Application of soluble protein B7-H5 in preparation of Crohn disease detection preparation

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of soluble B7-H5 in preparation of a Crohn disease detection reagent. According to the invention, the level of soluble B7-H5 (sB7-H5) in serum of a patient suffering from Crohn's disease (CD) is deeply analyzed by using an ELISA (Enzyme-Linked Immunosorbent Assay) technology. Studies find that sB7-H5 in serum of a CD patient is remarkably and highly expressed and is positively correlated with a disease activity index (CDAI). Further experimental verification shows that blocking of B7-H5 can promote polarization of inflammatory macrophages. Based on the discoveries, the invention provides the application of the serum B7-H5 as the molecular marker of the Crohn's disease to development of products for detection, prognosis or treatment of the Crohn's disease. The molecular marker can be in a soluble B7-H5 gene or protein form. The invention also shows that the B7-H5 can be used as a potential target for developing the medicine for preventing and treating the Crohn disease, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of soluble B7-H5 in the preparation of Crohn's disease detection reagents. Background Technology

[0002] Crohn's disease (CD) is characterized by transmural inflammation that can affect the entire digestive tract. Clinical manifestations are diverse, with abdominal pain, diarrhea, and weight loss being the most common, severely impacting patients' quality of life. Although traditional treatments (such as immunosuppressants and biologics) have improved the prognosis of some patients, 30% to 40% still face treatment failure or disease relapse, highlighting the urgent need to explore new therapeutic targets and precise diagnostic biomarkers.

[0003] Currently, the differential diagnosis of Crohn's disease relies on laboratory tests, endoscopy, and imaging examinations. Laboratory tests include blood tests (such as hemoglobin, white blood cell count, and C-reactive protein) and stool tests (such as blood, microorganisms, and calprotectin) to assess anemia, infection, and inflammation. Among endoscopy examinations, colonoscopy is a commonly used method for diagnosing Crohn's disease, allowing direct observation of the colon and terminal ileum mucosa and biopsy. Enteroscopy is suitable for observing the deep parts of the small intestine. Capsule endoscopy involves the patient swallowing a capsule with a built-in camera to capture images of the small intestine, but there is a risk of capsule retention. Imaging examinations include CT scans (especially CT enterography), MRI scans (magnetic resonance enterography), and ultrasound examinations to show the extent, severity, and complications of intestinal lesions. Diagnostic models based on multidimensional data improve diagnostic accuracy, but their application is complex; biomarker detections such as ANCA and ASCA have been studied, but their diagnostic value remains controversial. Therefore, there is an urgent need for highly specific and sensitive hematological diagnostic methods for clinical application.

[0004] B7-H5 (also known as B7-H5, PD-1H, Gi24, Dies1, SISP1, C10orf54, etc.) is a member of the B7 family, belonging to the transmembrane glycoprotein family, and plays a crucial role in tumor immunity and various inflammation-related diseases. Studies have found that B7-H5 exists in a soluble form, and its levels are significantly elevated in the serum of patients with gastric cancer, colorectal cancer, and lung cancer, demonstrating high diagnostic value. However, there are currently no reports on the application of B7-H5 in the detection and diagnosis of Crohn's disease. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an application of soluble B7-H5 levels as a molecular marker for Crohn's disease. It was found that the expression level of soluble B7-H5 in Crohn's disease was significantly higher than that in healthy controls, and it was positively correlated with the disease activity index (CDAI).

[0006] The technical solution provided by this invention is as follows:

[0007] To achieve the above objectives, the first aspect of this application provides an innovative application of soluble B7-H5 as a biomarker in the development of products for diagnosing Crohn's disease.

[0008] A second aspect of this application provides the use of a reagent for detecting the expression level of soluble B7-H5 in the preparation of a Crohn's disease diagnostic product. As a preferred embodiment, the reagent comprises primers, probes, and antibodies for detecting B7-H5.

[0009] A second aspect of this application provides a drug for treating Crohn's disease based on B7-H5 as a target. As a preferred embodiment, the reagent comprises a recombinant B7-H5 protein and an activating antibody.

[0010] Application of soluble protein B7-H5 in the preparation of Crohn's disease detection reagents.

[0011] Furthermore, the detection reagent is used to detect the expression level of soluble protein B7-H5 in peripheral blood serum.

[0012] Furthermore, the expression level of soluble protein B7-H5 in peripheral blood serum was detected using the ELISA method.

[0013] This invention also provides the application of soluble protein B7-H5 blocking antibodies in the preparation of anti-inflammatory drugs.

[0014] Furthermore, the anti-inflammatory drug is used to treat ulcerative colitis.

[0015] Furthermore, the anti-inflammatory drug is used to treat Crohn's disease.

[0016] Furthermore, the anti-inflammatory drug is used to negatively regulate macrophage activation.

[0017] Furthermore, the concentration of the soluble protein B7-H5 blocking antibody is 8~12µg / µL.

[0018] The present invention also provides a kit for detecting Crohn's disease, the kit comprising reagents for detecting the expression level of soluble protein B7-H5 in peripheral blood serum.

[0019] Furthermore, the reagent used to detect the expression level of soluble protein B7-H5 in peripheral blood serum includes an antibody that specifically binds to soluble protein B7-H5.

[0020] Beneficial effects

[0021] This application used an ELISA kit to detect the expression level of soluble B7-H5 in peripheral blood serum of healthy volunteers and newly diagnosed Crohn's disease patients. The results showed that the expression level of soluble B7-H5 in Crohn's disease was significantly higher than that in healthy controls, and it was positively correlated with the Disease Activity Index (CDAI). In diagnostic performance evaluation, soluble B7-H5 demonstrated excellent diagnostic value, with an AUC value of 0.894 (95% CI: 0.794-0.994), corresponding to a sensitivity of 90.7% and a specificity of 83.3%. Compared with the traditional marker C-reactive protein (CRP, with a maximum AUC value of 0.880, 95% CI: 0.793-0.968, corresponding to a sensitivity of 78.2% and a specificity of 83.3%), sB7-H5 has superior diagnostic efficacy. Further experimental validation showed that blocking B7-H5 can promote the polarization of inflammatory macrophages. Attached Figure Description

[0022] Figure 1 The expression of soluble B7-H5 in the peripheral blood of Crohn's disease patients and healthy volunteers;

[0023] Figure 2 ROC curve for assessing Crohn's disease activity using soluble B7-H5;

[0024] Figure 3 The changes in CD86 and F4 / 80 in macrophages after B7-H5 blockade are shown; where A is a scatter plot and B is a bar chart.

[0025] Figure 4 The images show the exacerbation of DSS-induced enteritis symptoms in mice after B7-H5 blockade. A shows the weight trend of mice in the Control group, DSS group, and DSS+B7-H5 mAb treatment, and B shows colon images of mice in the Control group, DSS group, and DSS+B7-H5 mAb treatment. Detailed Implementation

[0026] To help those skilled in the art gain a deeper understanding of the present application, the technical details of the present application will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described only cover a part of the present application, not all of it. Those skilled in the art can derive other feasible solutions based on these embodiments without creative effort, and all such solutions should be included within the protection scope of this application. Subsequently, this document will provide a comprehensive analysis of the present application through the organic combination of the accompanying drawings and embodiments.

[0027] Example 1

[0028] ELISA method for detecting the expression of soluble B7-H5 in peripheral blood of Crohn's disease patients and healthy volunteers

[0029] The B7-H5 ELISA kit used was purchased from Suzhou Xuguang Kexing Antibody Biotechnology Co., Ltd. (Catalog No.: XG-K3014).

[0030] I. Serum: Peripheral blood was collected using a coagulation tube, allowed to coagulate at room temperature for at least 30 minutes, and then centrifuged at 1000×g for 10 minutes to collect serum.

[0031] II. Preparation of Reagents and Samples

[0032] 1. Preparation of Dilution buffer working solution: Dilute each vial with 30ml of 1×PBS.

[0033] 2. Preparation of Washing buffer working solution: Calculate the total amount according to the actual usage, and dilute it 20 times with pure water (for example: take 1 ml of Washing buffer (20×) and 19 ml of pure water, and mix well).

[0034] 3. Dilution and use of human B7-H5 standard: Prepare within 2 hours prior to use. The kit provides two tubes of standard, 4 ng each; use one tube at a time.

[0035] (1) Prepare 4000pg / ml standard: Add 1000μl of Dilution buffer into the standard tube, cover it and repeatedly invert / shake to help dissolve, and let it stand for more than 10 minutes.

[0036] (2) Preparation of 2000pg / ml→31.25pg / ml standard: Prepare 8 Eppendorf tubes, add 500μl of Dilution buffer to each tube, and label them as 2000pg / ml, 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25pg / ml, and 0pg / ml respectively. Take 500μl of the 4000pg / ml standard and add it to the tube labeled 2000pg / ml. Mix well and then take 500μl of the standard and add it to the next tube.

[0037] 4. Preparation of Antibody-Bio antibody working solution: Calculate the total volume based on 100 μl required per well. Dilute with Dilution buffer at a ratio of 1:4000, mix gently, and prepare within 2 hours before use (e.g., take 1 μl of Antibody-Bio and 4000 μl of Dilution buffer, and mix thoroughly).

[0038] 5. Preparation of Avidin-HRP working solution: Calculate the total amount needed based on 100 μl per well. Dilute with Dilution buffer at a ratio of 1:6000, mix gently, and prepare within 1 hour before use (e.g., take 1 μl of Avidin-HRP and 6000 μl of Dilution buffer, and mix well).

[0039] 6. Researchers must estimate the concentration of the analyte in the sample and determine an appropriate dilution factor to ensure that the concentration of the analyte in the diluted sample is within the optimal detection range of the ELISA kit. Samples should be diluted with dilution buffer. A 2-fold dilution is recommended for breast cancer samples, a 5-fold dilution for cervical cancer samples, and undiluted for normal samples. Unknown samples require serial dilution to determine the optimal dilution factor.

[0040] III. Testing Steps

[0041] 1. Before use, mix all reagents thoroughly to avoid generating bubbles.

[0042] 2. Determine the required number of strips based on the number of test wells (standards, samples, and blanks).

[0043] 3. Add sample: Add 100 μl of standard (2000 pg / ml-0 pg / ml, add each concentration in sequence) or sample to each well, and incubate at 37℃ for 2 h.

[0044] 4. Washing the plate: Shake off the liquid in the wells, add 300 μl of washing buffer per well, wash the plate 3 times, discard the liquid in the wells, and pat dry on filter paper / absorbent paper.

[0045] 5. Add detection antibody: Add 100 μl of diluted Antibody-Bio per well, cover with sealing film, and incubate at 37°C for 1 hour.

[0046] 6. Washing the plate: Shake off the liquid in the wells, add 300 μl of washing buffer per well, wash the plate 3 times, discard the liquid in the wells, and pat dry on filter paper / absorbent paper.

[0047] 7. Add enzyme: Add 100 μl of Avidin-HRP working solution per well, cover with sealing film, and incubate at 37°C for 1 h.

[0048] 8. Washing the plate: Shake off the liquid in the wells, add 300 μl of washing buffer per well, wash the plate 6 times, discard the liquid in the wells, and pat dry on filter paper / absorbent paper.

[0049] 9. Color development: Add TMB, 100 μl / well, and incubate at 37°C in the dark for about 15 min.

[0050] 10. Terminate the reaction: Add 50 μl of stop solution per well.

[0051] 11. Reading the plate: Within 10 minutes after termination, read the value using a detection wavelength of 450nm.

[0052] 12. Calculate the average absorbance values ​​of the standards and samples, and plot a standard curve, with the standard concentration on the x-axis and the absorbance value on the y-axis. Calculate the concentration of the target antigen in the unknown sample based on the standard curve, which can be done using software or manually.

[0053] IV. Results

[0054] The expression level of soluble B7-H5 in peripheral blood serum of 18 healthy volunteers and 47 newly diagnosed Crohn's disease patients was detected by ELISA. The results showed that the expression level of soluble B7-H5 in Crohn's disease patients was significantly higher than that in healthy controls. Figure 1 ).

[0055] Example 2

[0056] Correlation between soluble B7-H5 expression levels and clinical characteristics of Crohn's disease patients

[0057] SPSS 22.0 statistical software was used to analyze the correlation between serum soluble B7-H5 expression levels and clinical characteristics such as patient gender, age, disease location, disease behavior, perianal lesions, history of intestinal surgery, fecal calprotectin, and fecal occult blood in Crohn's disease patients. The results are shown in Table 1. Soluble B7-H5 expression showed statistically significant differences among different CDAI score groups. However, no statistically significant differences were found in Crohn's disease expression among gender, age, disease location, disease behavior, perianal lesions, history of intestinal surgery, fecal calprotectin, and fecal occult blood groups. These results strongly confirm that soluble B7-H5 can serve as a potential biomarker for the diagnosis of ulcerative colitis, providing a crucial reference for clinical diagnosis.

[0058] Table 1. Relationship between sB7-H5 expression and clinicopathological features in CD patients.

[0059]

[0060] Example 3

[0061] Soluble B7-H5 as a biomarker for Crohn's disease assessment

[0062] This study aimed to evaluate the diagnostic value of soluble B7-H5 as a novel biomarker for Crohn's disease (CD) activity using GraphPad Prism 8.0 software and ROC curve analysis. To ensure a more comprehensive and clinically in-depth assessment, two reference groups were specifically included: the traditional biomarker CRP and the systemic immune inflammatory index (SII). This was done to: firstly, using CRP as the "gold standard" to assess whether B7-H5 is superior to or comparable to current clinical practice; and secondly, introducing SII to explore the intrinsic relationship between this specific immune molecule, B7-H5, and the overall immune inflammatory state of the body. Together, these three elements constitute a multi-level assessment framework, ranging from classical to cutting-edge research, and from specific pathways to the systemic level.

[0063] The results showed that ROC curves were constructed for three indicators—soluble protein B7-H5, SII, and CRP—to evaluate the diagnostic value of these indicators for Crohn's disease activity levels. The AUC value for soluble protein was 0.894 (95% CI: 0.794–0.994), with a sensitivity of 90.7% and a specificity of 83.3%. The AUC value for SII was 0.843 (95% CI: 0.745–0.941), with a sensitivity of 76.6% and a specificity of 88.9%. The highest AUC value for CRP was 0.880 (95% CI: 0.793–0.968), with a sensitivity of 78.2% and a specificity of 83.3%. The differential diagnostic value of sB7-H5 for Crohn's disease was evaluated using ROC curve analysis, and the optimal cutoff value was determined accordingly. Analysis showed that the Youden index was highest at a cutoff value of 208.1 ng / mL, indicating that the sensitivity and specificity of the detection were optimally balanced at this concentration. Based on these results, this study preliminarily suggests that serum sB7-H5 > 208.1 ng / mL as a potential laboratory indicator to support the diagnosis of Crohn's disease. Therefore, soluble protein B7-H5 is a promising potential biomarker for CD diagnosis. Figure 3 ).

[0064] Example 4

[0065] B7-H5 blockade promotes LPS-induced expression of CD86 and F4 / 80 in macrophages.

[0066] Reagents used: B7-H5 blocking antibody (BioXCell, USA, catalog number: #BE0310).

[0067] 1. RAW264.7 cells were digested with trypsin to prepare a single-cell suspension. After counting, the cells were seeded into 6-well culture plates. The cell density per well is generally 1 × 10⁻⁶ cells / well. 6 Approximately [number] cells / mL.

[0068] 2. RAW264.7 cells were pretreated with B7-H5 blocking antibody (10 µg / µl) for 2 hours, followed by the addition of LPS (100 ng / ml) and cultured in a 37°C, 5% CO2 incubator for stimulation.

[0069] 3.48 hours, digest cells to obtain cell suspension, 1300 r / min, 5 min.

[0070] 4. Discard the supernatant, add 1 ml of PBS and wash for 5 min at 1300 rpm.

[0071] 5. Discard the supernatant, add 400 μL of PBS, and mix well. Evenly pipette 100 μL into 4 flow cytometry tubes. The first tube serves as a blank control. Add 1 μL of CD86 fluorescent antibody to the second tube, 1 μL of F4 / 80 fluorescent antibody to the third tube, and 1 μL of both CD86 and F4 / 80 fluorescent antibodies to the fourth tube. Protect from light and incubate at 4°C for 30 min.

[0072] 6. Add 500 μL of PBS to each tube and centrifuge for 5 minutes at 1300 rpm. Discard the supernatant, then add 350 μL of PBS and mix thoroughly by pipetting.

[0073] 7. Flow cytometry assay.

[0074] 8. Results: Flow cytometry analysis revealed that LPS stimulation of RAW264.7 cells significantly increased the proportion of CD86 and F4 / 80 double-positive cells. Figure 3 In further experiments, the addition of a B7-H5 inhibitor to LPS stimulation resulted in a further increase in the proportion of CD86 and F4 / 80 double-positive cells compared to the LPS-only stimulation group (P<0.05). This result indicates that blocking B7-H5 can enhance macrophage activation and polarization. Given that previous studies have clearly established that abnormal activation and functional imbalance of macrophages are the core drivers of intestinal inflammation and tissue damage in Crohn's disease, and that one of the key mechanisms of action of existing therapies is to correct this imbalance, this study further proposes that B7-H5 may exert its inhibitory effect on the inflammatory response by negatively regulating macrophage activation.

[0075] Example 5

[0076] B7-H5 blockade exacerbated DSS-induced enteritis symptoms in mice.

[0077] Reagents used: sodium dextran sulfate (DSS, MCE, catalog number: HY-116282C), B7-H5 blocking antibody (BioXCell, USA, catalog number: #BE0310).

[0078] Experimental animals: SPF-grade C57 / BL black mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (SCXK (Shanghai) 2018-0003), and were quality inspected by Suzhou Xishan Biotechnology Co., Ltd. The breeding site was the Experimental Animal Center of Soochow University. The experimental black mice were male, 6-8 weeks old, and weighed approximately (21.45 ± 1.12) g.

[0079] 1. Twenty male C57 / BL mice were randomly divided into 3 groups: normal control group (Control group = 5 mice), DSS-induced colitis group (DSS group = 5 mice), and B7-H5 blocking group (DSS + B7-H5 mAb group = 5 mice).

[0080] 2. The Control group was fed with sterile ddH2O freely from day 1 to day 7; the DSS + PBS group was fed with 3% DSS freely and injected intraperitoneally with PBS from day 1 to day 7; the DSS + B7-H5 mAb group was fed with 3% DSS freely and injected intraperitoneally with B7-H5 mAb (200 μg / mouse).

[0081] 3. The body weight, bloody stool, and mental state of the mice were evaluated daily.

[0082] 4. Results: Compared with the Control group, the body weight of the mice began to decrease on the 3rd day after DSS-induced colitis, and then decreased significantly. After administration of B7-H5 mAb, the downward trend of the body weight of the mice was aggravated ( Figure 4 A). After the mice were sacrificed by cervical dislocation and dissected, the entire colon was obtained. As Figure 4 shown in B, compared with the Control group, the colon fragility in the DSS group increased, the colon length decreased sharply, and the granular stools in the colon lumen became less. After treatment with B7-H5 mAb, the colon length was further shortened, and there were no formed granular stools in the intestinal lumen. This result indicates that blocking B7-H5 exacerbates the symptoms of DSS-induced murine enteritis, further suggesting its potential in the treatment of Crohn's disease.

[0083] The above description is only the preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Application of soluble protein B7-H5 in the preparation of Crohn's disease detection reagents.

2. The application according to claim 1, characterized in that, The detection reagent is used to detect the expression level of soluble protein B7-H5 in peripheral blood serum.

3. The application according to claim 2, characterized in that, The expression level of soluble protein B7-H5 in peripheral blood serum was detected using the ELISA method.

4. Application of soluble protein B7-H5 blocking antibody in the preparation of anti-inflammatory drugs.

5. The application according to claim 4, characterized in that, The anti-inflammatory drug is used to treat ulcerative colitis.

6. The application according to claim 4, characterized in that, The anti-inflammatory drug is used to treat Crohn's disease.

7. The application according to claim 4, characterized in that, The anti-inflammatory drug is used to negatively regulate macrophage activation.

8. The application according to claim 4, characterized in that, The concentration of the soluble protein B7-H5 blocking antibody is 8~12µg / µL.

9. A kit for detecting Crohn's disease, characterized in that, The kit includes reagents for detecting the expression level of soluble protein B7-H5 in peripheral blood serum.

10. The application according to claim 9, characterized in that, The reagent used to detect the expression level of soluble protein B7-H5 in peripheral blood serum includes an antibody that specifically binds to soluble protein B7-H5.

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