Application of kit for detecting fibronectin in evaluating intestinal injury state

Abstract

The invention belongs to the technical field of application of biomarker detection kits, and particularly relates to application of a kit for detecting fibronectin in evaluating the intestinal injury state, and the kit comprises an antibody specifically binding to fibronectin; the kit is used for detecting the fibronectin content in an excrement sample, and the intestinal injury state is evaluated based on the fibronectin content. In conclusion, the invention provides the application of the kit for detecting fibronectin, which is based on the excrement sample, is accurate in early stage and is superior to the existing marker, in evaluating the intestinal injury state.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection kit application technology, and particularly relates to the application of a kit for detecting fibronectin in assessing intestinal injury status. Background Technology

[0002] Gut health is central to maintaining overall homeostasis, and its imbalance is closely related to various intestinal and systemic diseases. Accurate assessment of gut health, particularly the early identification and dynamic monitoring of intestinal barrier damage, is crucial in clinical diagnosis and health management. Currently, researchers have identified several in vitro biomarkers for assessing intestinal barrier damage, such as intestinal fatty acid-binding protein (I-FABP), serum endotoxin (LPS), diamine oxidase (DAO), and D-lactic acid. However, the detection of these indicators requires blood samples, making them unsuitable for continuous, dynamic health monitoring or home-based monitoring. In contrast, detecting biomarkers in fecal samples offers advantages such as simple and non-invasive sampling; furthermore, fecal biomarkers come into direct contact with intestinal tissue, directly reflecting local intestinal lesions, thus possessing high clinical application value.

[0003] Currently, fecal calprotectin, as a non-invasive biomarker, is widely used in the clinical monitoring of intestinal inflammation and disease. However, intestinal barrier damage involves multidimensional pathophysiological changes, including mechanical barrier disruption, immune dysregulation, and microecological disturbances. Fecal calprotectin, as a single indicator, cannot systematically reflect the actual state of intestinal barrier damage. Although studies have attempted to find new fecal biomarkers related to intestinal injury and establish relevant detection kits and methods, few have demonstrated superior diagnostic performance compared to calprotectin.

[0004] Fibronectin (FN) is an important extracellular matrix protein and a crucial structural basis for the integrity of tissue mechanical barriers. During wound healing, FN directly participates in the functional reconstruction of the mechanical barrier by clearing necrotic tissue, guiding cell migration, and promoting granulation tissue formation. On the one hand, existing diagnostic kits detect fibronectin (FN) levels in plasma to aid in the diagnosis of diseases such as hepatitis, cirrhosis, and rheumatoid arthritis, but these do not address the assessment of intestinal injury. On the other hand, some technologies have developed detection methods for fecal samples, such as the colorectal cancer-related protein combined detection kit disclosed in CN116660536A, which detects colorectal cancer-related indicators by measuring protein markers such as CAT and LTF in feces.

[0005] Existing technologies have significant shortcomings: First, plasma fibronectin testing requires invasive blood collection, leading to poor patient compliance, and its association with intestinal injury status has not been explored, failing to meet the needs of intestinal injury assessment. Second, the core target of fecal test kits such as CN116660536A is colorectal cancer-related proteins, not fibronectin, and their application focuses on cancer detection, not early assessment and severity determination of intestinal injury. Third, traditional fecal biomarkers (such as calprotectin) have insufficient early sensitivity and weak correlation with the degree of intestinal tissue damage, making it difficult to achieve early warning and accurate grading of intestinal injury. Fourth, fibronectin in fecal samples has not yet been addressed by existing technologies, leaving a gap in its application as a specific biomarker for intestinal injury, resulting in a current lack of intestinal injury assessment tools that are non-invasive, have early sensitivity, and are accurate. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for detecting fibronectin and its application in assessing intestinal injury status, in order to solve the above-mentioned problems.

[0007] To achieve the above objectives, the present invention provides the following solution: The application of a kit for detecting fibronectin in assessing intestinal injury status includes: the kit containing an antibody that specifically binds to fibronectin; using the kit to detect the fibronectin content in a fecal sample, and assessing the intestinal injury status based on the fibronectin content.

[0008] Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) kit containing fibronectin antibodies, a chemiluminescent kit containing fibronectin antibodies, an immunochromatographic assay kit containing fibronectin antibodies, or a flow cytometry kit containing fibronectin antibodies.

[0009] Preferably, the steps for detecting the fibronectin content in a fecal sample include: S1. Weigh 0.05g-0.2g of fecal sample into a microcentrifuge tube, add 1mL-2mL of lysis buffer, homogenize thoroughly, and then centrifuge to obtain fecal protein extract. S2. After diluting the fecal protein extract by 50 to 200 times, the content of fibronectin in the extract is detected using the kit.

[0010] Preferably, in step S1, the amount of fecal sample weighed is 0.1g, and the microcentrifuge tube is 2mL.

[0011] Preferably, the volume of the lysis solution added is 1.5 mL.

[0012] Preferably, in step S2, the fecal protein extract is diluted 100 times before testing.

[0013] Preferably, the intestinal injury status includes an early assessment of intestinal mucosal injury.

[0014] Preferably, the assessment is based on the positive correlation between the concentration of fibronectin in fecal samples and the degree of intestinal tissue damage.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: In the early stages of intestinal mucosal injury, fecal fibronectin concentration shows a significant increase, while blood fibronectin concentration does not change significantly. This characteristic allows it to accurately reflect early local intestinal damage, avoiding the limitation of blood markers in capturing local lesions. In a mouse model of intestinal injury, fecal fibronectin showed a significant increase on day 1, much earlier than the traditional fecal marker calprotectin on day 3. Its peak concentration was nearly 30 times higher than that of the healthy control group, more than 3 times that of calprotectin and less than 2 times that of blood fibronectin. This strong signal response significantly reduces the risk of missing early, low-grade damage, providing a critical time window for early intervention in intestinal injury.

[0016] Fecal fibronectin concentration showed a significant positive correlation with the degree of intestinal pathological damage (r=0.8224, p=0.0023), while the correlation between the traditional marker calprotectin and the degree of intestinal pathological damage was relatively weak (r=0.5992, p=0.0450). This indicates that fecal fibronectin can not only indicate the presence of intestinal damage but also accurately quantify its severity, providing an objective and reliable basis for developing personalized treatment plans in clinical practice. This addresses the problem that existing single markers cannot systematically reflect the actual state of intestinal barrier damage.

[0017] In terms of population classification efficacy, fecal fibronectin performed better. Clinical sample test results showed a significant difference in fecal fibronectin concentration between the intestinal injury disease group and the healthy control group. Receiver operating characteristic (ROC) curve analysis showed that its area under the curve (AUC=0.732) was higher than that of the classic marker calprotectin (AUC=0.705), indicating a stronger ability to distinguish between patients with intestinal injury and healthy individuals. This effectively improves diagnostic accuracy, reduces the probability of clinical misjudgment, and meets the clinical need for precise assessment tools.

[0018] This technical solution also boasts significant practical advantages. The test sample is feces, which, compared to blood samples, is non-invasive, simple to operate, and has high patient compliance, making it particularly suitable for continuous dynamic health monitoring or home-based monitoring scenarios. Furthermore, the test kit supports multiple types, including enzyme-linked immunosorbent assay (ELISA) kits, chemiluminescence immunoassay kits, immunochromatographic kits, and flow cytometry kits, adapting to different testing needs. It allows for dynamic tracking of intestinal damage status and treatment effects through continuous sample collection, providing a powerful tool for the prediction, prognosis, and monitoring of intestinal barrier damage, filling a gap in clinical applications in this field.

[0019] In summary, this invention provides a kit for detecting fibronectin based on fecal samples, which is accurate early and superior to existing biomarkers, and its application in assessing intestinal injury status. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 A shows the trend of fecal fibronectin concentration changes during disease progression in mice with intestinal injury; Figure 1 B is a graph showing the trend of blood fibronectin concentration changes during disease progression in mice with intestinal injury; Figure 1 C is a graph showing the trend of fecal calprotectin concentration changes during disease progression in mice with intestinal injury; Figure 2 A is a Spearman correlation analysis graph showing the relationship between histopathological scores of mice with intestinal injury and fecal fibronectin concentration. Figure 2 B is a Spearman correlation analysis plot of the histopathological score of mice with intestinal injury and the concentration of fecal calprotectin. Figure 3 A is a comparison of the concentration levels of fecal calprotectin in the intestinal injury disease group and the healthy control group; Figure 3 B is a comparison chart of the concentration levels of fecal fibronectin in the intestinal injury disease group and the healthy control group; Figure 3 C represents the receiver operating characteristic (ROC) curve and the corresponding area under the curve (AUC) of fecal calprotectin and fibronectin concentration levels on intestinal injury diseases. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1 This embodiment establishes a mouse model of intestinal barrier injury, detects and compares the dynamic changes in the concentrations of fibronectin (FN) in feces and calprotectin in blood, and verifies the early indicative value of fecal fibronectin for intestinal injury. The specific steps are as follows: S1. Establishment of the intestinal barrier mouse model and collection of fecal and blood samples. Forty C57BL / 6J mice were randomly divided into an intestinal barrier injury group (DSS) and a control group (NC), with 20 mice in each group (each mouse was housed in a single cage).

[0024] The DSS group received a 3% DSS aqueous solution for 7 days, while the NC group received distilled water. During the experiment, the weight changes, occult blood reaction in excrement, and morphological characteristics of each mouse were measured daily.

[0025] S2, Fecal Sample Collection During the experiment, fecal samples were collected from each mouse regularly every day and stored at -80℃. S3, Blood Sample Collection Blood samples were collected from 5 mice in the DSS group and NC group on days 1, 3, 5 and 7, respectively. Serum was separated and stored at -80℃.

[0026] S4. Detect the concentration levels of fibronectin and calprotectin in fecal samples. Weigh 0.1g of fecal sample into a 2mL microcentrifuge tube, place it at 4℃, add 1.5mL of lysis buffer, and homogenize thoroughly. Then, centrifuge at 8000×g for 5 minutes at 4℃ to obtain fecal protein supernatant; The supernatant was dispensed and stored using a pipette; After diluting the supernatant 100 times, take 100 μL to determine the concentration of fibronectin in the sample. The specific method should be performed according to the instructions of the fibronectin ELISA kit. Take another 100 μL of supernatant to determine the concentration of calprotectin in the sample, following the instructions of the calprotectin ELISA kit.

[0027] S5. Detect the concentration level of fibronectin in serum samples. Dilute the collected serum sample 100 times, take 100 μL of the diluted serum, and determine the concentration of fibronectin in the serum sample. The specific method should be performed according to the instructions of the fibronectin ELISA kit.

[0028] The results are as follows Figure 1 As shown, compared to the control group, the concentration of fecal fibronectin in mice with intestinal barrier damage was significantly increased on day 1 and remained at a high level thereafter. Figure 1A); however, the concentration of fibronectin in their blood did not show a significant increase compared to the control group ( Figure 1 B). The results showed that fecal fibronectin had a good indicative effect on early intestinal injury, while blood fibronectin failed to indicate local intestinal injury.

[0029] In addition, we measured the concentration level of calprotectin in feces. As a classic biomarker, calprotectin in the feces of mice with intestinal barrier injury showed a significant increase only on the third day after DSS stimulation. Figure 1 (C) This result also indicates that the early diagnostic value of fecal fibronectin concentration measurement is superior to that of the existing typical biomarker calprotectin. Furthermore, the peak concentration of fecal fibronectin in mice with intestinal barrier injury was nearly 30 times higher than that in the normal control group, while the peak concentration of fecal calprotectin changed only 3 times, and the peak concentration of blood fibronectin changed less than 2 times, indicating that fecal fibronectin responds most strongly to intestinal barrier injury.

[0030] Example 2 This embodiment establishes a mouse model of intestinal barrier injury to explore the correlation between fecal fibronectin and calprotectin concentrations and the degree of intestinal tissue pathological damage, and to verify the indicative advantage of fecal fibronectin in the degree of intestinal damage. The specific steps are as follows: S1. Establishment of the intestinal barrier mouse model and collection of fecal samples. Twelve C57BL / 6J mice were housed individually and given 3% DSS solution as drinking water for 7 days. During the experiment, the weight changes, occult blood reaction in excrement, and morphological characteristics of each mouse were measured daily.

[0031] S2, Fecal Sample Collection Fecal samples were collected from each mouse on days 1, 3, 5 and 7 of the experiment and stored at -80℃. S3. Tissue sample collection and pathological analysis On days 1, 3, 5, and 7, fecal samples were collected from each mouse. Colon tissue samples were then collected from three mice for histopathological observation and assessment of the degree of pathological damage.

[0032] S4. Detect the concentration levels of fibronectin and calprotectin in fecal samples. Weigh 0.1g of fecal sample into a 2mL microcentrifuge tube, place it at 4℃, add 1.5mL of lysis buffer, and homogenize thoroughly. Then, centrifuge at 8000×g for 5 minutes at 4℃ to obtain fecal protein supernatant; The supernatant was dispensed and stored using a pipette; After diluting the supernatant 100 times, take 100 μL to determine the concentration of fibronectin in the sample. The specific method should be performed according to the instructions of the fibronectin ELISA kit. Take another 100 μL of supernatant to determine the concentration of calprotectin in the sample, following the instructions of the calprotectin ELISA kit.

[0033] S5. Spearman correlation analysis was performed to compare the concentrations of fecal fibronectin and calprotectin with the degree of histopathological damage.

[0034] The results are as follows Figure 2 As shown, the concentration level of fibronectin in the feces of mice with intestinal barrier damage was strongly positively correlated with the degree of histopathological damage (r=0.8224, p=0.0023). Figure 2 A), while the correlation between fecal calprotectin concentration and the degree of histopathological damage was relatively weak (r=0.5992, p=0.0450). Figure 2 B). The results showed that the concentration level of fecal fibronectin was a better indicator of the degree of intestinal tissue damage than the existing biomarker calprotectin.

[0035] Example 3 This embodiment collects fecal samples from individuals with intestinal barrier damage (colorectal cancer patients) and healthy individuals, detects the concentrations of fibronectin and calprotectin in the feces, uses the ROC curve method to evaluate the classification efficacy of fecal fibronectin for intestinal damage, and compares it with the classic biomarker calprotectin to further verify the effectiveness of the present invention. The specific steps are as follows: S1. Collection of fecal samples from individuals with intestinal barrier damage In this embodiment, stool samples were collected from 20 colorectal cancer patients and 36 healthy volunteers. All stool samples were stored at -80°C before unified testing. S2. Detect the concentration levels of fibronectin and calprotectin in fecal samples. Weigh 0.1g of fecal sample into a 2mL microcentrifuge tube, place it at 4℃, add 1.5mL of lysis buffer, and homogenize thoroughly. Then, centrifuge at 8000×g for 5 minutes at 4℃ to obtain fecal protein supernatant; The supernatant was dispensed and stored using a pipette; After diluting the supernatant 100 times, take 100 μL to determine the concentration of fibronectin in the sample. The specific method should be performed according to the instructions of the fibronectin ELISA kit. Take another 100 μL of supernatant to determine the concentration of calprotectin in the sample, following the instructions of the calprotectin ELISA kit.

[0036] S3. The predictive efficacy of fecal fibronectin and calprotectin for intestinal barrier damage was evaluated using the ROC curve method.

[0037] The results are as follows Figure 3 As shown, compared with the healthy control group, the levels of calprotectin and fibronectin in the feces of patients in the intestinal barrier injury group were significantly increased. Figure 3 A and Figure 3 B). Furthermore, ROC analysis was used to evaluate the predictive power of calprotectin and fibronectin levels for intestinal barrier damage / health status using the area under the curve (AUC). The results showed ( Figure 3 C) The classification efficacy of fecal fibronectin for patients with intestinal injury (AUC=0.732) is higher than that of classic calprotectin (AUC=0.705), indicating that fecal fibronectin has a better ability to identify intestinal injury at the population level and has good potential for clinical application.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a kit for detecting fibronectin in the assessment of the state of injury of the intestine, characterized in that, The kit comprises an antibody specifically binding to fibronectin; the content of fibronectin in a fecal sample is detected by using the kit, and the intestinal injury state is evaluated based on the content of fibronectin. The kit is an enzyme-linked immunoassay kit containing fibronectin antibodies, a chemiluminescence kit containing fibronectin antibodies, an immunochromatography kit containing fibronectin antibodies, or a flow fluorescence detection kit containing fibronectin antibodies.

2. Use of a kit for detecting fibronectin according to claim 1 for assessing the state of intestinal damage, characterized in that: The step of detecting the content of fibronectin in the fecal sample comprises:

3. Use of a kit for detecting fibronectin according to claim 1 for assessing the state of intestinal damage, characterized in that: S1, weighing 0.05g-0.2g of the fecal sample into a microcentrifuge tube, adding 1mL-2mL of lysis solution, homogenizing, and then centrifuging to obtain a fecal protein extract; S2, diluting the fecal protein extract by 50-200 times, and then detecting the content of fibronectin in the fecal protein extract by using the kit. In the step S1, the weighing amount of the fecal sample is 0.1g, and the specification of the microcentrifuge tube is 2mL.

4. Use of a kit for detecting fibronectin according to claim 3 for assessing the state of intestinal damage, characterized in that: The adding volume of the lysis solution is 1.5mL.

5. Use of a kit for detecting fibronectin according to claim 3 for assessing the state of intestinal damage, characterized in that: In the step S2, the fecal protein extract is diluted by 100 times before detection.

6. Use of a kit for detecting fibronectin according to claim 3 for assessing the state of intestinal damage, characterized in that: The intestinal injury state comprises early state evaluation of intestinal mucosal injury.

7. Use of a kit for detecting fibronectin according to claim 1 for assessing the state of intestinal damage, characterized in that: The evaluation is based on the positive correlation between the concentration of fibronectin in the fecal sample and the degree of intestinal tissue injury.

8. Use of a kit for detecting fibronectin according to claim 1 for assessing the state of intestinal damage, characterized in that: ​

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