Application of protein biomarker GOLM1 in clinical diagnosis of Crohn's disease

Abstract

The application belongs to the technical field of clinical medicine, and discloses application of a reagent for detecting expression level of a protein biomarker GOLM1 in preparation of a clinical diagnosis kit for Crohn's disease. When the expression level of the protein biomarker GOLM1 in peripheral blood serum of a subject is increased, it indicates that the subject has Crohn's disease. In addition, the application also provides a kit for clinical diagnosis of Crohn's disease, a module for clinical diagnosis of Crohn's disease, and a method for screening drugs for treating Crohn's disease. The CD serum diagnostic marker provided by the application has high sensitivity and specificity, and can be used for early diagnosis of CD in clinic, so as to guide the treatment process of CD in clinic, and provide a new theoretical basis for further explaining the pathophysiological pathogenesis of CD.

Description

[0001] This application is a divisional application of patent application filed on September 23, 2022, with application number 202211168032.3 and invention title "Application of serum biomarkers in the clinical diagnosis of Crohn's disease". Technical Field

[0002] This invention belongs to the field of clinical medical technology, specifically relating to the application of a group of serum biomarkers in the clinical diagnosis of Crohn's disease. Background Technology

[0003] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease with an incompletely understood etiology. The prevailing view is that it primarily affects individuals with a genetic predisposition, leading to intestinal inflammation and damage due to an imbalance in intestinal immune homeostasis caused by various factors such as environment, diet, medications, and gut microbiota. IBD includes Crohn's disease (CD) and ulcerative colitis (UC). In recent years, the incidence of CD in my country has been increasing annually, with an incidence rate of 0.848 / 100,000 and a prevalence of 2.29 / 100,000, a 2.5-fold increase compared to 10 years ago. Clinical symptoms of CD mainly include abdominal pain, diarrhea, abdominal masses, and perianal fistulas. The lesions are multi-segmental and disseminated in a skip pattern, often leading to intestinal obstruction, stenosis, and perforation, resulting in severe intestinal disability. Due to its recurrent and chronic nature, the increasing incidence among young and middle-aged adults, and the lack of sensitive early diagnostic methods and effective treatments, CD (Chronic Crohn's disease) places a heavy burden on individuals, families, and society, hence its nickname "green cancer." Currently, the main clinical diagnostic methods for CD are endoscopic examination and biopsy histopathological diagnosis, which are highly invasive, causing significant pain and relatively high costs, making them inconvenient in clinical application. Imaging examinations have low diagnostic value for CD, and the specificity or sensitivity of serum and fecal biomarkers are also low. Early diagnosis and treatment greatly benefit the prognosis of CD patients; therefore, finding convenient and effective serum biomarkers is of great significance in the clinical diagnosis, treatment, and prognostic management of CD.

[0004] Current techniques assess disease activity in CD patients by detecting peripheral blood CRP levels. CRP is a non-specific serological marker of acute inflammation, primarily synthesized by hepatocytes. It is an acute real-phase reactant protein detectable in plasma when the body is infected or damaged by tissue. It is closely correlated with inflammatory factors and has a strong positive correlation with the clinical activity of CD, and can be used to differentiate IBD from other non-inflammatory gastrointestinal diseases. However, CRP is a non-specific inflammatory marker, rising in various inflammatory conditions, including infections and other diseases causing tissue inflammation and damage. Its specificity is low, and it can only be used to assess the level of systemic inflammatory response in patients with CD, and to some extent, to assess the disease activity, but it cannot provide accurate diagnosis of CD. Furthermore, some CD patients, including those with low BMI or isolated jejunal-ileal CD, may exhibit persistently low CRP levels even during clinically active phases.

[0005] In addition, existing technologies also diagnose Crohn's disease by detecting antimicrobial-associated antibodies (such as ASCA, Cbir1, and OMPC) in the peripheral blood of CD patients. Antipolysaccharide antibodies are a class of antibodies targeting the antigenic determinants of polysaccharides in the cell walls of microorganisms (bacteria and fungi). As specific serological markers associated with CD, they have been widely accepted by most clinicians. Among them, ASCA, a serum reactive antibody against mannan in the cell wall of *Saccharomyces cerevisiae*, was the first serological marker found to have high specificity for CD. Currently, the level of ASCA in the peripheral blood of IBD patients is measured clinically to assess disease behavior and disease activity. However, studies have shown that these antimicrobial-associated antibodies generally have shortcomings in the diagnosis of CD, including low sensitivity, low positive detection rate, and low specificity.

[0006] the term:

[0007] 1. Inflammatory bowel disease, abbreviated as IBD.

[0008] 2. Crohn's disease, abbreviated as CD.

[0009] 3. Serum amyloid-2, abbreviated as SAA2.

[0010] 4. Insulin-like growth factor binding protein-2, abbreviated as IGFBP2.

[0011] 5. Golgi Membrane Protein 1, abbreviated as GOLM1.

[0012] 6. Dipeptidyl peptidase-4, abbreviated as DPP4.

[0013] 7. Guanylate Binding Protein 1 (GBP1)

[0014] 8. “Biomarker”, which may be written as “marker” or “serological marker” in this document, includes molecular markers based on DNA, RNA, protein, carbohydrate or glycolipid. In this document, it refers to soluble protein molecular markers in serum that can be detected in peripheral serum samples of subjects or patients by standard methods (the methods described herein).

[0015] 9. “Sample” refers to a peripheral serum sample derived from a CD patient or a healthy subject, which can be obtained using known techniques. Summary of the Invention

[0016] The purpose of this invention is to provide a set of highly sensitive and specific serum diagnostic biomarkers for CD, for the early clinical diagnosis of CD, to guide the clinical treatment of CD, and to provide new theoretical basis for further explanation of the pathophysiological pathogenesis of CD.

[0017] In our previous work, we collected tissue samples from the ileum segments surgically removed from 30 CD patients, including inflamed and relatively normal tissues, such as the intestinal mucosa (MUC), submucosa-muscularis-serosa (SMS), paraintestinal adipose tissue (MAT), mesentery (MES), and mesenteric lymph nodes (MLN). We also collected relatively normal tissue samples from 5 non-CD patients (non-CD control, NC) (these 5 control patients had early-stage ascending colon tumors, underwent surgical removal of the ascending colon and terminal ileum, and their samples were backed up and subjected to immunopathological examination, verified by at least two professional pathologists to be relatively normal tissues without tumor invasion). We first performed proteomic sequencing and 16S rRNA sequencing analysis on these samples to characterize the protein and bacterial profiles of different tissue sites during the CD intestinal inflammation process. Subsequently, we constructed c-Fuzzy maps based on the changes in the relative expression levels of differentially expressed protein molecules and differentially expressed bacterial genes in different tissue sites of CD patients. Statistical analysis showed a strong correlation between P.C1 (protein cluster 1) and M.C1 (microbial cluster 1). Following correlation analysis and functional annotation of protein molecules in P.C1 and bacterial genes in M.C1, we identified a series of key differentially expressed protein molecules and key bacterial species that play important roles in the dissemination of intestinal inflammation to extraintestinal tissues and the translocation of bacterial flora to extraintestinal tissues during the development of CD intestinal inflammation.

[0018] Based on our previous research, we believe that during the development of intestinal inflammation in CD, the dysregulation of immune regulation and the disruption of physical barrier function in different tissues of the intestine and its appendages may be involved in the dissemination of intestinal inflammation to extraintestinal tissues and the translocation of bacterial flora during CD. Therefore, we hypothesized whether the aforementioned key protein molecules and key bacterial species could have diagnostic efficacy in the body fluid samples of CD patients. We collected peripheral serum and fecal samples from 30 CD patients and 30 healthy controls, and performed serum non-targeted proteomics sequencing and fecal 16S rRNA sequencing analysis. The results were compared with those from tissue proteomics and bacteriomics. We found that five of the aforementioned key protein molecules were differentially expressed in the peripheral serum of CD patients, including SAA2, DPP4, GOLM1, IGFBP2, and GBP1. Furthermore, these key protein molecules showed a good correlation with key bacterial species in feces.

[0019] Based on this, a first aspect of the present invention proposes the application of a set of serum biomarkers in the clinical diagnosis of Crohn's disease, said serum biomarkers including at least one of the protein molecules SAA2, DPP4, GOLM1, IGFBP2 or GBP1.

[0020] According to the application provided by the present invention, whether a subject has Crohn's disease can be determined by detecting the expression level of the serum biomarker in the peripheral blood serum of the subject.

[0021] Specifically, an elevated expression level of at least one of SAA2, GOLM1, IGFBP2, or GBP1 in a subject's peripheral blood serum indicates that the subject has Crohn's disease; a decreased expression level of DPP4 in a subject's peripheral blood serum indicates that the subject has Crohn's disease.

[0022] A second aspect of the invention provides a kit for the clinical diagnosis of Crohn's disease, the kit comprising reagents for detecting the expression levels of serum biomarkers, said serum biomarkers including at least one of the protein molecules SAA2, DPP4, GOLM1, IGFBP2, or GBP1.

[0023] Preferably, in the kit for clinical diagnosis of Crohn's disease provided by the present invention, the reagent for detecting the expression level of serum biomarkers includes: reagents for detecting the expression level of serum biomarkers based on RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, microarray or high-throughput sequencing.

[0024] More preferably, the reagent for detecting the expression level of the serum biomarker based on RT-PCR includes at least one pair of primers specifically amplifying the nucleic acid sequence of the serum biomarker; the reagent for detecting the expression level of the serum biomarker based on real-time quantitative PCR includes at least one pair of primers specifically amplifying the nucleic acid sequence of the serum biomarker; the reagent for detecting the expression level of the serum biomarker based on immunoassay includes at least one antibody specifically binding to the serum biomarker; the reagent for detecting the expression level of the serum biomarker based on in situ hybridization includes at least one probe hybridizing to the nucleic acid sequence of the serum biomarker; the reagent for detecting the expression level of the serum biomarker based on a microarray includes at least one protein microarray and one gene microarray; the protein microarray includes an antibody specifically binding to the serum biomarker, and the gene microarray includes a probe hybridizing to the nucleic acid sequence of the serum biomarker; the reagent for detecting the expression level of the serum biomarker based on high-throughput sequencing includes at least one primer and / or probe for detecting the transcriptional level of the serum biomarker.

[0025] A third aspect of the present invention provides a module for the clinical diagnosis of Crohn's disease, the module comprising a diagnostic indicator input module and a Crohn's disease status assessment module; the diagnostic indicator input module includes at least obtaining the expression level of a serum biomarker in the peripheral blood serum of a subject, the serum biomarker including at least one of the protein molecules SAA2, DPP4, GOLM1, IGFBP2, or GBP1; the Crohn's disease status assessment module includes at least inputting the expression level of the serum biomarker in the peripheral blood serum of the subject obtained by the diagnostic indicator input module into a diagnostic model to obtain a score value; comparing the obtained score value with a pre-set cutoff value of the diagnostic model, and outputting an assessment result of the subject's Crohn's disease status; the Crohn's disease status includes Crohn's disease-related statuses pre-set by the diagnostic model.

[0026] A fourth aspect of the present invention provides a method for screening drugs for treating Crohn's disease, comprising: in a test group, administering a test drug to a subject and detecting the expression level V1 of a serum biomarker in samples derived from the subject in the test group; in a control group, administering a blank control drug to the subject and detecting the expression level V2 of a serum biomarker in samples derived from the subject in the control group; comparing the detected serum biomarkers V1 and V2 to determine whether the test drug has efficacy in treating Crohn's disease; wherein the serum biomarker includes at least one of the protein molecules SAA2, DPP4, GOLM1, IGFBP2, or GBP1. Attached Figure Description

[0027] Figure 1 This study compares the expression levels of IGFBP2, DPP4, GBP1, SAA2, and GOLM1 in the peripheral serum of Crohn's disease patients and healthy controls. HC refers to healthy controls, and A-CD refers to patients with active Crohn's disease.

[0028] Figure 2 The ROC curve of SAA2 as a biomarker for diagnosing Crohn's disease is shown and compared with CRP.

[0029] Figure 3 The ROC curve of GBP1 as a biomarker for diagnosing Crohn's disease is shown and compared with CRP.

[0030] Figure 4 The ROC curve of IGFBP2 as a biomarker for diagnosing Crohn's disease is shown and compared with CRP.

[0031] Figure 5 The ROC curve of GOLM1 as a biomarker for diagnosing Crohn's disease is shown and compared with CRP.

[0032] Figure 6The ROC curve of DPP4 as a biomarker for diagnosing Crohn's disease is shown and compared with CRP.

[0033] Figure 7 The average ROC curves of SAA2, DPP4, IGFBP2, GOLM1, and GBP1 as biomarkers for diagnosing Crohn's disease are shown and compared with CRP.

[0034] Figure 8 This analysis examines the correlation between SAA2 and CRP expression levels in the peripheral serum of Crohn's disease patients.

[0035] Figure 9 This analysis examines the correlation between GBP1 and CRP expression levels in the peripheral serum of Crohn's disease patients.

[0036] Figure 10 This analysis examines the correlation between IGFBP2 and CRP expression levels in the peripheral serum of Crohn's disease patients.

[0037] Figure 11 This analysis examines the correlation between GOLM1 and CRP expression levels in the peripheral serum of Crohn's disease patients.

[0038] Figure 12 This study analyzed the correlation between DPP4 and CRP expression levels in the peripheral serum of Crohn's disease patients. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below. However, those skilled in the art will understand that many technical details have been provided in the various embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in the claims of this application can be implemented even without these technical details and with various variations and modifications based on the following embodiments.

[0040] 1. Discovery of biomarkers: Proteomics and bacteriomics sequencing analysis of the intestines and accessory organs of Crohn's disease patients and non-Crohn's disease patients.

[0041] 1.1 Sample Collection:

[0042] Patients aged >18 years were selected who had not used immunosuppressants, small molecule targeted drugs, glucocorticoids, antibiotics, or other special medications, had no other diseases affecting the pathophysiology of the intestine, and had no hypertension, diabetes, or other similar conditions. Informed consent was obtained from all patients beforehand. During the surgery, a professional surgeon collected intestinal mucosa, submucosa-muscularis-serosa, paraintestinal fat, mesentery, and mesenteric lymph nodes from inflamed and relatively normal sites in 30 Crohn's disease patients under sterile conditions. Also, in 5 non-Crohn's disease patients, relatively normal mucosa, submucosa-muscularis-serosa, paraintestinal fat, mesentery, and mesenteric lymph nodes from unaffected sites were collected. The samples were transported in liquid nitrogen and stored at -80°C for later use. All relatively normal tissue samples from non-CD patients underwent histopathological examination, and were verified by at least two professional pathologists to be non-disease-affected tissue.

[0043] 1.2 Tissue proteomics sequencing analysis:

[0044] ① 4-D proteomics sequencing analysis was performed on tissue samples from surgically removed intestines and their appendages in CD patients (including paired inflamed and relatively normal intestinal mucosa, muscle and serosa, peri-intestinal adipose tissue, mesenteric tissue, and mesenteric lymph node tissue) and tissue samples from surgically removed intestines and their appendages in relatively normal locations in non-CD patients (including intestinal mucosa, muscle and serosa, peri-intestinal adipose tissue, mesenteric tissue, and mesenteric lymph node tissue).

[0045] ② Compare the differences in protein expression profiles between inflammatory tissue samples from CD patients and tissue samples from non-CD patients in the same type of normal tissue, calculate the expression levels of differentially expressed proteins in the tissue samples, perform pathway enrichment analysis on the differentially expressed proteins, and perform functional annotation of the differentially expressed proteins based on databases (Nanostring and Import, etc.) to explore the changes in protein expression profiles in different tissues during CD intestinal inflammation and investigate the changes in biological functions of different tissues.

[0046] ③ Compare the differences in bacterial profiles between inflamed and relatively normal tissue samples from CD patients, and calculate the expression levels of differentially expressed proteins in the tissue samples to explore the influence of local intestinal inflammation on protein expression profiles during CD.

[0047] ④ Draw a protein interaction network diagram in different types of tissue samples from inflamed sites of CD patients, and analyze the key protein molecules and their interactions in different sites during intestinal inflammation in CD patients.

[0048] 1.3 Tissue bacterial omics sequencing analysis:

[0049] Describe the bacterial spectrum of different parts of the intestine and its accessory organs during the occurrence of enteritis in CD patients.

[0050] ① 16S rDNA bacterial omics sequencing analysis was performed on tissue samples from surgically removed intestines and their appendages in CD patients (including paired inflamed and relatively normal intestinal mucosa, muscular and serosa, peri-intestinal adipose tissue, mesenteric tissue, and mesenteric lymph node tissue) and tissue samples from surgically removed intestines and their appendages in relatively normal locations in non-CD patients (including intestinal mucosa, muscular and serosa, peri-intestinal adipose tissue, mesenteric tissue, and mesenteric lymph node tissue).

[0051] ② Compare the bacterial spectrum in the same type of tissue samples from inflammatory sites in CD patients and non-CD patients relative to normal sites, and calculate the abundance of differentially expressed bacterial species in the tissue samples to explore the changes in the bacterial spectrum of the intestine and its accessory organs during CD enteritis.

[0052] ③ Compare the differences in bacterial profiles between inflamed and relatively normal tissue samples from CD patients, and calculate the abundance of differentially expressed bacterial species in the tissue samples to explore the impact of local intestinal inflammation on the bacterial community profile during CD.

[0053] ④ The abundance of the above-mentioned differentially expressed bacterial species in different types of tissue samples at the site of inflammation in CD patients was compared longitudinally, and bioinformatics methods were used to calculate and simulate the possible sources of the formation of specific bacterial communities in different sites, so as to explore the translocation and spatial distribution of bacterial communities in the intestine and its accessory organs during CD.

[0054] ⑤ Draw a network diagram of microbial interactions in different types of tissue samples from inflamed sites of CD patients, and analyze the spatial distribution of key bacterial species in different sites during intestinal inflammation in CD patients.

[0055] 1.4 Study on the interaction between bacteria and host proteins:

[0056] ① Based on the abundance of bacterial flora and the expression level of proteins in different tissues of the intestine and its accessory organs of CD patients, plot the abundance / expression level change curves and compare them to find bacterial flora clusters and protein molecular clusters with similar abundance / expression level change trends.

[0057] ② Pathway enrichment analysis was performed on the above-mentioned bacterial and protein clusters to explore the changes in host function in different tissue sites they represent and their impact on bacterial populations.

[0058] ③Based on the abundance of bacterial flora and protein expression levels in different tissue samples from the intestines and accessory organs of CD patients, and existing databases, a network diagram of bacterial-protein molecular interactions was drawn to explore the key bacterial species and protein molecules that play a crucial role in different tissue sites during intestinal inflammation.

[0059] ④ A longitudinal comparison was made of key interacting protein molecules and bacterial species in different tissues of the intestine and its accessory organs in CD patients with inflammation, to explore the role of this interaction mode in the translocation of the microbiota during the occurrence of CD enteritis.

[0060] 1.5 Verify the enrichment of the above-mentioned key protein molecules and bacterial species in the peripheral serum and feces of CD patients:

[0061] Paired peripheral blood serum and stool samples were collected from 30 patients with active Crohn's disease (CD) and 30 healthy controls. Non-targeted proteomics techniques were used to detect key protein molecules identified in previous studies. 16S rRNA detection was used to analyze the bacterial community composition in the samples, detecting the abundance of key bacterial species identified in previous studies and evaluating their sensitivity and specificity as diagnostic biomarkers for CD. Clinical information of CD patients, such as age, sex, disease behavior, medication use, and comorbidities, was collected. Statistical methods were used to study the correlation between changes in the aforementioned key protein molecules and key bacterial species and patients' clinical symptoms or treatment methods. Ultimately, five differentially expressed proteins (e.g., [missing information]) were identified in the peripheral blood of CD patients. Figures 1-7 The key protein molecules shown in Table 1 below are those that have a good correlation with the corresponding fecal bacteria species.

[0062] Table 1

[0063]

[0064] 2. Validation of the diagnostic efficacy of novel biomarkers:

[0065] 2.1 Sample Collection:

[0066] Endoscopic examinations were performed on patients with Crohn's disease and non-Crohn's disease patients (or healthy subjects). In Crohn's disease patients, lesions are segmental or skip-pathogenic. Early-stage lesions appear as thrush-like ulcers, which subsequently enlarge and merge, forming longitudinal fissure-like ulcers along the long axis of the intestine, dividing the mucosa into a cobblestone-like appearance. Mucosal tissue from the lesion sites in the patient's intestine was collected and reviewed by at least two professional pathologists. The characteristic features of Crohn's disease lesions are non-caseating granulomas composed of epithelioid cells and multinucleated giant cells, fissure-like ulcers extending to the submucosa and even the muscle layer, accompanied by abundant lymphocyte aggregation, lymphangiectasia, and ganglionitis in the lamina propria and submucosa. Subjects can be distinguished as Crohn's disease patients or non-Crohn's disease patients based on the endoscopic and histopathological examination results.

[0067] 2.2 Collection and analysis of peripheral serum samples from subjects:

[0068] Objective: To obtain peripheral serum samples from 30 Crohn's disease (CD) patients and 30 healthy controls, and to detect the expression levels of five soluble protein molecules (SAA2, IGFBP2, GOLM1, DPP4, and GBP1) in the peripheral serum of CD patients using ELISA, and to compare the expression levels of the corresponding protein molecules in the peripheral serum samples of the control group. The controls were healthy subjects without Crohn's disease.

[0069] Experimental Method: Enzyme-Linked Immunosorbent Assay (ELISA): An ELISA plate was coated with pre-prepared antibodies corresponding to the target soluble protein molecules to form a solid-phase carrier. Pre-diluted standards and subject serum were added to the wells coated with the antibody, allowing the target soluble protein molecules (including SAA2, IGFBP2, GOLM1, DPP4, and GBP1) to bind to the immobilized antibodies. Unbound samples were washed away, and horseradish peroxidase-labeled detection antibodies were added. After incubation, the enzyme-labeled detection antibodies bound to the target protein molecules in the subject serum, thus immobilizing them on the microplate. Unbound enzyme-labeled antibodies were thoroughly washed away, and the plate was developed with the substrate TMB. TMB forms a blue cationic product under the catalysis of peroxidase; the blue color changes to yellow after acid is added to terminate the reaction. The color intensity is positively correlated with the concentration of the target soluble protein molecules in the sample. The OD value was measured at 450 nm using an ELISA reader to calculate the concentration of soluble protein molecules in the sample.

[0070] Data Analysis: The expression levels of target soluble protein molecules in peripheral blood serum of 30 Crohn's disease patients and 30 healthy controls (matched for age and sex) were detected and compared. ROC curve analysis was performed, and the results were compared with C-reactive protein (CRP), a marker used clinically to assess disease activity in Crohn's disease patients. The AUC values ​​of CRP, SAA2, GBP1, IGFBP2, GOLM1, and DPP4 were 0.890, 0.902, 0.913, 0.917, and 0.920, respectively.

[0071] Figure 2 Table 2 shows the ROC curve of SAA2 as a biomarker for diagnosing Crohn's disease, and compares it with CRP. The area under the curve is calculated.

[0072] Table 2 Figure 2 Area under the curve

[0073]

[0074] (Note: The test result variable: CRP has at least one knot between the positive and negative actual state groups. The statistic may be biased.)

[0075] a. Under nonparametric assumptions; b. Null hypothesis: real area = 0.5.

[0076] Figure 3 Table 3 shows the ROC curve of GBP1 as a biomarker for diagnosing Crohn's disease, and compares it with CRP. The area under the curve is calculated.

[0077] Table 3 Figure 3 Area under the curve

[0078]

[0079] (Note: The test result variable: CRP has at least one knot between the positive and negative actual state groups. The statistic may be biased.)

[0080] a. Under nonparametric assumptions; b. Null hypothesis: real area = 0.5.

[0081] Figure 4 Table 4 shows the ROC curve of IGFBP2 as a biomarker for diagnosing Crohn's disease, and compares it with CRP. The area under the curve is calculated.

[0082] Table 4 Figure 4 Area under the curve

[0083]

[0084] (Note: The test result variable: CRP has at least one knot between the positive and negative actual state groups. The statistic may be biased.)

[0085] a. Under nonparametric assumptions; b. Null hypothesis: real area = 0.5.

[0086] Figure 5 Table 5 shows the ROC curve of GOLM1 as a biomarker for diagnosing Crohn's disease, and compares it with CRP. The area under the curve is calculated.

[0087] Table 5 Figure 5 Area under the curve

[0088]

[0089] (Note: The test result variable: CRP has at least one knot between the positive and negative actual state groups. The statistic may be biased.)

[0090] a. Under nonparametric assumptions; b. Null hypothesis: real area = 0.5.

[0091] Figure 6 This is the ROC curve of DPP4 as a biomarker for diagnosing Crohn's disease, compared with CRP, and the area under the curve is calculated.

[0092] Table 6 Figure 6 Area under the curve

[0093]

[0094]

[0095] (Note: The test result variable: CRP has at least one knot between the positive and negative actual state groups. The statistic may be biased.)

[0096] a. Under nonparametric assumptions; b. Null hypothesis: real area = 0.5.

[0097] Figure 7 The average ROC curves of SAA2, DPP4, IGFBP2, GOLM1, and GBP1 as biomarkers for diagnosing Crohn's disease are shown and compared with CRP.

[0098] The correlation between target soluble protein molecules and CRP in peripheral serum of Crohn's disease patients was then compared. Figure 8 This analysis examines the correlation between SAA2 and CRP expression levels in the peripheral serum of Crohn's disease patients. Figure 9 This analysis examines the correlation between GBP1 and CRP expression levels in the peripheral serum of Crohn's disease patients. Figure 10 This analysis examines the correlation between IGFBP2 and CRP expression levels in the peripheral serum of Crohn's disease patients. Figure 11 This analysis examines the correlation between GOLM1 and CRP expression levels in the peripheral serum of Crohn's disease patients. Figure 12 This study analyzed the correlation between DPP4 and CRP expression levels in the peripheral serum of Crohn's disease patients. Statistical analysis showed that all correlations were significant (p < 0.01). Specifically, the correlation coefficients between SAA2 and CRP were 0.906, GOLM1 and CRP were 0.556, IGFBP2 and CRP were 0.137, and GBP1 and CRP were 0.411, all showing significant positive correlations. The correlation coefficient between DPP4 and CRP was -0.527, showing a significant negative correlation.

[0099] Based on the above research findings, this invention proposes that one or more of the protein molecules SAA2, DPP4, GOLM1, IGFBP2, or GBP1 can be used as serum biomarkers for the clinical diagnosis of Crohn's disease.

[0100] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. Application of reagents for detecting the expression level of the protein biomarker GOLM1 in the preparation of clinical diagnostic kits for Crohn's disease, wherein, The kit determines whether a subject has Crohn's disease by detecting the expression level of the protein biomarker GOLM1 in the subject's peripheral blood serum.

2. The application according to claim 1, characterized in that, An elevated level of the protein biomarker GOLM1 in the peripheral blood serum of a subject indicates that the subject has Crohn's disease.

3. The application according to claim 1, characterized in that, The reagents for detecting the expression level of the protein biomarker GOLM1 include: reagents for detecting the expression level of the protein biomarker GOLM1 based on immunoassay technology.

4. The application according to claim 3, characterized in that, The reagent used to detect the expression level of the protein biomarker GOLM1 based on immunoassay technology includes at least an antibody that specifically binds to the protein biomarker.

5. A module for the clinical diagnosis of Crohn's disease, characterized in that, Includes a diagnostic indicator input module and a Crohn's disease status assessment module; The diagnostic indicator input module includes at least obtaining the expression level of the protein biomarker GOLM1 in the peripheral blood serum of the subject; The Crohn's disease status assessment module includes at least the following steps: inputting the expression level of the protein biomarker GOLM1 in the peripheral blood serum of the subject obtained by the diagnostic indicator input module into the diagnostic model to obtain a score; comparing the obtained score with the cutoff value of the pre-set diagnostic model to output the assessment result of the subject's Crohn's disease status; the Crohn's disease status includes Crohn's disease-related statuses pre-set by the diagnostic model.

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