Application of Myelin Basic Protein Antibody in the Diagnosis of Neonatal Necrotizing Enterocolitis

Abstract

The present invention provides the application of myelin basic protein antibody in the diagnosis of neonatal necrotizing enterocolitis. Through a large number of studies and screenings, the present invention has obtained a specific biomarker for detecting NEC, and the biomarker is myelin basic protein antibody, including IgG type and / or IgM type. The content of the myelin basic protein antibody in the plasma of children with NEC is significantly higher than that of normal controls. Further analysis reveals that when the myelin basic protein antibody is used for diagnosing children with NEC, it has high sensitivity, specificity, and accuracy. Therefore, the myelin basic protein antibody can be used as a specific biomarker for the early prediction and confirmation of NEC, and can be used for the diagnosis of the disease and the evaluation of its progression.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and particularly relates to the application of myelin basic protein antibody in the diagnosis of neonatal necrotizing enterocolitis. Background Art

[0002] Neonatal necrotizing enterocolitis (NEC) is one of the common digestive diseases in the neonatal intensive care unit. In very low birth weight infants, the incidence of this disease is about 10% to 12%, but according to data from different research centers, its incidence ranges from 2% to 22%. Currently, the modified Bell staging is used to diagnose NEC, which divides NEC into NEC stage I (suspected NEC), NEC stage II, and NEC stage III. Among them, the case fatality rate of stage II and stage III NEC reaches 30%. Conservative treatment is generally adopted for stage I and stage II NEC, but it is prone to progress to stage III. Children in stage III are mainly treated by surgery, and the postoperative mortality rate is extremely high (75%-80%). Therefore, early diagnosis and monitoring of NEC are crucial for the diagnosis and treatment of NEC.

[0003] Myelin basic protein (MBP) is an autoimmune antigen. Research reports that it promotes the release of inflammatory factors (such as TNF-α, IL-4, etc.) through autoantibody - myelin basic protein antibody (MBP-Abs) and participates in the pathogenesis of multiple sclerosis. However, the application of MBP-Ab in the diagnosis of NEC has not been reported. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide the application of myelin basic protein antibody in the diagnosis of neonatal necrotizing enterocolitis. The myelin basic protein antibody can be used as a specific diagnostic marker for neonatal necrotizing enterocolitis and has good diagnostic effects on stage I, stage II, and stage III neonatal necrotizing enterocolitis.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions.

[0006] The first aspect of the present invention is to provide the application of myelin basic protein antibody as a biomarker in the preparation of a detection reagent for neonatal necrotizing enterocolitis.

[0007] The second aspect of the present invention is to provide the application of a reagent for detecting the content of myelin basic protein antibody in the preparation of a detection kit for neonatal necrotizing enterocolitis.

[0008] In some embodiments, the myelin basic protein antibody includes IgG type and / or IgM type.

[0009] In some embodiments, the myelin basic protein antibody comprises the amino acid sequence shown in SEQ ID NO.1.

[0010] In some embodiments, the neonatal necrotizing enterocolitis includes stage I neonatal necrotizing enterocolitis, stage II neonatal necrotizing enterocolitis, and / or stage III neonatal necrotizing enterocolitis.

[0011] In some embodiments, the detection methods include protein chip method, ELISA method, immunoblotting method, and flow cytometry.

[0012] The third aspect of the present invention is to provide a detection kit for neonatal necrotizing enterocolitis, which includes reagents for detecting the content of myelin basic protein antibody.

[0013] In some embodiments, the myelin basic protein antibody includes IgG type and / or IgM type.

[0014] In some embodiments, the myelin basic protein antibody comprises the amino acid sequence shown in SEQ ID NO.1.

[0015] In some embodiments, the kit includes reagents for detection by protein chip method, ELISA method, immunoblotting method, and flow cytometry.

[0016] The present invention has the following beneficial effects.

[0017] Through a large number of studies and screenings, the present invention has obtained a specific biomarker for detecting NEC, and the biomarker is myelin basic protein antibody, including IgG type and / or IgM type. The content of the myelin basic protein antibody in the plasma of children with NEC is significantly higher than that of the normal control. Further analysis found that when the myelin basic protein antibody is used for diagnosing children with NEC, the AUC value is 0.9005, the sensitivity corresponding to the optimal cut-off is 76.27%, and the specificity is 96.77%; when diagnosing children with NEC I, the AUC value is 0.8797, the sensitivity corresponding to the optimal cut-off is 73.07%, and the specificity is 96.77%; when diagnosing children with NEC II, the AUC value is 0.9165, the sensitivity corresponding to the optimal cut-off is 82.35%, and the specificity is 96.77%; when diagnosing children with NEC III, the AUC value is 0.9173, the sensitivity corresponding to the optimal cut-off is 70%, and the specificity is 100%. Therefore, the myelin basic protein antibody can be used as a specific biomarker for the early prediction and diagnosis of NEC, and can be used for the diagnosis of the disease and the evaluation of the disease progression. Description of the Drawings

[0018] Figure 1 To analyze the plasma of the control group and the necrotizing enterocolitis (NEC) group using an autoimmune chip.

[0019] Figure 2 To detect the plasma level of MBP-Abs in children with NEC and the ROC curve by enzyme-linked immunosorbent assay (ELISA) technology.

[0020] Figure 3 For the plasma level of MBP-Abs in children with NEC at each stage and the ROC curve. Specific implementation manner

[0021] In the following examples of the present invention, the experimental methods without specific conditions noted are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or equipment.

[0024] The following is described in conjunction with specific embodiments.

[0025] The MBP-Abs described in the present invention is a human myelin basic protein antibody.

[0026] In some of these embodiments, the MBP-Abs includes the amino acid sequence shown in SEQ ID NO.1 (see details at http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi?chain_id=7710).

[0027] SEQ ID NO.1: EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSYIHWVRQAPGKGLEWVAYISSYSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTPWWYWSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT。

[0028] In the following examples, it was first found through autoantibody chip analysis that the level of MBP-Abs in plasma was significantly increased in children with NEC. Next, the ELISA kit used was provided by Shanghai Zhenke Biochemical Co., Ltd., and the level of MBP-Abs was detected using the Human Myelin Basic Protein Antibody (MBP-Abs) ELISA Kit (product number: ZK-1993).

[0029] Example 1

[0030] 1. Samples and grouping

[0031] NEC group: According to the Bell staging criteria of the NEC revised edition in "Practical Neonatology" (Fourth Edition), 30 children with NEC diagnosed in Guangzhou First People's Hospital and Guangzhou Women and Children's Medical Center from January 2021 to May 2022 were selected, including children with NEC stage I, NEC stage II, and NEC stage III.

[0032] Control group: 30 non-NEC children (mainly children with physiological diarrhea, and children with inflammatory diseases, digestive tract malformations, and genetic metabolism were excluded) with the same corrected gestational age, gender, and birth weight as those in the NEC group during the same period were selected.

[0033] Fresh peripheral blood of all included children was collected in EDTA anticoagulant tubes, and plasma was centrifuged and stored in a -80°C refrigerator for autoantibody chip analysis of the level of autoimmune antibodies.

[0034] 2. Experimental method

[0035] Analysis of plasma autoimmune antibodies using an autoantibody chip: Plasma samples of children with NEC (n = 30) and children in the control group (n = 30) were collected, and an autoantigen microarray consisting of 120 autoantigens and 8 calibration proteins was used to screen for autoantigen microarrays in the plasma of children with NEC (detected by entrusting Guangzhou Yijin Biotechnology Co., Ltd.).

[0036] 3. Experimental Results

[0037] Figure 1 In A and B, they are the detection results of IgG MBP-Ab in children with NEC. Figure 1 In C and D, they are the detection results of IgM MBP-Ab in children with NEC. The results show that the levels of IgG MBP-Ab and IgM MBP-Ab in children with NEC are significantly higher than those in the control group (p < 0.0001), and they can be used as potential biomarkers for the detection of NEC.

[0038] Example 2

[0039] Samples were recollected for testing to verify the efficacy of MBP-Abs found in the above example in the diagnosis of NEC.

[0040] 1. Samples and Grouping

[0041] NEC Group: According to the Bell staging criteria of the revised version of NEC in "Practical Neonatology" (Fourth Edition), 59 children diagnosed with NEC in Guangzhou First People's Hospital and Guangzhou Women and Children's Medical Center from August 2022 to May 2024 were selected, including 26 cases in NEC stage I, 17 cases in NEC stage II, and 16 cases in NEC stage III.

[0042] Control Group: 31 non-NEC children (excluding children with inflammatory diseases, digestive tract malformations, and genetic metabolism) with corrected gestational age, gender, and birth weight matched to those in the NEC group during the same period were selected.

[0043] Using the method of calculating the sample size based on the area under the ROC curve in PASS software, and based on the relevant articles (PMID: 37715022) reporting on the research of diagnosing NEC, the areas under the ROC curve for diagnosing NEC stage I, diagnosing NEC stage II, and diagnosing NEC stage III are 0.94, 0.83, and 0.84 respectively to estimate the sample size of the present invention. Among them, the number of cases in NEC stage I is not less than 6, the number of cases in NEC stage II is not less than 12, the number of cases in NEC stage III is not less than 13, and the control group is not less than 6. The sample size included in this example is as follows: 26 cases in NEC stage I, 17 cases in NEC stage II, 16 cases in NEC stage III, and 31 cases in the control group, and the sample size meets the statistical requirements.

[0044] Fresh peripheral blood of all included children was collected into EDTA anticoagulant tubes, and the plasma was centrifuged and stored in a -80°C refrigerator for detecting the level of MBP-Abs by enzyme-linked immunosorbent assay (ELISA).

[0045] 2. Experimental Methods

[0046] Detect the levels of MBP-Abs (including IgG and IgM types) using an ELISA kit for human myelin basic protein antibody (MBP-Abs) (Shanghai Zhenke Biochemical, product number ZK-1993).

[0047] The specific detection steps are as follows:

[0048] 1) Place the kit at room temperature for 20 min to restore to room temperature. Take out the strips to be used from the foil bag, and seal and store the unused strips at 4°C.

[0049] 2) Set up the standard wells and sample wells according to the number of samples. Add different concentrations of standards to each standard well at 50 μl per well.

[0050] 3) Sample dilution: Dilute the serum / plasma samples of the children by 5 times, that is, add 10 μl of the children's serum / plasma samples to 40 μl of the sample diluent and mix well.

[0051] 4) Add samples: Add 50 μl of different concentrations of standards to each standard well, add 50 μl of the diluted serum / plasma samples to each sample well, and add 50 μl of ddH20 to the blank well.

[0052] 5) Add 100 μl of the detection antibody labeled with horseradish peroxidase (HRP) to each well of the standards and samples, seal the wells with a sealing film, and incubate in a 37°C incubator in the dark with tin foil for 60 min.

[0053] 6) Discard the liquid, pat dry on the absorbent paper, fill each well with washing solution, let stand for 1 min, discard the washing solution, and pat dry on the absorbent paper. Repeat the washing process 5 times.

[0054] 7) Add 50 μl of substrate A or B to each well, and incubate in a 37°C incubator in the dark with tin foil for 15 min.

[0055] 8) Add 50 μl of the termination solution to each well. Measure within 15 min and measure the OD value of each well at a wavelength of 450 nm.

[0056] 9) Draw a standard curve: Name the abscissa as the standard concentration and the ordinate as the OD value, draw the standard linear regression curve, and calculate the concentration values of each sample according to the curve equation.

[0057] 3. Experimental results

[0058] (1) Levels of MBP-Abs

[0059] Analyze the levels of MBP-Abs in the plasma of 59 NEC children and 31 control group by ELISA. The results show that the MBP-Abs in NEC children is significantly higher than that in the control group (p < 0.0001), asFigure 2 As shown in A. Further, NEC was divided into stage I, stage II, and stage III of NEC. It was found that the levels of MBP-Abs in the plasma of children with each stage of NEC were higher than those in the control group (p < 0.0001 for all), as Figure 3 shown in A.

[0060] (2) ROC curve analysis

[0061] Further ROC curve analysis was performed. It was found that when diagnosing children with NEC, the AUC value of MBP-Abs was 0.9005, the optimal cut-off corresponded to a sensitivity of 76.27% and a specificity of 96.77%, as Figure 2 shown in B.

[0062] According to the revised Bell staging, NEC was staged into stage I (suspected NEC), stage II, and stage III of NEC. It was found that when diagnosing children with stage I of NEC, the AUC value of MBP-Abs was 0.8797, the optimal cut-off corresponded to a sensitivity of 73.07% and a specificity of 96.77%; when diagnosing children with stage II of NEC, the AUC value of MBP-Abs was 0.9165, the optimal cut-off corresponded to a sensitivity of 82.35% and a specificity of 96.77%; when diagnosing children with stage III of NEC, the AUC value of MBP-Abs was 0.9173, the optimal cut-off corresponded to a sensitivity of 70% and a specificity of 100%, as Figure 3 shown in B.

[0063] Thus, it can be seen that MBP-Abs has high specificity and accuracy for NEC and each stage of NEC (stage I, stage II, and stage III), and can be used for the diagnosis of NEC and early NEC.

[0064] The diagnostic method based on MBP-Abs has the advantages of simple operation, non-invasive, high throughput, low cost, and no radiation hazards in the diagnosis and prediction of neonatal NEC compared with auxiliary diagnostic examinations such as abdominal X-ray plain film in clinical practice.

[0065] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered to be within the scope described in this specification.

[0066] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. Use of a reagent for detecting myelin basic protein antibodies in plasma in the preparation of a reagent for detecting neonatal necrotizing enterocolitis, characterized in that: The myelin basic protein antibodies include IgG type and IgM type.

2. Use of a reagent for detecting the content of myelin basic protein antibodies in plasma in the preparation of a detection kit for neonatal necrotizing enterocolitis, characterized in that: The myelin basic protein antibodies include IgG type and IgM type.

3. The use according to claim 1 or 2, characterized in that: The neonatal necrotizing enterocolitis includes stage I neonatal necrotizing enterocolitis, stage II neonatal necrotizing enterocolitis and / or stage III neonatal necrotizing enterocolitis.

4. The use according to claim 1 or 2, characterized in that: The detection methods include protein chip method, ELISA method, immunoblotting method and flow cytometry.

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