Use of serpinb10 protein in preparation of product for differential diagnosis of eosinophilic esophagitis and reflux esophagitis

Abstract

The application provides use of SERPINB10 protein as a diagnostic marker in preparation of a diagnostic kit for differentiating eosinophilic esophagitis from reflux esophagitis. The application also provides use of SERPINB10 protein in preparation of a product for differential diagnosis of eosinophilic esophagitis and reflux esophagitis. The application also provides use of a reagent for detecting SERPINB10 protein in preparation of a diagnostic kit for differentiating eosinophilic esophagitis from reflux esophagitis. When the integral optical density of SERPINB10 protein immunohistochemical detection of an esophageal tissue sample of a subject is greater than or equal to 41666, it shows that the patient has eosinophilic esophagitis, and otherwise, the patient has reflux esophagitis. The application establishes an objective, accurate and low-missed-diagnosis-rate EoE diagnosis and differential diagnosis system.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to a SERPINB10 protein, in particular, application of the SERPINB10 protein as a novel specific biomarker in preparation of a differential diagnosis product for eosinophilic esophagitis and reflux esophagitis. BACKGROUND

[0002] Eosinophilic esophagitis (EoE) is a chronic allergen-induced type 2 immune-mediated esophageal disease characterized by symptoms of esophageal dysfunction and esophageal infiltration dominated by eosinophils. Its diagnosis needs to meet the following standards: symptoms of esophageal dysfunction; esophageal biopsy finds at least 15 eosinophils per high-power field; and assessment and exclusion of non-eosinophilic esophagitis conditions that cause or potentially contribute to esophageal eosinophilia.

[0003] Reflux esophagitis (RE) is an important subtype of gastroesophageal reflux disease (GERD), characterized by endoscopic visible esophageal mucosal erosion or ulcer and other mucosal damage, mainly caused by gastric content reflux. It is usually graded and diagnosed by endoscopic Los Angeles classification, with Los Angeles grade B and above as the standard for erosive esophagitis. The typical symptoms of RE include heartburn and reflux. Its diagnosis needs to integrate multiple evidence, mainly including clinical symptom assessment, response to trial treatment of proton pump inhibitor (PPI), endoscopic observation of mucosal damage, and objective evidence provided by esophageal reflux monitoring. However, symptoms such as heartburn and reflux are not unique to RE and can also be seen in other diseases such as eosinophilic esophagitis. RE can be complicated with esophageal stenosis, which is often considered as a common cause of dysphagia, while EoE, as a rare disease, is often overlooked in the diagnosis of the cause of dysphagia.

[0004] The differential diagnosis of EoE and RE faces many challenges. There is significant overlap in clinical symptoms between the two diseases, both of which can present as dysphagia, retrosternal pain, reflux, and heartburn, making it difficult to accurately distinguish between the two based on clinical manifestations alone. Endoscopic findings such as erosion and stenosis can occur in both RE and EoE, further increasing the difficulty of differentiation. Histological examination also shows cross-over phenomena, as RE can also cause a small amount of esophageal eosinophilic infiltration. In addition, due to the fragility of the esophageal mucosa, the actual number of clinical biopsies is often lower than the international standard (≥5 pieces), and eosinophils often show aggregated distribution, with a significantly increased false negative rate of pathological sampling errors.

[0005] In recent years, the rapid development of mass spectrometry (MS) provides a new way to break through the above bottleneck. Based on mass spectrometry, proteomics technology can realize the systematic and unbiased analysis of protein expression abundance, post-translational modification and protein interaction under different disease conditions by directly identifying and quantifying amino acid sequences in the sample. With the introduction of automation and standardization technology in biological sample pretreatment, various sample types including formalin fixed paraffin embedded (FFPE) samples can be used for mass spectrometry analysis.

[0006] At the same time, the data acquisition strategy used in mass spectrometry technology is also gradually innovating. The data-independent acquisition (DIA) acquisition mode divides the mass charge ratio full range into continuous windows, and performs unbiased fragmentation on all peptide segments in each window, avoiding the loss of low-abundance peptide segments caused by ion intensity threshold screening, and significantly improving the coverage of the proteome. The DIA mode overcomes the bias and randomness defects of the traditional data-dependent acquisition (DDA) mode, and has great potential for clinical application transformation, and has been widely used in the exploration of biomarkers for various diseases such as autoimmune diseases and tumors.

[0007] Serine protease inhibitors (Serpins) are a superfamily of proteins with a conserved tertiary structure, and SERPINB10 protein is a member of the family.

[0008] In summary, it is urgent to develop an EoE pathological diagnosis strategy with strong specificity, high sensitivity, and not affected by the number of esophageal biopsy. This study explores the value of SERPINB10 protein in the differential diagnosis of EoE and RE, which is expected to provide a new direction for solving this clinical problem. SUMMARY

[0009] In view of the above technical problems in the prior art, the application provides application of SERPINB10 protein in preparation of a product for differential diagnosis of eosinophilic esophagitis and reflux esophagitis. The application of SERPINB10 protein in preparation of a product for differential diagnosis of eosinophilic esophagitis and reflux esophagitis solves the technical problems of the prior art that the diagnosis method has insufficient specificity in distinguishing the two diseases, depends on multiple tissue biopsies, and is easily affected by subjective factors.

[0010] The application provides use of a SERPINB10 protein as a diagnostic marker in preparation of a diagnostic kit for distinguishing eosinophilic esophagitis from reflux esophagitis.

[0011] Further, the kit contains Anti-SERPINB10 antibody.

[0012] The application also provides use of the SERPINB10 protein in preparation of a product for differential diagnosis of eosinophilic esophagitis and reflux esophagitis.

[0013] The SERPINB10 is a member of the serine protease inhibitor family; the eosinophilic esophagitis has the symptoms of esophageal dysfunction, esophageal eosinophil infiltration ≥ 15 / HPF, and exclusion of other non-eosinophilic esophagitis diseases causing esophageal eosinophilia; the reflux esophagitis is a subtype of gastroesophageal reflux disease, and has the prominent features of mucosal damage such as esophageal mucosal erosion or ulcer caused by reflux, and the Los Angeles B grade and above is the standard of erosive esophagitis.

[0014] Specifically, the diagnostic method of the application comprises the following steps: obtaining an esophageal tissue sample of a subject; detecting the expression level of the SERPINB10 protein in the sample; when the IOD value of the SERPINB10 protein immunohistochemical detection of the esophageal tissue is ≥ 41666, the eosinophilic esophagitis is determined to be positive (sensitivity 92.3%, specificity 61.29%), and vice versa, the reflux esophagitis is determined to be positive, wherein the detection uses Anti-SERPINB10 antibody and Image-Pro Plus software.

[0015] The application also provides use of a reagent for detecting the SERPINB10 protein in preparation of a diagnostic kit for distinguishing eosinophilic esophagitis from reflux esophagitis.

[0016] Further, the reagent for detecting the SERPINB10 protein contains Anti-SERPINB10 antibody.

[0017] Further, when the integral optical density of the SERPINB10 protein immunohistochemical detection of the esophageal tissue sample of the subject is ≥ 41666, the eosinophilic esophagitis is determined to be positive, and vice versa, the reflux esophagitis is determined to be positive.

[0018] The application also provides an in vitro diagnosis method of eosinophilic esophagitis, comprising the following steps: obtaining an esophageal tissue sample of a subject; detecting the expression level of SERPINB10 protein in the sample; when the integrated optical density (IOD value) of SERPINB10 protein immunohistochemical detection of the esophageal tissue sample of the subject is greater than or equal to 41666, it is determined that the subject is positive for eosinophilic esophagitis, otherwise, the subject is positive for reflux esophagitis.

[0019] Further, the sensitivity of the diagnosis method using SERPINB10 protein as a specific biomarker is 92.31%, and the specificity is 61.29%.

[0020] The application also provides a diagnosis system for differentiating eosinophilic esophagitis from reflux esophagitis for non-diagnostic and therapeutic purposes, comprising:

[0021] 1) a data processing module, which obtains an esophageal tissue sample, detects the expression level of SERPINB10 protein in the esophageal tissue, and then obtains the integrated optical density value (IOD) of SERPINB10 protein in the esophageal tissue by calculation;

[0022] 2) a sample evaluation module, which receives the integrated optical density value of SERPINB10 protein transmitted by the data processing module, and sets the threshold value of the integrated optical density value of SERPINB10 protein to 41666; when the integrated optical density value of SERPINB10 protein is greater than or equal to 41666, it is displayed that the patient has eosinophilic esophagitis, otherwise, the patient has reflux esophagitis.

[0023] The application preliminarily explores by DIA mass spectrometry scanning technology, and verifies by immunohistochemical staining, attempts to explore the tissue biomarker that can be used for auxiliary diagnosis of eosinophilic esophagitis, and finally determines the important value of SERPINB10 in the differential diagnosis of eosinophilic esophagitis and reflux esophagitis.

[0024] The application first finds that the expression level of SERPINB10 protein in the esophageal tissue of an EoE patient is significantly up-regulated compared with the RE group and the non-esophagitis control group, and is widely expressed in the nuclei and cytoplasm of esophageal epithelial cells and eosinophils. The expression level of SERPINB10 in the esophageal tissue of EoE is independent of the number of eosinophilic infiltration, and can be used as a relatively independent auxiliary diagnostic standard to enhance the accuracy of the existing pathological diagnostic standard, effectively avoiding the sampling error and false negative problem caused by the patchy distribution of eosinophils, and making up for the limitations of the existing tissue biopsy sampling. The above results show that SERPINB10 protein has great application prospect in the preparation of differential diagnosis products for eosinophilic esophagitis and reflux esophagitis.

[0025] Based on the above characteristics, SERPINB10 provides a new molecular basis and solution for establishing an objective, accurate, and low-missed diagnosis and differential diagnosis system for EoE.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] 1. The expression level of SERPINB10 protein in esophageal tissue is quantified by integral optical density value (IOD), which is significantly more objective than traditional manual interpretation. When the IOD cutoff value of SERPINB10 is 41666, the diagnostic sensitivity reaches 92.31%, which is high.

[0028] 2. The expression level of SERPINB10 protein in the esophageal tissue of the EoE group is significantly higher than that of the reflux esophagitis group and the healthy control group, and there is no significant difference between the reflux esophagitis group and the healthy control group, which indicates that the high expression of specificity has diagnostic value for differentiating EoE from RE.

[0029] 3. The expression level of SERPINB10 in the esophageal tissue of EoE is independent of the number of esophageal eosinophilic granulocyte infiltration, so it can be used as a relatively independent auxiliary diagnostic standard, which can to some extent avoid the missed diagnosis of EoE due to not taking the esophageal eosinophilic granulocyte infiltration site, reduce the trauma of multiple sampling, and reduce the missed diagnosis rate. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The proteinomic characteristics of EoE tissue and the composition of the biomarker exploration research team are shown.

[0031] Figure 2 is a statistical graph of esophageal tissue protein identification of EoE group and control group subjects; Figure 2A The number of esophageal tissue protein identification of EoE group and control group subjects and the proportion of sample protein missing value; Figure 2B The heat map of esophageal tissue expressed protein of EoE group and control group subjects; Figure 2C The principal component analysis graph of esophageal tissue expressed protein of EoE group and control group subjects.

[0032] Figure 3 is a statistical graph of esophageal expressed protein difference analysis of EoE group and control group subjects; Figure 3A The distribution graph of esophageal protein raw intensity value of EoE group and control group subjects in the exploration team; Figure 3B The volcano plot of esophageal differential expressed protein of EoE group and control group subjects in the exploration team.

[0033] Figure 4 is a result graph of differential expressed protein gene GO / KEGG enrichment analysis of EoE group and control group subjects.Figure 4A Bubble plot of GO enrichment analysis of up-regulated protein genes in esophageal tissue of EoE group in cohort E; Figure 4B Bubble plot of GO enrichment analysis of down-regulated protein genes in esophageal tissue of EoE group in cohort E; Figure 4C Bubble plot of KEGG enrichment analysis of up-regulated protein genes in esophageal tissue of EoE group in cohort E; Figure 4D Bubble plot of KEGG enrichment analysis of down-regulated protein genes in esophageal tissue of EoE group in cohort E.

[0034] Figure 5 Microscopic view of immunohistochemical staining of ALOX15 and SERPINB10 in esophageal tissue of an EoE group subject; A-C. H&E staining, ALOX15 immunohistochemical staining and SERPINB10 immunohistochemical staining (x200) of the same EoE group subject in sequence.

[0035] Figure 6 Statistical chart of semi-quantitative difference analysis of ALOX15 and SERPINB10 expression levels in EoE group, RE group and non-esophagitis group; A. Scatter plot of ALOX15 expression levels in EoE group, RE group and non-esophagitis group obtained by IHC semi-quantitative verification; B. Scatter plot of SERPINB10 expression levels in EoE group, RE group and non-esophagitis group obtained by IHC semi-quantitative verification. **P<0.01, ***P<0.001, ****P<0.0001, ns no significant difference.

[0036] Figure 7 Statistical chart of diagnostic performance analysis of ALOX15 and SERPINB10 expression levels for EoE; A. EoE diagnostic model based on ALOX15 and SERPINB10 protein expression levels; B. Scatter plot of ALOX15 expression levels in histologically confirmed EoE cases and suspected EoE cases of esophageal tissue obtained by IHC semi-quantitative verification; C. Scatter plot of SERPINB10 expression levels in histologically confirmed EoE cases and suspected EoE cases of esophageal tissue obtained by IHC semi-quantitative verification. **P<0.01, ****P<0.0001, ns no significant difference. DETAILED DESCRIPTION

[0037] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents, instruments, equipment, etc. mentioned in the present application can be commercially available.

[0038] Example 1

[0039] 1. Steps and methods

[0040] (1) Clinical sample collection:

[0041] In this study, 19 subjects in the EoE group were selected, and 15 subjects who underwent esophageal diverticulum surgery in the Department of Thoracic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, were added as a control group to form an exploration cohort. FFPE samples of biopsy or surgical tissues for pathological diagnosis were collected, and protein identification and screening were performed by DIA mass spectrometry scanning technology.

[0042] Another 39 subjects in the EoE group, 31 subjects in the RE group and 31 subjects in the non-esophagitis group were selected to form a verification cohort. FFPE samples of biopsy for pathological diagnosis were collected, and verification was performed by immunohistochemical staining technology.

[0043] The exclusion criteria are: (1) age <14 or >75 years old; (2) pathological indication of esophageal epithelial dysplasia; (3) use of drugs that may cause eosinophilia within 3 months before gastroscopy; (4) combined with severe past or present parasitic, fungal infection and severe stress state.

[0044] The above research subjects have been approved by the ethics review committee and meet the requirements of the national ethics review method.

[0045] The composition of the cohort is shown in the accompanying Figure 1 .

[0046] (2) DIA mode mass spectrometry analysis: FFPE samples of esophageal biopsy or surgical tissues of subjects in the exploration cohort were analyzed by DIA mass spectrometry scanning (technical support was provided by the mass spectrometry platform of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences),

[0047] The steps are briefly described as follows:

[0048] 1) FFPE sample pretreatment: Take the FFPE samples of EoE group subjects and esophageal diverticulum subjects in the exploration queue and place them in a -20°C refrigerator for 2 hours. Use a paraffin microtome to cut 15-20 paraffin sections with a thickness of 5 pm and collect them in a 1.5 ml EP tube. Immerse the paraffin sections in a xylene solution for dewaxing treatment, with a duration of 5 minutes. Centrifuge at room temperature at a speed of 12000g for 5 minutes, and discard the upper liquid. Add 1 ml of 100% anhydrous ethanol, incubate for 1 minute, centrifuge at room temperature at a speed of 12000g for 5 minutes, and discard the upper solution. Add 1 ml of 70% anhydrous ethanol, incubate for 1 minute, centrifuge at room temperature at a speed of 12000g for 5 minutes, discard the upper solution, and place it in a clean and dry environment for natural air drying. Add pre-cooled tissue lysis solution (containing 4% SDS, 1 mM DTT, 1x protease inhibitor, 100 mM Tris-HCl buffer, pH 8.0) to the previously treated sample, mix thoroughly, and incubate for 5 minutes. Centrifuge at a speed of 12000g for 10 minutes to remove the precipitate, and collect the supernatant. Use the acetone low-temperature precipitation method to enrich proteins, add pre-cooled acetone to a final concentration of 80%, and precipitate at -20°C for 2 hours. Centrifuge at a speed of 15000g for 15 minutes to collect the precipitate. Add NH4HCO3 to dissolve the protein precipitate, and use the BCA quantitative method to detect the protein concentration. After reducing treatment of the precipitated protein with DTT at 56°C water bath for 30 minutes, add iodoacetamide alkylate under light-proof conditions for 30 minutes. Add trypsin solution according to an enzyme-substrate ratio of 1:100, and incubate at 37°C for 16 hours to complete the enzyme digestion process. After desalting treatment, the peptide sample is vacuum dried by SpeedVac vacuum concentrator, and stored in a -80°C refrigerator before analysis.

[0049] 2) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) analysis: The sample to be tested was fully suspended in an acidic buffer system (0.1% formic acid), and after high-speed centrifugation, the clear supernatant was taken through the automatic sampler equipped on the Vanquish Neo liquid phase system, and was loaded onto a C18 capillary chromatographic column (inner diameter 75 μm, column length 25 cm). A constant flow mode (flow rate 300 nL / min) was set, and a multi-order linear gradient was used for separation and elution. The eluted peptide segments were ionized by a nano-ESI ion source on an Orbitrap Exploris 480 mass spectrometer, and MS data was acquired in DIA scanning mode. The specific setting parameters of the DIA method are as follows: the primary mass spectrum was detected at a resolution of 120,000, the mass scan range of the parent ion was 350-1300 m / z, the AGC target was set to Custom, and the maximum injection time was Custom. The secondary fragment ions were detected at a resolution of 30 000, the AGC target was Custom, and the HCD was set to 28%.

[0050] 3) Mass spectrometry data processing and bioinformatics analysis:

[0051] Spectronaut 17.0 was used to search the DIA data generated by mass spectrometry based on the Uniprot human protein database, with a protein level FDR less than 1%, Trypsin / P as the enzyme cutting method, a maximum of 2 missed cutting sites for trypsin, cysteine urea methylation as the fixed modification, and oxidation and protein acetylation as the variable modification. Finally, the protein identification and quantification results were compared between the two groups, and proteins with a FC greater than 2 and an FDR less than 0.05 were defined as differentially expressed proteins. The up-regulated and down-regulated differential proteins in the EoE group were subjected to visual GO (Gene Ontology) functional annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis.

[0052] (3) Immunohistochemical staining: The esophageal FFPE samples of each group of subjects in the verification cohort were placed in a refrigerator at -20°C for about 2 hours, and a paraffin tissue section with a thickness of 4 pm was cut using a paraffin microtome; the cut paraffin tissue section was fixed on a glass slide and placed in an oven at 60°C for about 1 hour; the paraffin section was sequentially immersed in xylene I and xylene II for 15 minutes each; the paraffin section was sequentially placed in anhydrous ethanol I, anhydrous ethanol II for 5 minutes each; the paraffin section was sequentially placed in 85% ethanol, 75% ethanol for 5 minutes each, and rinsed with distilled water for 3 times, 5 minutes each time; the section was placed in a repair box, and was completely immersed in citrate buffer (pH 6.0) in a microwave oven for antigen repair, and the buffer should not be evaporated too much during the process; after the repair was completed, the section was naturally cooled, and was washed with PBS buffer (pH 7.4) on a shaker for 3 times, 5 minutes each time; it was immersed in 3% H2O2 solution, and was treated at room temperature in the dark for 25 minutes, and was washed with PBS buffer as before for 3 times; after the section was dried, the tissue area was marked using a hydrophobic group, and 10% sheep serum was added for blocking to ensure complete coverage of the sample, and the blocking was performed at room temperature for 30 minutes; the blocking solution was gently shaken off, and the pre-prepared primary antibody diluent was added to the section, and the wet box was placed in a 4°C incubator overnight; after the section was incubated at 4°C, the primary antibody diluent was shaken off, and was washed with PBS solution (pH 7.4) for 3 times, 5 minutes each time; after the section was dried, the universal secondary antibody was added to cover the tissue, and was incubated at room temperature for 50 minutes; the secondary antibody on the surface of the section was gently shaken off, and was washed with PBS solution (pH 7.4) for 3 times, 5 minutes each time, and the prepared DAB color developing solution was added to the section, and the staining time was observed under a microscope; when obvious brownish yellow color appeared under the microscope, the color development was stopped by flushing with tap water, and the color development time was recorded; the tissue section was stained with hematoxylin for 3 minutes, the excess dye was flushed with running water, 1% hydrochloric acid alcohol was added for 10 seconds, and the section was flushed with running water and then treated with ammonia water for blue recovery; the section was sequentially placed in 75% ethanol, 85% ethanol for 6 minutes each, anhydrous ethanol I, anhydrous ethanol II for 6 minutes each, xylene I for 5 minutes for dehydration and transparency treatment, and was naturally dried, and was then sealed with neutral resin; high-power lens observation was performed on the section under an optical microscope, and since the esophageal biopsy tissue was extremely small, 3 fields of view were randomly selected for image acquisition on each section, and the tissue on each section should be as much as possible to fill the field of view, and the integrated optical density (IOD) value of each image was quantified using Image-Pro Plus software, and the average value of multiple fields of view was taken for each sample.

[0053] 2. Results

[0054] (1) Through database searching based on Uniprot human protein database, a total of 9622 proteins were identified from the DIA data generated in this study. Figure 2 shows the number of protein expression and the proportion of missing values in the esophageal tissue of the two groups of subjects (EoE group and esophageal diverticulum control group) in the exploration cohort. Figure 2A The number of protein identification and the proportion of missing values in the esophageal tissue of the EoE group and the control group; Figure 2B The heat map of protein expression in the esophageal tissue of the EoE group and the control group; Figure 2C The principal component analysis (PCA) plot of protein expression in the esophageal tissue of the EoE group and the control group.

[0055] At the same time, the correlation analysis between samples showed that the intra-group correlation of protein identification results in the esophageal tissue of the two groups of subjects was good, and the principal component analysis (PCA) visualization results showed that there were differences between the protein expression patterns of the esophageal tissue of the EoE group and the control group (15 esophageal diverticulum subjects as the control group) in the exploration cohort.

[0056] (2) The protein identification results of the esophageal tissue of the two groups of subjects (EoE group and esophageal diverticulum control group) in the exploration cohort were analyzed for differences. We defined FC>2 and FDR<0.05 as the standard for significant differences, and screened out a total of 2299 differentially expressed proteins between the two groups, including 1849 significantly up-regulated proteins in the EoE group and 450 significantly down-regulated proteins in the EoE group, as shown in Figure 3. Figure 3A The distribution of raw intensity values of esophageal proteins in the EoE group and the control group in the exploration cohort; Figure 3B The volcano plot of esophageal differentially expressed proteins in the EoE group and the control group in the exploration cohort. We used P-value (representing difference significance) and fold change (FC, representing difference magnitude) as the main indicators to screen candidate biomarkers from the comparison of protein groups between EOE patients and control group. The following table shows the top 20 most potential candidate biomarkers ranked in descending order of FC value.

[0057]

[0058] (3) In order to further clarify the specific functions of the above differentially expressed proteins, we performed enrichment analysis on the 1849 up-regulated protein genes and 450 down-regulated protein genes in the esophageal tissue of the EoE group in the exploration cohort, and the results are shown in Figure 4. Figure 4A Bubble chart of GO enrichment analysis of up-regulated differentially expressed protein genes in the esophageal tissue of the EoE group in the exploration cohort;Figure 4B Figure 2 is a bubble chart of GO enrichment analysis of down-regulated protein genes in esophageal tissues of subjects in the EoE group of the discovery cohort; Figure 4C Figure 3 is a bubble chart of KEGG enrichment analysis of up-regulated protein genes in esophageal tissues of subjects in the EoE group of the discovery cohort; Figure 4D Figure 4 is a bubble chart of KEGG enrichment analysis of down-regulated protein genes in esophageal tissues of subjects in the EoE group of the discovery cohort.

[0059] (4) The IOD of the same magnification under the condition of full field of view was used to compare the expression of two proteins (ALOX15 and SERPINB10, wherein ALOX15 has been confirmed to be highly expressed specifically in esophageal tissues of EoE children patients) in esophageal tissues of three groups of subjects in the validation cohort. It was observed under a microscope that SERPINB10 could be colored in the nuclei and cytoplasm of esophageal epithelial cells and eosinophils, as shown in Figure 5. Figure 5 .

[0060] (5) The semi-quantitative analysis result of protein expression confirmed that the expression level of SERPINB10 in the esophagus of subjects in the EoE group in the validation cohort was significantly higher than that in the RE group (P = 0.0007) and the non-esophagitis group (P < 0.0001), but there was no significant difference in the expression level of SERPINB10 in the esophagus between the RE group and the non-esophagitis group (P = 0.836), as shown in Figure 6. Figure 6 .

[0061] (6) The present application also constructed an EoE diagnosis model based on the expression level of SERPINB10 protein, and drew a ROC curve to preliminarily evaluate the diagnosis performance of the model for identifying EoE and non-EoE diseases (the combination of the RE group and the non-esophagitis group), and the relative expression level of the protein corresponding to the maximum Youden index was taken as the optimal cutoff value. When the IOD value of SERPINB10 was 41666, the sensitivity was 92.31% and the specificity was 61.29%.

[0062] The above evaluation used the validation cohort, which included a total of 101 people. The validation cohort was divided into 39 EOE and 62 non-EOE groups, and the non-EOE group was the combination of the RE group and the non-esophagitis group. When the maximum Youden index was used in the model, 36 of the 39 EOE patients met the model positive, and 3 were false negatives; 38 of the 62 non-EOE patients met the model negative, and 24 were false positives.

[0063]

[0064] The subjects in the validation cohort were divided into histologically confirmed cases (≥ 15 eosinophils / HPF) and suspected cases (< 15 eosinophils / HPF) according to the number of esophageal eosinophil infiltration, and group difference analysis was performed. It was found that the expression level of SERPINB10 in the esophageal tissue of the two groups (EoE group in the validation cohort was divided into confirmed cases and suspected cases according to the number of eosinophil infiltration) showed no significant difference (P = 0.080), indicating that the expression level of SERPINB10 in the esophageal tissue of EoE can be used as a relatively independent auxiliary diagnostic standard, and to a certain extent, it can avoid the situation of missed diagnosis of EoE due to not taking the esophageal eosinophil infiltration site. The above is shown in detail in the following Figure 7 .

Claims

1. The use of SERPINB10 protein as a diagnostic marker in the preparation of a diagnostic kit to differentiate between eosinophilic esophagitis and reflux esophagitis: when the integrated optical density of SERPINB10 protein in the esophageal tissue sample of the subject is ≥41666, it is determined to be positive for eosinophilic esophagitis; otherwise, it is positive for reflux esophagitis.

2. The use according to claim 1, characterized in that, The kit contains Anti-SERPINB10 antibody.

3. Application of SERPINB10 protein in the preparation of a diagnostic product for the differential diagnosis of eosinophilic esophagitis and reflux esophagitis: When the integrated optical density of SERPINB10 protein in the esophageal tissue sample of the subject is ≥41666 by immunohistochemical detection, it is determined to be positive for eosinophilic esophagitis; otherwise, it is positive for reflux esophagitis.

4. The use of reagents for detecting SERPINB10 protein in the preparation of diagnostic kits for differentiating between eosinophilic esophagitis and reflux esophagitis: When the integrated optical density of SERPINB10 protein in the esophageal tissue sample of the subject is ≥41666 by immunohistochemical detection, it is determined to be positive for eosinophilic esophagitis; otherwise, it is positive for reflux esophagitis.

5. The use according to claim 4, characterized in that, The reagent for detecting SERPINB10 protein contains an anti-SERPINB10 antibody.

6. A diagnostic system for differentiating eosinophilic esophagitis from reflux esophagitis for non-therapeutic and non-clinical purposes, characterized in that, include: 1) Data processing module: acquire esophageal tissue samples, detect the expression level of SERPINB10 protein in esophageal tissue, and then calculate the integrated optical density value of SERPINB10 protein in esophageal tissue. 2) Sample evaluation module: Receives the integrated optical density value of SERPINB10 protein transmitted by the data processing module. The threshold for the integrated optical density value of SERPINB10 is set to 41666. When the integrated optical density value of SERPINB10 protein is ≥41666, it indicates that the patient has eosinophilic esophagitis; otherwise, the patient has reflux esophagitis.

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