Early warning marker combination and application and product for multiple trauma

Abstract

The application provides a multiple trauma early warning marker combination and application and product, and belongs to the biomarker field.The application provides a marker combination for multiple trauma early warning, which comprises Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and through detection and interpretation of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum, multiple trauma combined with MODS and prognosis can be predicted.The detection reagent for Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 detection can be used for preparing a kit for predicting multiple trauma combined with MODS and prognosis, and is simple, efficient and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of biomarkers, and relates to multiple trauma, specifically to a combination of early warning biomarkers for multiple trauma and their applications and products. Background Technology

[0002] Multiple trauma is a common emergency condition, typically involving injuries to two or more anatomical sites. It is often accompanied by massive hemorrhage, shock, and organ dysfunction. Severe multiple trauma patients, if not treated promptly, frequently develop multiple organ dysfunction syndrome (MODS), a leading cause of death in trauma patients. Because the condition of patients with multiple trauma and MODS changes rapidly, monitoring their condition and assessing their prognosis are crucial. Traditional monitoring methods relying on the individual experience of clinicians lack scientific rigor and accuracy.

[0003] Eosinophil chemokine (Eotaxin), also known as chemokine-11 (CCL11), is a member of the monocyte chemotactic protein family of CC chemokines. Eotaxin exhibits strong chemotactic activity towards eosinophils, basophils, and Th2 lymphocytes. Th2 cytokines, such as IL-4, IL-10, IL-13, complement factors, and immune complexes, can induce eosinophils, T cells, B cells, macrophages, endothelial cells, fibroblasts, epithelial cells, chondrocytes, microglia, keratinocytes, and smooth muscle cells to produce Eotaxin. The Eotaxin gene is expressed in various tissues, including the heart, lungs, kidneys, lymph nodes, thymus, and intestines. Eotaxin is also present in human lung epithelial cells, pleural mesothelial cells, bronchial respiratory tract epithelial cells, and smooth muscle cells. Eotaxin can rapidly cross the blood-brain barrier and be transported from the bloodstream to the brain. An age-related increase in eotaxin is associated with cognitive impairments in executive function, episodic memory, and semantic memory; therefore, this chemokine is known as the "endogenous cognitive decline chemokine" (ECDC) or the "accelerating brain aging chemokine" (ABAC). In patients with schizophrenia, an increase in eotaxin is associated not only with impaired cognitive function but also with the form of thought disorder.

[0004] Interferon-gamma inducible protein 10 (IP-10) is a pro-inflammatory chemokine secreted by various cell types. IP-10 activates T lymphocytes (Th1), NK cells, macrophages, dendritic cells, and B cells. Changes in IP-10 expression levels are associated with inflammatory diseases, including infectious diseases, angiogenesis, immune dysfunction, and tumor development. Mature human IP-10 shares 68% amino acid sequence identity with mouse and cytokine cells. IP-10 exerts its biological effects by binding to CXCR3, a seven-transmembrane G protein-coupled receptor expressed paracrinely or autocrinely on activated T and B lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages. IP-10 induction is primarily dependent on the C-terminal region of CXCR3, which is crucial for IP-10 ligand-induced CXCR3 internalization, chemotaxis, and calcium mobilization. IP-10's potent chemotactic activity on activated lymphocytes enables it to modulate innate and adaptive immunity, induce tissue damage, and regulate tumorigenesis. IP-10 is a pleiotropic molecule capable of exerting effective biological functions, including promoting chemotaxis in CXCR3+ cells, inducing apoptosis, regulating cell growth and proliferation, and angiogenesis in infectious and inflammatory diseases and cancer.

[0005] Interleukins (ILs) are a class of cytokines secreted by immune cells (such as T cells and macrophages), primarily involved in immune regulation, inflammatory responses, and hematopoiesis. At least 38 interleukin members have been identified, including IL-1, IL-2, IL-6, IL-10, and IL-23. Among them, interleukin-18 (IL-18) is a pro-inflammatory cytokine belonging to the IL-1 superfamily, mainly produced by immune cells such as macrophages and dendritic cells. It participates in anti-infection, autoimmune diseases, and tumor regulation by activating immune responses, and is closely related to the occurrence and development of inflammatory diseases. IL-18 binds to its receptor IL-18Rα, activating the NF-κB and MAPK signaling pathways, promoting the release of inflammatory factors such as interferon-gamma (IFN-γ), and enhancing the activity of T cells and natural killer (NK) cells. In the early stages of infection, IL-18 synergistically enhances antiviral and antibacterial immune responses with IL-12.

[0006] Monocyte chemotactic protein 1 (MCP-1) is a cytokine that exhibits chemotactic activity towards monocytes, but not neutrophils. Primarily secreted by leukocytes and stromal cells in the hematopoietic microenvironment, it can also bind to the surface of endothelial cells, playing a crucial role in the entire inflammatory process and activating corresponding inflammatory transcription factors. In vivo and in vitro experiments have confirmed that MCP-1 possesses chemotactic activity towards monocytes, activating monocytes and macrophages, increasing intracytoplasmic Ca2+ concentration, producing and releasing superoxide anions, releasing lysozyme, upregulating the expression of monocyte and macrophage adhesion molecules such as the integrin family β2 group and α4 molecules, and the production of cytokines IL-1 and IL-6. Activated macrophages can inhibit tumor cell growth. MCP is also a chemotactic and activator of basophils, particularly strongly stimulating basophil degranulation and histamine release.

[0007] Monocyte chemokine 4 (MCP-4), a member of the chemokine family, is primarily responsible for attracting monocytes, eosinophils, and T cells to sites of inflammation. The role of MCP-4 in immune regulation, inflammatory responses, and certain diseases has been extensively studied. MCP-4 is a chemokine that recruits different types of immune cells, especially monocytes, eosinophils, and memory T cells, by binding to corresponding chemokine receptors (such as CCR2 and CCR3). Therefore, it plays a crucial role in regulating the migration and localization of immune cells. MCP-4 plays a role in various inflammation-related diseases, including asthma, allergic diseases, and rheumatoid arthritis. Its elevated expression levels in these diseases indicate that MCP4 is involved in the regulation of pathological immune responses and inflammation. MCP4 is mainly secreted by activated immune cells, endothelial cells, and certain tissue cells. It participates in chronic and acute inflammatory responses by regulating the migration of immune cells to sites of inflammation.

[0008] Du Qirong et al. found that the area under the ROC curve of serum HMGB-1, CK, Mb combined with APACHE II score in diagnosing MODS was 0.958, which was higher than that of a single indicator, suggesting that the combined diagnosis of multiple traumas complicated with MODS in the emergency department has good value. The study also analyzed the predictive value of serum HMGB-1, CK, Mb combined with APACHE II score in predicting mortality in patients with MODS, and found that the combined prediction was higher than the diagnostic value of each indicator alone, suggesting that the combined diagnosis has good value in prognostic evaluation of patients with multiple traumas complicated with MODS in the emergency department, making up for the limitations of a single indicator, and serological indicators can be dynamically monitored. The disadvantage of this technique is that it requires the measurement of four indicators, which is cumbersome and costly (Du Qirong, Pan Shuming, Li Ming, et al. Value of serum markers combined with acute physiology and chronic health score in diagnosis and prognosis of multiple traumas complicated with multiple organ dysfunction syndrome [J]. Chinese Journal of Clinical Physicians, 2022).

[0009] Chinese patent CN2024100411303 discloses an inflammatory factor composition, model, and kit for early warning of MODS. The inflammatory factor composition includes IL6, IL8, IFN-γ, STNF-RII, BLC, and IL1RA, which can accurately and rapidly predict MODS caused by cytokine storms in the early stages of severe trauma, helping to improve the success rate of treatment for severe trauma. ROC curve analysis was used to analyze the early warning efficacy of the six selected target cytokines for MODS: the standardized concentration values ​​of the six target cytokines (IL6, IL8, IFN-γ, STNF-RII, BLC, and IL1RA) from the subjects were input into the target MODS risk assessment model. ROC curves were plotted based on the standard concentrations of six target cytokines (IL6, IL8, IFN-γ, STNFRII, BLC, and IL1RA) in the subjects. The results showed that the area under the ROC curve for these six target cytokines was 0.9722, indicating a high area under the ROC curve, good specificity and sensitivity, and suggesting that the combined predictive efficacy of these six factors is good, meaning the model has good early warning efficacy for MODS. However, this technique requires the measurement of six indicators, which is a complex and costly process. Summary of the Invention

[0010] This invention addresses the problems of existing technologies, such as the need to measure a large number of indicators, cumbersome processes, and high costs, by providing a combination of biomarkers for early warning of multiple trauma, along with their applications and products. This invention provides a biomarker combination for early warning of multiple trauma, including Eotaxin, MCP-4, IP-10, IL-18, and MCP-1. This invention predicts multiple trauma complicated with MODS by detecting and interpreting Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in serum. The detection reagents for Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 can be used to prepare kits for predicting multiple trauma complicated with MODS, which is simple, efficient, and cost-effective.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] On one hand, the present invention provides a combination of biomarkers for early warning of multiple traumas, including Eotaxin, MCP-4, IP-10, IL-18 and MCP-1; wherein Eotaxin is the serum concentration level of eosinophil chemokine, MCP-4 is the serum concentration level of monocyte chemoattractant protein 4, IP-10 is the serum concentration level of interferon-γ-induced protein 10, IL-18 is the serum concentration level of interleukin-18, and MCP-1 is the serum concentration level of monocyte chemoattractant protein 1.

[0013] On the other hand, the present invention provides the application of the detection reagent of the above-described biomarker composition in the preparation of a kit for predicting multiple traumatic events complicated with MODS.

[0014] Preferably, the detection reagent is used for the quantitative or semi-quantitative detection of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0015] Preferably, the quantitative or semi-quantitative method includes any one or more of ELISA detection, chemiluminescence detection, Dot blot detection, Western blot detection, immunochromatography, and immunohistochemistry.

[0016] Preferably, the prediction of multiple traumatic events combined with MODS is achieved through the interpretation of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0017] Preferably, the critical values ​​of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 are 221.48 ng / L, 158.43 ng / L, 201.86 ng / L, 89.66 ng / L and 594.23 ng / L, respectively.

[0018] When Eotaxin ≥ 221.48 ng / L, MCP-4 ≥ 158.43 ng / L, IP-10 ≥ 201.86 ng / L, IL-18 ≥ 89.66 ng / L and MCP-1 ≥ 594.23 ng / L, it is predicted to be multiple trauma combined with MODS.

[0019] When Eotaxin < 221.48 ng / L, MCP-4 < 158.43 ng / L, IP-10 < 201.86 ng / L, IL-18 < 89.66 ng / L, and MCP-1 < 594.23 ng / L, it is predicted to be multiple traumatic non-MODS.

[0020] Preferably, the kit is used for detection and includes the following steps:

[0021] Sample collection, sample pretreatment, determination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and interpretation of results.

[0022] On the other hand, the present invention provides a kit for predicting multiple traumatic events complicated with MODS, the kit comprising all reagents for quantitative or semi-quantitative detection of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0023] Preferably, the kit includes Eotaxin antibody, MCP-4 antibody, IP-10 antibody, IL-18 antibody, MCP-1 antibody, magnetic beads, acridine ester, and quantitative or semi-quantitative detection reagents.

[0024] Preferably, the quantitative or semi-quantitative detection reagent includes any one or more of the following: magnetic bead preservation solution, acrid ester preservation solution, sample processing solution, pre-activation solution, activation solution, magnetic bead cleaning solution, and magnetic bead sealing solution.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention detects and interprets Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in serum. When Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 are all greater than or equal to a critical value, it is determined to be multiple trauma combined with MODS; when Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 are all less than the critical value, it is determined to be non-MODS. The detection reagents for Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 can be used to prepare kits for predicting multiple trauma combined with MODS.

[0027] 2. This invention can predict multiple traumatic events complicated with MODS by detecting and interpreting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum. It requires fewer detection items, is more efficient, and reduces costs. Attached Figure Description

[0028] Figure 1 ROC curves for predicting multiple traumatic events complicated with MODS using serum Eotaxin, MCP-4, IP-10, IL-18, and MCP-1. Detailed Implementation

[0029] Unless otherwise specified, all raw materials used in this invention are commercially available products and their sources are not specifically limited.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or the manufacturer's recommendations. All reagents or instruments without specified manufacturers are commercially available conventional products. To better illustrate this invention, numerous specific details are given in the following detailed embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques are found in many publications, such as *Molecular Cloning: A Laboratory Manual (4th Edition)* (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al. Current Protocols in Molecular Biology It is also described in Greene Publishing Association and Wiley-Interscience.

[0031] Research on cohorts and data acquisition methods

[0032] (1) General Information

[0033] This prospective study included 148 patients diagnosed with severe multiple injuries at Peking University People's Hospital. 78 patients with multiple injuries with multiple lesions (MODS) were assigned to the MODS group, and 70 patients without MODS served as the control group (non-MODS group). In the MODS group, there were 40 males and 38 females, aged 21-54 years (mean 39.18 ± 10.32 years). Causes of injury were: traffic accidents (35 cases), violent injuries (23 cases), and other causes (20 cases). Number of injured sites: ≤3 in 42 cases, >3 in 36 cases. In the control group, there were 39 males and 31 females, aged 23-55 years (mean 38.57 ± 9.65 years). Causes of injury were: traffic accidents (30 cases), violent injuries (23 cases), and other causes (17 cases). Number of injured sites: ≤3 in 40 cases, >3 in 30 cases. There were no statistically significant differences in gender, age, body mass index, cause of injury, and number of injured sites between the MODS group and the control group (P>0.05), indicating that they were comparable. Specific statistical information is shown in Table 1.

[0034] Table 1. Statistics and Explanation of Research Cohort Data

[0035]

[0036] Note: "-" indicates that there is no corresponding data here.

[0037] Inclusion criteria: ① Meeting the diagnostic criteria for severe multiple injuries as outlined in the "Expert Consensus on Multiple Trauma Cases and Diagnosis (2013 Edition)"; ② Injury Severity Score (ISS score) > 16 points. Exclusion criteria: ① Having other infectious diseases; ② Having received treatment prior to consultation.

[0038] In this invention, all patients and their families provided informed consent and signed informed consent forms. This study was reviewed and approved by the Ethics Committee of Peking University People's Hospital.

[0039] (2) Blood collection and analysis methods

[0040] Five mL of fasting venous blood was collected from all subjects in heparin anticoagulant tubes. After centrifugation, the supernatant was separated and stored at -80°C for later analysis. The concentrations of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in serum were determined using immunological methods.

[0041] (3) Data processing

[0042] SPSS 25.0 statistical software was used for data analysis and processing. Count data are expressed as number of cases or percentages, and comparisons between groups are expressed using the x-axis. 2 Tests were performed; normally distributed measurement data were expressed as mean ± standard deviation, and t-tests were used for comparisons between two groups. Pearson correlation was used for correlation analysis; receiver operating characteristic (ROC) curves were used for predictive value analysis; and multivariate logistic regression analysis was used to analyze influencing factors. A p-value < 0.05 was considered statistically significant.

[0043] Example 1: A method for assessing the risk of multiple traumatic events and MODS

[0044] I. Serum marker detection

[0045] The determination of serum indicators includes, but is not limited to, ELISA and chemiluminescence methods, among which the chemiluminescence method includes the following acridinium ester chemiluminescence scheme:

[0046] (1) Preparation of basic solution

[0047] 1.1 Preparation of magnetic bead preservation solution: 79.8% 0.01M phosphate buffer, 10% fetal bovine serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20 by volume fraction;

[0048] 1.2 Preparation of acridinium ester preservation solution: 69.9% by volume of 0.01M phosphate buffer, 30% glycerol and 0.1% Proclin 300, and then add 1% by mass of BSA.

[0049] 1.3 Sample processing solution preparation: Add 1% BSA, 0.1% Triton X-100 and 0.3% Proclin 300 to 0.01M phosphate buffer at pH 7.4.

[0050] 1.4 Preparation of pre-activation solution: Add 0.08 mol / L hydrogen peroxide and 0.02 mol / L nitric acid to purified water and mix well.

[0051] 1.5. Preparation of activating solution: Add 1.0 mol / L sodium hydroxide and 4.5 g / L Triton 100 to purified water.

[0052] 1.6 Preparation of magnetic bead cleaning solution: Prepare 0.2M MES buffer and add 0.02% (v / v) Proclin300.

[0053] 1.7 Preparation of magnetic bead blocking solution: 0.01M phosphate buffer (99.4% by volume) and 0.6% Tween 20, then add 0.5% BSA (by mass).

[0054] (2) Preparation of antibodies coated with magnetic beads

[0055] Eotaxin-coated antibody (purchased from abcam, catalog number ab133604) was used for Eotaxin assay; MCP-4-coated antibody (purchased from abcam, catalog number ab224593) was used for MCP-4 assay; IP-10-coated antibody (purchased from abcam, catalog number ab283681) was used for IP-10 assay; interleukin-18-coated antibody (purchased from abcam, catalog number ab245697) was used for interleukin-18 assay; and MCP-1-coated antibody (purchased from abcam, catalog number ab9858) was used for MCP-1 assay.

[0056] 2.1 Take 200 μL of magnetic beads (EM1-100 / 40 (high carboxyl) magnetic microspheres, catalog number 23710087) and add them to a 2 mL centrifuge tube. Perform magnetic separation for 3 min and then discard the supernatant.

[0057] 2.2 Add 400 μL of magnetic bead cleaning solution to the centrifuge tube, shake to mix, magnetically separate to remove the supernatant, and wash twice.

[0058] 2.3 Weigh a certain amount of EDC and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml EDC solution; weigh a certain amount of NHS and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml NHS solution.

[0059] 2.4 Add 100 μL of EDC solution and 100 μL of NHS solution to the centrifuge tube, and shake to mix.

[0060] 2.5 Place the two centrifuge tubes on a mixer and activate for 30 minutes. Adjust the speed appropriately to ensure the liquid flows steadily downwards when inverted.

[0061] 2.6 After activation, wash twice with 2 volumes of 0.02M MES buffer and remove the supernatant.

[0062] 2.7 Add 120 μg of coated antibody to a centrifuge tube, bring the volume to 300 μL with 0.01 M phosphate buffer, and couple for 3 h.

[0063] 2.8 Wash twice with 600 μL of magnetic bead blocking solution and remove the supernatant.

[0064] 2.9 Add 600 μL of magnetic bead blocking solution to the centrifuge tube, shake to mix for 30 min, and discard the supernatant.

[0065] 2.10 Wash twice with 600 μL of magnetic bead preservation solution, remove the supernatant, and then transfer to 30 mL of magnetic bead preservation solution to obtain the working solution of magnetic bead coating.

[0066] (3) Preparation of acridine ester labeled antibody

[0067] Eotaxin-labeled antibody (purchased from abcam, catalog number ab226143) was used for Eotaxin assay; MCP-4-labeled antibody (purchased from abcam, catalog number ab206405) was used for MCP-4 assay; IP-10-labeled antibody (purchased from abcam, catalog number ab307997) was used for IP-10 assay; interleukin-18-labeled antibody (purchased from abcam, catalog number ab207324) was used for interleukin-1 assay; and MCP-1-labeled antibody (purchased from abcam, catalog number ab214819) was used for MCP-1 assay.

[0068] 3.1 Take 200 μg of labeled antibody and dissolve it in 0.01 M phosphate buffer. Add acridine ester (9 mg / mL NSP-SA-NHS, dissolved in DMSO). Add more antibody to make the final concentration of labeled antibody 2 mg / mL. The final mass ratio of antibody to acridine ester is 1:10.

[0069] 3.2. React at 25°C in the dark for 3 hours on a constant temperature shaker.

[0070] 3.3 Add lysine (acridinium ester: lysine = 1:140, molar concentration ratio), block the reaction, and react for 20 min.

[0071] 3.4 After the reaction was completed, the buffer solution was replaced using a 50KD dialysis bag. The dialysis buffer was 0.01M phosphate buffer. Dialysis was performed 4 times in total, with each dialysis lasting 3 hours.

[0072] 3.5 After dialysis, add glycerol until the final antibody concentration is 0.5 mg / mL, and set aside for use.

[0073] 3.6. Acridinium ester was diluted with acridinium ester preservation solution to a final antibody concentration of 8 μg / mL to obtain acridinium ester-labeled antibody working solution.

[0074] (4) Sample preprocessing steps:

[0075] Mix 10 μL of the test sample with 190 μL of sample processing solution.

[0076] (5) Detection and calculation in the sample to be tested

[0077] The detection was performed using a fully automated chemiluminescence analyzer, and the specific operation is as follows:

[0078] 5.1 Take 100 μL of the pretreated sample, add 50 μL of the magnetic bead coating working solution, mix well, incubate at 37℃ for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen complex.

[0079] 5.2 Add 50 μL of acridine ester labeled antibody working solution to the reaction vessel containing the magnetic bead-antigen complex, mix well, incubate at 37°C for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex.

[0080] 5.3 Add 100 μL of pre-excitation solution and 100 μL of excitation solution to the reaction vessel containing the magnetic bead-antigen-detection antibody complex, mix thoroughly, and then measure the maximum luminescence intensity.

[0081] 5.4. Based on the luminescence intensity measured from the standard samples, a standard curve is fitted. The results are then analyzed using the standard curve (R² of the standard curve). 2 The concentration levels of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in the sample were calculated (all values ​​were greater than 0.98).

[0082] II. Measurement Results and Analysis

[0083] (1) Comparison of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 levels between the MODS group and the control group

[0084] Serum levels of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in the MODS group were significantly higher than those in the control group (without MODS), with statistically significant differences (P<0.05), as shown in Table 2.

[0085] Table 2 Comparison of indicators between the two groups of patients

[0086]

[0087] Note: "-" indicates that there is no corresponding data here; "*" indicates that there is a significant difference compared with the group without MODS (P<0.05).

[0088] (2) Combined prediction of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 for multiple trauma with MODS

[0089] The ROC curve of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 combined for predicting multiple traumatic events with MODS is shown in the figure. Figure 1 As shown, when the optimal cutoff value for serum eotaxin was 221.48 ng / L, the area under the ROC curve (AUC) for predicting MODS was 0.745 (95% CI: 0.652–0.818), with a sensitivity of 76.72% and a specificity of 89.17%; when the optimal cutoff value for serum MCP-4 was 158.43 ng / L, the AUC for predicting MODS was 0.793 (95% CI: 0.724–0.842), with a sensitivity of 74.53% and a specificity of 91.64%; when the optimal cutoff value for IP-10 was 201.86 ng / L, the AUC for predicting MODS was 0.754 (95% CI: 0.664–0.824), with a sensitivity of 73.46% and a specificity of 92.23%; the optimal cutoff value for IL-18 was 89.66 ng / L. At a concentration of 1 ng / L, the AUC for predicting MODS was 0.725 (95% CI: 0.641-0.789), with a sensitivity of 69.84% and a specificity of 90.69%. When the optimal cutoff value of MCP-1 was 594.23 ng / L, the AUC for predicting MODS was 0.761 (95% CI: 0.662-0.840), with a sensitivity of 77.94% and a specificity of 91.76%. The combined AUC for predicting MODS using Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 was 0.965 (95% CI: 0.927-0.983), with a sensitivity of 91.12% and a specificity of 94.52%. It can be seen that the AUC of the combined application of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 was significantly higher than the AUC of their individual applications.

[0090] (3) Comparison of various indicators among patients with different prognoses in the MODS group

[0091] In the MODS group, the serum levels of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in deceased patients were all higher than those in surviving patients, with significant differences (P < 0.05), as shown in Table 3.

[0092] Table 3 Comparison of patient indicators in the group with MODS

[0093]

[0094] Note: "-" indicates that there is no corresponding data here; "*" indicates that there is a significant difference compared with the survival group (P<0.05).

[0095] (4) Serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 combined predict mortality in patients with MODS

[0096] Following the analytical method described above, the AUC for mortality in patients with MODS due to the combined levels of serum Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 was 0.821 (sensitivity and specificity were 72.15% and 90.24%, respectively), which was significantly higher than the AUC predicted by each indicator alone: ​​0.736, 0.753, 0.746, 0.721, and 0.742 (P < 0.05).

[0097] Comparative Example 1: Evaluation of the effect of IL-6 replacing IL-18

[0098] The study cohort remained unchanged, but serum IL-6 levels were measured. The method of Example 1 was followed, except that the antibody in the chemiluminescence detection was replaced with an IL-6 antibody (including a coating antibody and a labeled antibody). Specifically, the IL-6 coating antibody was purchased from Abcam, catalog number ab11449; the IL-6 labeled protein antibody was purchased from Abcam, catalog number ab9324. All other procedures and analytical methods were the same.

[0099] Based on the experimental data of Eotaxin, MCP-4, IP-10, and MCP-1 in Example 1, the area under the ROC curve (AUC) of the combined IL-6 for diagnosing MODS was 0.908, which was higher than the AUC of the individual indicators (0.745, 0.793, 0.754, 0.761, and 0.728, respectively (P < 0.05)). Its diagnostic sensitivity and specificity were 82.57% and 91.98%, respectively, but significantly lower than the combined effect of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in this invention.

[0100] Comparative Example 2: Evaluation of the effect of C-reactive protein replacing MCP-1

[0101] The study cohort remained unchanged, but serum CRP levels were measured. The method of Example 1 was followed, except that the antibody in the chemiluminescence detection was replaced with a CRP antibody (including a coating antibody and a labeled antibody). Specifically, the CRP coating antibody was purchased from Abcam, catalog number ab185558; the CRP labeled antibody was purchased from Abcam, catalog number ab211631. All other procedures and analytical methods were the same.

[0102] Based on the experimental data of Eotaxin, MCP-4, IP-10, and IL-18 in Example 1, the area under the ROC curve for predicting multiple traumatic events with MODS by combining C-reactive protein was 0.916, which was higher than the AUC for each indicator alone (0.745, 0.793, 0.754, 0.725, and 0.757, respectively (P < 0.05)). Its diagnostic sensitivity and specificity were 83.15% and 93.03%, respectively, but significantly lower than the combined effect of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 in this invention.

[0103] As can be seen from Comparative Examples 1 and 2, when IL-18 is replaced by IL-6 or MCP-1 is replaced by CRP, the predictive effect of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 is not as good as that of the present invention. The combination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 is more effective in predicting multiple traumatic events combined with MODS.

[0104] Validation example: Combined assessment of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 for multiple traumatic events complicated with MODS.

[0105] In this validation example, an additional cohort of 20 patients was selected, who were prospectively collected critically ill patients in the intensive care unit. All of them met the inclusion criteria of this invention (but did not meet the exclusion criteria). Among them, there were 10 patients with multiple trauma and MODS and 10 patients with multiple trauma but without MODS. The serum levels of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in the above 20 patients were detected and analyzed according to the method in Example 1. The results are shown in Table 4. The criteria for diagnosis are as follows: when Eotaxin ≥ 221.48 ng / L, MCP-4 ≥ 158.43 ng / L, IP-10 ≥ 201.86 ng / L, IL-18 ≥ 89.66 ng / L, and MCP-1 ≥ 594.23 ng / L, it is diagnosed as multiple trauma combined with MODS; when Eotaxin < 221.48 ng / L, MCP-4 < 158.43 ng / L, IP-10 < 201.86 ng / L, IL-18 < 89.66 ng / L, and MCP-1 < 594.23 ng / L, it is diagnosed as multiple trauma without MODS; other conditions are considered uncertain. It can be seen that using the combination of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 as biomarkers provided by this invention for diagnosis, within the validation sample range, the accuracy rate for classifying MODS patients and non-MODS patients is 80%, demonstrating significant effectiveness.

[0106] Table 4. Detection and analysis of 20 patients

[0107]

[0108] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a detection reagent for a biomarker composition for early warning of multiple trauma in the preparation of a kit for predicting multiple trauma combined with MODS, characterized in that... ； The biomarker combination consists of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1; where Eotaxin is the serum concentration level of eosinophil chemokine, MCP-4 is the serum concentration level of monocyte chemoattractant protein 4, IP-10 is the serum concentration level of interferon-γ-induced protein 10, IL-18 is the serum concentration level of interleukin-18, and MCP-1 is the serum concentration level of monocyte chemoattractant protein 1. The detection reagents described herein are used for the quantitative or semi-quantitative analysis of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1. The quantitative or semi-quantitative method mentioned is chemiluminescence detection; The kit includes Eotaxin antibody, MCP-4 antibody, IP-10 antibody, IL-18 antibody, MCP-1 antibody, magnetic beads, acridine ester, and quantitative or semi-quantitative detection reagents.

2. The application according to claim 1, characterized in that, The critical values ​​for Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 are 221.48 ng / L, 158.43 ng / L, 201.86 ng / L, 89.66 ng / L, and 594.23 ng / L, respectively. When Eotaxin ≥ 221.48 ng / L, MCP-4 ≥ 158.43 ng / L, IP-10 ≥ 201.86 ng / L, IL-18 ≥ 89.66 ng / L and MCP-1 ≥ 594.23 ng / L, it is predicted to be multiple trauma combined with MODS. When Eotaxin < 221.48 ng / L, MCP-4 < 158.43 ng / L, IP-10 < 201.86 ng / L, IL-18 < 89.66 ng / L, and MCP-1 < 594.23 ng / L, it is predicted to be multiple traumatic non-MODS.

3. The application according to any one of claims 1-2, characterized in that, When the kit is used for detection, it includes the following steps: Sample collection, sample pretreatment, determination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and interpretation of results.

4. A kit for predicting multiple traumatic events complicated with MODS, characterized in that, The kit includes all reagents for quantitative or semi-quantitative detection of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1; The kit includes Eotaxin antibody, MCP-4 antibody, IP-10 antibody, IL-18 antibody, MCP-1 antibody, magnetic beads, acridine ester, and quantitative or semi-quantitative detection reagents.

5. The reagent kit according to claim 4, characterized in that, The quantitative or semi-quantitative detection reagents include any one or more of the following: magnetic bead preservation solution, acrid ester preservation solution, sample processing solution, pre-activation solution, activation solution, magnetic bead cleaning solution, and magnetic bead blocking solution.

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