reflecting cholestasis, such as alkaline phosphatase (ALP) elevated to more than 1.5 times the upper limit of normal and / or glutamyl transferase (GGT) persistently elevated to more than 3 times the upper limit of normal; (2) positive serum antimitochondrial antibodies (AMA) or AMA-M2 type positive (titer > 1:40) reflecting immune abnormalities; (3) liver histopathological evidence consistent with PBC (non-suppurative cholangitis). However, the above laboratory indicators have certain limitations, such as GGT is easily affected by factors such as alcohol, drugs, and obesity. Although AMA is a highly specific serological antibody for PBC and can be detected in approximately 95% of PBC patients, AMA titer is not correlated with the activity and severity of PBC. In addition, a recent study found that more than 80% of AMA patients without elevated serum ALP levels had histological features of PBC, suggesting that there are some undiagnosed PBC patients among patients who are AMA-positive but have normal ALP levels.

[0004] Currently, the gold standard for diagnosing PBC is still liver tissue biopsy. However, this is an invasive procedure with risks such as puncture site bleeding, liver damage, and abdominal infection, which is not easily accepted by patients. Therefore, liver histological examination is usually only necessary for diagnosis when AMA is negative or the patient has atypical PBC biochemical characteristics.

[0005] In summary, for the early diagnosis of PBC, clinical screening for other highly accurate, easily disseminated, and non-invasive markers is needed. In addition to blood, urine, as a filtered component of plasma, contains information not only from the urinary system but also from other organs after plasma filtration. Therefore, urine can also accumulate and reflect a range of changes in the body to a certain extent. Using proteomics technology to systematically analyze and identify protein molecules in urine at high throughput allows for the study of their biological functions. There are more than 1,500 types of proteins in the urine of healthy people, so the quantitative and qualitative changes in urine may be of great significance for disease diagnosis. Urine has the following advantages over blood: 1) Sample collection is simple, non-invasive, and easy to obtain continuously, and can be used as a low-cost clinical test; 2) Certain protein or peptide metabolites are quickly metabolized into urine through the kidneys after entering the blood. At this time, they may not be detected in the blood, but may be detected in the urine; 3) Compared with blood, proteins and peptides in urine are more stable, less prone to degradation, and easy to transport and store; 4) The proteins in blood and tissue fluid are complex and numerous, and urine is easier to observe changes in low-abundance proteins, making it more suitable for mass spectrometry analysis. Summary of the Invention

[0006] In order to solve the above technical problems, the present application provides the application of urine protein as a diagnostic marker for primary biliary cholangitis.

[0007] This application is implemented through the following technical solutions:

[0008] The present application relates to the use of a urine protein detection product in the preparation of a product for diagnosing primary biliary cholangitis.

[0009] The inventors first used liquid chromatography-mass spectrometry to identify 194 urine proteins that were significantly differentially expressed between PBC patients and normal controls, of which 109 were upregulated in PBC patients and 85 were downregulated.

[0010] Secondly, combined with GO analysis and signal pathway enrichment, 10 differentially expressed proteins related to immune response were screened out, namely: osteopontin (OPN), receptor activity-modifying protein 3 (RAMP3), CD44, activin receptor type 1B protein (ACVR1B), chemokine CXCL12 protein, CD74, Wnt1-induced signaling pathway protein (CCN4), calcium-binding protein (S100A8), lysosomal associated membrane protein 3 (LAMP3), and phosphoinositide-3-kinase catalytic subunit delta peptide (PIK3Cd).

[0011] Finally, combining proteomic analysis and correlation analysis, it was found that OPN, RAMP3, S100A8 were most significantly correlated with clinical parameters such as ALT, AST, ALP, GGT, TBA, IgM, IgG, and AMA; experimental demonstration showed that the levels of OPN, RAMP3, and S100A8 proteins in the urine of patients with primary biliary cholangitis were significantly higher than those in healthy people.

[0012] It is worth noting that when the detection product is used to prepare a product for diagnosing primary biliary cholangitis, the urine protein detected by the detection product includes one or a combination of several of OPN, RAMP3, and S100A8.

[0013] OPN, RAMP3, and S100A8 are all present in urine. Choosing urine as a detection sample is non-invasive and has the advantages of being simple and easy to obtain.

[0014] Optionally, the product for diagnosing primary biliary cholangitis includes a kit.

[0015] The product for diagnosing primary biliary cholangitis provided in the present application comprises a detection agent for urine protein, wherein the urine protein comprises one or a combination of several of OPN, RAMP3, and S100A8.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] This application uses urine as the test sample, which is non-invasive and easier for patients to accept than blood collection. It is of great significance for the early non-invasive diagnosis of PBC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the embodiments of the present application, constitute a part of this application, and do not constitute a limitation on the embodiments of the present invention.

[0019] Figure 1 is a flow chart of conducting a biomarker study of PBC patients in urine in the embodiment;

[0020] Figure 2 2. ...

[0021] Figure 3 This is a volcano plot for urine protein identification in PBC patients and healthy controls in the embodiment;

[0022] Figure 4 This is a diagram of the biological processes in which differential urine proteins are mainly involved in the GO biological function analysis in the embodiment;

[0023] Figure 5This is a diagram showing the main cellular locations of differentially expressed urinary proteins analyzed by GO biological functions in the embodiment;

[0024] Figure 6 This is a chart showing the main molecular functions of differentially expressed urinary proteins in the GO biological function analysis in the embodiment;

[0025] Figure 7 This is a KEGG analysis diagram of differentially expressed proteins in the embodiment;

[0026] Figure 8 1 is a correlation analysis diagram of differentially expressed proteins in the embodiment;

[0027] Figure 9 1 is a graph showing the concentration of OPN protein in urine detected by ELISA in the embodiment, wherein A is the OPN standard curve, and B is a schematic diagram showing the expression of OPN protein in urine of a normal group and PBC patients;

[0028] Figure 10 : is a graph of RAMP3 protein concentration in urine detected by ELISA experiment in the embodiment, wherein A is a RAMP3 standard curve, and B is a schematic diagram of RAMP3 protein expression in urine of a normal group and PBC patients;

[0029] Figure 11 1 is a graph showing the concentration of S100A8 protein in urine detected by ELISA in the embodiment, wherein A is the S100A8 standard curve, and B is a schematic diagram showing the expression of S100A8 protein in urine of a normal group and PBC patients. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] It should be noted that, in the absence of any conflict, the embodiments and features of the embodiments of the present invention may be combined with each other. It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to in conjunction with each other.

[0032] Urinary proteomics research on biomarkers primarily involves two phases: 1) Discovery: Sample preparation precedes liquid chromatography-tandem mass spectrometry (LC-MS), where data analysis identifies potential target proteins. 2) Validation: Enzyme-linked immunosorbent assay (ELISA) is a commonly used validation method. LC-MS utilizes chromatographic separation of proteins of varying molecular weight based on their retention times on a liquid chromatography column, separating peptide ion fragments by mass-to-charge ratio to generate spectra. Statistical methods are then used to identify differentially expressed proteins. Bioinformatics analysis is then used to select target proteins that significantly impact signaling pathways, molecular functions, and biological processes, and validation is performed using ELISA.

[0033] In this example, urine samples were collected from PBC patients and healthy subjects, and urine proteomics technology was used to analyze the differences in urine proteomics between PBC patients and healthy subjects, thereby determining urine biomarkers related to disease diagnosis in PBC patients.

[0034] 1. Research subjects

[0035] 1.1 This example included 30 patients who were hospitalized or treated in the outpatient clinic of West China Hospital of Sichuan University from January 2022 to December 2022, and met the diagnostic criteria for PBC, including 9 patients diagnosed by liver puncture biopsy; 20 healthy controls were included through the physical examination center during the same period.

[0036] 1.2 Diagnostic criteria

[0037] ① Biochemical evidence of cholestasis with elevated ALP and GGT, and imaging studies excluding extrahepatic or intrahepatic bile duct obstruction; ② Positive serum AMA / AMA-M2; ③ Histopathological evidence of chronic non-suppurative destructive cholangitis and interlobular bile duct destruction. A diagnosis is made if two of these criteria are met.

[0038] 1.3 Inclusion and Exclusion Criteria

[0039] Inclusion criteria: ① meet the diagnostic criteria for PBC; ② liver biopsy histology is consistent with the pathological characteristics of PBC; ③ general information, laboratory data, and pathological data are complete.

[0040] Exclusion criteria: ① Patients with autoimmune hepatitis, primary sclerosing cholangitis, or other non-immune liver damage, such as drug-induced hepatitis, alcoholic liver disease, and inherited metabolic liver disease; ② Patients with secondary liver damage due to failure of other important organs; or patients with severe renal insufficiency or massive proteinuria.

[0041] 2. Sample collection

[0042] 2.1 Clinical data collection

[0043] The patients' basic information (such as name, gender, age, etc.) and complete clinical and laboratory data were collected through the HIS system of West China Hospital of Sichuan University, such as medical history, family history, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), serum glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBiL), albumin (ALB), immunoglobulin M (IgM), immunoglobulin G (IgG), anti-mitochondrial antibodies (AMA), anti-gp210 antibodies, anti-sp100 antibodies, liver biopsy pathology and other examination results.

[0044] 2.2 Clinical sample collection and preservation

[0045] All eligible patients and healthy subjects had clean, interrupted urine collected from a urine cup on the first day of hospitalization or the day of their visit. The urine was then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected into clean EP tubes, labeled, and stored in a -80°C freezer. Prior to testing, the labeled urine sample was removed, thawed, and thoroughly mixed.

[0046] 2.3 Urine sample pretreatment (protein extraction, concentration determination and drying)

[0047] Urine samples from PBC patients with liver biopsy (nine cases) were selected as the protein profiling experimental group, and urine samples from healthy individuals with complete clinical data (seven cases) were selected as the protein profiling control group. First, urine was pretreated to extract urine protein. The specific steps were as follows:

[0048] ① Rapidly rewarm the pre-packaged urine sample at 37°C and centrifuge at 800g for 5 minutes;

[0049] ② Take 100 μl of sample and add 8 M urea to make up to 500 μl;

[0050] ③ Add 100 μl of 100 mM DTT solution, mix well, and incubate at 37°C for 4 h;

[0051] ④ Add 30 μl of 1 M IAM solution, mix well, and incubate at 37°C for 30 min;

[0052] ⑤ Add 400 μl of 30k FASP replacement solution, centrifuge and collect the filtrate, repeat three times;

[0053] ⑥ Add 100 μl of urea to the ultrafiltration tube, centrifuge and collect the filtrate, repeat twice;

[0054] ⑦ Add 50mM ammonium bicarbonate, centrifuge and collect the filtrate;

[0055] ⑧Add 2μg of Trypsin to each sample and enzymatically hydrolyze at 37℃ for 16h.

[0056] ⑨ Centrifuge and collect the filtrate, then use the kit to determine the peptide concentration.

[0057] ⑩ Use C18 desalting columns to dry and purify peptide fragments.

[0058] 3. Technical route

[0059] 3.1 Baseline data of subjects included in urine proteomics

[0060] The baseline data of the experimental and control groups were analyzed by urine proteomics and SPSS statistics.

[0061] Among the PBC patients, there was 1 male patient and 8 female patients with an average age of (51.33±11.39) years. The laboratory test indicators were ALP (361.67±240.00) U / L, GGT (460.22±354.11) U / L, TB (15.34±4.91) umol / L, DB (7.46±3.60) umol / L, TBA (26.12±24.48) umol / L, ALT (77.33±62.37) U / L, AST (90.33±57.98) U / L, ALB (46.74±15.77) g / L, Cr (58.89±10.42) umol / L, IgM (4973.33±3395.99) mg / L, and IgG (18.11±6.83) g / L.

[0062] In the normal control group, there were 2 males and 5 females with an average age of (34.71±6.85) years. The laboratory test indicators were ALP (58.57±9.71) U / L, GGT (19.70±10.08) U / L, TB (9.67±4.63) μmol / L, DB (5.24±2.36) μmol / L, TBA (2.77±1.85) μmol / L, ALT (19.13±372.21) U / L, AST (23.00±6.85) U / L,

[0063] ALB (48.31±6.05) g / L, Cr (56.40±7.43) μmol / L, IgM (807.14±48.21) mg / L, and IgG (9.39±1.25) g / L were not statistically significant between the two groups in terms of gender, age, TBA, ALB, and Cr (P ≥ 0.05). However, ALP, GGT, TB, DB, ALT, AST, IgM, and IgG were significantly different between the two groups (P < 0.05). (See Table 1.)

[0064] Table 1: Baseline information

[0065]

[0066]

[0067] 3.2 Urine protein spectrum and data processing

[0068] Urinary protein was extracted from the experimental and control groups and analyzed by protein profiling. Spectronaut version 14.8 database search software was used to obtain qualitative and quantitative protein data. Missing values ​​were counted for each sample and protein. Data were median-normalized and then log2-normalized. Data with missing values ​​greater than 50% were removed and filled. The coefficient of variation was calculated, and data with large fluctuations in the coefficient of variation (>30%) were removed. Finally, standardized urine protein profiling data were obtained for subsequent statistical analysis.

[0069] The steps of high performance liquid chromatography-electrospray ion trap mass spectrometry analysis are as follows:

[0070] ① The sample was loaded onto a packed column (2 cm * 150 μm) and separated using a capillary column (15 cm * 150 μm), both filled with C18 reversed-phase particles (1.9 μm).

[0071] ②The dried peptide was re-dissolved with mobile phase A;

[0072] ③ Then elute and separate with mobile phase B gradient solution at a flow rate of 600nL / min for 120min;

[0073] ④ The liquid phase separation gradient is as follows: 10 min 6% B → 15 min 9-14% B → 50 min 14-30% B → 30 min 30-40% B → 3 min 40-95% B → 7 min 95% B → 1 min 95-6% B → 4 min 6% B;

[0074] ⑤ After ionization through the interface, the ion fragments are separated according to the mass-to-charge ratio by the Orbitrap mass analyzer of the tandem mass spectrometer, and the mass spectrum is obtained by the detector.

[0075] 3.3 Bioinformatics analysis of differentially expressed proteins

[0076] Bioinformatics methods were used to perform principal component analysis on the protein matrix data. The Panther database was used to identify and analyze urine proteins. Principal component analysis and volcano plots were performed on the resulting proteins using DAVID Bioinformatics Resources to identify statistically significantly upregulated and downregulated proteins in the urine of PBC patients.

[0077] This example found that the intra-group repeatability and the inter-group variability were good. Figure 2 As shown. The Limma software package was used to perform hypothesis testing on the data between the two groups, and the BH method was used to correct the p-value. The corrected P-value is also called FDR. The log2 logarithm of the quantitative ratio FC (Fold Change) between the two groups was calculated as the horizontal axis, and the negative logarithm of FDR -log10 was used as the vertical axis to obtain the volcano plot, as shown in the figure below. Figure 3 shown. Figure 3 In the figure, UP (red) represents upregulation, NoChange (grey) represents no significant change, and DOWN (blue) represents downregulation.

[0078] In this example, a fold change of 2 and a corrected p-value of <0.01 were selected as screening conditions. A total of 194 statistically significant and differentially expressed urine proteins were obtained, of which 109 were significantly upregulated and 85 were significantly downregulated. The top 50 upregulated and downregulated proteins are listed in Tables 2 and 3, respectively.

[0079] Table 2: Upregulated differentially expressed proteins (top 50)

[0080]

[0081]

[0082] Table 3: Down-regulated differentially expressed proteins (top 50)

[0083]

[0084]

[0085] 3.4 GO analysis of the top 50 significantly upregulated differentially expressed proteins

[0086] Gene Ontology analysis is used to describe molecular functions, cellular locations, and biological processes involved.

[0087] In this example, GO analysis was performed on the top 50 differentially upregulated genes, mainly including three independent ontologies: cellular component (CC), molecular function (MF), and biological process (BP).

[0088] GO biological function analysis found that the biological processes (BP) in which the significant differential urinary proteins were mainly involved included: receptor-mediated endocytosis, immune response, complement activation, classical pathway, regulation of immune response, Fc-gamma receptor signaling pathway involved in phagocytosis, innate immune response, Fc-epsilon receptor signaling pathway, complement activation, signal transduction, retinahomeostasis, B cell receptor signaling pathway, protein phosphorylation, phagocytosis, recognition, phagocytosis, engulfment, inflammatory response, such as Figure 4 shown.

[0089] GO biological function analysis found that the main cellular locations (CC) of significant differential urinary proteins were: extracellular exosome, plasma membrane, extracellular region, extracellular space, membrane, blood microparticle, cytosol, nucleus, cell surface, integral component of plasma membrane, Golgi membrane, external side of plasma membrane, cytoplasm, Golgi apparatus, endoplasmic reticulum, such as Figure 5 shown.

[0090] GO biological function analysis found that the major molecular functions (MF) of the significant differential urinary proteins were as follows: antigen binding, ATP binding, serine-type endopeptidase activity, ubiquitin protein ligase binding, identical protein binding, endopeptidase inhibitor activity, cysteine-type endopeptidase inhibitor activity, zinc ion binding, signaling receptor binding, serine-type endopeptidase inhibitor activity, protein serine / threonine kinase activity, kinase activity, drug binding, calcium ion binding, and amyloid-beta binding. Figure 6 shown.

[0091] 3.5 Pathway metabolic pathway annotation of the top 50 significantly upregulated differentially expressed proteins

[0092] Pathway analysis of proteins in organisms can clearly elucidate their functional mechanisms and identify the metabolic and signaling pathways that play important roles in physiological activities. KEGG Pathway can be used to screen for immune-related proteins, perform cluster analysis using Cluster 3.0 software, and generate enrichment maps using JAVA Treeview software.

[0093] In this example, the top 50 significantly upregulated differentially expressed proteins in urine were subjected to KEGG analysis and enrichment, and the more important pathway results are described as follows: Regulation of actin cytoskeleton, Focal adhesion, Signaling pathways regulating pluripotency of stem cells Focal adhesion, Wnt signaling pathway, Rap1 signaling pathway, PI3K-Akt signaling pathway, ECM-receptor interaction, VEGF signaling pathway, Notch signaling pathway, Hedgehog signaling pathway, ErbB signaling pathway, Metabolic pathway, Leukocyte transendothelial migration, Toll-like receptor signaling pathway (toll-like receptor signaling pathway), Thyroid hormone signaling pathway (thyroid hormone signaling pathway), Chemokine signaling pathway (chemokine signaling pathway), such as Figure 7 shown.

[0094] Previous studies have shown that immune response is closely related to the occurrence and development of PBC. Figure 6The enrichment number in each group was at the top, and the proteins related to immune response were screened at the same time, and 10 proteins of interest were selected for the next correlation analysis. These 10 proteins are: osteopontin (OPN), also known as secreted phosphoprotein 1 (SPP1), receptor activity-modifying protein 3 (RAMP3), CD44, activin receptor type 1B protein (ACVR1B), chemokine CXCL12 protein, CD74, Wnt1-induced signaling pathway protein (CCN4), calcium binding protein (S100A8), lysosomal associated membrane protein 3 (LAMP3), and phosphoinositide-3-kinase catalytic subunit delta peptide (PIK3Cd).

[0095] 3.6 Correlation analysis of differentially expressed proteins

[0096] To further study the correlation between urine protein and clinical indicators, this example again performed Pearson correlation analysis on osteopontin (OPN), receptor activity-modifying protein 3 (RAMP3), CD44, activin receptor type 1B protein (ACVR1B), chemokine CXCL12 protein, CD74, Wnt1-induced signaling pathway protein (CCN4), calcium-binding protein (S100A8), lysosomal associated membrane protein 3 (LAMP3), and phosphoinositide-3-kinase catalytic subunit delta peptide (PIK3Cd). The analysis results showed that 3 of the proteins were most significantly correlated with clinical parameters such as ALT, AST, ALP, GGT, TBA, IgM, IgG, and AMA. Figure 8 The three proteins identified were osteopontin (OPN), receptor activity-modifying protein 3 (RAMP3), and calcium-binding protein (S100A8). This suggests that these three proteins may have potential as biomarkers for the clinical diagnosis of PBC.

[0097] 3.7 ELSA Experimental Results and Analysis

[0098] (1) Further sample expansion for urine protein ELISA validation. Urine samples were collected from 30 PBC patients and 20 normal controls. Comparison of general information of the validation cohort subjects: There was no statistically significant difference in general information (age, gender) between the PBC group and the normal control group (see Table 4).

[0099] Table 4: Urine samples used in ELISA experiments

[0100]

[0101] (2) ELISA validation: ELISA kits were used to detect the protein expression of OPN, RAMP3, and S100A8 in urine protein of PBC patients and healthy controls. SPSS 23.0 software was used for statistical analysis. Data were tested for normality. All continuous variables with normal distribution were expressed as mean ± standard deviation, those with skewed distribution were expressed as median and interquartile range, and categorical variables were described as frequency or percentage. Student's t-test or Mann-Whitney U test was used for comparison between groups. Pearson correlation test was used. Graphs were prepared using GraphPad prism8 software.

[0102] The results suggest that: OPN in PBC patients (such as Figure 9 As shown), RAMP3 (as Figure 10 as shown) and S100A8 (as Figure 11 The relative expression level of WT and WT cells was significantly higher than that of the normal control group (p < 0.01), which was basically consistent with the expression trend of mass spectrometry results.

[0103] The ELISA test steps are as follows:

[0104] (1) Dilute the standard sample according to the kit instructions, set up standard wells, blank wells and sample wells respectively, and do not add sample or enzyme-labeled reagent to the white wells.

[0105] (2) The detection process of S100A8 and OPN is as follows:

[0106] a. Add 100 μl of standard or sample to each well and incubate at 37°C for 90 minutes.

[0107] b. Pour off the liquid in the well, add 100 μl of biotinylated antibody / antigen working solution, and incubate at 37°C for 60 minutes.

[0108] c. Shake off the solution in the wells, add washing solution to wash, let it stand for 30 seconds and then discard the suspension. Repeat this three times and pat dry with absorbent paper.

[0109] d. Add 100 μl of enzyme conjugate working solution, incubate at 37°C for 30 minutes, and then wash five times.

[0110] e. Add 90 μl of substrate solution and incubate at 37°C for about 15 minutes.

[0111] f. Add 50 μl of stop solution and immediately measure the OD value at a wavelength of 450 nm. Draw a standard curve, calculate the target protein content in the sample based on the sample OD value, and analyze the data.

[0112] (3) The RAMP3 testing process is as follows:

[0113] a. Add 50 μl of standard or sample to each well and incubate at 37°C for 30 minutes.

[0114] b. Wash five times, add 50 μl of enzyme-labeled reagent, and incubate at 37°C for 30 minutes.

[0115] c. Wash five times, add 50 μl each of color developing solution A and B, and develop color at 37°C for 10 minutes.

[0116] d. Add 50 μl of stop solution and immediately measure the OD value at a wavelength of 450 nm. Draw a standard curve, calculate the target protein content in the sample based on the sample OD value, and analyze the data.

[0117] This study utilizes proteomics technology to investigate biomarkers in urine for PBC patients, a method more readily accepted by patients compared to blood. This study eliminates high-abundance proteins that interfere with detection and maximizes the enrichment of low-abundance proteins, providing comprehensive urine proteome information. Finally, combining proteomic analysis with relevant literature research, three new urine protein markers were identified, which are of great significance for the early, noninvasive diagnosis of PBC patients.

[0118] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of this application in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a urine protein detection product in the preparation of a product for diagnosing primary biliary cholangitis, characterized in that: The urine protein includes one or a combination of RAMP3 and S100A8.

2. The use according to claim 1, characterized in that: The product for diagnosing primary biliary cholangitis includes a kit.

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