Use of ITGA6 in preparation of a diagnostic reagent or kit for sepsis

Abstract

The application relates to the technical field of biological medicine, and provides application of ITGA6 in preparation of a sepsis diagnosis and condition monitoring reagent or kit. The reagent or kit is used for diagnosing and / or condition monitoring sepsis by detecting the ITGA6 level in blood plasma. The application detects the ITGA6 level in blood plasma by an ELISA method to diagnose and condition monitor sepsis. ITGA6 is used as a biomarker, has high sensitivity and high specificity, effectively increases the accuracy of sepsis diagnosis, and assists in evaluating the severity and prognosis monitoring of the condition, and has the advantages of simple operation, no intervention, high flux, low cost, no radiation and the like in the diagnosis of sepsis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of ITGA6 in the preparation of reagents or kits for the diagnosis and monitoring of sepsis. Background Technology

[0002] Sepsis is a syndrome characterized by multiple organ dysfunction caused by infection and is a leading cause of death in intensive care unit (ICU) patients. Treatment principles for sepsis include anti-infection therapy, fluid resuscitation, and multi-organ support therapy. While attention to the diagnosis and treatment of sepsis continues to increase, the etiology and pathogenesis of sepsis in ICU patients remain incompletely understood. Currently, sepsis is considered a complex disease based on infection and mediated by dysregulation of both innate and adaptive immune systems. Therefore, providing an accurate diagnostic reagent or kit for sepsis and its monitoring is urgently needed in clinical practice, enabling clinicians to accurately diagnose and monitor the condition, thereby improving the prognosis of sepsis patients. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides the application of ITGA6 in the preparation of reagents or kits for the diagnosis and monitoring of sepsis, with the aim of solving the problems mentioned in the background art.

[0004] ITGA6 (human integrin-α6) belongs to the integrin protein family. ITGA6 can be used as a surface marker for hematopoietic stem cells (HSCs) and has the potential to generate long-term multi-lineage differentiation.

[0005] This invention provides the application of ITGA6 in the preparation of reagents or kits for the diagnosis and monitoring of sepsis, wherein the reagents or kits diagnose and / or monitor sepsis by detecting the level of ITGA6 in plasma.

[0006] Furthermore, the ITGA6 level was negatively correlated with the SOFA score in patients with sepsis.

[0007] Furthermore, the ITGA6 level was reduced in the plasma of patients with sepsis.

[0008] Furthermore, the ITGA6 level was reduced in the plasma of patients with septic shock.

[0009] Furthermore, the ITGA6 level was reduced in the plasma of non-surviving sepsis patients at 28 days.

[0010] Furthermore, the reagent or kit is used to detect the level of ITGA6 in plasma by ELISA.

[0011] Furthermore, the reagent or kit is used to detect the expression level and / or concentration of ITGA6 protein in plasma by ELISA.

[0012] Furthermore, the reagent or kit is used to detect the concentration of ITGA6 protein in plasma as <22.22 pg / ml by ELISA, which indicates that the patient is a sepsis patient.

[0013] This invention offers the following technical advantages: It uses methods such as ELISA to detect ITGA6 levels in plasma for the diagnosis and monitoring of sepsis. ITGA6, as a biomarker, exhibits high sensitivity and specificity. This effectively increases the accuracy of sepsis diagnosis and assists in assessing disease severity and prognostic monitoring. Furthermore, it offers advantages in sepsis diagnosis, including simple operation, non-invasiveness, high throughput, low cost, and no radiation risks or other potential hazards. Attached Figure Description

[0014] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0015] Figure 1 In Example 1 of this invention, Oscar deep proteomic sequencing analysis revealed that the relative expression level of ITGA6 in plasma was significantly reduced in the sepsis group and was correlated with disease severity and prognosis, wherein:

[0016] Figure 1 A in the diagram represents the signaling pathways enriched by KEGG: the top 20 signaling pathways with significantly enriched differentially expressed proteins in the sepsis group and the control group, among which the hematopoietic lineage pathway is one of the significantly enriched pathways;

[0017] Figure 1 B in the diagram is a heatmap of differential protein expression in the hematopoietic lineage pathway, where the relative expression of ITGA6 showed the greatest difference between the sepsis group and the control group.

[0018] Figure 1 C in the figure represents the statistical graph of the relative expression level of ITGA6 in the plasma of the sepsis group and the control group. The relative expression level of ITGA6 was significantly lower in the sepsis group. **** indicates P < 0.0001.

[0019] Figure 1 D in the figure is a statistical graph of the relative expression level of ITGA6 in plasma of the non-septic shock group and the septic shock group. The relative expression level of ITGA6 was significantly reduced in the septic shock group. *** indicates P < 0.001.

[0020] Figure 1 E in the figure is a statistical graph showing the correlation between the relative expression level of ITGA6 and the SOFA score in sepsis patients. The relative expression level of ITGA6 was negatively correlated with the SOFA score (P < 0.0001).

[0021] Figure 2This is the analysis result of Example 2 of the present invention, which uses ELISA to verify the effect of plasma ITGA6 levels on the diagnosis of sepsis and the monitoring of its condition, wherein:

[0022] Figure 2 In the figure, A is a statistical graph of the plasma ITGA6 levels in the sepsis group and the control group. The ITGA6 level was significantly lower in the sepsis group. **** indicates P < 0.0001.

[0023] Figure 2 B in the figure represents the ROC curve analysis results of the effect of plasma ITGA6 on the diagnosis of sepsis;

[0024] Figure 2 C in the figure is a statistical graph of the plasma ITGA6 level in the non-septic shock group and the septic shock group. The ITGA6 level was significantly lower in the septic shock group, ** indicates P=0.0081;

[0025] Figure 2 D in the figure is a statistical graph showing the correlation between the plasma ITGA6 level and SOFA score in sepsis patients. The ITGA6 level and SOFA score were significantly negatively correlated (P < 0.0001).

[0026] Figure 2 E in the figure is a statistical graph of ITGA6 levels in plasma of the survivor group and the non-survivor group. The ITGA6 level was significantly lower in the non-survivor group. * indicates P=0.0266. Detailed Implementation

[0027] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0029] This invention provides the application of ITGA6 in the preparation of reagents or kits for the diagnosis and monitoring of sepsis, wherein the reagents or kits diagnose and / or monitor sepsis by detecting the level of ITGA6 in plasma.

[0030] In some embodiments, ITGA6 levels are negatively correlated with SOFA scores in patients with sepsis.

[0031] In some embodiments, ITGA6 levels were reduced in the plasma of patients with sepsis (P < 0.0001).

[0032] In some embodiments, ITGA6 levels were reduced in the plasma of patients with septic shock, P=0.0081.

[0033] In some embodiments, ITGA6 levels were reduced in the plasma of non-surviving sepsis patients at 28 days, P=0.0266.

[0034] In some embodiments, the reagent or kit detects the level of ITGA6 in plasma using an ELISA (enzyme-linked immunosorbent assay).

[0035] In some embodiments, the reagent or kit detects the expression level and / or concentration of ITGA6 protein in plasma using an ELISA method.

[0036] In some embodiments, a patient is diagnosed with sepsis if the concentration of ITGA6 protein in plasma is <22.22 pg / ml by ELISA.

[0037] Example 1:

[0038] 1. Sample Collection and Grouping: Sepsis Group: Suspected sepsis patients were diagnosed according to the Sepsis diagnostic criteria (Sepsis-3). Seventeen patients diagnosed with sepsis at the First Affiliated Hospital of Nanchang University between January and June 2024 were collected. Control Group: Twelve patients who were age- and weight-matched to sepsis patients during the same period, without infectious diseases, hematological diseases, or major congenital diseases, were collected. Plasma from both the sepsis and control groups was collected and stored at -80°C for deep protein sequencing to identify plasma protein biomarkers associated with sepsis.

[0039] 2. By analyzing the significant differences in proteins between the sepsis group (n=17) and the control group (n=12) using Oscar Deep Blood Protein (Shanghai Zhongke New Life Biotechnology Co., Ltd.), the sepsis patients were further divided into the sepsis non-shock group and the sepsis shock group (more severe condition).

[0040] Analysis results as follows Figure 1 As shown, the results indicated that significantly different proteins between the sepsis group and the control group were enriched in signaling pathways such as hematopoietic cell lineage (…). Figure 1 (A in the original text); ITGA6 is the most significantly differentiated protein in the hematopoietic cell lineage signaling pathway ( ). Figure 1 (B in the text); ITG6 levels in the plasma of sepsis patients were significantly reduced ( Figure 1 The C in the group was significantly reduced, and the reduction was more pronounced in the septic shock group. Figure 1 (D in the text); SOFA score correlation analysis showed that the expression level of ITGA6 in the plasma of sepsis patients was significantly negatively correlated with the SOFA score (D in the text). Figure 1The SOFA score (E in the original text) is a tool used to assess the severity of sepsis in patients; a higher SOFA score indicates a more severe sepsis condition.

[0041] Example 2:

[0042] 1. Sample Collection and Grouping: Sepsis Group: Patients suspected of having sepsis were diagnosed according to the Sepsis diagnostic criteria (Sepsis-3). Twenty-six patients diagnosed with sepsis at the First Affiliated Hospital of Nanchang University between July 2024 and February 2025 were collected. Control Group: Twenty-nine patients matched for age and weight with sepsis patients during the same period, without infectious diseases, hematological disorders, or major congenital diseases, were collected. Plasma from both the sepsis group and the control group was stored at -80°C.

[0043] 2. The level of ITGA6 in plasma was detected using the human anti-human ITGA6 ELISA kit (catalog number: ZK-15684, Shanghai Zhenke Biotechnology Co., Ltd.). The specific detection steps are as follows:

[0044] (1) Place the kit at room temperature for 20 minutes to allow it to return to room temperature. Remove the strips to be used from the foil bag. Seal and store the unused strips at 4°C.

[0045] (2) Set up standard wells and sample wells according to the number of samples. Add different concentrations of standard to each standard well at 50 μL.

[0046] (3) Sample dilution: Dilute the plasma 5 times, i.e., 10 μL of plasma, add 40 μL of diluent and mix well.

[0047] (4) Sample addition: Add 50 μL of different concentrations of standard to each standard well, add 50 μL of diluted serum / plasma sample to each sample well, and add to the blank well. 50ul.

[0048] (5) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard wells and sample wells, seal the wells with a sealing film, and incubate in a 37°C incubator with aluminum foil in the dark for 60 min.

[0049] (6) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0050] (7) Add 50 μL each of substrate A (2% hydrogen peroxide) and substrate B (0.5% TMB colorimetric solution) to each well and incubate in a constant temperature oven at 37°C with aluminum foil in the dark for 15 min.

[0051] (8) Add 50 μL of stop solution to each well and measure within 15 min. Measure the OD value of each well at a wavelength of 450 nm.

[0052] (9) The horizontal axis is named the standard concentration and the vertical axis is named the OD value. Plot the linear regression curve of the standard and calculate the concentration value of each sample according to the curve equation.

[0053] Results analysis:

[0054] The plasma levels (concentrations) of ITGA6 in 26 sepsis patients and 29 control patients were analyzed using ELISA. The results are as follows: Figure 2 As shown in Figure A, the results indicate that the plasma ITGA6 level was significantly lower in the sepsis group compared to the control group.

[0055] The ROC curve analysis results are as follows: Figure 2 As shown in B, the results indicate that ITGA6 has an AUC of 0.90 in diagnosing sepsis patients. The optimal cutoff value of 22.22 pg / ml corresponds to a sensitivity of 88.46% and a specificity of 89.66%. This demonstrates that ITGA6 < 22.22 pg / ml has extremely high sensitivity and specificity in diagnosing sepsis.

[0056] like Figure 2 As shown in C, sepsis patients were further divided into a non-septic shock group and a septic shock group. The results showed that, compared with the non-septic shock group, the plasma ITGA6 level in the septic shock group was significantly lower; Figure 2 As shown in D, a correlation analysis was performed between plasma ITGA6 levels and SOFA scores in sepsis patients. The results showed that ITGA6 levels were negatively correlated with SOFA scores in sepsis patients. This indicates that plasma ITGA6 can assess the severity of sepsis and provides important guidance for the clinical treatment of sepsis.

[0057] like Figure 2 As shown in E, sepsis patients were divided into a survival group and a non-survival group based on their 28-day survival status. The results showed that the plasma ITGA6 level was significantly lower in the non-survival group compared to the survival group, indicating that the plasma ITGA6 level has a predictive effect on the prognosis of sepsis.

[0058] In summary, detecting ITGA6 levels in plasma using methods such as ELISA is effective for the diagnosis and disease monitoring of sepsis. ITGA6, as a biomarker, exhibits high sensitivity and specificity, effectively increasing the accuracy of sepsis diagnosis and aiding in the assessment of disease severity and prognosis. Furthermore, it offers advantages in sepsis diagnosis, including simple operation, non-invasiveness, high throughput, low cost, and no radiation risks or other potential hazards.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of ITGA6 in the preparation of sepsis diagnostic kits, characterized by: The kit diagnoses sepsis by detecting the level of ITGA6 in plasma.

2. The application as described in claim 1, characterized in that: The ITGA6 level was reduced in the plasma of patients with sepsis.

3. The application as described in claim 2, characterized in that: The kit detects the concentration of ITGA6 protein in plasma using an ELISA method.

4. The application as described in claim 3, characterized in that: The kit was used to detect that the concentration of ITGA6 protein in plasma was <22.22 pg / ml by ELISA, which indicated that the patient was a sepsis patient.

Images