Application of myelin basic protein antibody in diagnosis of neonatal necrotizing enterocolitis

Abstract

The invention provides application of a myelin basic protein antibody in diagnosis of neonatal necrotizing enterocolitis. A specific biomarker for detecting NEC is obtained through a large amount of research and screening, and the biomarker is a myelin sheath basic protein antibody including an IgG type and / or an IgM type. The content of the myelin basic protein antibody in NEC child patient plasma is obviously higher than that of a normal control. Further analysis shows that the myelin basic protein antibody has high sensitivity, specificity and accuracy when being used for diagnosing NEC sick children. Therefore, the myelin basic protein antibody can be used as a specific biomarker for early prediction and definite diagnosis of NEC, and can be used for diagnosis and progression evaluation of diseases.

Description

Technical Field

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

[0002] Necrotizing enterocolitis (NEC) is one of the common gastrointestinal diseases in neonatal intensive care units. The incidence of the disease is about 10% to 12% in extremely low birth weight infants, but according to data from different research centers, its incidence is between 2% and 22%. Currently, the revised version of Bell's staging system is used to diagnose NEC, which is divided into NEC stage I (suspected NEC), NEC stage II, and NEC stage III, of which stage II and stage III NEC cases have an associated mortality rate of 30%. NEC stage I and II are generally treated conservatively, but they are prone to progress to stage III. Children in stage III are mainly treated with 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, and studies have reported that it participates in the pathogenesis of multiple sclerosis by promoting the release of inflammatory factors (such as TNF-α, IL-4, etc.) through autoantibodies - myelin basic protein antibodies (MBP-Abs). However, MBP-Ab has not been reported in the diagnosis of NEC. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide the use of myelin basic protein antibodies in the diagnosis of neonatal necrotizing enterocolitis. Myelin basic protein antibodies can be used as specific diagnostic markers for neonatal necrotizing enterocolitis, and have a good diagnostic effect on stage I, stage II and stage III neonatal necrotizing enterocolitis.

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

[0006] The first aspect of the present invention is to provide the use 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 use of a reagent for detecting the content of myelin basic protein antibodies in the preparation of a detection kit for neonatal necrotizing enterocolitis.

[0008] In some embodiments, the MBP antibody comprises 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 comprises stage I neonatal necrotizing enterocolitis, stage II neonatal necrotizing enterocolitis, and / or stage III neonatal necrotizing enterocolitis.

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

[0012] The third aspect of the present invention is to provide a neonatal necrotizing enterocolitis detection kit, comprising a reagent for detecting the content of myelin basic protein antibodies.

[0013] In some embodiments, the MBP antibody comprises 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 protein chip, ELISA, immunoblotting, and flow cytometry.

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

[0017] The present invention has obtained a specific biomarker for detecting NEC through a large amount of research and screening, and the biomarker is a 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 to diagnose children with NEC, the AUC value is 0.9005, the best limit corresponds to a sensitivity of 76.27%, and the specificity is 96.77%; when diagnosing children with NECI, the AUC value is 0.8797, the best limit corresponds to a sensitivity of 73.07%, and the specificity is 96.77%; when diagnosing children with NECII, the AUC value is 0.9165, the best limit corresponds to a sensitivity of 82.35%, and the specificity is 96.77%; when diagnosing children with NECIII, the AUC value is 0.9173, the best limit corresponds to a sensitivity of 70%, and the specificity is 100%. Therefore, myelin basic protein antibodies can be used as specific biomarkers for early prediction and diagnosis of NEC and can be used for the diagnosis and progression assessment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 The ELISA technique was used to detect the plasma levels of MBP-Abs in children with NEC and the ROC curve was used.

[0020] Figure 3 Figure 3 shows the plasma levels of MBP-Abs in children with different NEC stages and the ROC curve. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. 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 "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0024] The following is a description with reference to specific embodiments.

[0025] The MBP-Abs described in the present invention are human myelin basic protein antibodies.

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

[0027] SEQ ID NO.1: EISEVQLVESGGGLVQPGGSLRLSCAASGFNFSSSYIHW VRQAPGKGLEWVAYISSYSGYTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTPWWYWSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT.

[0028] In the following examples, the autoantibody chip analysis first found that the MBP-Abs level in plasma was significantly increased in children with NEC. The ELISA kit used next was provided by Shanghai Zhenke Biochemical, and the MBP-Abs level was detected using the human myelin basic protein antibody (MBP-Abs) ELISA detection kit (Cat. No.: ZK-1993).

[0029] Example 1

[0030] 1. Samples and Grouping

[0031] NEC group: According to the Bell staging criteria of the NEC revised version of Practical Neonatology (4th 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 NECI stage, NEC stage II and NEC stage III.

[0032] Control group: 30 non-NEC infants (mainly infants with physiological diarrhea, and excluding those with inflammatory diseases, gastrointestinal malformations and genetic metabolism) who were hospitalized during the same period and matched with the NEC group in terms of corrected gestational age, gender and birth weight were selected.

[0033] Fresh peripheral blood was collected from all included children in EDTA anticoagulant tubes, and plasma was collected by centrifugation and stored in a -80°C refrigerator for analysis of autoimmune antibody levels using autoantibody chip.

[0034] 2. Experimental Methods

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

[0036] 3. Experimental results

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

[0038] Example 2

[0039] The samples were re-collected for testing to verify the efficacy of the MBP-Abs found in the above examples in diagnosing NEC.

[0040] 1. Samples and Grouping

[0041] NEC group: According to the Bell staging standard of the NEC revised version of "Practical Neonatology" (4th 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 children in stage NECI, 17 children in stage NECII, and 16 children in stage NECIII.

[0042] Control group: 31 non-NEC infants who were hospitalized during the same period and matched with the NEC group in terms of corrected gestational age, gender, and birth weight (infants with inflammatory diseases, gastrointestinal malformations, and genetic metabolism were excluded).

[0043] The method of calculating the sample size using the area under the ROC curve in the PASS software was based on the relevant article on the diagnosis of NEC research (PMID: 37715022). The areas under the ROC curve for diagnosing NEC stage I, diagnosing NEC stage II, and diagnosing NEC stage III were 0.94, 0.83, and 0.84, respectively. The sample size of the present invention was estimated, wherein NEC stage I was not less than 6 cases, NEC stage II was not less than 12 cases, NEC stage III was not less than 13 cases, and the control group was not less than 6 cases. The sample size included in this embodiment is as follows: 26 cases in NECI stage, 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 was collected from all included children in EDTA anticoagulant tubes, and plasma was collected by centrifugation and stored in a -80°C refrigerator for enzyme-linked immunosorbent assay (ELISA) to detect MBP-Abs levels.

[0045] 2. Experimental Methods

[0046] The level of MBP-Abs (including IgG and IgM types) was detected using human myelin basic protein antibody (MBP-Abs) ELISA detection kit (Shanghai Zhenke Biochemical, catalog number ZK-1993).

[0047] The specific detection steps are as follows:

[0048] 1) Place the test kit at room temperature for 20 minutes, return to room temperature, take out the strips to be used from the tin foil bag, and seal the unused strips and store them at 4°C.

[0049] 2) According to the number of samples, set standard wells and sample wells, and add 50ul of standards of different concentrations to each well of the standard wells.

[0050] 3) Sample dilution: dilute the patient's serum / plasma sample 5 times, i.e., add 40ul of sample diluent to 10ul of the patient's serum / plasma sample and mix well.

[0051] 4) Sample addition: Add 50ul of standards of different concentrations to the standard wells, add 50ul of diluted serum / plasma samples to the sample wells, and add 50ul of ddH20 to the blank wells.

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

[0053] 6) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.

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

[0055] 8) Add 50ul of stop solution to each well, measure within 15 minutes, and obtain the OD value of each well at a wavelength of 450nm.

[0056] 9) Draw a standard curve: the horizontal axis is the standard concentration, the vertical axis is the OD value, draw a linear regression curve of the standard, and calculate the concentration value of each sample according to the curve equation.

[0057] 3. Experimental results

[0058] (1) Levels of MBP-Abs

[0059] The levels of MBP-Abs in the plasma of 59 NEC children and 31 controls were analyzed by ELISA. The results showed that the MBP-Abs in NEC children were significantly higher than those in the control group (p<0.0001). Figure 2 As shown in A. NEC was further divided into NEC stage I, NEC II and NEC stage III. The results showed that the MBP-Abs levels in the plasma of children with NEC at all stages were higher than those in the control group (p < 0.0001). Figure 3 As shown in A.

[0060] (2) ROC curve analysis

[0061] Further ROC curve analysis showed that the AUC value of MBP-Abs in diagnosing NEC in children was 0.9005, the best limit corresponding to a sensitivity of 76.27%, and a specificity of 96.77%. Figure 2 As shown in B.

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

[0063] It can be seen from this 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] Compared with clinical abdominal X-ray and other auxiliary diagnostic examinations, the MBP-Abs-based diagnostic method has the advantages of simple operation, non-invasiveness, high throughput, low cost, and no radiation hazards in the diagnosis and prediction of neonatal NEC.

[0065] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, 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.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of myelin basic protein antibody as a biomarker in the preparation of neonatal necrotizing enterocolitis detection reagents.

2. Application of a reagent for detecting the content of myelin basic protein antibodies in the preparation of a detection kit for neonatal necrotizing enterocolitis.

3. The use according to claim 1 or 2, characterized in that: The myelin basic protein antibody includes IgG type and / or IgM type.

4. The use according to claim 3, characterized in that The myelin basic protein antibody comprises the amino acid sequence shown in SEQ ID NO.

1.

5. 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.

6. 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.

7. A detection kit for neonatal necrotizing enterocolitis, characterized in that: Includes reagents for detecting myelin basic protein antibody levels.

8. The detection kit according to claim 7, characterized in that The myelin basic protein antibody includes IgG type and / or IgM type.

9. The detection kit according to claim 8, characterized in that The myelin basic protein antibody comprises the amino acid sequence shown in SEQ ID NO.

1.

10. The detection kit according to any one of claims 7 to 9, characterized in that: The kit comprises reagents for protein chip method, ELISA method, immunoblotting method and flow cytometry detection.

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