Use of anti-myosin autoantibodies in the preparation of a diagnostic product for necrotizing enterocolitis in newborns

Abstract

The application discloses application of anti-myosin autoantibody in preparation of a diagnosis product for neonatal necrotizing enterocolitis. The application determines that IgG and IgM type anti-myosin autoantibody in peripheral blood of a child with neonatal necrotizing enterocolitis (NEC) is significantly increased in early stage of the disease; the application finds that the anti-myosin autoantibody can be used as an early prediction and diagnosis marker, and is used for diagnosis of NEC; the marker has high diagnosis efficiency for early NEC, and provides an effective basis for early prediction and diagnosis of NEC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of antimyosin autoantibodies in the preparation of diagnostic products for neonatal necrotizing enterocolitis. Background Technology

[0002] Necrotizing enterocolitis (NEC) is a common and fatal gastrointestinal disease in premature newborns, especially those with very low birth weight, with a mortality rate as high as 15% to 30%. According to the Bell-NEC staging system, NEC can be divided into stages I-III. Early NEC often presents with abdominal distension and bloody stools, lacking specific symptoms and making early diagnosis and timely intervention difficult. Once NEC develops, it progresses very rapidly, easily leading to life-threatening conditions such as intestinal necrosis and perforation. Therefore, early diagnosis and treatment are crucial for improving the clinical efficacy and prognosis of NEC.

[0003] The diagnosis of NEC primarily relies on imaging examinations, such as X-rays and ultrasound. Detecting characteristic imaging changes, such as pneumothorax in the intestinal wall and portal vein, is of significant diagnostic value, but the sensitivity is low. By the time these typical manifestations appear, the disease has progressed to NEC stage II-III. Atypical imaging findings can easily lead to missed diagnoses or misdiagnoses. Serological markers are also commonly used to assess the severity and treatment efficacy of NEC. Inflammatory markers such as serum amyloid-beta (SAA) and trefoil factor 3 (TFF3) are correlated with disease severity and have high value in monitoring NEC, and can also be used for diagnosis and prognostic assessment. Intestinal fatty acid-binding protein (I-FABP) has better diagnostic value for advanced stages (stages II-III) than for early stages (stage I). Calprotectin mainly reflects the state of acute inflammatory cell activation and has limited diagnostic value for NEC.

[0004] Premature infants and very low birth weight infants often experience intestinal developmental delays, leading to intestinal mucosal damage and even necrosis, as well as intestinal inflammation, due to multiple factors such as pathogenic microorganism invasion and inappropriate feeding. These are prominent pathological features of non-cancerous choriocarcinoma (NCC). Severe intestinal inflammation and mucosal damage inevitably induce the production of autoantibodies. However, the correlation between the production of autoantibodies and NEC remains unclear. Anti-myosin antibodies are common in myocarditis and dilated cardiomyopathy and can be used for early diagnosis of cardiovascular diseases. However, the relationship between anti-myosin autoantibodies and NEC has not been reported. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes anti-myosin autoantibody as an early predictive and diagnostic biomarker for necrotizing enterocolitis (NEC) in newborns, and the application of anti-myosin autoantibody detection reagents in the preparation of diagnostic products for NEC. This invention discovers that the plasma anti-myosin autoantibody content is significantly increased in children diagnosed with NEC. Anti-myosin autoantibody can serve as an early predictive and diagnostic biomarker for NEC, exhibiting high sensitivity and specificity. It can provide timely diagnosis of early NEC symptoms such as bloody stools and abdominal distension, and has a good effect in the differential diagnosis of NEC from other digestive system diseases. It can identify NEC earlier, thus enabling early intervention, and can provide an effective basis for early diagnosis and early warning of NEC in children, significantly improving the clinical efficacy and prognosis of NEC.

[0006] According to a first aspect of the present invention, an early predictive and diagnostic biomarker for necrotizing enterocolitis in newborns is provided, said biomarker being an antimyosin autoantibody.

[0007] In some embodiments of the present invention, the autoantibody is an antibody produced by the body's immune system when it mistakenly uses components of its own tissues or organs as antigens.

[0008] In this invention, the inventors verified the effectiveness and accuracy of antimyosin autoantibodies as a predictive and diagnostic marker for neonatal necrotizing enterocolitis through a large amount of data and experiments. The AUC value was 0.8856, the optimal cutoff value was 14.68 ng / mL, corresponding to a sensitivity of 81.58% and a specificity of 76.93%.

[0009] According to a second aspect of the present invention, the application of an antimyosin autoantibody detection reagent in the preparation of diagnostic products for neonatal necrotizing enterocolitis is proposed.

[0010] In some embodiments of the present invention, the product includes test strips, test chips, or reagent kits.

[0011] In this invention, those skilled in the art can prepare it into other diagnostic products for neonatal necrotizing enterocolitis according to actual usage needs, including but not limited to the test strips, test chips or reagent kits involved in the embodiments of this invention.

[0012] In some embodiments of the present invention, the antimyosin autoantibody detection reagent is used to perform any of the following methods: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, indirect immunofluorescence assay, dot immunogold filtration assay, Western blotting, and flow cytometry.

[0013] In some specific embodiments of the present invention, the verification method for the antimyosin autoantibody detection reagent is enzyme-linked immunosorbent assay (ELISA).

[0014] In some embodiments of the present invention, the antimyosin autoantibody includes IgG and IgM type antimyosin autoantibodies.

[0015] In some embodiments of the present invention, the newborn is a premature newborn with a corrected gestational age of <44 weeks.

[0016] In some embodiments of the present invention, the newborn is a premature newborn with a gestational age of 27 to 36 weeks.

[0017] In some embodiments of the present invention, the test sample for the product is selected from blood or tissue fluid.

[0018] In some specific embodiments of the present invention, the sample is plasma from peripheral blood.

[0019] In some embodiments of the present invention, the product also includes a control sample.

[0020] In some embodiments of the present invention, the control sample is peripheral blood of a healthy newborn or a product of peripheral blood after processing or treatment.

[0021] In some embodiments of the present invention, the product of the peripheral blood after processing or treatment is peripheral blood after the removal of red blood cells.

[0022] In some embodiments of the present invention, the criteria for determining the diagnostic product for neonatal necrotizing enterocolitis are as follows:

[0023] Using the level of anti-myosin autoantibodies in healthy newborns as a control, if the level of anti-myosin autoantibodies in the tested sample is higher than that in the control, there is a risk of neonatal necrotizing enterocolitis; otherwise, there is no risk of neonatal necrotizing enterocolitis.

[0024] In this invention, all experimental results are analyzed using statistical methods. The t-test is used to assess the differences between the two groups, and P<0.05 is used to indicate statistical significance. All P values ​​are two-tailed.

[0025] In some embodiments of the present invention, the criteria for determining the diagnostic product for neonatal necrotizing enterocolitis are as follows:

[0026] When the level of antimyosin autoantibody in the sample is higher than 14.68 ng / mL, there is a risk of neonatal necrotizing enterocolitis; otherwise, there is no risk of neonatal necrotizing enterocolitis.

[0027] In some embodiments of the present invention, when antimyosin autoantibodies are used as predictive and diagnostic markers for stages I, II, and III of neonatal necrotizing enterocolitis (NEC), the AUC value for stage I NEC is 0.9457 (95% CI: 0.8727 to 1.000), with the optimal cutoff corresponding to a sensitivity of 88.23% and a specificity of 84.62%; the AUC value for stage II NEC is 0.8322 (95% CI: 0.6697 to 0.9947), with the optimal cutoff corresponding to a sensitivity of 72.73% and a specificity of 76.93%; and the AUC value for stage III NEC is 0.8423 (95% CI: 0.6824 to 1.000), with the optimal cutoff corresponding to a sensitivity of 80% and a specificity of 76.93%.

[0028] In this invention, the inventors compared the expression levels of antimyosin autoantibodies at different NEC stages with those of the control group and found that the levels of antimyosin autoantibodies at different NEC stages were higher than those in the control group.

[0029] A third aspect of the present invention provides a diagnostic product for neonatal necrotizing enterocolitis, the product comprising an antimyosin autoantibody detection reagent.

[0030] The beneficial effects of this invention are:

[0031] 1. This invention is the first to identify antimyosin autoantibodies as biomarkers for the identification and diagnosis of neonatal necrotizing enterocolitis (NEC), providing a valid basis for the early diagnosis and warning of NEC.

[0032] 2. This invention uses plasma samples for detection, which has the advantages of simple operation, non-invasiveness, high throughput and low cost. Furthermore, this invention has found that the detection of antimyosin autoantibodies can have high sensitivity and specificity in the diagnosis of NEC stage I, thus enabling the differentiation between NEC patients and non-NEC patients at the NEC stage I. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0034] Figure 1 The above are the IgG detection results in plasma samples from the NEC and non-NEC groups of the abdominal distension group screened using a human autoimmune antigen chip in this embodiment of the invention.

[0035] Figure 2 The above are the IgM detection results in plasma samples from the NEC and non-NEC groups of the abdominal distension group screened using human autoimmune antigen chips in this embodiment of the invention.

[0036] Figure 3 This invention provides a comparison of the levels of antimyosin autoantibodies in plasma samples from a control group and a group diagnosed with NEC, detected by enzyme-linked immunosorbent assay (ELISA).

[0037] Figure 4 The ROC curves of antimyosin autoantibodies against plasma samples from the control group and the group diagnosed with NEC are shown in this embodiment of the invention.

[0038] Figure 5 This invention provides a comparison of the levels of antimyosin autoantibodies in plasma samples from the control group and the confirmed NEC group at stages I, II, and III of neonatal necrotizing enterocolitis, detected by enzyme-linked immunosorbent assay (ELISA).

[0039] Figure 6 The ROC curves of antimyosin autoantibodies against plasma samples from three groups of neonatal necrotizing enterocolitis (NEC) in stages I, II, and III, in an embodiment of the present invention, are shown. Detailed Implementation

[0040] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0041] In this embodiment of the invention, all samples tested were obtained from Guangzhou Women and Children's Medical Center. The collection of all experimental tissue and blood samples was authorized by the Ethics Committee of Guangzhou Women and Children's Medical Center and obtained the consent of the patients.

[0042] In the analysis of the experimental results in this embodiment of the invention, a two-tailed t-test was used to assess the differences between the two groups, and P<0.05 indicated statistical significance.

[0043] In this embodiment of the invention, "AUC" refers to the area under the ROC curve, which is between 0.1 and 1. It is used to evaluate the quality of the classifier. The closer the AUC is to 1, the better the discrimination effect of the classifier.

[0044] Plasma sample collection and grouping

[0045] In this embodiment, the diagnosis of "necrotizing enterocolitis (NEC) in newborns" is based on "Practical Neonatology" (4th Edition), and is divided into stages I, II, and III according to the revised Bell-NEC staging system, as shown in Table 1 below:

[0046] Table 1. Bell Staging for Necrotizing Enterocolitis (Revised Version)

[0047]

[0048]

[0049] Plasma samples were collected from different population groups at Guangzhou Women and Children's Medical Center for use in subsequent experiments. Newborns whose mothers had a history of autoimmune diseases, children with digestive tract malformations, and children with inherited metabolic disorders were excluded. Blood was collected using anticoagulation, followed by centrifugation to separate the plasma, and the samples were then cryopreserved. Specific collection procedures are as follows:

[0050] Abdominal distension group: Newborns with unexplained abdominal distension (premature infants with corrected gestational age <44 weeks) admitted to the neonatal department were selected, and their plasma samples were collected for subsequent experiments.

[0051] Based on the subsequent final diagnosis, the newborns were divided into two groups: the abdominal distension NEC group (24 cases, which were further divided into NEC stage I (10 cases), stage II (9 cases), and stage III (5 cases) according to the Bell-NEC staging criteria) and the abdominal distension non-NEC group (26 cases). The gestational age of the newborns in both groups was 27-36 weeks, and the birth weight was 900-1950g. There was no significant difference in gestational age and birth weight between the two groups.

[0052] Confirmed NEC group: 38 cases of confirmed NEC patients hospitalized in the neonatal department with similar gestational age and weight as mentioned above (17 cases of NEC stage I, 11 cases of stage II, and 10 cases of stage III) were selected as the confirmed NEC group, and their plasma samples were collected for subsequent experiments.

[0053] Control group: Thirteen non-NEC newborns with matched gestational age and weight were randomly selected as the control group, and their plasma samples were collected for subsequent experiments.

[0054] Human autoimmune antigen microarray screening and differential expression analysis of autoantibodies

[0055] This embodiment uses a human autoimmune antigen chip to screen and analyze differentially expressed autoantibodies in samples from the abdominal distension group (NEC group and non-NEC group) obtained in the above embodiment. The specific process is as follows:

[0056] Samples from the abdominal distension NEC group (24 cases, including 10 cases of NEC stage I, 9 cases of stage II, and 5 cases of stage III) and the abdominal distension non-NEC group (26 cases) were screened using a human autoimmune antigen microarray (from Guangzhou Yijin Biotechnology Co., Ltd.). In this embodiment, an autoantigen microarray composed of 48 autoantigens and 8 calibration proteins was used to screen for autoantibodies, including dsDNA, MAG, GAD1, myosin, and other autoantibodies (including IgG and IgM antibodies). Raw data and calibration proteins served as negative controls. After subtracting the negative controls, the data were normalized using the RLM method, and the results were analyzed for inter-group differences using M-statistics.

[0057] Figure 1 and Figure 2 The cluster analysis plot shows the differentially expressed autoantibodies, where Myosin represents the expression level of anti-myosin autoantibody. Figure 1 The study showed differences in the expression levels of IgG antibodies against myosin autoantibodies between the abdominal distension NEC group and the abdominal distension non-NEC group. Figure 2 The study showed differences in the expression levels of antimyosin autoantibodies IgM antibodies between the bloating NEC group and the bloating non-NEC group, including IgG (P = 0.0037) and IgM (P < 0.0001).

[0058] It was found that the expression level of antimyosin autoantibodies in the plasma of children with abdominal distension (NEC) was significantly higher than that in children without NEC, including IgG (P=0.0037) and IgM (P<0.0001), indicating a significant difference in the expression level of antimyosin autoantibodies between the two groups. This suggests that detecting the expression level of antimyosin autoantibodies can effectively identify neonatal necrotizing enterocolitis.

[0059] Enzyme-linked immunosorbent assay (ELISA) for the detection of antimyosin autoantibodies

[0060] To further verify the diagnostic efficacy of antimyosin autoantibodies for necrotizing enterocolitis (NEC) in newborns, this embodiment used enzyme-linked immunosorbent assay (ELISA) to detect the levels of antimyosin autoantibodies in samples from the confirmed NEC group and the control group in the above embodiment. The specific process is as follows:

[0061] The levels of anti-myosin autoantibodies in plasma samples from the confirmed NEC group and control group collected in the above examples were detected using a commercially available ELISA kit. The detection method was performed according to the instructions in the ELISA kit. The ELISA kit used in this example is the human myosin heavy chain autoantibody (MYHA) ELISA kit (purchased from Shanghai Zhenke Biotechnology Co., Ltd.).

[0062] The results of anti-myosin autoantibody expression levels in plasma samples from each group are as follows: Figure 3 As shown, the plasma samples of NEC patients showed significantly higher levels of MYHA than those of the control group (P<0.0001).

[0063] This embodiment uses ROC curves to evaluate the diagnostic efficacy of antimyosin autoantibodies for NEC. The results are as follows: Figure 4 As shown, the AUC was 0.8856; the optimal cutoff value for the AUC curve was 14.68 ng / mL, corresponding to a sensitivity of 81.58% and a specificity of 76.93%. Therefore, anti-myosin autoantibodies can effectively diagnose necrotizing enterocolitis in newborns, and a level of anti-myosin autoantibodies above 14.68 ng / mL can effectively predict the risk of neonatal necrotizing enterocolitis.

[0064] The diagnostic value of antimyosin autoantibodies in the early diagnosis of necrotizing enterocolitis in newborns.

[0065] This embodiment further verifies the predictive effect of antimyosin autoantibodies in early neonatal necrotizing enterocolitis (NEC). The specific process is as follows:

[0066] The above-mentioned confirmed NEC group was divided into three groups: I, II, and III according to the Bell-NEC staging. The expression levels of antimyosin autoantibodies in the three staging groups were distinguished by the NEC I, II, and III staging criteria. The diagnostic efficacy of antimyosin autoantibodies in the three staging groups was evaluated by ROC curves.

[0067] The results of anti-myosin autoantibody expression levels in plasma samples from each group are as follows: Figure 5 As shown, the antimyosin autoantibodies in all three stages of the NEC diagnosis group were higher than those in the control group, and the differences were statistically significant.

[0068] The ROC curve evaluation results are as follows: Figure 6As shown, the AUC value of antimyosin autoantibody for predicting NEC stage I was 0.9457 (95% CI: 0.8727 to 1.000), the AUC value for predicting NEC stage II was 0.8322 (95% CI: 0.6697 to 0.9947), and the AUC value for predicting NEC stage III was 0.8423 (95% CI: 0.6824 to 1.000).

[0069] It was found that the levels of antimyosin autoantibodies in all three stages of NEC differed significantly from those in the control group. Furthermore, the levels of antimyosin autoantibodies were significantly elevated in NEC stage I, effectively distinguishing NEC stage I from the non-NEC control, with an AUC of 0.9457. Therefore, antimyosin autoantibodies have good diagnostic value for NEC, especially in differentiating between NEC stage I and non-NEC, providing a reference for early clinical diagnosis and differentiation of NEC.

[0070] In summary, this invention screened for significantly elevated anti-myosin autoantibody expression levels in the plasma of neonatal necrotizing enterocolitis (NEC) infants using a human autoimmune antigen chip. Enzyme-linked immunosorbent assay (ELISA) was used in samples from an independent NEC-diagnosed group to verify that the expression level of anti-myosin autoantibody in the NEC-diagnosed group was significantly higher than that in the control group, effectively diagnosing NEC (AUC: 0.8856). The optimal cutoff value was 14.68 ng / mL, corresponding to a sensitivity of 81.58% and a specificity of 76.93%. Notably, a significant increase in anti-myosin autoantibody was found in NEC stage I cases, with an AUC of 0.9457 (95% CI: 0.8727). The result of the above verification experiments (1.000) suggests that it can be used to differentiate between NEC and non-NEC cases in the early stage, providing an effective basis for the early diagnosis and warning of NEC. The results of the above verification experiments prove that antimyosin autoantibodies can be used as diagnostic markers for NEC plasma, especially suitable for the early diagnosis of NEC and non-NEC, providing a strong reference indicator for the early diagnosis and treatment of clinical NEC.

[0071] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of antimyosin autoantibody detection reagent in the preparation of diagnostic products for neonatal necrotizing enterocolitis; The antimyosin autoantibody is an IgG and IgM type antimyosin autoantibody.

2. The application according to claim 1, characterized in that, The products include test strips, test chips, or reagent kits.

3. The application according to claim 1, characterized in that, The newborns mentioned are premature newborns with a corrected gestational age of <44 weeks.

4. The application according to claim 1, characterized in that, The test sample for the product was blood.

5. The application according to claim 1, characterized in that, The product also includes a control sample.

6. The application according to claim 1, characterized in that, The diagnostic criteria for the aforementioned neonatal necrotizing enterocolitis diagnostic product are as follows: Using the level of anti-myosin autoantibodies in healthy newborns as a control, if the level of anti-myosin autoantibodies in the tested sample is higher than that in the control, there is a risk of neonatal necrotizing enterocolitis; otherwise, there is no risk of neonatal necrotizing enterocolitis.

7. The application according to claim 1, characterized in that, The diagnostic criteria for the aforementioned neonatal necrotizing enterocolitis diagnostic product are as follows: When the level of antimyosin autoantibody in the sample is higher than 14.68 ng / mL, there is a risk of neonatal necrotizing enterocolitis; otherwise, there is no risk of neonatal necrotizing enterocolitis.

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