Marker combination for diagnosing neonatal necrotizing enterocolitis and application thereof
By detecting a combination of CC chemokine ligand 20, heparin-binding epidermal growth factor, angiotensin, and aldosterone markers in neonatal plasma, the problem of early and accurate diagnosis of neonatal necrotizing enterocolitis has been solved, achieving highly sensitive and specific diagnostic results and supporting clinical treatment decisions.
Patent Information
- Application Number
- CN202511670736.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2026-02-17
AI Technical Summary
Existing technologies make it difficult to diagnose neonatal necrotizing enterocolitis accurately in the early stages, leading to misdiagnosis and delayed treatment. Traditional methods lack sensitivity and specificity, and cannot effectively determine the severity and prognosis of the disease.
The concentrations of four biomarkers—CC chemokine ligand 20, heparin-binding epidermal growth factor, angiotensin, and aldosterone—in plasma samples were detected using a double-antibody sandwich method or PCR. The data analysis module was then used to determine the presence and severity of NEC.
It achieves highly sensitive and specific diagnosis of neonatal necrotizing enterocolitis, enabling early identification of the disease and assessment of its severity, providing a basis for clinical intervention and treatment decisions.
Smart Images
Figure CN121540892A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of molecular biology technology, and in particular to a combination of biomarkers for diagnosing necrotizing enterocolitis in newborns and their applications. Background Technology
[0002] Necrotizing enterocolitis (NEC) of the newborn is the most common and fatal gastrointestinal disease in premature infants; the incidence of NEC in very low birth weight infants is approximately 7%, with a mortality rate reaching 10% to 30%. NEC is a devastating disease that not only causes problems such as local abdominal perforation but also leads to severe systemic sepsis and systemic inflammatory response syndrome. Its morbidity and mortality rates are extremely high. Surviving infants may suffer from sequelae such as short bowel syndrome, intestinal stenosis, and neurodevelopmental abnormalities, all of which result in a severe family and socioeconomic burden. Therefore, the prevention and treatment of NEC are of great significance for improving neonatal survival rates and quality of life.
[0003] Active prevention, early identification, and standardized treatment are key to reducing the incidence of NEC, improving the survival rate of children with NEC, and enhancing their prognosis. With the rapid development of medical technology, the clinical diagnosis of NEC now mainly includes the identification of clinical manifestations, elevated inflammatory markers, and abdominal imaging examinations. Clinical manifestations primarily involve the early identification of abdominal and systemic infection symptoms and signs, including abdominal distension, rectal bleeding, and systemic inflammatory response syndrome. However, the identification of clinical manifestations varies from person to person and is greatly influenced by experience. Different diseases can present with the same clinical manifestations; for example, the early symptoms of food protein-induced allergic enterocolitis are indistinguishable from those of NEC, thus easily confusing clinical judgment. Elevated inflammatory markers such as CRP, PCT, and IL-6 can, to some extent, indicate a diagnosis of NEC, but these markers are nonspecific and easily elevated by other factors or diseases. Furthermore, inflammatory markers have poor sensitivity, making early identification of NEC difficult and easily leading to misdiagnosis and delayed treatment. While abdominal imaging has high specificity, it has poor sensitivity. Children with significant changes on imaging are already at NEC stage II or higher. Moreover, abdominal imaging is difficult to perform in relatively under-resourced primary hospitals. Therefore, all of these methods are unsuitable for early identification and intervention of NEC, and cannot determine the severity and prognosis of the disease.
[0004] Therefore, traditional diagnostic and identification methods still need improvement. Summary of the Invention
[0005] Based on this, one or more embodiments of this application provide a combination of biomarkers for diagnosing neonatal necrotizing enterocolitis that combines high sensitivity and high specificity, and their application.
[0006] According to a first aspect of the embodiments of this application, a combination of biomarkers for diagnosing neonatal necrotizing enterocolitis is provided, the biomarker combination comprising CC chemokine ligand 20, heparin-binding epidermal growth factor, angiotensin, and aldosterone.
[0007] In some embodiments, necrotizing enterocolitis of the newborn is diagnosed by detecting the concentration of the combination of biomarkers in the sample to be tested.
[0008] In some embodiments, the sample to be tested includes a plasma sample.
[0009] In some embodiments, the concentration of the biomarker combination is detected by a double-antibody sandwich method and / or PCR.
[0010] According to a second aspect of the embodiments of this application, the use of the combination of markers for diagnosing neonatal necrotizing enterocolitis as described above in the preparation of reagents and / or kits for diagnosing neonatal necrotizing enterocolitis is provided.
[0011] According to a third aspect of the embodiments of this application, a kit for diagnosing neonatal necrotizing enterocolitis is provided, comprising reagents for detecting the above-described combination of markers for diagnosing neonatal necrotizing enterocolitis.
[0012] In some embodiments, the reagents include standards, coated antibodies, and enzyme-labeled antibodies.
[0013] In some embodiments, the reagent further includes one or more of sample extraction reagent, sample washing solution, sample diluent, colorimetric reagent, and stop solution.
[0014] In some embodiments, the enzyme-labeled antibody is a horseradish peroxidase-labeled antibody.
[0015] According to a fourth aspect of the embodiments of this application, an apparatus for diagnosing necrotizing enterocolitis in newborns is provided, comprising:
[0016] The data acquisition module is used to acquire the concentration of the above-mentioned biomarker combination in the sample to be tested; and,
[0017] A data analysis template is used to determine whether the sample to be tested is a neonatal necrotizing enterocolitis sample based on the concentration of the biomarker combination.
[0018] Compared with traditional technologies, this application has the following advantages:
[0019] This application identified four biomarkers associated with neonatal necrotizing enterocolitis through research screening. Based on these biomarkers, it is possible to effectively determine whether neonatal necrotizing enterocolitis has occurred and its severity, with high sensitivity and specificity, which can help in the clinical diagnosis and prognosis of neonatal necrotizing enterocolitis. Attached Figure Description
[0020] To more clearly illustrate the technical solutions of the specific embodiments of this application, the drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings.
[0021] Figure 1 ROC curves of the biomarkers in this application and necrotizing enterocolitis in newborns. Detailed Implementation
[0022] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.
[0023] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.
[0024] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").
[0025] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.
[0026] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.
[0027] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.
[0028] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.
[0029] In this application, "diagnosis" includes aspects such as auxiliary diagnosis, recurrence risk assessment, assessment of cancer risk and degree, and prognosis.
[0030] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.
[0031] Some embodiments of this application provide a combination of biomarkers for diagnosing neonatal necrotizing enterocolitis, the biomarker combination including CC chemokine ligand 20 (CCL20), heparin-bound epidermal growth factor (HBEGF), angiotensin (Ang), and aldosterone (ALD).
[0032] This application identified four biomarkers associated with neonatal necrotizing enterocolitis through research screening. Based on these biomarkers, it is possible to effectively determine whether neonatal necrotizing enterocolitis has occurred and its severity, with high sensitivity and specificity, which can help in the clinical diagnosis and prognosis of neonatal necrotizing enterocolitis.
[0033] In some embodiments, neonatal necrotizing enterocolitis is diagnosed by detecting the concentration of a combination of markers in a test sample.
[0034] In some embodiments, the sample to be tested includes a plasma sample.
[0035] It should be noted that samples should be extracted and used as soon as possible after collection. If testing cannot be performed immediately, samples can be temporarily stored at -20°C, but the number of freeze-thaw cycles should be minimized.
[0036] In some embodiments, the concentration of the biomarker combination is detected by a double-antibody sandwich assay and / or PCR. It is understood that the methods used to detect biomarker concentrations are not limited to the specific methods described above, and other methods commonly used in the art may be employed.
[0037] In some embodiments, when detecting the concentration of the biomarker combination using a double-antibody sandwich assay, the sample to be tested does not contain NaN3. It is understood that NaN3 can affect the activity of horseradish peroxidase; therefore, NaN3 in the sample to be tested must be removed during sample collection, or a sample free of NaN3 must be selected for testing.
[0038] Some embodiments of this application also provide the use of the combination of markers for diagnosing neonatal necrotizing enterocolitis as described above in the preparation of reagents and / or kits for diagnosing neonatal necrotizing enterocolitis.
[0039] Some embodiments of this application also provide a kit for diagnosing neonatal necrotizing enterocolitis, comprising reagents for detecting the above-described combination of markers for diagnosing neonatal necrotizing enterocolitis.
[0040] In some embodiments, reagents are included for detecting the combination of markers described above for diagnosing necrotizing enterocolitis in newborns.
[0041] In some of these embodiments, the reagents include standards, coated antibodies, and enzyme-labeled antibodies.
[0042] In some embodiments, the standards include CCL20 standards, HBEGF standards, Ang standards, and ALD standards.
[0043] In some specific examples, the concentration of the CCL20 standard was 80 pg / mL.
[0044] In some specific examples, the concentration of the HBEGF standard was 480 pg / mL.
[0045] In some specific examples, the concentration of the Ang standard was 4800 pg / mL.
[0046] In some specific examples, the concentration of the ALD standard was 320 pg / mL.
[0047] In some embodiments, the reagents also include one or more of the following: sample extraction reagent, sample washing solution, sample diluent, colorimetric reagent, and stop solution.
[0048] It is understood that in some other specific examples, the kit described above may not include any of the reagents in the sample extraction reagent, sample washing solution, sample diluent, colorimetric reagent, and stop solution, and the excluded reagents may reasonably be obtained from external sources.
[0049] In some of these embodiments, the enzyme-labeled antibody is a horseradish peroxidase-labeled antibody.
[0050] Some embodiments of this application also provide an apparatus for diagnosing necrotizing enterocolitis in newborns, comprising:
[0051] The data acquisition module is used to acquire the concentration of the above-mentioned biomarker combination in the sample to be tested; and,
[0052] A data analysis template for determining whether a sample is a neonatal necrotizing enterocolitis sample based on the concentration of a combination of biomarkers.
[0053] In some embodiments, determining whether a test sample is a sample of necrotizing enterocolitis of the newborn based on the concentration of a combination of markers includes:
[0054] The concentration of the biomarker combination is scored, and the scoring results are used to determine whether it is neonatal necrotizing enterocolitis and its severity.
[0055] In some embodiments, the scoring criteria include:
[0056] Let the concentration of CCL20 be X1. If X1 < 40 pg / mL, score 0 points; if 40 ≤ X1 < 50 pg / mL, score 1 point; if 50 ≤ X1 < 60 pg / mL, score 2 points; if 60 ≤ X1 < 70 pg / mL, score 3 points; if X1 ≥ 70 pg / mL, score 4 points.
[0057] Let the concentration of HBEGF be X2. If X2 < 240 pg / mL, score 0 points; if 240 ≤ X2 < 250 pg / mL, score 1 point; if 250 ≤ X2 < 260 pg / mL, score 2 points; if 260 ≤ X2 < 270 pg / mL, score 3 points; if 270 ≤ X2 < 280 pg / mL, score 4 points; if X2 ≥ 280 pg / mL, score 5 points.
[0058] Let the concentration of Ang be X3. If X3 < 2400 pg / mL, score 0 points; if 2400 ≤ X3 < 2500 pg / mL, score 1 point; if 2500 ≤ X3 < 2600 pg / mL, score 2 points; if 2600 ≤ X3 < 2700 pg / mL, score 3 points; if 2700 ≤ X3 < 2800 pg / mL, score 4 points; if X3 ≥ 2800 pg / mL, score 5 points.
[0059] Let the concentration of ALD be X4. If X4 < 160 pg / mL, score 0 points; if 160 ≤ X4 < 170 pg / mL, score 1 point; if 170 ≤ X4 < 180 pg / mL, score 2 points; if 180 ≤ X4 < 1900 pg / mL, score 3 points; if X4 ≥ 190 pg / mL, score 4 points.
[0060] Calculate the sum of CCL20, HBEGF, Ang, and ALD scores, X. If X ≤ 5, the likelihood of NEC is low; if 5 < X ≤ 8, mild NEC is considered; if 8 < X ≤ 12, moderate NEC is considered; and if X > 13, severe NEC is considered. Surgical treatment is recommended, as there is a very high risk of intestinal stenosis and short bowel syndrome.
[0061] The present application will be further described below with reference to specific embodiments and comparative examples, but these should not be construed as limiting the scope of protection of the present application. Unless otherwise specified, the raw materials involved in the following specific embodiments are all commercially available, the instruments used are all commercially available, and the processes involved are conventionally selected by those skilled in the art unless otherwise specified.
[0062] Example 1
[0063] (1) Indicator Screening: Clinical studies have found that newborns with NEC were not satisfied after being fed using the traditional passive feeding method via gavage, and some showed increased anxiety and restlessness. Therefore, it is speculated that the occurrence of NEC may be a stress response related to the passive feeding method. Based on this, the inventors conducted stress hormone tests on the plasma of the normal group and the disease group. The test indicators included adrenaline, noradrenaline, dopamine, isoproterenol, serotonin, renin, angiotensin, aldosterone, corticotropin-releasing hormone, adrenocorticotropic hormone, glucocorticoids, thyrotropin-releasing hormone, thyroid-stimulating hormone, thyroxine, CRP, PCT, HBEGF, EREG, AREG, CXCL8, IL1β, CCL20, CCL4, CCL3, IL8 and IL6. The results showed that, compared with the normal group, the levels of CCL20, HBEGF, angiotensin, and aldosterone were significantly elevated in the disease group, with p values ≤ 0.005. The average levels of each indicator in the normal group and the disease group are shown in Table 1 (unit: pg / mL).
[0064] Table 1
[0065]
[0066] (2) CCL20 was determined by a double-antibody sandwich method (using kit MM-8534102 manufactured by Jiangsu Enzyme Immunosorbent Assay Co., Ltd.): Microplates were coated with purified CC chemokine ligand 20 (CCL20) antibody to prepare a solid-phase antibody. CCL20 was added sequentially to the wells coated with the monoclonal antibody, followed by binding with HRP-labeled CCL20 antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The color intensity was positively correlated with the CCL20 concentration in the sample. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the CCL20 concentration in the sample was calculated using a standard curve.
[0067] The test kit includes: 1 bottle of 30x concentrated wash buffer (20mL), 1 bottle of enzyme-labeled reagent (6mL), 1 strip of 12-well enzyme-labeled coated plate (12-well), 1 bottle of sample diluent (6mL), 1 bottle of chromogenic reagent A (6mL), 1 bottle of chromogenic reagent B (6mL), 1 bottle of stop solution (6mL), 1 bottle of standard (0.5mL), 1 bottle of standard diluent (1.5mL), 1 instruction manual, 2 sealing films, and 1 sealing bag.
[0068] Standard dilution: Provide CCL20 standard with a concentration of 80 pg / mL, and use standard dilution solution to prepare CCL20 standard with concentrations of 40 pg / mL, 20 pg / mL, 10 pg / mL, 5 pg / mL and 2.5 pg / mL.
[0069] Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent; all other steps are the same), standard wells, and sample wells. Add 50 μL of standard sample to the standard wells of the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, followed by 10 μL of the sample to be tested (final sample dilution is 5-fold). When adding samples, place them at the bottom of the wells, avoiding contact with the well walls, and gently shake to mix.
[0070] Absorbance determination: After sealing the plate with the sealing film, incubate at 37°C for 30 minutes; dilute the 30-fold concentrated washing buffer 30 times with distilled water and set aside; carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing buffer, let stand for 30 seconds and discard, repeat this 5 times, and pat dry; add 50 μL of enzyme-labeled reagent to each well, except for the blank well; seal the plate with the sealing film and incubate at 37°C for 30 minutes; fill with washing buffer, let stand for 30 seconds and discard, repeat this 5 times, and pat dry; add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, gently shake to mix, and develop color at 37°C in the dark for 10 minutes; add 50 μL of stop solution to each well to stop the reaction; zero the plate using the blank well, and measure the absorbance of each well sequentially at a wavelength of 450 nm.
[0071] Concentration calculation: Plot a standard curve on graph paper with the concentration of the standard as the x-axis and absorbance as the y-axis. Find the corresponding concentration based on the absorbance of the sample and the standard curve, and then multiply it by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration and absorbance of the standard, substitute the absorbance of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.
[0072] Scoring: The baseline is half the concentration of the standard, i.e., 40 pg / mL. A concentration less than 40 pg / mL is scored as 0 points, 40 ≤ X1 < 50 pg / mL is scored as 1 point, 50 ≤ X1 < 60 pg / mL is scored as 2 points, 60 ≤ X1 < 70 pg / mL is scored as 3 points, and X1 ≥ 70 pg / mL is scored as 4 points.
[0073] (3) The procedure for measuring heparin-bound EGF-like growth factor (HBEGF) is basically the same as that for CCL20, except that the concentration of the standard is different. Specifically, HBEGF standards with concentrations of 240 pg / mL, 120 pg / mL, 60 pg / mL, 30 pg / mL and 15 pg / mL are obtained by diluting the 480 pg / mL standard.
[0074] Scoring: The baseline is half the concentration of the standard, i.e., 240 pg / mL. A concentration less than 240 pg / mL is scored as 0 points, 240 ≤ X2 < 250 pg / mL is scored as 1 point, 250 ≤ X2 < 260 pg / mL is scored as 2 points, 260 ≤ X2 < 270 pg / mL is scored as 3 points, 270 ≤ X2 < 280 pg / mL is scored as 4 points, and X2 ≥ 280 pg / mL is scored as 5 points.
[0075] (4) The procedure for determining angiotensin (Ang) is basically the same as that for CCL20, except that the concentration of the standard is different. Specifically, Ang standards with concentrations of 2400 pg / mL, 1200 pg / mL, 600 pg / mL, 300 pg / mL and 150 pg / mL are obtained by diluting the 4800 pg / mL standard.
[0076] Scoring: The baseline is half the concentration of the standard, i.e., 2400 pg / mL. A concentration less than 2400 pg / mL is scored as 0 points, 2400 ≤ X3 < 2500 pg / mL is scored as 1 point, 2500 ≤ X3 < 2600 pg / mL is scored as 2 points, 2600 ≤ X3 < 2700 pg / mL is scored as 3 points, 2700 ≤ X3 < 2800 pg / mL is scored as 4 points, and X3 ≥ 2800 pg / mL is scored as 5 points.
[0077] (5) The procedure for determining aldosterone (ALD) is basically the same as that for CCL20, except that the concentration of the standard is different. Specifically, ALD standards with concentrations of 160 pg / mL, 80 pg / mL, 40 pg / mL, 20 pg / mL and 10 pg / mL are obtained by diluting the 320 pg / mL standard.
[0078] Scoring: The baseline is half the concentration of the standard, i.e., 160 pg / mL. A concentration less than 160 pg / mL is scored as 0 points, 160 ≤ X4 < 170 pg / mL is scored as 1 point, 170 ≤ X4 < 180 pg / mL is scored as 2 points, 180 ≤ X4 < 190 pg / mL is scored as 3 points, and X4 ≥ 190 pg / mL is scored as 4 points.
[0079] (6) The scores of the above four indicators are totaled. If the total score is ≤5, the possibility of NEC is low; if 5 < X ≤ 8, mild NEC is considered; if 8 < X ≤ 12, moderate NEC is considered; if X > 13, severe NEC is considered. Surgical treatment is recommended, as there is a very high probability of intestinal stenosis and short bowel syndrome.
[0080] (7) Sample testing:
[0081] Blood samples were collected from 11 NEC patients and 7 normal control group patients from Shenzhen Children's Hospital. After centrifugation at 3000 rpm, plasma was collected. The concentrations of CCL20, HBEGF, Ang, and ALD in the plasma samples were detected according to the methods described in steps (2) to (6) above. The average of the two test results was used as the final result, as shown in Table 2. Scoring was performed according to the scoring criteria, as shown in Table 3. Among them, NEC-C had the most severe clinical manifestations and ascites, and also scored the highest.
[0082] Table 2 (Unit: pg / mL)
[0083]
[0084] Table 3
[0085]
[0086] Based on the above results, ROC curves for the four indicators and the diagnosis of NEC were plotted, as follows: Figure 1 As shown in Table 4, the statistical results of AUC values are as follows.
[0087] Table 4
[0088]
[0089] Based on the above results, this application demonstrates that by detecting the concentrations of four indicators in plasma samples and scoring them individually, the sum of the scores can effectively assess the disease status and symptom severity. Therefore, the biomarker combination proposed in this application can provide a new direction for the clinical diagnosis of NEC, helping clinicians decide on the timing of surgery and predict disease prognosis, facilitating timely intervention and treatment.
[0090] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0091] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.
Claims
1. A marker combination for diagnosing necrotizing enterocolitis in a neonate, characterized in that, The biomarker combination includes CC chemokine ligand 20, heparin-binding epidermal growth factor, angiotensin, and aldosterone.
2. The marker combination for diagnosing neonatal necrotizing enterocolitis according to claim 1, characterized in that, Necrotizing enterocolitis of the newborn can be diagnosed by detecting the concentration of the combination of markers in the sample.
3. The marker combination for diagnosing neonatal necrotizing enterocolitis according to claim 2, characterized in that, The samples to be tested include plasma samples.
4. The diagnostic marker combination for necrotizing enterocolitis of the newborn according to any one of claims 1 to 3, characterized in that, The concentration of the biomarker combination was detected by a double-antibody sandwich method and / or PCR.
5. The use of the combination of markers for diagnosing neonatal necrotizing enterocolitis as described in any one of claims 1 to 4 in the preparation of reagents and / or kits for diagnosing neonatal necrotizing enterocolitis.
6. A reagent kit for diagnosing necrotizing enterocolitis in newborns, characterized in that, The reagent includes a combination of markers for diagnosing necrotizing enterocolitis of the newborn as claimed in any one of claims 1 to 4.
7. The kit for diagnosing necrotizing enterocolitis in newborns according to claim 6, characterized in that, The reagents include standards, coated antibodies, and enzyme-labeled antibodies.
8. The kit for diagnosing necrotizing enterocolitis in newborns according to claim 7, characterized in that, The reagents also include one or more of the following: sample extraction reagent, sample washing solution, sample diluent, colorimetric reagent, and stop solution.
9. The kit for diagnosing necrotizing enterocolitis in newborns according to claim 7, characterized in that, The enzyme-labeled antibody is a horseradish peroxidase-labeled antibody.
10. A device for diagnosing necrotizing enterocolitis in newborns, characterized in that, include: The data acquisition module is used to acquire the concentration of the biomarker combination according to any one of claims 1 to 4 in the sample to be tested; and, A data analysis template is used to determine whether the sample to be tested is a neonatal necrotizing enterocolitis sample based on the concentration of the biomarker combination.