Multi-trauma early warning marker combination, application and product

Abstract

The invention provides a multi-trauma early warning marker combination as well as application and a product thereof, and belongs to the field of biomarkers. The invention provides a marker combination for early warning of multiple wounds, the marker combination comprises Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, the Eotaxin, the MCP-4, the IP-10, the IL-18 and the MCP-1 in serum are detected and interpreted, and the multiple wounds combined with MODS and prognosis are predicted. The detection reagent for detecting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 can be used for preparing a kit for predicting multiple wounds with MODS and prognosis, is simple and efficient, and reduces the cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomarkers, and relates to multiple trauma, in particular to a multiple trauma early warning marker combination and application and product. BACKGROUND

[0002] Multiple injuries belong to common diseases in emergency department, which generally refers to trauma occurring in two or more anatomical sites of a patient, and is often accompanied by massive hemorrhage, shock and organ dysfunction. If severe multiple injury patients are not treated in time, they will often develop multiple organ dysfunction syndrome (MODS), which is one of the important reasons for the death of trauma patients. Since the condition of multiple injury patients combined with MODS changes rapidly, monitoring the condition of the patient and prognosis evaluation are key links, and the traditional monitoring relying on the personal experience of clinicians is not scientific and accurate.

[0003] Eotaxin, also known as chemokine-11 (CCL11), belongs to the monocyte chemotactic protein member in the CC chemokine family. Eotaxin has a strong chemotactic effect on 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. Eotaxin gene is expressed in various tissues, including heart, lung, kidney, lymph node, thymus and intestinal tract. Eotaxin also exists in human lung epithelial cells, pleural mesothelial cells, bronchial respiratory epithelial cells and smooth muscle cells. Eotaxin can rapidly transport from blood to brain through the blood-brain barrier. Age-related increase in Eotaxin is associated with cognitive impairment in executive function, episodic memory and semantic memory, so this chemokine is called "endogenous cognitive degradation chemokine" (ECDC) or "accelerated brain aging chemokine" (ABAC). In patients with schizophrenia, the increase of Eotaxin is not only related to impaired cognitive function, but also related to the form of thought disorder.

[0004] Interferon gamma inducible protein 10 (IP-10) is a proinflammatory chemokine secreted by a variety of cells. 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 has 68% amino acid sequence identity with mouse and mouse. IP-10 exerts its biological effects by binding to CXCR3, a seven-transmembrane G protein-coupled receptor that is expressed mainly on activated T, B lymphocytes, natural killer (NK), dendritic cells, and macrophages in a paracrine or autocrine manner. IP-10 induction is mainly dependent on the carboxy-terminal region of CXCR3, which is essential for IP-10 ligand-induced CXCR3 internalization, chemotaxis, and calcium mobilization. The strong chemotactic effect of IP-10 on activated lymphocytes enables it to regulate innate and adaptive immunity, induce tissue damage, and regulate tumor formation. IP-10 is a pleiotropic molecule that can exert potent biological functions, including promoting chemotactic activity of 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, macrophages, etc.), mainly involved in immune regulation, inflammatory response and hematopoietic function. At present, at least 38 interleukin members have been found, such as IL-1, IL-2, IL-6, IL-10, IL-23, etc. Among them, interleukin 18 (IL-18) is a proinflammatory cytokine, which belongs to the IL-1 superfamily member, mainly produced by immune cells such as macrophages and dendritic cells. It participates in anti-infection, autoimmune disease and tumor regulation by activating immune response, and is closely related to the occurrence and development of inflammatory diseases. IL-18 binds to receptor IL-18Rα, activates NF-κB and MAPK signaling pathways, promotes the release of inflammatory factors such as interferon gamma (IFN-γ), and enhances the activity of T cells and natural killer (NK) cells. In the early stage of infection, IL-18 enhances the anti-viral and anti-bacterial immune response in cooperation with IL-12.

[0006] Monocyte chemoattractant protein 1 (MCP-1) is a cytokine that has chemotactic effects on monocytes but not on neutrophils, mainly secreted by leukocytes and stromal cells in the hematopoietic microenvironment, and can also bind to the surface of endothelial cells, playing a key role in the entire inflammatory process, and can activate the corresponding inflammatory transcription factors. Both in vivo and in vitro experiments have confirmed that MCP-1 has chemotactic activity on monocytes, activates monocytes and macrophages, increases the concentration of Ca2+ in the cytoplasm, produces and releases superoxide anions, and releases lysozyme, up-regulates the expression of adhesion molecules such as the integrin family β2 group and α4 molecules in monocytes and macrophages, and the production of cytokines IL-1 and IL-6, activated macrophages can inhibit the growth of tumor cells. MCP is a chemotactic agent and activator of basophils, especially in stimulating basophil degranulation and histamine release.

[0007] Monocyte chemoattractant protein 4 (MCP-4) is a member of the chemokine family, mainly responsible for attracting monocytes, eosinophils, and T cells to inflammatory sites. The role of MCP-4 in immune regulation, inflammatory response, and certain diseases has been extensively studied. MCP-4 is a chemokine that can recruit 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 key role in regulating the migration and localization of immune cells. MCP-4 plays a role in various inflammation-related diseases, including asthma, allergic diseases, rheumatoid arthritis, etc. Its expression level is elevated in these diseases, indicating that MCP4 is involved in the regulation of pathological immune response 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 at the inflammatory site.

[0008] Ducheng et al. found that the area under the ROC curve of serum HMGB-1, CK, Mb combined with APACHE II score for the diagnosis of MODS was 0.958, which was higher than that of single indicator diagnosis, suggesting that combined diagnosis of emergency multiple injuries combined with MODS has good value. The study also analyzed the predictive value of serum HMGB-1, CK, Mb combined with APACHE II score for MODS patients, and found that the combined prediction was higher than that of each single indicator diagnosis, suggesting that combined diagnosis has good value for the prognosis evaluation of emergency multiple injuries combined with MODS, which makes up for the limitations of single indicator, and serological indicators can be dynamically monitored. The disadvantage of this technology is that it needs to measure 4 kinds of index data, the process is cumbersome, and the cost is high (Ducheng, Pan Shuming, Li Ming, et al. Serum markers combined with acute physiology and chronic health score in the diagnosis and prognosis of multiple injuries combined with multiple organ dysfunction syndrome [J]. Chinese Clinicians Journal, 2022).

[0009] Chinese patent CN2024100411303 provides an inflammatory factor composition, model and kit for early warning of MODS. Among them, the inflammatory factor composition includes: IL6, IL8, IFN gama, STNFRII, BLC, IL1RA, which can accurately and quickly predict MODS caused by inflammatory factor storm in early stage of severe trauma, and help to improve the success rate of treatment of severe trauma. ROC curve analysis of the early warning performance of the selected 6 target cytokines to MODS: the standard concentration values of the 6 target cytokines IL_6, IL_8, IFN_gama, STNF_RII, BLC and IL_1RA of the subject are brought into the target MODS risk assessment model. Based on the standard concentration values of the 6 target cytokines IL6, IL8, IFNgama, STNFRII, BLC and IL1RA of the subject, the ROC curve is drawn, and the result shows that the area under the ROC curve of the 6 target cytokines is 0.9722, the area under the ROC curve is high, and the specificity and sensitivity are good, which indicates that the combined prediction performance of the 6 factors is good, that is, the model has good MODS early warning performance. The technology determines that 6 index data need to be measured, the process is complicated, and the cost is high. SUMMARY

[0010] The present application provides a multi-trauma early warning marker combination and application and product to solve the problem of the prior art that a large number of indexes need to be measured, the process is complicated, and the cost is high. The present application provides a marker combination for early warning of multiple trauma, which comprises Eotaxin, MCP-4, IP-10, IL-18 and MCP-1. The present application predicts multiple trauma combined with MODS by detecting and interpreting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum. The detection reagent for detecting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 can be used to prepare a kit for predicting multiple trauma combined with MODS, which is simple and efficient and reduces the cost.

[0011] To achieve the above object, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides a marker combination for early warning of multiple trauma, which comprises Eotaxin, MCP-4, IP-10, IL-18 and MCP-1. The Eotaxin is the concentration level of serum eosinophil chemotactic factor, the MCP-4 is the concentration level of serum monocyte chemotactic protein 4, the IP-10 is the concentration level of serum interferon gamma-induced protein 10, the IL-18 is the concentration level of serum interleukin 18, and the MCP-1 is the concentration level of serum monocyte chemotactic protein 1.

[0012] In another aspect, the present application provides use of the detection reagent of the marker composition as described above in the preparation of a kit for predicting multiple trauma combined with MODS.

[0013] Preferably, the detection reagent is used for the quantification or semi-quantification of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0014] Preferably, the quantification or semi-quantification method comprises any one or more of ELISA detection, chemiluminescence detection, Dot blot detection, Western blot detection, immunochromatography and immunohistochemical detection.

[0015] Preferably, the prediction of multiple trauma combined with MODS is achieved by the interpretation of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0016] 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. 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 trauma without MODS.

[0017] Preferably, the kit comprises the following steps when used for detection: Sample collection, sample pretreatment, determination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and interpretation of results.

[0018] In another aspect, the present application provides a kit for predicting multiple trauma combined with MODS, which comprises all reagents for quantitatively or semi-quantitatively detecting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

[0019] Preferably, the kit comprises Eotaxin antibody, MCP-4 antibody, IP-10 antibody, IL-18 antibody, MCP-1 antibody, magnetic beads, acridinium ester, and quantitative or semi-quantitative detection reagent.

[0020] Preferably, the quantitative or semi-quantitative detection reagent comprises any one or more of magnetic bead storage solution, acridinium ester storage solution, sample processing solution, pre-priming solution, priming solution, magnetic bead cleaning solution, and magnetic bead sealing solution.

[0021] Compared with the prior art, the present application has the following beneficial effects: 1. The present application detects and interprets Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum, and judges that multiple trauma combined with MODS when Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 are all greater than or equal to the critical value, and judges that non-MODS when Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 are all less than the critical value, and the detection reagent for Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 detection can be used to prepare a kit for predicting multiple trauma combined with MODS; 2. The present application can realize the prediction of multiple trauma combined with MODS by detecting and interpreting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum, which has less detection items, high efficiency and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 ROC curve for predicting multiple trauma combined with MODS by serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1. DETAILED DESCRIPTION

[0023] Unless otherwise specified, the raw materials used in the present application are ordinary commercially available products, and their sources are not specifically limited.

[0024] To make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. If specific conditions are not specified in the examples, the conditions are performed according to conventional conditions or the conditions recommended by the manufacturers. If the manufacturers of all reagents or instruments are not specified, the reagents or instruments are all conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following detailed description of the embodiments. The specific examples described herein are intended to be illustrative only and are not intended to be limiting in any way. Furthermore, in the following description, the description of well-known structures and techniques is omitted so as not to unnecessarily obscure the concept of the present application. Such structures and techniques are described in many publications, for example, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Sambrook, et al., 2012; Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 2018; Stryer, L., Biochemistry (Fifth Edition), W. H. Freeman and Company, 2002; and Gait, M. J., Oligonucleotide Synthesis: A Practical Approach, IRL Press, 1984. Current Protocols in Molecular Biology

[0025] Study cohort and data collection methods (1) General information Prospectively collected 148 cases of patients with severe multiple injuries diagnosed in Peking University People's Hospital as research objects, of which 78 cases with MODS were taken as MODS group and 70 cases without MODS were taken as control group (non-MODS group). In the MODS group, there were 40 males and 38 females, with an age of 21-54 years, an average of (39.18±10.32) years, 35 cases of traffic injury, 23 cases of violent injury, and 20 cases of other injuries; the number of injury sites was ≤3 in 42 cases and >3 in 36 cases. In the control group, there were 39 males and 31 females, with an age of 23-55 years, an average of (38.57±9.65) years, 30 cases of traffic injury, 23 cases of violent injury, and 17 cases of other injuries; the number of injury sites was ≤3 in 40 cases and >3 in 30 cases. There was no statistically significant difference in gender, age, body mass index, cause of injury, and number of injury sites between the MODS group and the control group (P>0.05), and the specific statistical information is shown in Table 1.

[0026] Table 1. Statistics and explanation of the study cohort data

[0027] Note: "-" indicates that there is no corresponding data here.

[0028] Inclusion criteria: ① meet the diagnostic criteria of severe multiple injury patients in Multiple Trauma Case Report and Diagnosis: Expert Consensus Opinion (2013 Edition); ② Injury Severity Score (ISS score)>16 points. Exclusion criteria: ① suffering from other infectious diseases; ② receiving treatment before visiting the doctor.

[0029] ​All patients and family members in the present application are informed and signed the informed consent, and the study is approved by the ethics committee of Peking University People's Hospital.

[0030] (2) Blood collection and analysis method Collect 5 mL of fasting venous blood of all study subjects in a heparin anticoagulation tube, separate the supernatant after centrifugation, and store it in a-80℃ refrigerator for testing; the concentration levels of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in serum are determined by immunological method.

[0031] (3) Data processing SPSS25.0 statistical software is used for data analysis and processing. The number of cases or percentage is used for count data, and χ 2 test is used for comparison between groups; the measurement data conforming to the normal distribution are expressed by mean ± standard deviation, and t test is used for comparison between two groups. Pearson correlation is used for correlation analysis; receiver operating characteristic (ROC) curve is used for predictive value analysis; and multi-factor logistic regression analysis is used for influencing factors. P<0.05 is considered to have statistically significant difference.

[0032] Example 1: A method for assessing the risk of multiple trauma and MODS I. Serum index detection The determination of the indexes in serum includes but is not limited to ELISA method and chemiluminescence method, wherein the chemiluminescence method includes the following acridan ester chemiluminescence scheme: (1) Preparation of base solution 1.1 Preparation of magnetic bead storage solution: 79.8% 0.01M phosphate buffer solution, 10% calf serum, 10% glycerol, 0.1% Proclin300 and 0.1% Tween 20 by volume fraction; 1.2 Preparation of acridan ester storage solution: 69.9% 0.01M phosphate buffer solution and 30% glycerol and 0.1% Proclin300 by volume fraction, and then adding 1% BSA by mass fraction.

[0033] 1.3 Preparation of sample treatment solution: 0.01M phosphate buffer solution with pH 7.4, adding 1% BSA, 0.1% Triton X-100 and 0.3% Proclin300 by mass fraction.

[0034] 1.4 Preparation of pre-priming solution: adding 0.08 mol / L hydrogen peroxide and 0.02 mol / L nitric acid in purified water and mixing well.

[0035] 1.5 Preparation of priming solution: adding 1.0 mol / L sodium hydroxide and 4.5 g / L Triton 100 in purified water.

[0036] 1.6, Magnetic bead washing solution preparation: 0.2M MES buffer is prepared, and 0.02% (v / v) Proclin300 is added.

[0037] 1.7, Magnetic bead blocking solution preparation: 99.4% by volume 0.01M phosphate buffer and 0.6% Tween 20 are added, and 0.5% by mass BSA is added.

[0038] (2) Preparation of magnetic bead-coated antibody Eotaxin-coated antibody (purchased from abcam, item number ab133604) is used for the determination of Eotaxin, MCP-4-coated antibody (purchased from abcam, item number ab224593) is used for the determination of MCP-4, IP-10-coated antibody (purchased from abcam, item number ab283681) is used for the determination of IP-10, interleukin 18-coated antibody (purchased from abcam, item number ab245697) is used for the determination of interleukin 18, and MCP-1-coated antibody (purchased from abcam, item number ab9858) is used for the determination of MCP-1.

[0039] 2.1, 200μL of magnetic beads (EM1-100 / 40 (high carboxyl) magnetic microspheres, item number 23710087) are taken into a 2mL centrifuge tube, magnetically separated for 3min, and then the supernatant is removed.

[0040] 2.2, 400μL of magnetic bead washing solution is added to the centrifuge tube, shaken and mixed, magnetically separated to remove the supernatant, and washed twice.

[0041] 2.3, A certain amount of EDC is weighed, a certain volume of 0.02M MES buffer is added, and a 50mg / ml EDC solution is prepared; a certain amount of NHS is weighed, a certain volume of 0.02M MES buffer is added, and a 50mg / ml NHS solution is prepared.

[0042] 2.4, 100μL of EDC solution and 100μL of NHS solution are added to the centrifuge tube, and shaken and mixed.

[0043] 2.5, Two centrifuge tubes are placed on a shaker for activation for 30min. The rotation speed is adjusted appropriately so that the liquid can flow steadily when inverted.

[0044] 2.6, After activation is complete, 2 volumes of 0.02M MES buffer are used for washing twice, and the supernatant is removed.

[0045] 2.7, 120μg of coated antibody is added to the centrifuge tube, and 0.01M phosphate buffer is added to a volume of 300μL, and coupled for 3h.

[0046] 2.8, Wash 2 times with 600 μL of magnetic bead blocking solution, remove supernatant.

[0047] 2.9, Add 600 μL of magnetic bead blocking solution to the centrifuge tube, shake for 30 min, and remove the supernatant.

[0048] 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 magnetic bead coating working solution.

[0049] (3) Preparation of acridan ester labeled antibody Eotaxin labeled antibody (purchased from abcam, ab226143) was used for the determination of Eotaxin, MCP-4 labeled antibody (purchased from abcam, ab206405) was used for the determination of MCP-4, IP-10 labeled antibody (purchased from abcam, ab307997) was used for the determination of IP-10, interleukin 18 labeled antibody (purchased from abcam, ab207324) was used for the determination of interleukin 18, and MCP-1 labeled antibody (purchased from abcam, ab214819) was used for the determination of MCP-1.

[0050] 3.1, Dissolve 200 ug of labeled antibody in 0.01M phosphate buffer, add acridan ester (9mg / mL NSP-SA-NHS, dissolved in DMSO), add antibody to make the final concentration of labeled antibody 2mg / mL, and the final mass ratio of antibody to acridan ester is 1:10; 3.2, React on a constant temperature shaker at 25°C for 3h in the dark; 3.3, Add lysine (acridan ester:lysine = 1:140, molar concentration ratio) for blocking, and react for 20 min.

[0051] 3.4, After the reaction is completed, use a 50KD dialysis bag for buffer replacement, and the dialysis buffer is 0.01M phosphate buffer, and dialysis is performed for 4 times, 3 hours each time.

[0052] 3.5, After dialysis is completed, add glycerol to make the final concentration of antibody 0.5mg / mL, and wait for use.

[0053] 3.6, Dilute the acridan ester with acridan ester preservation solution to a final concentration of 8ug / mL of antibody to obtain the acridan ester labeled antibody working solution.

[0054] (4) Sample pretreatment steps: Mix 10 μL of the sample to be tested with 190 μL of sample treatment solution.

[0055] (5) Detection and calculation of the sample to be tested The detection is carried out in a full-automatic chemiluminescence analyzer, and the specific operation is as follows: 5.1, take 100 μL of the pretreated sample, add 50 μL of magnetic bead coating working solution, mix and incubate at 37°C for 5 min, then separate by magnetism, wash to remove unbound substances, and remove the supernatant to obtain a magnetic bead-antigen complex; 5.2, add 50 μL of acridine lipid-labeled antibody working solution to the reaction cup containing the magnetic bead-antigen complex, mix and incubate at 37°C for 5 min, then separate by magnetism, wash to remove unbound substances, and remove the supernatant to obtain a magnetic bead-antigen-detection antibody complex; 5.3, add 100 μL of pre-activation solution and 100 μL of activation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix thoroughly, and then measure the maximum luminescence intensity; 5.4, according to the luminescence intensity detected by the standard, a standard curve is fitted, and the concentrations of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in the sample are calculated through the standard curve (the R 2 of the standard curve is greater than 0.98).

[0056] II. Determination results and analysis (1) Comparison of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 levels between MODS group and control group The serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 levels in the MODS group were significantly higher than those in the control group (no MODS group), and the difference was significant (P<0.05), as shown in Table 2.

[0057] Table 2 Comparison of indicators between two groups of patients

[0058] Note: “-” indicates that there is no corresponding data here; “*” indicates that there is a significant difference compared with the no MODS group (P<0.05).

[0059] (2) Combined prediction of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 for multiple trauma with MODS The ROC curve of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 for combined prediction of multiple trauma with MODS is as follows Figure 1The area under the ROC curve (AUC) of serum Eotaxin for predicting MODS was 0.745 (95% CI: 0.652-0.818) with a sensitivity of 76.72% and a specificity of 89.17% at the optimal cut-off value of 221.48 ng / L. The AUC of serum MCP-4 for predicting MODS was 0.793 (95% CI: 0.724-0.842) with a sensitivity of 74.53% and a specificity of 91.64% at the optimal cut-off value of 158.43 ng / L. The AUC of IP-10 for predicting MODS was 0.754 (95% CI: 0.664-0.824) with a sensitivity of 73.46% and a specificity of 92.23% at the optimal cut-off value of 201.86 ng / L. The AUC of IL-18 for predicting MODS was 0.725 (95% CI: 0.641-0.789) with a sensitivity of 69.84% and a specificity of 90.69% at the optimal cut-off value of 89.66 ng / L. The AUC of MCP-1 for predicting MODS was 0.761 (95% CI: 0.662-0.840) with a sensitivity of 77.94% and a specificity of 91.76% at the optimal cut-off value of 594.23 ng / L. The AUC of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 combined for predicting MODS 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 Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 combined was significantly higher than that of each cytokine alone.

[0060] (3) Comparison of various indicators in patients with different prognosis in the MODS group In the MODS group, the serum levels of Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 were significantly higher in patients who died than in patients who survived (P<0.05), as shown in Table 3.

[0061] Table 3 Comparison of various indicators in patients with MODS

[0062] Note: “-” indicates that there is no corresponding data here; “*” indicates a significant difference compared with the survival group (P<0.05).

[0063] (4) Combined prediction of serum Eotaxin, MCP-4, IP-10, IL-18, and MCP-1 for death in patients with MODS According to the above analysis method, the AUC of serum Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 combined for the prediction of death in patients with MODS was 0.821 (sensitivity and specificity were 72.15% and 90.24%, respectively), which was significantly higher than the AUC of each indicator alone: ​​0.736, 0.753, 0.746, 0.721 and 0.742 (P < 0.05).

[0064] Comparative Example 1: Evaluation of the effect of replacing IL-18 with IL-6 The study cohort remained unchanged, and serum IL-6 index determination was added; the method of Example 1 was followed, except that the antibody in the chemiluminescence detection was replaced with IL-6 antibody (including coating antibody and 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. The other procedures and analysis methods were the same.

[0065] Based on the experimental data of Eotaxin, MCP-4, IP-10 and MCP-1 in Example 1, the area under the ROC curve (AUC) for the diagnosis of MODS in combination with IL-6 was 0.908, which was higher than the AUC of each indicator alone (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 inferior to the combined effect of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in the present invention.

[0066] Comparative Example 2: Evaluation of the effect of replacing MCP-1 with C-reactive protein The study cohort remained unchanged, and serum CRP index determination was added; 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. The other procedures and analysis methods were the same.

[0067] 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 trauma with MODS combined with C-reactive protein was 0.916, which was higher than the AUC of 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 inferior to the combined effect of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in the present invention.

[0068] From the comparative examples 1 and 2, it can be seen that IL-18 is replaced by IL-6 or MCP-1 is replaced by CRP in the combination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and the predictive effect is not as good as the present application. The combination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 has more significant effect in predicting multiple trauma combined with MODS.

[0069] Verification: Combination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 in evaluating multiple trauma combined with MODS In this verification example, another 20 people were selected as the research cohort, which were critically ill patients in the intensive care unit, all of which met the inclusion criteria (did not meet the exclusion criteria) of the present application. Among them, 10 were multiple trauma combined with MODS and 10 were multiple trauma without MODS. According to the method of Example 1, the serum levels of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 of the above 20 patients were detected and analyzed, and the results are shown in Table 4. Among them, the judgment criteria are: 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 judged 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 judged as multiple trauma without MODS; otherwise, it is considered as uncertain. It can be seen that using the combination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 provided by the present application as a marker to determine, the accuracy of dividing MODS patients and non-MODS patients in the verification sample range is 80%, which has obvious effect.

[0070] Table 4 Detection and analysis of 20 patients

[0071] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A marker combination for early warning of multiple trauma, characterized in that: Including Eotaxin, MCP-4, IP-10, IL-18 and MCP-1; the Eotaxin is the concentration level of eosinophil chemotactic factor in serum, the MCP-4 is the concentration level of monocyte chemoattractant protein 4 in serum, the IP-10 is the concentration level of interferon gamma-induced protein 10 in serum, the IL-18 is the concentration level of interleukin 18 in serum, and the MCP-1 is the concentration level of monocyte chemoattractant protein 1 in serum.

2. Use of the detection reagent of the marker composition according to claim 1 in the preparation of a kit for predicting multiple trauma complicated with MODS.

3. The use according to claim 2, characterized in that The detection reagent is used for the quantitative or semi-quantitative detection of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

4. The use according to claim 3, characterized in that 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 detection.

5. The use according to claim 2, characterized in that The prediction of multiple trauma complicated with MODS is achieved by interpreting Eotaxin, MCP-4, IP-10, IL-18 and MCP-1.

6. The use according to claim 5, characterized in that The critical values ​​of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1 were 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, multiple trauma combined with MODS was predicted; 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 was predicted to be polytrauma non-MODS.

7. The use according to any one of claims 2 to 6, characterized in that: The kit comprises the following steps when used for detection: Sample collection, sample pretreatment, determination of Eotaxin, MCP-4, IP-10, IL-18 and MCP-1, and interpretation of results.

8. A kit for predicting multiple trauma combined 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.

9. The kit according to claim 8, characterized in that The kit comprises Eotaxin antibody, MCP-4 antibody, IP-10 antibody, IL-18 antibody, MCP-1 antibody, magnetic beads, acridinium ester, and quantitative or semi-quantitative detection reagent.

10. The kit according to claim 9, characterized in that The quantitative or semi-quantitative detection reagents include any one or more of magnetic bead preservation solution, acridinium ester preservation solution, sample processing solution, pre-excitation solution, excitation solution, magnetic bead cleaning solution and magnetic bead blocking solution.

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