WO2022206585A1 - 诊断先天性巨结肠的抗体标志物及其应用 - Google Patents
诊断先天性巨结肠的抗体标志物及其应用 Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
Definitions
- the invention belongs to the technical field of gene therapy, and particularly relates to an antibody marker for diagnosing Hirschsprung's disease and its application.
- Hirschsprung disease is a birth defect disease with abnormal enteric nerve development in children.
- the pathological mechanism is the migration and differentiation of enteric neural crest cells into enteric neurons. It is one of the common congenital intestinal diseases in children.
- the early manifestations of Hirschsprung's disease are vomiting, abdominal distension, diarrhea, etc., which can clinically cause neonatal death, recurrent enteritis after surgery, refractory constipation and other complications, which seriously affect the growth and development and quality of life of children.
- Timely diagnosis and treatment of Hirschsprung's disease can reduce the risk of Hirschsprung's colitis and obtain a good prognosis.
- the diagnosis of the disease requires postoperative pathological sections of the diseased tissue.
- the main methods of preoperative diagnosis are barium enema, rectal biopsy, and rectal manometry to determine whether to implement "radical resection of megacolon".
- barium enema is the most important diagnostic method. The principle is that there is stenosis and proximal dilatation of the intestine in children with Hirschsprung's congenital. However, this method can only diagnose children with typical intestinal morphological changes, the sensitivity needs to be improved, and the diagnostic accuracy is about 80%.
- Rectal biopsy is to directly take rectal tissue to detect the absence of ganglion cells, with high accuracy, but the sampling site has an impact on the results.
- This method is invasive and very expensive. In general, this method is used in children whose barium enema is not obvious or not suitable, such as when the child has necrotizing enterocolitis (NEC).
- NEC enterocolitis
- Barium enema can lead to intestinal perforation, this time is not suitable for diagnosis with barium enema, only consider rectal biopsy.
- Rectal manometry is to determine the abnormal innervation of the enteric nerve by detecting the lack of relaxation of the internal anal sphincter. It is only an auxiliary diagnostic method, with many false positives and false negatives, and cannot be used for independent detection.
- the purpose of the present invention is to provide a diagnostic marker for Hirschsprung's disease and its application, and to provide a new accurate and sensitive detection method for the diagnosis of Hirschsprung's disease.
- the present invention adopts the following technical solutions to realize:
- a first aspect of the present invention provides a combination of diagnostic markers for Hirschsprung's disease.
- the diagnostic marker combination comprises at least two selected from the group consisting of anti-NMDAR antibodies, anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies, and anti-nRNP/Sm antibodies.
- the diagnostic marker combination comprises at least three, or at least four selected from the group consisting of: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm Antibody.
- the diagnostic marker combination comprises two, three, four or five selected from the group consisting of anti-NMDAR antibodies, anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies, anti- nRNP/Sm antibody.
- the diagnostic marker combination comprises an anti-NMDAR antibody, and at least one, at least two, at least one of an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, an anti-nRNP/Sm antibody Three or four antibodies.
- the diagnostic marker combination comprises an anti-NMDAR antibody, and one, two, three or four of an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody Antibody.
- the antibody is an autoantibody.
- the anti-NMDAR antibody is an anti-NMDAR autoantibody.
- the anti-single-stranded DNA antibody is an anti-single-stranded DNA autoantibody.
- the anti-double-stranded DNA antibody is an anti-double-stranded DNA autoantibody.
- the anti-SSA antibody is an anti-SSA autoantibody.
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm autoantibody.
- the anti-NMDAR antibody is an anti-NMDAR IgG antibody.
- the anti-NMDAR autoantibody is an anti-NMDAR IgG autoantibody.
- the anti-single-stranded DNA antibody is an anti-single-stranded DNA IgG antibody.
- the anti-single-stranded DNA autoantibody is an anti-single-stranded DNA IgG autoantibody.
- the anti-double-stranded DNA antibody is an anti-double-stranded DNA IgG antibody.
- the anti-double-stranded DNA autoantibody is an anti-double-stranded DNA IgG autoantibody.
- the anti-SSA antibody is an anti-SSA IgG antibody.
- the anti-SSA autoantibody is an anti-SSA IgG autoantibody.
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm IgG antibody. In some embodiments, the anti-nRNP/Sm autoantibody is an anti-nRNP/Sm IgG autoantibody.
- the second aspect of the present invention provides a diagnostic product for Hirschsprung's disease.
- the diagnostic product comprises a detection agent for detecting at least one diagnostic marker selected from the group consisting of: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/ Sm antibody.
- the diagnostic product comprises a detection agent for detecting at least two, at least three, at least four or five diagnostic markers selected from the group consisting of: anti-NMDAR antibodies, Stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody.
- the detection agent is used to detect one, two, three, four or five diagnostic markers selected from the group consisting of: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti- SSA antibody, anti-nRNP/Sm antibody.
- the detection agent comprises an anti-NMDAR antibody detection agent, and at least one, at least two, an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody, At least three or four detection agents.
- the detection agent comprises an anti-NMDAR antibody detection agent, and one, two, three or four of anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies and anti-nRNP/Sm antibodies detection agent.
- the diagnostic product is a reagent, kit or test strip.
- the antibody is an autoantibody.
- the anti-NMDAR antibody is an anti-NMDAR autoantibody.
- the anti-single-stranded DNA antibody is an anti-single-stranded DNA autoantibody.
- the anti-double-stranded DNA antibody is an anti-double-stranded DNA autoantibody.
- the anti-SSA antibody is an anti-SSA autoantibody.
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm autoantibody.
- the anti-NMDAR antibody is an anti-NMDAR IgG antibody.
- the anti-NMDAR autoantibody is an anti-NMDAR IgG autoantibody.
- the anti-single-stranded DNA antibody is an anti-single-stranded DNA IgG antibody.
- the anti-single-stranded DNA antibody is an IgG autoantibody against single-stranded DNA.
- the anti-double-stranded DNA antibody is an anti-double-stranded DNA IgG antibody.
- the anti-double-stranded DNA antibody is an IgG autoantibody against double-stranded DNA.
- the anti-SSA antibody is an anti-SSA IgG antibody.
- the anti-SSA autoantibody is an anti-SSA IgG autoantibody.
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm IgG antibody.
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm IgG autoantibody.
- the detection is a quantitative detection.
- the detection agent is a quantitative detection agent.
- the quantitative detection agent is used to perform any one of the following methods: radioimmunoassay, indirect immunofluorescence, dot immunogold diafiltration, biological mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
- the detection agent includes an antigen.
- the antigen includes at least one, at least two, at least three of NMDAR protein, ssDNA, dsDNA, SSA protein, nRNP/Sm protein (selected from nRNP, Sm protein, and the complex of Sm protein and nRNP) species, at least four or five.
- the antigens include one, two, three, and four of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein (selected from nRNP, Sm protein, and the complex of Sm protein and nRNP). species or five.
- the antigen of anti-NMDAR antibody is NMDAR protein; the antigen of anti-single-stranded DNA antibody is ssDNA; the antigen of anti-double-stranded DNA antibody is dsDNA; the antigen of anti-SSA antibody is SSA protein; the antigen of anti-nRNP/Sm antibody is nRNP /Sm protein (selected from nRNP, Sm protein, and a complex of Sm protein and nRNP).
- the antigen is conjugated to a solid support.
- the solid support is selected from test tubes, EP tubes, multi-well plates, wells of micro-reaction plates and microspheres.
- the detection agent further comprises an anti-human Ig antibody.
- the quantitative detection agent further includes anti-human Ig antibody.
- the anti-human Ig antibody is an anti-human IgG antibody.
- the anti-human Ig antibody is conjugated with a signal substance.
- the sample to be detected by the detection agent is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample; preferably at least one of blood, plasma, serum ; preferably at least one of intestinal tissue, intestinal cells or intestinal cell lysates.
- the third aspect of the present invention provides the use of the diagnostic product or detection agent of the second aspect of the present invention in the preparation of Hirschsprung's disease diagnostic reagents, kits or test strips.
- the diagnostic marker found in the present invention has the advantages of simple operation, no intervention, high throughput and low cost in the diagnosis of congenital diseases in children, and overcomes the shortcomings of plasma-free diagnosis in the prior art.
- the diagnostic sensitivity and specificity of this method are good: the AUC value of anti-NMDAR antibody alone is 0.9046, the optimal limit corresponds to a sensitivity of 93.75% and a specificity of 81.58%.
- the AUC value of anti-ssDNA antibody alone for diagnosing Hirschsprung's disease was 0.9167; the best cutoff corresponds to a sensitivity of 74.63% and a specificity of 96.88%.
- the AUC value for the detection of dsDNA antibody alone was 0.8313, and the best limit corresponds to a sensitivity of 96.88% and a specificity of 69.23%; the AUC value for the detection of anti-SSA antibody alone was 0.7756; the best limit corresponds to a sensitivity of 62.50% and a specificity of 69.23%. 74.36%.
- the AUC value of the anti-nRNP/Sm antibody alone was 0.8165, and the best cutoff corresponds to a sensitivity of 100% and a specificity of 64.10%.
- the present invention provides an effective method for clinical diagnosis of Hirschsprung disease.
- the markers When the markers are combined for detection, their detection performance, such as AUC value, sensitivity and specificity are further improved.
- AUC value of combined detection of anti-NMDAR antibody and anti-ssDNA antibody for the diagnosis of Hirschsprung's disease is 0.9351
- the optimal cutoff corresponds to a sensitivity of 82.05% and a specificity of 93.75%.
- the diagnostic effect is significantly better than that of single index detection, which provides a more reliable method for clinical diagnosis of Hirschsprung's disease.
- Figure 1 shows the diagnostic marker anti-NMDAR antibody in the plasma of children with Hirschsprung's disease screened by chip.
- Figure 2 shows the microarray screening the diagnostic marker anti-single-chain DNA antibody in the plasma of children with Hirschsprung's disease.
- Figure 3 shows the microarray screening the diagnostic marker anti-double-stranded DNA antibody in the plasma of children with Hirschsprung's disease.
- Figure 4 shows the microarray screening the diagnostic marker anti-SSA antibody in the plasma of children with Hirschsprung's disease.
- Figure 5 shows the microarray screening for the diagnostic marker anti-nRNP/Sm antibody in the plasma of children with Hirschsprung's disease.
- FIG. 6 shows the detection of anti-NMDAR antibodies in Hirschsprung's disease tissue (HSCR) and control colon tissue (DC).
- HCR Hirschsprung's disease tissue
- DC control colon tissue
- FIG 7 shows the detection of anti-NMDAR antibodies in the plasma of children with Hirschsprung's disease (HSCR), disease controls (DC) and healthy children (HC).
- HSCR Hirschsprung's disease
- DC disease controls
- HC healthy children
- Figure 8 is the ROC curve analysis of the diagnostic effect of anti-NMDAR antibodies in plasma in Hirschsprung's disease.
- FIG 9 shows the detection of anti-single-chain DNA antibodies in plasma of children with Hirschsprung's disease (HSCR), disease controls (DC) and healthy children (HC).
- HSCR Hirschsprung's disease
- DC disease controls
- HC healthy children
- Figure 10 is the ROC curve analysis of the diagnostic effect of anti-single-stranded DNA antibodies in plasma in Hirschsprung's disease.
- FIG 11 shows the detection of anti-double-stranded DNA antibodies in Hirschsprung's disease tissue (HSCR) and control colon tissue (DC).
- HCR Hirschsprung's disease tissue
- DC control colon tissue
- Figure 12 shows the detection of anti-double-stranded DNA antibodies in plasma of children with Hirschsprung's disease (HSCR), disease controls (DC) and healthy children (HC).
- HCR Hirschsprung's disease
- DC disease controls
- HC healthy children
- Figure 13 shows the ROC curve analysis of the diagnostic effect of anti-double-stranded DNA antibodies in plasma in Hirschsprung's disease.
- Figure 14 shows the detection of anti-SSA antibodies in plasma of children with Hirschsprung's disease (HSCR), disease controls (DC) and healthy children (HC).
- HSCR Hirschsprung's disease
- DC disease controls
- HC healthy children
- Fig. 15 is the ROC curve analysis of the diagnostic effect of anti-SSA antibodies in plasma in Hirschsprung's disease.
- Figure 16 shows the detection of anti-nRNP/Sm antibodies in plasma of children with Hirschsprung's disease (HSCR), disease controls (DC) and healthy children (HC).
- HSCR Hirschsprung's disease
- DC disease controls
- HC healthy children
- Figure 17 shows the ROC curve analysis of the diagnostic effect of anti-nRNP/Sm antibodies in plasma in Hirschsprung's disease.
- Figure 18 is the ROC curve analysis of the combined detection of anti-NMDAR antibody and anti-single-chain DNA antibody in plasma.
- the present invention relates to diagnostic markers, diagnostic marker combinations, diagnostic products, detection kits, and the application of detection agents in the preparation of Hirschsprung's disease diagnostic reagents, kits or test strips.
- the present invention finds for the first time that anti-NMDAR antibodies, anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies and anti-nRNP/Sm antibodies (especially autoantibodies) can be used alone or in combination as diagnostic markers for Hirschsprung's disease, filling in The diagnosis of Hirschsprung's disease, especially the diagnosis of plasma, has been eliminated.
- marker refers to a molecule to be used as a target for analysis of a patient's experimental sample.
- autoantibody refers to antibodies directed against one's own tissues, organs, cells and cellular components. There may be low titers of autoantibodies in normal human blood.
- the autoantibodies in the present invention are antibodies that mistakenly target specific tissues or organs of the body and damage the tissues or organs.
- the present invention found that certain autoantibodies can cause damage to the body and can be diagnostic markers for Hirschsprung's disease.
- the anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody are autoantibodies.
- the anti-NMDAR antibody is an autoantibody, and is an anti-NMDAR IgG antibody.
- the anti-single-stranded DNA antibody is an autoantibody, and is an IgG antibody against single-stranded DNA.
- the anti-double-stranded DNA antibody is an autoantibody, and is an IgG antibody against double-stranded DNA.
- the anti-SSA antibody is an autoantibody, and is an anti-SSA IgG antibody.
- the anti-nRNP/Sm antibody is an autoantibody, and is an anti-nRNP/Sm IgG antibody.
- the expression analysis method for various diagnostic markers is not particularly limited.
- relative amounts or concentrations can also be determined. More specifically, for example, the amount, concentration or activity of the above-mentioned antibodies and the like in a blood sample can be measured.
- the detection agent is used to perform any of the following methods: radioimmunoassay, indirect immunofluorescence, dot immunogold diafiltration, biological mass spectrometry, Western blotting and ELISA.
- the biological mass spectrometry includes time-of-flight mass spectrometry such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MASS) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MASS). (TOF-MASS). From the TOF-MASS graph, the concentration or amount of the antibody can be grasped by molecular weight peaks, other fragment peaks, their intensities, and the like.
- the antibody selectively bound to the protein chip can also be detected using a secondary antibody having a labeling group that can bind to each antibody.
- an immunoassay method is preferable as a method for performing the above-mentioned expression analysis.
- Immunoassays have high sensitivity and precision, and can detect even slight changes in antibody concentrations in test samples such as blood.
- the expression analysis of the antibody according to the present invention is performed by enzyme-linked immunosorbent assay (ELISA)
- ELISA enzyme-linked immunosorbent assay
- the diagnostic kit for Hirschsprung's disease according to the present invention can be suitably used.
- the detection agent is an antigen.
- the detection agent is a quantitative detection agent.
- the detection agent or quantitative detection agent is an NMDAR protein.
- the detection agent or quantitative detection agent is ssDNA.
- the detection agent or quantitative detection agent is dsDNA.
- the detection agent or quantitative detection agent is an SSA protein.
- the detection agent or quantitative detection agent is selected from at least one of nRNP, Sm protein, and a complex of Sm protein and nRNP.
- the antigens include NMDAR protein, ssDNA, dsDNA, SSA protein, nRNP/Sm protein (selected from nRNP, Sm protein, and the complex of Sm protein and nRNP) at least one, at least two, at least three, at least four or five of the
- the antigens include one, two, three, or three of NMDAR protein, ssDNA, dsDNA, SSA protein, nRNP/Sm protein (selected from nRNP, Sm protein, and the complex of Sm protein and nRNP) protein, Four or five.
- the antigen includes ssDNA and optionally NMDAR protein.
- Antigen fragments comprising epitopes recognized by antibodies may be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 in length , 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 nucleotides.
- the fragment can also be 5, 10, 15, 20, 25, 50, 75, 100, 150, 200 or 250 and specific antigens (NMDAR protein, SSA protein, nRNP, Sm protein, and complexes of Sm protein and nRNP)
- the full length is between one amino acid less.
- the fragment may also be between 5, 10, 15, 20, 25, 50, 75, 100, 150, 200 or 250 and one nucleotide less than the full length of the ssDNA or dsDNA.
- these epitopes are characterized in advance so that antibodies against a given antigen are known to recognize the epitope. Methods of epitope mapping are well known in the art.
- An "epitope" is a site on an antigen, such as the antigens disclosed herein, that is recognized by an antibody.
- the detection agent or quantitative detection agent can also be a fragment peptide, denatured product, or modified product of an antigenic substance (at least one of NMDAR protein, SSA protein, nRNP, Sm protein, and the complex of Sm protein and nRNP). of at least one peptide.
- a denatured substance of an antigenic substance refers to a denatured substance that can specifically bind to the above-mentioned antibody, obtained by physical treatment such as heating, freezing, and ultraviolet rays, or chemical treatment such as surfactant and denaturing agent. For example, denatured products obtained by treatment with SDS and DTT can be mentioned.
- the modified product refers to a modified product obtained by modifying one or more amino acids and can specifically bind to the above-mentioned (auto)antibody.
- a modified product obtained by treatment with glutaraldehyde can be mentioned.
- the above modification can be compared with the antigen (at least one of NMDAR protein, SSA protein, nRNP, Sm protein, and the complex of Sm protein and nRNP), which has one or several amino acid residue mutations, Substitutions, deletions and/or additions, as long as they can specifically bind to the above-mentioned (auto)antibodies.
- the modified substance has more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% , more than 99% sequence identity.
- the detection agent or quantitative detection agent can also be at least one of fragments, denatures and modifications of antigenic substances (at least one of ssDNA and dsDNA).
- a denatured substance of an antigenic substance refers to a denatured substance that can specifically bind to the above-mentioned antibody, obtained by physical treatment such as heating, freezing, and ultraviolet rays, or chemical treatment such as surfactant and denaturing agent.
- denatured products obtained by treatment with SDS and DTT can be mentioned.
- the modified product refers to a modified product obtained by modifying one or more nucleotides and which can specifically bind to the aforementioned antibody.
- a modified product obtained by treatment with glutaraldehyde can be mentioned.
- the above modification can be a mutation, substitution, deletion and/or addition of one or several nucleic acid bases compared with the antigen (at least one of ssDNA and dsDNA), as long as it can be specific to the above antibody combine.
- the modified substance has more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% compared with the original antigen sequence sequence identity above.
- the antigen (at least one of ssDNA, dsDNA, NMDAR protein, SSA protein, nRNP, Sm protein, and a complex of Sm protein and nRNP) is conjugated to a solid support.
- the solid support is selected from the group consisting of test tubes, EP tubes, multi-well plates, wells of microtiter plates, and microspheres.
- solid support means that the carrier material is predominantly non-liquid solid, allowing precise and trackable localization of nucleic acids on the carrier material.
- the solid phase support can be selected from materials such as polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel and the like.
- a preferred solid phase carrier is an ELISA plate. It may contain 16, 32, 48, 64, 96 or more pore positions.
- microsphere may be spherical, near-spherical, cubic, polyhedral or irregular in shape.
- the diameter of the microspheres is preferably 10 nm ⁇ 1 mm, such as 100 nm, 500 nm, 1 ⁇ m, 10 ⁇ m, 100 ⁇ m, 500 ⁇ m; preferably 400 nm ⁇ 10 ⁇ m.
- the surface of the microspheres has specific binding properties for the substance of interest (target or analyte) to be assayed.
- the microspheres are preferably magnetic beads, the components of which contain a magnetic substance.
- the magnetic substance can be a metal (elemental metal or alloy), a non-metal, or a composite formed by a metal and a non-metal.
- Metals such as iron, AlNiCo metal, etc.; non-metals such as ferrite non-metals (preferably Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles); composites formed by metals and non-metals such as NdFeB rubber magnets composite material.
- the surface of the microsphere is modified with one or more active functional groups, and the active functional groups include one or more of -OH, -COOH, -NH 2 , -CHO, and -SO 3 H.
- the coated antigens and antibodies are conjugated or bound to the microspheres by physical adsorption or direct chemical conjugation (eg, bridging by a bridge).
- suitable techniques for forming bridges include, for example, covalent attachment, adsorption, non-covalent interactions, or combinations thereof.
- glutaraldehyde fixation N-hydroxysuccinimide (NHS) chemistry, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NHS chemistry to achieve direct bridging.
- Suitable means for indirect bridging include, for example, bridging via peptides, proteins, antibodies, linkers, or combinations thereof.
- indirect bridging to the solid support is via streptavidin and biotin.
- the detection agent further comprises an anti-human Ig antibody.
- the anti-human Ig antibody is an anti-human IgG antibody.
- An anti-human Ig antibody refers to an antibody against human Ig protein
- an anti-human IgG antibody refers to an antibody against human IgG protein.
- the anti-human Ig antibody is conjugated with a signal substance.
- the anti-human Ig antibody is not labeled with a signal substance
- the detection agent further comprises a second antibody to the anti-human Ig antibody labeled with the signal substance.
- Substances and the like that non-specifically bind to the above-mentioned diagnostic marker of the present invention are washed and removed with a buffer or the like, and then the above-mentioned second antibody is allowed to act.
- the diagnostic marker (auto) antibody binds to the second antibody and the above-mentioned antigen substance (on the solid support), respectively, the second antibody can be detected by a method corresponding to the signal substance.
- antibodies used as detection agents or quantitative detection agents may be of the IgA, IgD, IgG, IgE or IgM isotype or in the form of a single domain, such as a single domain antibody from camelid.
- the antibody used as a quantitative detection agent is an IgG antibody.
- the buffer comprises, for example, one or more of the following components: phosphate buffer, NaCl, EDTA, Pluronic F-127, sodium azide, sorbitol, sulfhydryl-modified bovine serum, or any combination thereof , variants or equivalents.
- the signal substance can provide the substance of the detected signal.
- the signal substance is independently selected from chromophores, digoxigenin-labeled probes, electron-dense substances, colloidal gold, or enzymes. any one or more. These tags are listed in the non-limiting section below:
- Enzymes that produce a detectable signal such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
- Chromophores such as fluorophores, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
- a detectable group such as its molecular size is sufficient to induce a modification detectable in its physical and/or chemical properties; such detection can be achieved by optical methods (eg diffraction, surface plasmon resonance, surface variation and contact variation angle) or Physical methods (such as atomic force spectroscopy and tunneling) are implemented.
- optical methods eg diffraction, surface plasmon resonance, surface variation and contact variation angle
- Physical methods such as atomic force spectroscopy and tunneling
- Electron dense substances such as radioactive molecules (eg 32 P, 35 S or 125 I).
- the signal substance is an acridine-based chemiluminescent substance.
- acridine-based chemiluminescent species include acridine esters and acridine sulfonamides.
- acridine-based chemiluminescent species include acridine ester AE-NHS, acridine ester DMAE-NHS, acridine ester Me-DMAE-NHS, acridine ester NSP-DMAE-NHS, acridinium salt NSP- SA, acridinium salt NSP-SA-NHS, acridinium hydrazide NSP-SA-ADH, etc.
- any biological sample containing the above diagnostic marker antibodies or autoantibodies can be used as a sample to be tested, including, but not limited to, serum, plasma, whole blood, saliva, feces, urine, semen, sweat, tears and body tissue.
- the sample to be detected by the detection agent is at least one of blood, plasma, serum, tissue, cells, tissue or cell lysate samples.
- tissue or cell lysate may also be used interchangeably with the terms “lysate”, “lysed sample”, “tissue or cell extract” and the like, to refer to a sample and/or biological sample material comprising lysed tissue or cells , i.e. where the structural integrity of a tissue or cell has been disrupted.
- the material is typically treated with enzymes and/or chemical agents to dissolve, degrade or disrupt the cell walls and membranes of such tissue or cells.
- enzymes and/or chemical agents to dissolve, degrade or disrupt the cell walls and membranes of such tissue or cells.
- the diagnostic kit of the present invention preferably contains a normal control sample and a Hirschsprung's disease control sample. If these samples are attached to the kit, the same experiment is performed on these samples, and the measured values are compared with the results of the test samples, whereby the presence or absence of Hirschsprung's disease in the test subject can be more objectively determined.
- the concentration or amount of the antibody contained in the sample is obtained indirectly from the intensity of the coloration.
- the obtained measured value can be converted into relative or absolute concentration, amount, activity and the like by a calibration curve or the like.
- the present invention also relates to a method for diagnosing Hirschsprung's disease, comprising quantitatively detecting antibody markers in a sample to be tested, wherein the elevated anti-NMDAR antibody level, the elevated anti-single-chain DNA antibody level, the elevated anti- At least one of double-stranded DNA antibody levels, elevated anti-SSA antibody levels, elevated anti-nRNP/Sm antibody levels are indicative of Hirschsprung's disease.
- the term "indicative" when used in the context of antibodies to which embodiments of the present invention are used includes that the antibodies whose presence or absence is determined by embodiments of the present invention are typically present in subjects with Hirschsprung's disease. "Usually present” means that the antibody is frequently associated with Hirschsprung's disease. "Frequently correlated” includes a probability of greater than 50%, preferably greater than 60%, more preferably greater than 70%, even more preferably greater than 80% and particularly preferably greater than 90% or 95%.
- the ideal scenario for diagnosis is a situation in which a single event or process causes various diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types.
- diagnosis without biochemical markers is 100% specific and with 100% sensitivity.
- biochemical markers can be used to assess, for example, the presence or severity of a disease with some probability or predictive value. Therefore, in routine clinical diagnosis, various clinical symptoms and biological markers are usually considered comprehensively to diagnose, treat and control the underlying disease.
- the presence or absence of Hirschsprung's disease is then determined from the result of the obtained expression analysis. That is, when Hirschsprung's disease is onset or the symptoms are more severe, the concentration, amount or activity of the antibody according to the present invention in the blood is increased.
- the expression level is high, it can be determined as positive, and if the expression level is low, it can be determined as negative.
- the boundary between positive and negative, ie the cutoff value can be changed. Therefore, at a stage where there is no general standard, it is necessary for the practitioner of the method of the present invention to determine the expression analysis method and cutoff value in advance through a preliminary experiment or the like, and then perform the measurement.
- the cut-off value of the above-mentioned antibodies for diagnosing Hirschsprung can be determined by those skilled in the art according to the actual situation. The determination of the cut-off value is a routine technical means in the art.
- Those skilled in the art can select a cutoff value, and test the detection accuracy (such as detection rate, precision rate, false positive rate, false negative rate, sensitivity, specificity, etc.) The effect is to increase or decrease the cut-off value until a better or optimal detection effect is achieved.
- the amount, concentration, activity and other indicators indicating the presence or expression of the diagnostic marker antibody are increased by 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, the diagnosis or auxiliary diagnosis is Hirschsprung's disease or Suspected Hirschsprung's disease. In some embodiments, the amount, concentration, activity, etc.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the amount, concentration, activity, etc. of the antibody combination which indicate the presence or expression thereof, are increased by 0.2 times, 0.3 times, 0.4 times, 0.5 times, 1 times, 1.5 times, 2 times, and 2.5 times. , 3 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more, the diagnosis or auxiliary diagnosis is Hirschsprung's disease or suspected Hirschsprung's disease.
- the subject is a patient under the age of 18, eg, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 age. Or infants such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months old.
- the samples used in the present invention all come from the Guangzhou Women and Children's Medical Center, and the collection of all experimental tissue and blood samples is authorized by the Ethics Committee of the Guangzhou Women and Children's Medical Center and consented by the patients.
- Statistical analysis was used for the test results of the present invention, t test was used to evaluate the difference between the two groups, p ⁇ 0.05 was used to indicate statistical significance, and all p values were used two-sided test. Statistical analysis was performed using R and Graphpad 8.0 software.
- the "Hirschsprung's disease” of the present invention is also called Hirschsprung's disease, which is caused by the lack of ganglion cells in the colon leading to continuous spasm of the intestinal tract, stagnation of feces in the proximal colon, and hypertrophy and dilation of the proximal colon. It is common in children.
- Short-segment lesions are located in the proximal and middle rectum, not more than 6.5cm away from the anal canal; common-type lesions are located in the proximal rectum or distal rectosigmoid colon, about 9cm away from the anal canal; long-segment lesions extend to the sigmoid or descending colon; full-colon type The lesions spread to the entire colon and terminal ileum, within 30cm from the ileocecal valve.
- N-methyl-D-aspartate receptor (NMDAR) of the present invention is a subtype of the ionotropic glutamate receptor and plays an important physiological role in the development of the nervous system; anti-N-methyl-D- An aspartate receptor (NMDAR) antibody refers to an antibody raised against an N-methyl-D-aspartate receptor (NMDAR) protein, preferably an IgG antibody.
- the ssDNA in the present invention refers to the single-stranded DNA in the nucleus;
- the anti-single-stranded DNA (ssDNA) antibody refers to the antibody produced against the single-stranded DNA in the nucleus, preferably an IgG antibody.
- the dsDNA in the present invention refers to the double-stranded DNA in the nucleus;
- the anti-double-stranded DNA (dsDNA) antibody refers to the antibody produced against the double-stranded DNA in the nucleus, preferably an IgG antibody.
- the SSA of the present invention is an RNase-resistant cytoplasmic ribonucleoprotein; the anti-SSA antibody is an antibody produced against SSA in the nucleus, preferably an IgG antibody.
- nRNP nuclear ribonucleoprotein
- Sm and nRNP are different antigenic sites in the same molecular complex (nRNP/Sm), and the two antigens are related, so the positive anti-Sm antibody is often accompanied by Positive anti-nRNP antibody.
- Anti-nRNP/Sm antibody is an anti-nuclear antibody, usually an autoantibody.
- the "ROC curve” in the present invention is a curve of changes in specificity (false positive rate) and sensitivity (true positive rate), which reflects the diagnostic ability of the binary classifier.
- the ratio of true positive rate to false positive rate change is greater than 1, away from the 45 degree straight line.
- the "AUC" in the present invention refers to the area under the ROC curve, which is between 0.1 and 1, and is used to evaluate the quality of the classifier. The closer it is to 1, the better the classifier. If it is greater than 0.5, it can be considered as a diagnostic marker for diseases. .
- the plasma of children with Hirschsprung's disease is screened by human neuroimmune chip and antigen chip, and the plasma of children with other enteropathy and healthy groups is used as a control to obtain anti-N-methyl- D-aspartate receptor (NMDAR) antibody, anti-single-stranded DNA (ssDNA) antibody highly expressed in plasma of children with Hirschsprung's disease, anti-double-stranded DNA (dsDNA) highly expressed in plasma of children with Hirschsprung's disease Antibody, anti-SSA antibody highly expressed in plasma of children with Hirschsprung's disease, anti-nRNP/Sm antibody highly expressed in plasma of children with Hirschsprung's disease.
- NMDAR N-methyl- D-aspartate receptor
- ssDNA anti-single-stranded DNA
- dsDNA anti-double-stranded DNA
- Antibody anti-SSA antibody highly expressed in plasma of children with Hirschsprung's disease
- anti-nRNP/Sm antibody highly expressed in plasma of children with Hirschsprung's disease.
- solid support is selected from test tubes, EP tubes, multi-well plates, micro-reaction plate wells and microspheres.
- sample to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample .
- detection reagents in the preparation of Hirschsprung's disease diagnostic reagents, kits or test strips; the detection reagents include quantitative detection reagents for anti-single-stranded DNA antibodies; optionally, the anti-single-stranded DNA antibodies are IgG antibodies against single-stranded DNA.
- the detection agent further comprises a quantitative detection agent of anti-NMDAR antibody; optionally, the anti-NMDAR antibody is an anti-NMDAR IgG antibody.
- the detection agent is an antigen
- the antigen includes ssDNA and optionally NMDAR protein.
- solid support is selected from test tubes, EP tubes, multi-well plates, micro-reaction plate wells and microspheres.
- sample to be detected by the detection agent is blood, plasma, serum, tissue, cell, tissue or cell lysate sample. at least one.
- solid support is selected from test tubes, EP tubes, multi-well plates, micro-reaction plate wells and microspheres.
- sample to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample .
- Radioimmunoassay Indirect Immunofluorescence, Dot Immunogold Diafiltration, Biological Mass Spectrometry, Western Blotting and ELISA.
- solid support is selected from test tubes, EP tubes, multi-well plates, micro-reaction plate wells and microspheres.
- sample to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample .
- the quantitative detection agent is an antigenic substance selected from nRNP, Sm protein, and a complex of Sm protein and nRNP.
- solid support is selected from test tubes, EP tubes, multi-well plates, wells of micro-reaction plates and microspheres.
- sample to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample .
- anti-NMDAR antibodies Anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies, and anti-nRNP/Sm antibodies as diagnostic markers for Hirschsprung's disease and their applications
- a diagnostic marker combination of Hirschsprung's disease it is characterised in that the diagnostic marker combination comprises at least two selected from the following: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti- SSA antibody, anti-nRNP/Sm antibody.
- diagnostic marker combination comprising an anti-NMDAR antibody, and an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody. at least one of.
- diagnostic marker combination according to 3, wherein the diagnostic marker combination further comprises at least one of an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody.
- diagnostic marker combination according to 1, wherein the diagnostic marker combination comprises an anti-NMDAR antibody, an anti-single-stranded DNA antibody and an anti-double-stranded DNA antibody.
- diagnostic marker combination comprising three kinds of antibodies: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, and anti-SSA antibody or anti-nRNP /Sm antibody.
- diagnostic marker combination comprises at least four selected from the group consisting of: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, and anti-SSA antibody , Anti-nRNP/Sm antibody.
- diagnostic marker combination comprises: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody.
- diagnostic marker combination according to 9, wherein the diagnostic marker combination comprises: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody and anti-nRNP/Sm antibody.
- the diagnostic marker combination comprises an anti-NMDAR antibody, an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody and an anti-nRNP/Sm antibody.
- NMDAR antibody anti-single-stranded DNA antibody, anti-nRNP/Sm antibody combination; (8). Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody combination; (9). Anti-single-stranded DNA antibody Chain DNA antibody, anti-double-stranded DNA antibody, anti-nRNP/Sm antibody combination; (10). Anti-single-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody combination; (11). NMDAR antibody, anti-single-chain DNA antibody , anti-double-stranded DNA antibody, anti-SSA antibody combination; (12).
- NMDAR antibody anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-nRNP/Sm antibody combination; (13). NMDAR antibody, anti-single-stranded DNA antibody , anti-SSA antibody, anti-nRNP/Sm antibody combination; (14). Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody combination; (15). Anti-NMDAR antibody, anti-single chain Combination of DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody; (16). Combination of anti-NMDAR antibody and anti-dsDNA antibody; (17).
- Combination of anti-NMDAR antibody and anti-SSA antibody ( 18). Combination of anti-NMDAR antibody and anti-nRNP/Sm antibody; (19). Combination of anti-dsDNA antibody and anti-SSA antibody; (20). Combination of anti-dsDNA antibody and anti-nRNP/Sm antibody; (21). Antibody Combination of SSA antibody, anti-nRNP/Sm antibody; (22). Combination of anti-NMDAR antibody, anti-dsDNA antibody, and anti-SSA antibody; (23). Combination of anti-NMDAR antibody, anti-dsDNA antibody, and anti-nRNP/Sm antibody; ( 24). Combination of anti-NMDAR antibody, anti-SSA antibody and anti-nRNP/Sm antibody; (25). Combination of anti-dsDNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody; (26). Anti-NMDAR antibody and anti-dsDNA antibody , a combination of anti-SSA antibody and anti-nRNPSm antibody.
- a diagnostic product for Hirschsprung's disease comprising a detection agent, the detection agent comprising: an anti-NMDAR antibody, an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody A detection agent for at least one of the antibodies.
- the detection agent comprises: a detection agent for at least two antibodies among anti-NMDAR antibodies, anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies, and anti-nRNP/Sm antibodies .
- detection agents for the at least two kinds of antibodies comprise: detection agents for anti-NMDAR antibodies, and anti-single-stranded DNA antibodies, anti-double-stranded DNA antibodies, anti-SSA antibodies, anti- A detection agent for at least one of the nRNP/Sm antibodies.
- the detection agent comprises: detection agents of anti-NMDAR antibody and anti-single-chain DNA antibody.
- the detection agent further comprises: a detection agent for at least one antibody selected from the group consisting of anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody and anti-double-stranded DNA antibody.
- the detection agent comprises a detection agent selected from the following three kinds of antibodies: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, and anti-SSA antibody or Anti-nRNP/Sm antibody.
- the detection agent comprises a detection agent selected from the following three types of antibodies: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, and anti-nRNP/Sm Antibody.
- the detection agent comprises: at least four of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody Antibody detection reagents.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody.
- Combination of NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, and anti-nRNP/Sm antibody ( 13). Combination of NMDAR antibody, anti-single-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody; (14). Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody Combination; (15). Combination of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody; (16).
- Combination of anti-NMDAR antibody and anti-dsDNA antibody ( 17). Combination of anti-NMDAR antibody and anti-SSA antibody; (18). Combination of anti-NMDAR antibody and anti-nRNP/Sm antibody; (19). Combination of anti-dsDNA antibody and anti-SSA antibody; (20).
- Anti-dsDNA antibody the combination of anti-nRNP/Sm antibody; (21). The combination of anti-SSA antibody and anti-nRNP/Sm antibody; (22). The combination of anti-NMDAR antibody, anti-dsDNA antibody and anti-SSA antibody; (23).
- Anti-NMDAR antibody Combination of anti-dsDNA antibody, anti-nRNP/Sm antibody; (24).
- Anti-NMDAR antibody Combination of anti-NMDAR antibody, anti-SSA antibody, anti-nRNP/Sm antibody; (25). Anti-dsDNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody (26). Combination of anti-NMDAR antibody, anti-dsDNA antibody, anti-SSA antibody, and anti-nRNPSm antibody; (27). Anti-NMDAR antibody; (28). Anti-single-stranded DNA antibody; (29). Anti-double-stranded DNA antibody; (30). Anti-SSA antibody; (31) Anti-nRNP/Sm antibody.
- the anti-NMDAR antibody is an anti-NMDAR IgG antibody; the anti-single-stranded DNA The antibody is an IgG antibody against single-stranded DNA; the anti-double-stranded DNA antibody is an IgG antibody against double-stranded DNA; the anti-SSA antibody is an anti-SSA IgG antibody; the anti-nRNP/Sm antibody is an anti-nRNP/Sm antibody IgG antibodies.
- the detection agent comprises an antigen
- the antigen comprises at least one of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises at least two of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises NMDAR protein, and at least one, at least two, at least three or four of ssDNA, dsDNA, SSA protein, nRNP/Sm protein .
- the antigen further comprises at least one, at least two or three of dsDNA, SSA protein, and nRNP/Sm protein.
- the diagnostic product of 33, wherein the antigen comprises NMDAR protein, ssDNA and dsDNA.
- the antigen further comprises at least one of SSA protein or nRNP/Sm protein.
- the diagnostic product according to 33, wherein the antigen comprises NMDAR protein, ssDNA or dsDNA, SSA protein or nRNP/Sm protein.
- the diagnostic product according to 33, wherein the antigen comprises NMDAR protein, ssDNA or dsDNA, and nRNP/Sm protein.
- the diagnostic product according to 33, wherein the antigens comprise four kinds selected from the group consisting of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the diagnostic product according to 42, wherein the antigen comprises NMDAR protein, ssDNA or dsDNA, SSA protein, nRNP/Sm protein.
- the diagnostic product according to 42, wherein the antigen comprises NMDAR protein, ssDNA, dsDNA, nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- Combination of NMDAR protein and SSA protein (18) . Combination of NMDAR protein and nRNP/Sm protein; (19). Combination of dsDNA and SSA protein; (20). Combination of dsDNA and nRNP/Sm protein; (21). Combination of SSA protein and nRNP/Sm protein; ( 22). Combination of NMDAR protein, dsDNA, SSA protein; (23). Combination of NMDAR protein, dsDNA, nRNP/Sm protein; (24). Combination of NMDAR protein, SSA protein, nRNP/Sm protein; (25). Combination of dsDNA, SSA protein, nRNP/Sm protein; (26).
- NMDAR protein Combination of NMDAR protein, dsDNA, SSA protein, nRNPSm protein; (27). NMDAR protein; (28). Single-stranded DNA; (29). Double-stranded DNA; (30). SSA protein; (31) nRNP/Sm protein.
- the diagnostic product according to 47, wherein the solid support is selected from the group consisting of test tubes, EP tubes, multi-well plates, wells of micro-reaction plates and microspheres.
- the detection sample to which the detection agent is applicable is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample.
- the diagnostic product according to 52, wherein the detection sample to which the detection agent is applicable is at least one of blood, plasma, and serum samples; or intestinal tissue, intestinal cells or intestinal cell lysis at least one of them.
- detection agent in the preparation of Hirschsprung's disease diagnostic reagent, test kit or test strip, wherein the detection agent comprises: anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti- A detection agent for at least one of SSA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises: at least two antibodies in an anti-NMDAR antibody, an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody detection agent.
- the detection agent for the at least two antibodies comprises: a detection agent for an anti-NMDAR antibody, and an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, an anti-nRNP antibody A detection agent for at least one of the /Sm antibodies.
- the detection agent comprises: detection agents of anti-NMDAR antibody and anti-single-chain DNA antibody.
- the detection agent further comprises: a detection agent for at least one antibody selected from the group consisting of an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody and anti-double-stranded DNA antibody.
- the detection agent further comprises: a detection agent for at least one antibody of anti-SSA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises a detection agent selected from the following three kinds of antibodies: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, and anti-SSA antibody or anti- nRNP/Sm antibody.
- the detection agent comprises a detection agent selected from the following three types of antibodies: anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, and anti-nRNP/Sm antibody .
- the detection agent comprises: at least four kinds of antibodies in an anti-NMDAR antibody, an anti-single-stranded DNA antibody, an anti-double-stranded DNA antibody, an anti-SSA antibody, and an anti-nRNP/Sm antibody detection agent.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody or anti-double-stranded DNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody.
- the detection agent comprises: detection agents of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody and anti-nRNP/Sm antibody.
- Combination of NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, and anti-nRNP/Sm antibody ( 13). Combination of NMDAR antibody, anti-single-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody; (14). Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody Combination; (15). Combination of anti-NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody; (16).
- Combination of anti-NMDAR antibody and anti-dsDNA antibody (17) ). Combination of anti-NMDAR antibody and anti-SSA antibody; (18). Combination of anti-NMDAR antibody and anti-nRNP/Sm antibody; (19). Combination of anti-dsDNA antibody and anti-SSA antibody; (20). Anti-dsDNA antibody, Combination of anti-nRNP/Sm antibodies; (21). Combination of anti-SSA antibodies and anti-nRNP/Sm antibodies; (22). Combinations of anti-NMDAR antibodies, anti-dsDNA antibodies, and anti-SSA antibodies; (23). Anti-NMDAR antibodies, Combination of anti-dsDNA antibody, anti-nRNP/Sm antibody; (24).
- Anti-NMDAR antibody Combination of anti-NMDAR antibody, anti-SSA antibody, anti-nRNP/Sm antibody; (25). Anti-dsDNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody Combination; (26). Combination of anti-NMDAR antibody, anti-dsDNA antibody, anti-SSA antibody, anti-nRNPSm antibody; (27). Anti-NMDAR antibody; (28). Anti-single-stranded DNA antibody; (29). Anti-double-stranded DNA Antibody; (30). Anti-SSA antibody; (31) Anti-nRNP/Sm antibody.
- the anti-NMDAR antibody is an anti-NMDAR IgG antibody
- the anti-single-stranded DNA antibody is an anti-single-stranded DNA IgG antibody
- the anti-double-stranded DNA antibody is The DNA antibody is an anti-double-stranded DNA IgG antibody
- the anti-SSA antibody is an anti-SSA IgG antibody
- the anti-nRNP/Sm antibody is an anti-nRNP/Sm IgG antibody.
- detection agent is used to perform any one of the following methods: radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, biological mass spectrometry, immunoassay Blotting and ELISA.
- the detection agent comprises an antigen
- the antigen comprises at least one of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises at least two of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises NMDAR protein, and at least one, at least two, at least three or four of ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises NMDAR protein and ssDNA.
- the antigen further comprises one or more of dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA and dsDNA.
- the antigen further comprises one or both of SSA protein or nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA or dsDNA, SSA protein or nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA or dsDNA, and nRNP/Sm protein.
- antigens include four selected from the group consisting of NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA or dsDNA, SSA protein, and nRNP/Sm protein.
- antigen comprises NMDAR protein, ssDNA, dsDNA, nRNP/Sm protein.
- the antigen comprises NMDAR protein, ssDNA, dsDNA, SSA protein, and nRNP/Sm protein.
- Combination of NMDAR protein and SSA protein (18) . Combination of NMDAR protein and nRNP/Sm protein; (19). Combination of dsDNA and SSA protein; (20). Combination of dsDNA and nRNP/Sm protein; (21). Combination of SSA protein and nRNP/Sm protein; ( 22). Combination of NMDAR protein, dsDNA, SSA protein; (23). Combination of NMDAR protein, dsDNA, nRNP/Sm protein; (24). Combination of NMDAR protein, SSA protein, nRNP/Sm protein; (25). Combination of dsDNA, SSA protein, nRNP/Sm protein; (26).
- NMDAR protein Combination of NMDAR protein, dsDNA, SSA protein, nRNPSm protein; (27). NMDAR protein; (28). Single-stranded DNA; (29). Double-stranded DNA; (30) SSA protein; (31) nRNP/Sm protein.
- solid support is selected from the group consisting of test tubes, EP tubes, multi-well plates, wells of micro-reaction plates and microspheres.
- the detection sample to which the detection agent is applicable is at least one of blood, plasma, serum, tissue, cell, tissue or cell lysate sample.
- the detection sample to which the detection agent is applicable is at least one of blood, plasma, and serum samples; or intestinal tissue, intestinal cells or intestinal cell lysates at least one of them.
- Plasma samples were divided into Hirschsprung's disease children group (37 cases), other enteropathy control group (18 cases), healthy children group (30 cases), the age ranged from 3 months to 3 years old, and 3/4 of the sex was male , disease and controls were age- and sex-matched. All samples were obtained from Guangzhou Women's and Children's Medical Center, and the healthy children group was the remaining blood samples after physical examination of healthy children.
- the blood collection method is anticoagulation blood collection, centrifugation to separate plasma, and frozen samples.
- Colon tissue samples were from the group of children with Hirschsprung's disease (36 cases), which were surgically resected lesions.
- Other enteropathy control group including 11 cases of colon tissue with anal stricture and intestinal stricture fistula).
- the chips are all provided by Guangzhou Yijin Biotechnology Co., Ltd. and contain the IgG detection of more than 100 kinds of autoimmune antibodies.
- the original data, after subtracting the negative control, are normalized by the RLM method, and the results are obtained by M statistics. between-group difference analysis.
- NMDAR N-methyl-D-aspartate receptor
- ssDNA anti-single-stranded DNA
- dsDNA anti-double-stranded DNA
- N-methyl-D-aspartate receptor is a protein specifically expressed in neural tissue, and autoantibodies to this protein have been reported in encephalitis, but not in peripheral enteric nervous system diseases.
- the intestinal tissue of the diseased segment of the megacolon patient was collected (HSCR, 36 cases).
- DC intestinal disease colon tissue
- the anti-N-methyl-D-aspartate receptor in the tissue was detected by enzyme-linked immunosorbent assay (ELISA).
- NMDAR human N-methyl-D-aspartate receptor
- ELISA enzyme-linked immunosorbent assay
- N-methyl-D-aspartate receptor (NMDAR) autoantibodies In order to verify the diagnostic effect of anti-N-methyl-D-aspartate receptor (NMDAR) autoantibodies on Hirschsprung's disease, we collected data from Hirschsprung's disease patients (HSCR, 37 cases) and controls with other intestinal diseases. (DC, including 18 cases of plasma samples from children with anal stenosis and intestinal stenosis) and healthy children's control (HC, 30 cases), use enzyme-linked immunosorbent assay (ELISA) to detect anti-N-methyl-D- Aspartate receptor (NMDAR) autoantibody level, the detection kit is human N-methyl D-aspartate receptor antibody (NMDAR-Ab) enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Zhenke Biotechnology) Technology Co., Ltd).
- ELISA enzyme-linked immunosorbent assay
- N-methyl-D-aspartate receptor (NMDAR) autoantibodies in Hirschsprung's patients were significantly higher than those in the intestinal disease control group (p ⁇ 0.05) and the healthy child control group (p ⁇ 0.01), and also higher than the control group (p ⁇ 0.01) in which intestinal disease was combined with healthy children.
- NMDAR anti-N-methyl-D-aspartate receptor
- Figure 8 shows the ROC curve used to evaluate the diagnostic effect of anti-N-methyl-D-aspartate receptor (NMDAR) autoantibodies on Hirschsprung's disease.
- the AUC value was 0.9046, and the optimal cutoff corresponded to a sensitivity of 93.75% and a specificity of 81.58%.
- the specificity is 78.95%. It can be seen that anti-N-methyl-D-aspartate receptor (NMDAR) autoantibodies can effectively diagnose Hirschsprung's disease, and show a good effect on the screening of Hirschsprung's disease.
- NMDAR anti-N-methyl-D-aspartate receptor
- Figure 10 shows the ROC curve used to evaluate the diagnostic effect of anti-single-stranded DNA (ssDNA) antibody on Hirschsprung's disease, the AUC is 0.9167; the best cutoff corresponds to a sensitivity of 74.63% and a specificity of 96.88%.
- ssDNA anti-single-stranded DNA
- Double-stranded DNA is a neural tissue-specific expression protein, and its autoantibodies have been reported in encephalitis, but not in peripheral enteric nervous system diseases.
- the anti-double-stranded DNA (dsDNA) antibody is indeed derived from the enteric nerve tissue of the diseased segment of the megacolon patient, the intestinal tissue of the diseased segment of the megacolon patient (HSCR, 36 cases), and other intestinal disease colon tissues (DC, 11 cases of colon tissue including anal stenosis and intestinal stricture fistula)
- the level of anti-double-stranded DNA (dsDNA) antibody in the tissue was detected by enzyme-linked immunosorbent assay (ELISA), and the detection kit was human anti-double-stranded DNA (dsDNA) antibody enzyme.
- Figure 13 shows the ROC curve used to evaluate the diagnostic effect of anti-double-stranded DNA (dsDNA) antibodies on Hirschsprung's disease.
- the AUC value was 0.8313, and the best cutoff corresponded to a sensitivity of 96.88% and a specificity of 69.23%.
- anti-double-stranded DNA (dsDNA) antibodies can effectively diagnose Hirschsprung's disease.
- Figure 15 shows the ROC curve used to evaluate the diagnostic effect of anti-SSA antibodies on Hirschsprung's disease.
- the AUC was 0.7756; the best cutoff corresponds to a sensitivity of 62.50% and a specificity of 74.36%.
- anti-SSA antibody can effectively diagnose Hirschsprung's disease.
- Figure 17 shows the ROC curve used to evaluate the diagnostic effect of anti-nRNP/Sm antibody on Hirschsprung's disease.
- the AUC value was 0.8165, and the optimal cutoff corresponds to a sensitivity of 100% and a specificity of 64.10%. It can be seen that anti-nRNP/Sm antibody can effectively diagnose Hirschsprung's disease.
- anti-NMDAR antibody anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, and anti-nRNP/Sm antibody was further detected, and the diagnostic effect of the combined application on Hirschsprung's disease.
- ELISA enzyme-linked immunosorbent assay
- the results are shown in Figure 18.
- the logistic regression method was used to evaluate the effect of the combined NMDAR and ssDNA indicators in diagnosing Hirschsprung's disease.
- the AUC was 0.9351; the best cutoff corresponds to a sensitivity of 82.05% and a specificity of 93.75%. It can be seen that the combination of NMDAR and ssDNA antibody can better diagnose Hirschsprung's disease.
- the same batch data and the same logistic regression method were used to evaluate the effect of other combined antibody indicators in diagnosing Hirschsprung's disease.
- the results showed that the combination with AUC>0.9167 also included: 1. Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody Combination; 2. Combination of anti-single-stranded DNA antibody and anti-SSA antibody; 3.
- Combination of anti-single-stranded DNA antibody and anti-nRNP/Sm antibody 4. Combination of anti-NMDAR antibody, anti-single-stranded DNA antibody and anti-double-stranded DNA antibody; 5 .NMDAR antibody, anti-single-stranded DNA antibody, anti-SSA antibody combination; 6. NMDAR antibody, anti-single-stranded DNA antibody, anti-nRNP/Sm antibody combination; 7. Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody Combination; 8. Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-nRNP/Sm antibody combination; 9.
- Anti-single-chain DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody combination 10. NMDAR antibody, anti-single chain DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody combination; 11. NMDAR antibody, anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-nRNP/Sm antibody combination; 12. NMDAR antibody, anti-single-stranded DNA antibody, anti- SSA antibody, anti-nRNP/Sm antibody combination; 13. Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody combination; 14.
- Anti-NMDAR antibody anti-single-stranded DNA antibody, anti-double-stranded Combination of DNA antibody, anti-SSA antibody, anti-nRNP/Sm antibody.
- the AUC of the five marker combinations also reached more than 0.93. It can be seen that the above-mentioned combinations of 1 to 14 all showed unexpected synergistic effects higher than the highest AUC value (0.9167) in their single index.
- anti-NMDAR antibody + anti-dsDNA antibody for example, anti-NMDAR antibody + anti-dsDNA antibody; anti-NMDAR antibody + anti-SSA antibody; anti-NMDAR antibody + anti-nRNP/Sm antibody; anti-dsDNA antibody + anti-SSA antibody; anti-dsDNA antibody + anti-nRNP/Sm antibody; anti-SSA antibody + anti-nRNP/Sm antibody; anti-NMDAR antibody + anti-dsDNA antibody + anti-SSA antibody; anti-NMDAR antibody + anti-dsDNA antibody + anti-SSA antibody; anti-NMDAR antibody + anti-dsDNA antibody + anti-nRNP/Sm antibody; anti-NMDAR antibody + anti-SSA antibody + anti-nRNP/Sm antibody; anti-dsDNA antibody + anti-SSA antibody; anti-NMDAR antibody + anti-dsDNA antibody + anti-nRNP/Sm antibody; anti-NMDAR antibody + anti-SSA antibody + anti-nRNP/Sm antibody; anti-dsDNA antibody + anti-
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Abstract
先天性巨结肠的诊断标志物组合，先天性巨结肠的诊断产品及其在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。先天性巨结肠的诊断标志物组合包含选自抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的2-5种抗体。先天性巨结肠的诊断产品包含抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的1-5种抗体的检测剂。
Description
本发明属于基因治疗技术领域，具体涉及诊断先天性巨结肠的抗体标志物及其应用。
先天性巨结肠(Hirschsprung disease，HSCR)是一种儿童肠神经发育异常的出生缺陷疾病，病理机制为肠神经嵴细胞迁移和分化成肠神经元发生障碍，导致肠神经缺乏而发生持续性痉挛，是小儿常见的先天性肠道疾病之一。先天性巨结肠的早期表现为呕吐、腹胀、腹泻等，临床上会造成新生儿死亡或手术后反复肠炎、难治性便秘等并发症，严重影响患儿的生长发育和生活质量。
先天性巨结肠的及时诊治可以减少先天性巨结肠肠炎发生的危险，获得良好的预后。该疾病确诊需要术后病变组织的病理切片。术前诊断方法主要为钡剂灌肠，直肠活检、直肠测压，以判断是否要实施“巨结肠根治术”。目前，钡灌肠为最重要的诊断方法，原理为先天性巨结肠患儿的肠道存在无神经节段的狭窄、近端的扩张，钡灌肠后可见扩张和狭窄段而诊断为巨结肠。但该方法只能诊断出有典型肠道形态改变的患儿，灵敏度需要提高，诊断的准确度80％左右。直肠活检是直接取直肠组织，检测是否有神经节细胞的缺失，准确率高，但取样部位对结果有影响。该方法为介入有创型，且价格非常高，一般情况下该方法应用于钡灌肠不明显，或不适用的患儿，比如当患儿有坏死性小肠结肠炎(NEC)这种可能的时候，钡灌肠会导致肠穿孔，这种时候不适合用钡灌肠去诊断，才考虑用直肠活检。直肠测压是通过检测肛门内括约肌的松弛缺乏判定肠神经的支配异常，仅为辅助诊断方法，假阳性和假阴性都较多，不能用于单独检测。
发明内容
为克服上述现有技术的缺点，本发明的目的在于提供先天性巨结肠的诊断标志物及其应用，为先天性巨结肠的诊断提供了一种新的准确、灵敏的检测途径。
为了实现上述目的，本发明采用以下技术方案予以实现：
本发明的第一个方面，提供一种先天性巨结肠的诊断标志物组合。所述诊断标志物组合包含选自以下的至少二种：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
在一些实施方式中，所述诊断标志物组合包含选自以下的至少三种、或至少四种：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
在一些实施方式中，所述诊断标志物组合包含选自以下的二种、三种、四种或五种：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
在一些实施方式中，所述诊断标志物组合包含抗NMDAR抗体，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种、至少两种、至少三种抗体或四种抗体。优选的，所述诊断标志物组合包含抗NMDAR抗体，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的一种、两种、三种抗体或四种抗体。
在一些实施方式中，所述抗体为自身抗体。优选的，抗NMDAR抗体是抗NMDAR自身抗体。优选的，抗单链DNA抗体是抗单链DNA自身抗体。优选的，抗双链DNA抗体是抗双链DNA自身抗体。优选的，抗SSA抗体是抗SSA自身抗体。优选的，抗nRNP/Sm抗体是抗nRNP/Sm自身抗体。
在一些实施方式中，所述抗NMDAR抗体为抗NMDAR的IgG抗体。优选的，所述抗NMDAR自身抗体为抗NMDAR的IgG自身抗体。
在一些实施方式中，所述抗单链DNA抗体为抗单链DNA的IgG抗体。优选的，所述抗单链DNA自身抗体为抗单链DNA的IgG自身抗体。
在一些实施方式中，所述抗双链DNA抗体为抗双链DNA的IgG抗体。优选的，所述抗双链DNA自身抗体为抗双链DNA的IgG自身抗体。
在一些实施方式中，所述抗SSA抗体为抗SSA的IgG抗体。优选的，所述抗SSA自身抗体为抗SSA的IgG自身抗体。
在一些实施方式中，所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG抗体。在一些实施方式中，所述抗nRNP/Sm自身抗体为抗nRNP/Sm的IgG自身抗体。
本发明的第二个方面，提供一种先天性巨结肠的诊断产品。所述诊断产品包含检测剂，所述检测剂用于检测包含选自以下的至少一种诊断标志物：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
在一些实施方式中，所述诊断产品包含检测剂，所述检测剂用于检测包含选自以下的至少二种、至少三种、至少四种或五种诊断标志物：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。优选的，所述检测剂用于检测包含选自以下的一种、二种、三种、四种或五种诊断标志物：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
在一些实施方式中，所述检测剂包含抗NMDAR抗体的检测剂，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体中的至少一种、至少二种、至少三种或四种的检测剂。优选的，所述检测剂包含抗NMDAR抗体的检测剂，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体中的一种、二种、三种或四种的检测剂。
在一些实施方式中，所述诊断产品为试剂、试剂盒或试纸条。
在一些实施方式中，所述抗体为自身抗体。优选的，抗NMDAR抗体是抗NMDAR自身抗体。优选的，抗单链DNA抗体是抗单链DNA自身抗体。优选的，抗双链DNA抗体是抗双链DNA自身抗体。优选的，抗SSA抗体是抗SSA自身抗体。优选的，抗nRNP/Sm抗体是抗nRNP/Sm自身抗体。
在一些实施方式中，所述抗NMDAR抗体为抗NMDAR的IgG抗体。优选的，所述抗NMDAR自身抗体为抗NMDAR的IgG自身抗体。
在一些实施方式中，所述抗单链DNA抗体为抗单链DNA的IgG抗体。优选的，所述抗单链DNA抗体为抗单链DNA的IgG自身抗体。
在一些实施方式中，所述抗双链DNA抗体为抗双链DNA的IgG抗体。优选的，所述抗双链DNA抗体为抗双链DNA的IgG自身抗体。
在一些实施方式中，所述抗SSA抗体为抗SSA的IgG抗体。优选的，所述抗SSA自身抗体为抗SSA的IgG自身抗体。
在一些实施方式中，所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG抗体。优选的，所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG自身抗体。
在一些实施方式中，所述检测是定量检测。优选的，所述检测剂为定量检测剂。优选的，定量检测剂用于执行如下方法中的任意一项：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
在一些实施方式中，所述检测剂包括抗原。优选的，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白(选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)中的至少一种、至少二种、至少三种、至少四种或五种。优选的，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白(选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)中的一种、二种、三种、四种或五种。优选的，抗NMDAR抗体的抗原是NMDAR蛋白；抗单链DNA抗体的抗原是ssDNA；抗双链DNA抗体的抗原是dsDNA；抗SSA抗体的抗原是SSA蛋白；抗nRNP/Sm抗体的抗原是nRNP/Sm蛋白(选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)。
在一些实施方式中，所述抗原缀合于固相支持物上。优选的，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
在一些实施方式中，所述检测剂还包括抗人Ig抗体。优选的，所述定量检测剂还包括抗人Ig抗体。
在一些实施方式中，所述抗人Ig抗体为抗人IgG抗体。
在一些实施方式中，所述抗人Ig抗体缀合有信号物质。
在一些实施方式中，所述检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种；优选为血液、血浆、血清中的至少一种；优选为肠道组织、肠道细胞或肠道细胞裂解物中的至少一种。
本发明的第三个方面，提供本发明第二方面所述的诊断产品或检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
本发明的有益效果为：
(1)先天性巨结肠患儿表现为呕吐、腹胀、便秘、肠炎等，此类症状的患儿群体很大，其中为先天性巨结肠的儿童占比又很少，需要一个简单而有效的筛选方法。本发明发现的诊断标志物在儿童先天性疾病诊断中具有操作简单、无介入、通量高、成本低的优势，克服了现有技术中无血浆诊断的缺点。
(2)本方法诊断的灵敏度和特异性较好：单独检测抗NMDAR抗体的AUC值为0.9046，最佳界限对应灵敏性为93.75％，特异性为81.58％。单独检测抗ssDNA抗体用于诊断先天性巨结肠的AUC值为0.9167；最佳界限对应灵敏性为74.63％，特异性为96.88％。单独检测dsDNA抗体的AUC值为0.8313，最佳界限对应灵敏性为96.88％，特异性为69.23％；单独检测抗SSA抗体的AUC值为0.7756；最佳界限对应灵敏性为62.50％，特异性为74.36％。 单独检测抗nRNP/Sm抗体的AUC值为0.8165，最佳界限对应灵敏性为100％，特异性为64.10％。本发明为临床上诊断先天性巨结肠疾病提供了有效的方法。
(3)将标志物进行联合检测时，其检测性能，如AUC值、灵敏性和特异性进一步提升，例如联合检测抗NMDAR抗体和抗ssDNA抗体用于诊断先天性巨结肠的AUC值为0.9351，最佳界限对应灵敏性为82.05％，特异性为93.75％，诊断效果显著优于单个指标检测的效果，为临床上诊断先天性巨结肠疾病提供了更为可靠的方法。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为芯片筛选先天性巨结肠患儿血浆中的诊断标记物抗NMDAR抗体。
图2为芯片筛选先天性巨结肠患儿血浆中的诊断标记物抗单链DNA抗体。
图3为芯片筛选先天性巨结肠患儿血浆中的诊断标记物抗双链DNA抗体。
图4为芯片筛选先天性巨结肠患儿血浆中的诊断标记物抗SSA抗体。
图5为芯片筛选先天性巨结肠患儿血浆中的诊断标记物抗nRNP/Sm抗体。
图6为先天性巨结肠病变组织(HSCR)及对照结肠组织(DC)中抗NMDAR抗体的检测。
图7为先天性巨结肠患儿(HSCR)、疾病对照(DC)及健康儿童(HC)的血浆中抗NMDAR抗体的检测。
图8为ROC曲线分析血浆中抗NMDAR抗体在先天性巨结肠中的诊断效果。
图9为先天性巨结肠患儿(HSCR)、疾病对照(DC)及健康儿童(HC)的血浆中抗单链DNA抗体的检测。
图10为ROC曲线分析血浆中抗单链DNA抗体在先天性巨结肠中的诊断效果。
图11为先天性巨结肠病变组织(HSCR)及对照结肠组织(DC)中抗双链DNA抗体的检测。
图12为先天性巨结肠患儿(HSCR)、疾病对照(DC)及健康儿童(HC)的血浆中抗双链DNA抗体的检测。
图13为ROC曲线分析血浆中抗双链DNA抗体在先天性巨结肠中的诊断效果。
图14为先天性巨结肠患儿(HSCR)、疾病对照(DC)及健康儿童(HC)的血浆中抗SSA抗体的检测。
图15为ROC曲线分析血浆中抗SSA抗体在先天性巨结肠中的诊断效果。
图16为先天性巨结肠患儿(HSCR)、疾病对照(DC)及健康儿童(HC)的血浆中抗nRNP/Sm抗体的检测。
图17为ROC曲线分析血浆中抗nRNP/Sm抗体在先天性巨结肠中的诊断效果。
图18为血浆中抗NMDAR抗体和抗单链DNA抗体联合检测的ROC曲线分析。
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。 本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明涉及诊断标志物、诊断标志物组合、诊断产品、检测试剂盒、以及检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。本发明首次发现抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体以及抗nRNP/Sm抗体(尤其是自身抗体)可单独或联合作为先天性巨结肠的诊断标志物，填补了先天性巨结肠诊断特别是血浆诊断的空白。
本文使用的术语“标志物”或“生化标志物”指要用作分析患者实验样品的靶标的分子。
术语“自身抗体”是指针对自身组织、器官、细胞及细胞成分的抗体。正常人体血液中可以有低滴度的自身抗体，本发明所述自身抗体是错误地以机体特定组织或器官为靶目标并损伤组织或器官的抗体。本发明发现，某些自身抗体可对身体产生损伤并且能够成为先天性巨结肠的诊断标志物。
在一些实施例中，所述抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体为自身抗体。
在一些实施方式中，所述抗NMDAR抗体为自身抗体，并且是抗NMDAR的IgG抗体。
在一些实施方式中，所述抗单链DNA抗体为自身抗体，并且是抗单链DNA的IgG抗体。
在一些实施方式中，所述抗双链DNA抗体为自身抗体，并且是抗双链DNA的IgG抗体。
在一些实施方式中，所述抗SSA抗体为自身抗体，并且是抗SSA的IgG抗体。
在一些实施方式中，所述抗nRNP/Sm抗体为自身抗体，并且是抗nRNP/Sm的IgG抗体。
在本发明涉及的判定方法中，对于针对各种诊断标志物的表达分析法不作特别限定。例如，不限于测定血液样品中这些抗体的绝对量或浓度，也可以测定相对的量或浓度。更具体地说，例如可以测定上述抗体等在血液样品中的量、浓度或活性。这些方法在本领域是公知的，作为示例，在一些实施方式中，所述检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
作为上述检出方法，所述生物质谱法可以举出例如基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MASS)、表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MASS)等飞行时间质谱(TOF-MASS)。由TOF-MASS图，通过分子量峰、其它的片段峰、它们的强度等，可以掌握抗体的浓度或量。此外，与ELISA的情况同样地，也可以使用具有标记基团且可以与各抗体结合的二次抗体来检出与蛋白质芯片选择性地结合的抗体。
在这些方法中，作为用于进行上述表达分析的方法，优选免疫测定法。免疫测定法的灵敏度和精度高，即使血等检测样本中的抗体浓度稍微变化也可以检出。通过酶联免疫吸附法(ELISA)进行本发明涉及的抗体的表达分析时，可以合适地使用本发明涉及的先天性巨结肠的诊断用试剂盒。
在一些实施方式中，所述检测剂为抗原。优选的，所述检测剂为定量检测剂。
在一些实施方式中，所述检测剂或定量检测剂是NMDAR蛋白。
在一些实施方式中，所述检测剂或定量检测剂是ssDNA。
在一些实施方式中，所述检测剂或定量检测剂是dsDNA。
在一些实施方式中，所述检测剂或定量检测剂是SSA蛋白。
在一些实施方式中，所述检测剂或定量检测剂选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物的至少一种。
在一些实施方式中，根据选取的诊断标志物的不同，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白(选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)中的至少一种、至少二种、至少三种、至少四种或五种。优选的，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白(选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)蛋白中的一种、二种、三种、四种或五种。优选的，所述抗原包括ssDNA以及任选的NMDAR蛋白。
包含被抗体识别的表位的抗原片段的长度可至少为5、6、7、8、9、10、11、12、13、14、15、20、25、30、35、40、45、50、75、100、150、200、250、300、350、400、450、500、750或1000个核苷酸。该片段也可为5、10、15、20、25、50、75、100、150、200或250和比抗原(NMDAR蛋白、SSA蛋白、nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物)全长少一个氨基酸之间。优选的，该片段也可为5、10、15、20、25、50、75、100、150、200或250和比ssDNA或dsDNA全长少一个核苷酸之间。通常，提前表征这些表位使得已知给定抗原的抗体识别该表位。表位作图的方法为本领域熟知。“表位”为抗原例如本文公开的抗原上被抗体识别的位点。
显然，所述检测剂或定量检测剂还可以为抗原物质(NMDAR蛋白、SSA蛋白、nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物中至少一种)的片段肽、变性物、修饰物中的至少一种肽。抗原物质的变性物指的是通过实施加热、冷冻、紫外线等物理性处理或应用表面活性剂、变性剂等化学性处理而得到的、可以与上述抗体特异性地结合的变性物。例如，可以举出通过SDS、DTT处理而得到的变性物。修饰物指的是对1个以上氨基酸进行修饰而得到的、可以与上述(自身)抗体特异性地结合的修饰物。例如，可以举出用戊二醛处理而得到的修饰物。优选的，上述修饰物可以是与抗原(NMDAR蛋白、SSA蛋白、nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物中至少一种)相比，具有1个或几个氨基酸残基的突变、置换、缺失和/或添加，只要能与上述(自身)抗体特异性地结合。优选的，修饰物与原抗原序列相比具有百分之70％以上，75％以上、80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上的序列同一性。
显然，所述检测剂或定量检测剂还可以为抗原物质(ssDNA、dsDNA中至少一种)的片段、变性物、修饰物中的至少一种。抗原物质的变性物指的是通过实施加热、冷冻、紫外线等物理性处理或应用表面活性剂、变性剂等化学性处理而得到的、可以与上述抗体特异性地结合的变性物。例如，可以举出通过SDS、DTT处理而得到的变性物。修饰物指的是对1个以上核苷酸进行修饰而得到的、可以与上述抗体特异性地结合的修饰物。例如，可以举出用戊二醛处理而得到的修饰物。优选的，上述修饰物可以是与抗原(ssDNA、dsDNA中至少一种)相比，具有1个或几个核酸碱基的突变、置换、缺失和/或添加，只要能与上述抗体特异性地结合。优选的，修饰物与原抗原序列相比具有70％以上，75％以上、80％以上、85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上的序列同一性。
在一些实施方式中，所述抗原(ssDNA、dsDNA，NMDAR蛋白、SSA蛋白、nRNP、Sm蛋白，以及Sm蛋白 和nRNP的复合物中至少一种)缀合于固相支持物上。
在一些实施方式中，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
术语“固相支持物”表示载体材料以非液体坚固性为主，从而允许核酸在载体材料上的精确和可追踪的定位。固相支持物可以选用聚苯乙烯、塑料、纤维素、聚丙烯酰胺、聚乙烯聚丙烯、交联葡聚糖、玻璃、硅橡胶、琼脂糖凝胶等材质。优选的固相载体为酶标板。其含有的孔位可以为16、32、48、64、96或更多。
在本发明中，术语“微球”可以为球体、近球体、立方体、多面体或不规则形状。微球的直径优选为10nm～1mm，例如100nm、500nm、1μm、10μm、100μm、500μm；优选为400nm～10μm。
微球的表面上具有对于待测定的感兴趣物质(靶标或分析物)的特异性结合特性。
微球优选为磁珠，其成分中含有磁性物质。磁性物质可以为金属(金属单质或合金)、非金属，或金属与非金属所形成的复合物。金属例如铁、铝镍钴金属等；非金属例如铁氧体非金属(优选为Fe
2O
3或Fe
3O
4磁性纳米粒子)；金属与非金属所形成的复合物例如钕铁硼橡胶磁复合材料。
所述微球的表面修饰有一种或多种活性功能基团，所述活性功能基团包括-OH、-COOH、-NH
2、-CHO、以及-SO
3H中的一种或多种。在一些实施方式中，包被的抗原和抗体通过物理吸附或直接化学缀合(例如通过桥接物进行桥接)与所述微球缀合或结合。具体的，用于构成桥接物的合适技术包括，例如，共价附接、吸附、非共价相互作用或其组合。在一些实施方案中，可以通过戊二醛固定、N-羟基琥珀酰亚胺(NHS)化学法或1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)NHS化学法来实现直接桥接。用于间接桥接的合适方式包括，例如，通过肽、蛋白、抗体、接头或其组合的桥接。在一些实施方案中，至固体支持物的间接桥接是经由链霉抗生物素蛋白和生物素。
在一些实施方式中，所述检测剂还包括抗人Ig抗体。
在一些实施方式中，所述抗人Ig抗体为抗人IgG抗体。
抗人Ig抗体是指针对人Ig蛋白的抗体，抗人IgG抗体是指针对人IgG蛋白的抗体。
在一些实施方式中，所述抗人Ig抗体缀合有信号物质。
在另外一些实施方式中，抗人Ig抗体并未标记信号物质，且所述检测剂还包括标记有信号物质的抗人Ig抗体的第二抗体。用缓冲液等将与本发明上述的诊断标志物非特异性地结合的物质等洗涤除去后，使上述第二抗体进行作用。其中，当诊断标志物(自身)抗体分别结合第二抗体和上述(固相支持物上的)抗原物质时，第二抗体可用对应于信号物质的方法检出。
在本发明中，用作检测剂或定量检测剂的抗体可以是IgA、IgD、IgG、IgE或IgM同种型或单结构域形式，诸如来自骆驼科动物的单结构域抗体。在一些实施方案中，用作定量检测剂的抗体是IgG抗体。
所述缓冲液包含例如下列成分中的一种或多种：磷酸盐缓冲液、NaCl、EDTA、普朗尼克F-127、叠氮化钠、山梨糖醇、巯基修饰的牛血清或其任何组合、变体或等同物。
在本发明中，信号物质能够提供被检测的信号的物质，在一些实施方式中，所述信号物质独立的选自发色团、地高辛标记探针、电子致密物质、胶体金或酶中的任一种或多种。下面非限定部分列出这些标记：
·产生可检测信号的酶，如通过比色法、荧光和发光来检测，如辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶 和葡萄糖-6-磷酸脱氢酶。
·发色团，如荧光、量子点、荧光微球、发光化合物和染料。
·具有能被电子显微镜或通过其电特性，如传导性、电流分析、电压测量和电阻等检测的电子密度的基团。
·可检测基团，如其分子大小足以诱导在其物理和/或化学特性上可检测的修饰；这种检测可通过光学方法(如衍射、表面胞质团共振，表面变异和接触变异角度)或物理方法(如原子力谱学和隧道效应)实现。
·电子致密物质，如放射性分子(如
32P，
35S或
125I)。
在一些实施方式中，所述信号物质为吖啶类化学发光物质。
在一些实施方式中，吖啶类化学发光物质包括吖啶酯和吖啶磺酰胺。
在一些实施方式中，吖啶类化学发光物质包括吖啶酯AE-NHS、吖啶酯DMAE-NHS、吖啶酯Me-DMAE-NHS、吖啶酯NSP-DMAE-NHS、吖啶盐NSP-SA、吖啶盐NSP-SA-NHS、吖啶酰肼NSP-SA-ADH等。
只要包含上述诊断标志物抗体或自身抗体的任何生物样品都可以作为待检测样本，包括，但不限于，血清、血浆、全血、唾液、粪便、尿液、精液、汗液、泪液和身体组织。在一些较为优选的实施方式中，所述检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
本文使用的“组织或细胞裂解物”也可与“裂解物”、“裂解的样品”、“组织或细胞提取物”等用语通用，表示包含裂解的组织或细胞的样品和/或生物样品材料，即其中组织或细胞的结构完整性已经被破坏。为了释放细胞或组织样品的内容物，通常用酶和/或化学试剂处理所述材料，以溶解、降解或破坏这样的组织或细胞的细胞壁和细胞膜。熟练的技术员非常熟悉用于得到裂解物的适当方法。该过程被术语“裂解”包括。
作为本发明的诊断用试剂盒，其优选含有正常对照样品、先天性巨结肠对照样品。若试剂盒附带这些样品，则对这些样品进行同样的实验，将其测定值和受检样品的结果进行比较，由此可以更客观地判定受检者的先天性巨结肠的有无。
样品中含有的抗体的浓度或量是通过显色强度等间接得到的。可以通过校准曲线等将得到的测定值换算为相对或绝对浓度、量、活性等。
本发明还涉及一种诊断先天性巨结肠的方法，包括对待检样本中的抗体标志物进行定量检测，其中升高的抗NMDAR抗体水平、升高的抗单链DNA抗体水平、升高的抗双链DNA抗体水平、升高的抗SSA抗体水平、升高的抗nRNP/Sm抗体水平中的至少一种是先天性巨结肠的指征。
当在本发明实施方案应用的抗体的语境下使用的术语“指征”包括本发明实施方案测定其存在或缺失的抗体通常存在于患有先天性巨结肠的受试者中。“通常存在”意指该抗体经常与先天性巨结肠相关。“经常相关”包括大于50％的可能性，优选大于60％，更优选大于70％，甚至更优选大于80％并特别优选大于90％或95％。
诊断的理想场景是这样的情形，其中单一事件或过程会造成各种疾病。在所有其它情况下，正确的诊断可能非常困难，尤其当疾病的病因学不能完全理解时，如在许多癌症类型的情况下。如熟练的技术人员将明白的，对于给定的多因子病，没有生化标志物的诊断是100％特异性且同100％灵敏度。相反地，可使用生化标志物来以某种可能性或预测值评估例如疾病的存在与否或严重性。因此，在常规的临床诊断中，通常综合考虑各种临床症状和生物学 标志物来诊断、治疗和控制潜在的疾病。
在本发明方法中，接着由所得到的表达分析结果判定先天性巨结肠发病的有无。即，若先天性巨结肠发病、或其症状越重，则血液中的本发明涉及的抗体的浓度、量或活性越高。由此，基于本发明涉及的抗体的表达分析结果，若其表达量多则可以判断为阳性，若其表达量少则可以判断为阴性。
实际上，根据先天性巨结肠的定义、严重程度、本发明涉及的(自身)抗体的表达分析方法，可以改变阳性与阴性的边界，即截断值(cutoff value)。因此，在没有一般基准的阶段，本发明方法的实施者有必要在通过预实验等事先确定表达分析方法和截断值后进行测定。上述抗体用于诊断巨结肠的截断值可由本领域技术人员根据实际情况检测得出。所述截断值的确定是本领域的常规技术手段。本领域技术人员可以选定一个截断值，并测试选定所述截断值检测的准确率(如检出率、检准率、假阳性率、假阴性率、灵敏度、特异性等)，根据检测效果上调或者下调所述截断值，直至达到较佳或最佳的检测效果。
在一些实施方式中，所述诊断标志物抗体相对于健康组或其他肠病组，其量、浓度、活性等指示其存在或表达的指标升高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。在一些实施方式中，抗NMDAR抗体的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。在一些实施方式中，抗单链DNA抗体的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。在一些实施方式中，抗双链DNA抗体的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。在一些实施方式中，抗SSA抗体的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。在一些实施方式中，抗nRNP/Sm抗体的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。当联合检测所述抗体标志物时，抗体组合的量、浓度、活性等指示其存在或表达的指标提高0.2倍、0.3倍、0.4倍、0.5倍、1倍、1.5倍、2倍、2.5倍、3倍、4倍、4.5倍、5倍、6倍、7倍、8倍、9倍、10倍以上，诊断或辅助诊断为先天性巨结肠或者疑似先天性巨结肠。
在一些实施方式中，受试者为18岁以下的患者，例如17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1岁。或者是婴儿，例如1、2、3、4、5、6、7、8、9、10、11个月龄的婴儿。
以下将结合实施例对本发明的实施方案进行详细描述。
本发明所采用的样品均来来源于广州市妇女儿童医疗中心，所有实验用组织和血液样本的采集均获得广州市妇女儿童医疗中心伦理委员会授权和病人同意。
本发明试验结果均采用统计学分析，t检验用来评估两组间的差异，p<0.05用于表示统计学显著性，所有p值均使用双侧检验。统计分析采用R和Graphpad 8.0软件进行。
本发明所述“先天性巨结肠疾病”又称希尔施普龙病，是由于结肠缺乏神经节细胞导致肠管持续痉挛，粪便淤滞于近端结肠，近端结肠肥厚、扩张，是小儿常见的先天性肠道疾病之一，其中对于先天性巨结肠疾病而言，按照严重程度递增，临床上可分为短段型，普通型，长段型，全结肠型。短段型病变位于直肠近、中段，距肛管不超过6.5cm；普通型病变位于直肠近端或直肠乙状结肠远端，距肛管约9cm；长段型病变延至乙状结肠或降结肠；全结肠型病变波及全部结肠及回肠末端，距回盲瓣30cm以内。
本发明所述N-甲基-D-天冬氨酸受体(NMDAR)是离子型谷氨酸受体的一个亚型，对神经系统发育发挥重要生理作用；抗N-甲基-D-天冬氨酸受体(NMDAR)抗体是指针对N-甲基-D-天冬氨酸受体(NMDAR)蛋白产生的抗体，优选为IgG抗体。
本发明所述ssDNA是指细胞核中的单链DNA；抗单链DNA(ssDNA)抗体是指针对细胞核中的单链DNA产生的抗体，优选为IgG抗体。
本发明所述dsDNA是指细胞核中的双链DNA；抗双链DNA(dsDNA)抗体是指针对细胞核中的双链DNA产生的抗体，优选为IgG抗体。
本发明所述SSA是一种耐RNA酶的胞浆内的核糖核蛋白；抗SSA抗体是针对细胞核中的SSA产生的抗体，优选为IgG抗体。
本发明所述的nRNP((nuclear ribonucleoprotein)是核糖核蛋白。Sm和nRNP是同一分子复合物(nRNP/Sm)中的不同抗原位点，两种抗原具有相关性，故抗Sm抗体阳性常伴有抗nRNP抗体阳性。抗nRNP/Sm抗体属于抗核抗体，通常是一种自身抗体。
本发明所述“ROC曲线”是特异性(假阳性率)和敏感性(真阳性率)变化的曲线，反应了二分类器的诊断能力。一个好的分类器真阳性率与假阳性率的变化比值是大于1的，远离45度直线。
本发明所述“AUC”是指ROC曲线下面积，介于0.1和1之间，用于评价分类器的好坏，越接近1分类器越好，大于0.5可考虑用作疾病的诊断标志物。
本发明通过人神经免疫芯片和抗原芯片筛选先天性巨结肠患儿的血浆，用其它肠病和健康组患儿血浆做对照，得到先天性巨结肠患儿血浆高表达的抗N-甲基-D-天冬氨酸受体(NMDAR)抗体、先天性巨结肠患儿血浆高表达的抗单链DNA(ssDNA)的抗体、先天性巨结肠患儿血浆高表达的抗双链DNA(dsDNA)抗体、先天性巨结肠患儿血浆高表达的抗SSA抗体、先天性巨结肠患儿血浆高表达的抗nRNP/Sm抗体。并在独立的样品中用酶联免疫(ELISA)的方法验证了先天性巨结肠患者的上述标志物的表达水平显著高于健康组和其他疾病对照组，对其进行了进一步效果验证。ROC曲线分析证明所述方法特异性和灵敏性高，能够准确，便捷高效诊断先天性巨结肠。
关于本发明的具体实施方式更详细的描述如下。
一、关于抗NMDAR自身抗体作为先天性巨结肠诊断标志物的应用
1.抗NMDAR自身抗体的定量检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
2.根据1所述的应用，其特征在于，所述抗NMDAR自身抗体为抗NMDAR自身IgG抗体。
3.根据1所述的应用，其特征在于，所述定量检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
4.根据1所述的应用，其特征在于，所述定量检测剂为NMDAR。
5.根据4所述的应用，其特征在于，所述NMDAR缀合于固相支持物上。
6.根据5所述的应用，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
7.根据4～6任一项所述的应用，其特征在于，所述定量检测剂还包括抗人Ig抗体。
8.根据7所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
9.根据7所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
10.根据1～6、8、9任一项所述的应用，所述定量检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
二、关于抗单链DNA抗体作为先天性巨结肠诊断标志物的应用
1.检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用；所述检测剂包括抗单链DNA抗体的定量检测剂；可选的，所述抗单链DNA抗体为抗单链DNA的IgG抗体。
2.根据1所述的应用，其特征在于，所述检测剂还包括抗NMDAR抗体的定量检测剂；可选的，所述抗NMDAR抗体为抗NMDAR的IgG抗体。
3.根据1或2所述的应用，其特征在于，所述检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
4.根据2所述的应用，其特征在于，所述检测剂为抗原，所述抗原包括ssDNA以及任选的NMDAR蛋白。
5.根据4所述的应用，其特征在于，所述抗原缀合于固相支持物上。
6.根据5所述的应用，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
7.根据4～6任一项所述的应用，其特征在于，所述检测剂还包括抗人Ig抗体。
8.根据7所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
9.根据7所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
10.根据1、2、4～6、8、9任一项所述的应用，所述检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
三、关于抗双链DNA抗体作为先天性巨结肠诊断标志物的应用
1.抗双链DNA抗体的定量检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
2.根据1所述的应用，其特征在于，所述抗双链DNA抗体为抗双链DNA的IgG抗体。
3.根据1所述的应用，其特征在于，所述定量检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
4.根据1所述的应用，其特征在于，所述定量检测剂为dsDNA。
5.根据4所述的应用，其特征在于，所述dsDNA缀合于固相支持物上。
6.根据5所述的应用，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
7.根据4～6任一项所述的应用，其特征在于，所述定量检测剂还包括抗人Ig抗体。
8.根据7所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
9.根据7所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
10.根据1～6、8、9任一项所述的应用，所述定量检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
四、关于抗SSA自身抗体作为先天性巨结肠诊断标志物的应用
1.抗SSA自身抗体的定量检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
2.根据1所述的应用，其特征在于，所述抗SSA自身抗体为抗SSA自身IgG抗体。
3.根据1所述的应用，其特征在于，所述定量检测剂用于执行如下任一方法：
放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
4.根据1所述的应用，其特征在于，所述定量检测剂为SSA蛋白。
5.根据4所述的应用，其特征在于，所述SSA蛋白缀合于固相支持物上。
6.根据5所述的应用，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
7.根据4～6任一项所述的应用，其特征在于，所述定量检测剂还包括抗人Ig抗体。
8.根据7所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
9.根据7所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
10.根据1～6、8、9任一项所述的应用，所述定量检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
五、关于抗nRNP/Sm抗体作为先天性巨结肠诊断标志物的应用
1.抗nRNP/Sm抗体的定量检测剂在制备先天性巨结肠诊断试剂、试纸条或试剂盒中的应用。
2.根据1所述的应用，所述抗nRNP/Sm抗体为IgG抗体。
3.根据1所述的应用，所述定量检测剂用于执行如下方法中的任意一项：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
4.根据1所述的应用，所述定量检测剂为抗原物质，所述抗原物质选自nRNP、Sm蛋白，以及Sm蛋白和nRNP的复合物。
5.根据4所述的应用，所述抗原物质缀合于固相支持物上。
6.根据5所述的应用，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
7.根据4～6任一项所述的应用，其特征在于，所述定量检测剂还包括抗人Ig抗体。
8.根据7所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
9.根据7所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
10.根据1～6、8、9任一项所述的应用，所述定量检测剂所检测的待检样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中至少一种。
六、关于抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体作为先天性巨结肠诊断标志物及其应用
1.一种先天性巨结肠的诊断标志物组合，其特征在于，所述诊断标志物组合包含选自以下的至少两种：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
2.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含抗NMDAR抗体，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种。
3.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含抗NMDAR抗体和抗单链DNA抗体。
4.根据3所述的诊断标志物组合，其特征在于，所述诊断标志物组合还包含抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种。
5.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含抗NMDAR抗体、抗单链DNA抗体和抗双链DNA抗体。
6.根据5所述的诊断标志物组合，其特征在于，所述诊断标志物组合还包含抗SSA抗体、抗nRNP/Sm抗体中的至少一种。
7.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含三种抗体：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗SSA抗体或抗nRNP/Sm抗体。
8.根据7所述的诊断标志物组合，其特征在于，所述三种抗体为：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗nRNP/Sm抗体。
9.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含选自以下的至少四种：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体。
10.根据9所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、抗SSA抗体以及抗nRNP/Sm抗体。
11.根据9所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体以及抗nRNP/Sm抗体。
12.根据1所述诊断标志物组合，其特征在于，所述诊断标志物组合包含抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体。
13.根据1所述的诊断标志物组合，其特征在于，所述诊断标志物组合选自以下(1)～(26)之一：
(1).抗NMDAR抗体、抗单链DNA抗体组合；(2).抗单链DNA抗体、抗双链DNA抗体组合；(3).抗单链DNA抗体、抗SSA抗体组合；(4).抗单链DNA抗体、抗nRNP/Sm抗体组合；(5).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体组合；(6).NMDAR抗体、抗单链DNA抗体、抗SSA抗体组合；(7).NMDAR抗体、抗单链DNA抗体、抗nRNP/Sm抗体组合；(8).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体组合； (9).抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体组合；(10).抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；(11).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体组合；(12).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体组合；(13).NMDAR抗体、抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；(14).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；(15).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(16).抗NMDAR抗体、抗dsDNA抗体的组合；(17).抗NMDAR抗体、抗SSA抗体的组合；(18).抗NMDAR抗体、抗nRNP/Sm抗体的组合；(19).抗dsDNA抗体、抗SSA抗体的组合；(20).抗dsDNA抗体、抗nRNP/Sm抗体的组合；(21).抗SSA抗体、抗nRNP/Sm抗体的组合；(22).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体的组合；(23).抗NMDAR抗体、抗dsDNA抗体、抗nRNP/Sm抗体的组合；(24).抗NMDAR抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(25).抗dsDNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(26).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体、抗nRNPSm抗体的组合。
14.根据1-13任一所述的诊断标志物组合，其特征在于，所述抗体为自身抗体。
15.一种先天性巨结肠的诊断产品，所述产品包含检测剂，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
16.根据15所述的诊断产品，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少两种抗体的检测剂。
17.根据16所述的诊断产品，其特征在于，所述至少两种抗体的检测剂包含：抗NMDAR抗体的检测剂，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
18.根据15所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体和抗单链DNA抗体的检测剂。
19.根据18所述的诊断产品，其特征在于，所述检测剂还包含：抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
20.根据15所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体和抗双链DNA抗体的检测剂。
21.根据20所述的诊断产品，其特征在于，所述检测剂还包含：抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
22.根据15所述的诊断产品，其特征在于，所述检测剂包含选自以下三种抗体的检测剂：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗SSA抗体或抗nRNP/Sm抗体。
23.根据15所述的诊断产品，其特征在于，所述检测剂包含选自以下三种抗体的检测剂：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗nRNP/Sm抗体。
24.根据15所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少四种抗体的检测剂。
25.根据24所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链 DNA抗体以及抗nRNP/Sm抗体的检测剂。
26.根据24所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、抗SSA抗体以及抗nRNP/Sm抗体的检测剂。
27.根据15所述的诊断产品，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体的检测剂。
28.根据15所述的诊断产品，其特征在于，所述检测剂选自以下(1)～(31)之一的检测剂：
(1).抗NMDAR抗体、抗单链DNA抗体的组合；(2)抗单链DNA抗体、抗双链DNA抗体的组合；(3).抗单链DNA抗体、抗SSA抗体的组合；(4).抗单链DNA抗体、抗nRNP/Sm抗体的组合；(5).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体的组合；(6).NMDAR抗体、抗单链DNA抗体、抗SSA抗体的组合；(7).NMDAR抗体、抗单链DNA抗体、抗nRNP/Sm抗体的组合；(8).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体的组合；(9).抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体的组合；(10).抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(11).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体的组合；(12).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体的组合；(13).NMDAR抗体、抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(14).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(15).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(16).抗NMDAR抗体、抗dsDNA抗体的组合·；(17).抗NMDAR抗体、抗SSA抗体的组合；(18).抗NMDAR抗体、抗nRNP/Sm抗体的组合；(19).抗dsDNA抗体、抗SSA抗体的组合；(20).抗dsDNA抗体、抗nRNP/Sm抗体的组合；(21).抗SSA抗体、抗nRNP/Sm抗体的组合；(22).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体的组合；(23).抗NMDAR抗体、抗dsDNA抗体、抗nRNP/Sm抗体的组合；(24).抗NMDAR抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(25).抗dsDNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(26).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体、抗nRNPSm抗体的组合；(27).抗NMDAR抗体；(28).抗单链DNA抗体；(29).抗双链DNA抗体；(30).抗SSA抗体；(31)抗nRNP/Sm抗体。
29.根据15-28任一所述的诊断产品，其特征在于，所述抗体为自身抗体。
30.根据15-28任一所述的诊断产品，其特征在于，所述检测剂为定量检测剂。
31.根据15-28任一所述的诊断产品，或1-14任一所述的诊断标志物组合，其特征在于，所述抗NMDAR抗体是抗NMDAR的IgG抗体；所述抗单链DNA抗体为抗单链DNA的IgG抗体；所述抗双链DNA抗体为抗双链DNA的IgG抗体；所述抗SSA抗体为抗SSA的IgG抗体；所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG抗体。
32.根据15-28任一所述的诊断产品，其特征在于，所述检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
33.根据15-28任一所述的诊断产品，其特征在于，所述检测剂包括抗原，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少一种。
34.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少两种。
35.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白，以及包括ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少一种、至少两种、至少三种或四种。
36.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白和ssDNA。
37.根据36所述的诊断产品，其特征在于，所述抗原还包括dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少一种、至少两种或三种。
38.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA和dsDNA。
39.根据38所述的诊断产品，其特征在于，所述抗原还包括SSA蛋白或nRNP/Sm蛋白中的至少一种。
40.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、SSA蛋白或nRNP/Sm蛋白。
41.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、以及nRNP/Sm蛋白。
42.根据33所述的诊断产品，其特征在于，所述抗原包括选自NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的四种。
43.根据42所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、SSA蛋白、nRNP/Sm蛋白。
44.根据42所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、nRNP/Sm蛋白。
45.根据33所述的诊断产品，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、以及nRNP/Sm蛋白。
46.根据33所述的诊断产品，其特征在于，所述抗原选自以下(1)～(31)之一：
(1).NMDAR蛋白、ssDNA的组合；(2).ssDNA、dsDNA的组合；(3).ssDNA、SSA蛋白的组合；(4).ssDNA、nRNP/Sm蛋白的组合；(5).NMDAR蛋白、ssDNA、dsDNA的组合；(6).NMDAR蛋白、ssDNA、SSA蛋白的组合；(7).NMDAR蛋白、ssDNA、nRNP/Sm蛋白的组合；(8).ssDNA、dsDNA、SSA蛋白的组合；(9).ssDNA、dsDNA、nRNP/Sm蛋白的组合；(10).ssDNA、SSA蛋白、nRNP/Sm蛋白的组合；(11).NMDAR蛋白、ssDNA、dsDNA、SSA蛋白的组合；(12).NMDAR蛋白、ssDNA、dsDNA、nRNP/Sm蛋白的组合；(13).NMDAR蛋白、ssDNA、SSA蛋白、nRNP/Sm蛋白的组合；(14).ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(15).NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(16).NMDAR蛋白、dsDNA的组合；(17).NMDAR蛋白、SSA蛋白的组合；(18).NMDAR蛋白、nRNP/Sm蛋白的组合；(19).dsDNA、SSA蛋白的组合；(20).dsDNA、nRNP/Sm蛋白的组合；(21).SSA蛋白、nRNP/Sm蛋白的组合；(22).NMDAR蛋白、dsDNA、SSA蛋白的组合；(23).NMDAR蛋白、dsDNA、nRNP/Sm蛋白的组合；(24).NMDAR蛋白、SSA蛋白、nRNP/Sm蛋白的组合；(25).dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(26).NMDAR蛋白、dsDNA、 SSA蛋白、nRNPSm蛋白的组合；(27).NMDAR蛋白；(28).单链DNA；(29).双链DNA；(30).SSA蛋白；(31)nRNP/Sm蛋白。
47.根据33-46任一所述的诊断产品，其特征在于，所述抗原缀合于固相支持物上。
48.根据47所述的诊断产品，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
49.根据15-48任一所述的诊断产品，其特征在于，所述检测剂还包括抗人Ig抗体。
50.根据49所述的诊断产品，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
51.根据49或50所述的诊断产品，其特征在于，所述抗人Ig抗体缀合有信号物质。
52.根据15-51任一所述的诊断产品，其特征在于，所述检测剂所适用的检测样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中的至少一种。
53.根据52所述的诊断产品，其特征在于，所述检测剂所适用的检测样本为血液、血浆、血清样品中的至少一种；或为肠道组织、肠道细胞或肠道细胞裂解物中的至少一种。
54.15-53任一的诊断产品在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
55.检测剂在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
56.根据55所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少两种抗体的检测剂。
57.根据55所述的应用，其特征在于，所述至少两种抗体的检测剂包含：抗NMDAR抗体的检测剂，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
58.根据55所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体和抗单链DNA抗体的检测剂。
59.根据58所述的应用，其特征在于，所述检测剂还包含：抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
60.根据55所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体和抗双链DNA抗体的检测剂。
61.根据60所述的应用，其特征在于，所述检测剂还包含：抗SSA抗体、抗nRNP/Sm抗体中的至少一种抗体的检测剂。
62.根据55所述的应用，其特征在于，所述检测剂包含选自以下三种抗体的检测剂：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗SSA抗体或抗nRNP/Sm抗体。
63.根据55所述的应用，其特征在于，所述检测剂包含选自以下三种抗体的检测剂：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、以及抗nRNP/Sm抗体。
64.根据55所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少四种抗体的检测剂。
65.根据64所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体以及抗nRNP/Sm抗体的检测剂。
66.根据64所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体或抗双链DNA抗体、抗SSA抗体以及抗nRNP/Sm抗体的检测剂。
67.根据55所述的应用，其特征在于，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体的检测剂。
68.根据55所述的应用，其特征在于，所述检测剂选自以下(1)～(31)之一的检测剂：
(1).抗NMDAR抗体、抗单链DNA抗体的组合；(2)抗单链DNA抗体、抗双链DNA抗体的组合；(3).抗单链DNA抗体、抗SSA抗体的组合；(4).抗单链DNA抗体、抗nRNP/Sm抗体的组合；(5).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体的组合；(6).NMDAR抗体、抗单链DNA抗体、抗SSA抗体的组合；(7).NMDAR抗体、抗单链DNA抗体、抗nRNP/Sm抗体的组合；(8).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体的组合；(9).抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体的组合；(10).抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(11).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体的组合；(12).NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体的组合；(13).NMDAR抗体、抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(14).抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(15).抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(16).抗NMDAR抗体、抗dsDNA抗体的组合；(17).抗NMDAR抗体、抗SSA抗体的组合；(18).抗NMDAR抗体、抗nRNP/Sm抗体的组合；(19).抗dsDNA抗体、抗SSA抗体的组合；(20).抗dsDNA抗体、抗nRNP/Sm抗体的组合；(21).抗SSA抗体、抗nRNP/Sm抗体的组合；(22).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体的组合；(23).抗NMDAR抗体、抗dsDNA抗体、抗nRNP/Sm抗体的组合；(24).抗NMDAR抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(25).抗dsDNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合；(26).抗NMDAR抗体、抗dsDNA抗体、抗SSA抗体、抗nRNPSm抗体的组合；(27).抗NMDAR抗体；(28).抗单链DNA抗体；(29).抗双链DNA抗体；(30).抗SSA抗体；(31)抗nRNP/Sm抗体。
69.根据55-68任一所述的应用，其特征在于，所述抗体为自身抗体。
70.根据55-68任一所述的应用，其特征在于，所述检测剂为定量检测剂。
71.根据55-68任一所述的应用，其特征在于，所述抗NMDAR抗体是抗NMDAR的IgG抗体；所述抗单链DNA抗体为抗单链DNA的IgG抗体；所述抗双链DNA抗体为抗双链DNA的IgG抗体；所述抗SSA抗体为抗SSA的IgG抗体；所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG抗体。
72.根据55-68任一所述的应用，其特征在于，所述检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
73.根据55-68任一所述的应用，其特征在于，所述检测剂包括抗原，所述抗原包括NMDAR蛋白、ssDNA、 dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少一种。
74.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少两种。
75.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白，以及包括ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的至少一种、至少两种、至少三种或四种。
76.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白和ssDNA。
77.根据76所述的应用，其特征在于，所述抗原还包括dsDNA、SSA蛋白、nRNP/Sm蛋白中的一种或多种。
78.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA和dsDNA。
79.根据78所述的应用，其特征在于，所述抗原还包括SSA蛋白或nRNP/Sm蛋白中的一种或两种。
80.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、SSA蛋白或nRNP/Sm蛋白。
81.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、以及nRNP/Sm蛋白。
82.根据73所述的应用，其特征在于，所述抗原包括选自NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白中的四种。
83.根据82所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA或dsDNA、SSA蛋白、nRNP/Sm蛋白。
84.根据82所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、nRNP/Sm蛋白。
85.根据73所述的应用，其特征在于，所述抗原包括NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、以及nRNP/Sm蛋白。
86.根据73所述的应用，其特征在于，所述抗原选自以下(1)～(31)之一：
(1).NMDAR蛋白、ssDNA的组合；(2).ssDNA、dsDNA的组合；(3).ssDNA、SSA蛋白的组合；(4).ssDNA、nRNP/Sm蛋白的组合；(5).NMDAR蛋白、ssDNA、dsDNA的组合；(6).NMDAR蛋白、ssDNA、SSA蛋白的组合；(7).NMDAR蛋白、ssDNA、nRNP/Sm蛋白的组合；(8).ssDNA、dsDNA、SSA蛋白的组合；(9).ssDNA、dsDNA、nRNP/Sm蛋白的组合；(10).ssDNA、SSA蛋白、nRNP/Sm蛋白的组合；(11).NMDAR蛋白、ssDNA、dsDNA、SSA蛋白的组合；(12).NMDAR蛋白、ssDNA、dsDNA、nRNP/Sm蛋白的组合；(13).NMDAR蛋白、ssDNA、SSA蛋白、nRNP/Sm蛋白的组合；(14).ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(15).NMDAR蛋白、ssDNA、dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(16).NMDAR蛋白、dsDNA的组合；(17).NMDAR蛋白、SSA蛋白的组合；(18).NMDAR蛋白、nRNP/Sm蛋白的组合；(19).dsDNA、SSA蛋白的组合；(20).dsDNA、nRNP/Sm蛋白的组合；(21).SSA蛋白、nRNP/Sm蛋白的组合；(22).NMDAR蛋白、dsDNA、SSA蛋白的组合；(23).NMDAR蛋白、dsDNA、nRNP/Sm蛋白的组合；(24).NMDAR蛋白、SSA蛋白、nRNP/Sm蛋白的组合；(25).dsDNA、SSA蛋白、nRNP/Sm蛋白的组合；(26).NMDAR蛋白、dsDNA、SSA蛋白、nRNPSm蛋白的组合；(27).NMDAR蛋白；(28).单链DNA；(29).双链DNA；(30).SSA蛋白； (31)nRNP/Sm蛋白。
87.根据73-86任一所述的应用，其特征在于，所述抗原缀合于固相支持物上。
88.根据87所述的应用，其特征在于，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
89.根据55-88任一所述的应用，其特征在于，所述检测剂还包括抗人Ig抗体。
90.根据89所述的应用，其特征在于，所述抗人Ig抗体为抗人IgG抗体。
91.根据89或90所述的应用，其特征在于，所述抗人Ig抗体缀合有信号物质。
92.根据55-91任一所述的应用，其特征在于，所述检测剂所适用的检测样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中的至少一种。
93.根据92所述的应用，其特征在于，所述检测剂所适用的检测样本为血液、血浆、血清样品中的至少一种；或为肠道组织、肠道细胞或肠道细胞裂解物中的至少一种。
下面将结合实施例对本发明的试试方案进行详细描述。
实施例1.血浆和组织样本采集和分组
血浆样品分为先天性巨结肠患儿组(37例)，其它肠病对照组(18例)，健康儿童组(30例)，年龄介乎3个月到3岁，性别3/4为男性，疾病和对照组年龄和性别匹配。所有样品来源于广州市妇女儿童医疗中心，健康儿童组为健康儿童体检后剩余血样。采血方式为抗凝采血，离心分离血浆，冻存样品。结肠组织样品为先天性巨结肠患儿组(36例)，为手术切除的病变组织。其它肠病对照组(包括肛门狭窄和肠狭窄造瘘的结肠组织共11例)。
实施例2.人自免抗原芯片筛选和差异表达的自身抗体分析
先天性巨结肠(HSCR)患儿的血浆5例，健康组(HC)及其它肠病(DC)组患儿血浆各5例，其中四份用于人自免抗原芯片的筛选，剩余一份用于人神经免疫芯片筛选。所述芯片均由广州易锦生物技术有限公司提供，包含了超过100种自身免疫抗体的lgG检测，原始数据，减去阴性对照以后，用RLM方法进行归一化处理，得到结果用M统计做的组间差异分析。
图1显示了差异的自身抗体的聚类分析图，从中可以看到抗N-甲基-D-天冬氨酸受体(NMDAR)抗体在先天性巨结肠患儿血浆中显著高于对照组(p＝0.018)。
图2显示了差异的自身抗体的聚类分析图，从中可以看到抗单链DNA(ssDNA)抗体在先天性巨结肠患儿血浆中显著高于对照组(p＝0.019)。
图3显示了差异的自身抗体的聚类分析图，从中可以看到抗双链DNA(dsDNA)抗体在先天性巨结肠患儿血浆中显著高于对照组(p＝0.033)。
图4显示了差异的自身抗体的聚类分析图，从中可以看到抗SSA抗体在先天性巨结肠患儿血浆中显著高于对照组(p＝0.046)。
图5显示了差异的自身抗体的聚类分析图，从中可以看到抗nRNP/Sm抗体在先天性巨结肠患儿血浆中显著高于对照组(p＝0.003)。
实施例3.先天性巨结肠病变组织和血浆中抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体检测以及ROC曲线分 析
N-甲基-D-天冬氨酸受体(NMDAR)为神经组织特异性表达蛋白，该蛋白的自身抗体在脑炎中有报道，但在外周的肠神经系统疾病中没有报道过。为了验证抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体确实来源于巨结肠病人病变段的肠神经组织，收集巨结肠病人病变段的肠道组织(HSCR，36例)，以及其它肠道疾病结肠组织(DC，包括肛门狭窄和肠狭窄造瘘的结肠组织共11例)，用酶联免疫(ELISA)检测组织中抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体的水平，检测试剂盒为人N-甲基-D-天冬氨酸受体(NMDAR)抗体酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。图6所示结果发现，先天性巨结肠病变组织中N-甲基-D-天冬氨酸受体(NMDAR)的自身抗体水平显著高于其它肠病的结肠组织，此结果证明先天性巨结肠组织中确实有较高的抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体。
为了验证抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体对先天性巨结肠的诊断效果，我们收集来源于先天性巨结肠病人(HSCR，37例)、其它肠疾病对照(DC，包括肛门狭窄和肠狭窄造瘘儿童的血浆样品共18例)、健康儿童对照(HC，30例)的血浆，用酶联免疫(ELISA)检测血浆中抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体的水平，检测试剂盒为人N-甲基D-天冬氨酸型受体抗体(NMDAR-Ab)酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。结果如图7显示，先天性巨结肠病人的血浆中抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体显著高于肠疾病对照组(p<0.05)和健康儿童对照组(p<0.01)，也高于肠疾病与健康儿童合并的对照组(p<0.01)。
图8显示ROC曲线用于评价抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体对先天性巨结肠的诊断效果。AUC值为0.9046，最佳界限对应灵敏性为93.75％，特异性为81.58％。用于先天性巨结肠病筛选时，灵敏性为100％时，特异性为78.95％。由此可见，抗N-甲基-D-天冬氨酸受体(NMDAR)自身抗体可以有效诊断先天性巨结肠，对先天性巨结肠的筛选，表现出很好的效果。
实施例4.先天性巨结肠血浆中抗单链DNA(ssDNA)抗体检测以及ROC曲线分析
为了验证抗单链DNA(ssDNA)抗体对先天性巨结肠的诊断效果，我们收集来源于巨结肠病人(HSCR，37例)、其它肠疾病对照(DC，包括肛门狭窄和肠狭窄造瘘儿童的血浆样品共18例)、健康儿童对照(HC，30例)的血浆，用酶联免疫(ELISA)检测血浆中抗单链DNA(ssDNA)抗体的水平，检测试剂盒为人抗单链DNA(ssDNA)抗体(ssDNA-Ab)酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。结果如图9显示巨结肠病人的血浆中抗单链DNA(ssDNA)抗体显著高于肠疾病对照组(p<0.01)和健康儿童对照组(p<0.01)，也高于肠疾病与健康儿童合并的对照组(p<0.01)。
图10显示ROC曲线用于评价抗单链DNA(ssDNA)抗体对先天性巨结肠的诊断效果，AUC为0.9167；最佳界限对应灵敏性为74.63％，特异性为96.88％。由此可见，抗单链DNA(ssDNA)抗体可以有效诊断先天性巨结肠。
实施例5.先天性巨结肠病变组织和血浆中抗双链DNA(dsDNA)抗体检测以及ROC曲线分析
双链DNA(dsDNA)为神经组织特异性表达蛋白，该蛋白的自身抗体在脑炎中有报道，但在外周的肠神经系统疾病中没有报道过。为了验证抗双链DNA(dsDNA)抗体确实来源于巨结肠病人病变段的肠神经组织，收集巨 结肠病人病变段的肠道组织(HSCR，36例)，以及其它肠道疾病结肠组织(DC，包括肛门狭窄和肠狭窄造瘘的结肠组织共11例)，用酶联免疫(ELISA)检测组织中抗双链DNA(dsDNA)抗体的水平，检测试剂盒为人抗双链DNA(dsDNA)抗体酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。图11所示结果发现，先天性巨结肠病变组织中抗双链DNA(dsDNA)抗体水平显著高于其它肠病的结肠组织(p<0.01)，此结果证明先天性巨结肠组织中确实有较高的抗双链DNA(dsDNA)抗体。
为了验证抗双链DNA(dsDNA)抗体对先天性巨结肠的诊断效果，我们收集来源于巨结肠病人(HSCR，37例)、其它肠疾病对照(DC，包括肛门狭窄和肠狭窄造瘘儿童的血浆样品共18例)、健康儿童对照(HC，30例)的血浆，用酶联免疫(ELISA)检测血浆中抗双链DNA(dsDNA)抗体的水平，检测试剂盒为人抗双链DNA(dsDNA)抗体(dsDNA-Ab)酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。结果如图12显示巨结肠病人的血浆中抗双链DNA(dsDNA)抗体显著高于健康儿童对照组(p<0.01)，与肠疾病对照组无显著差异，也高于肠疾病与健康儿童合并的对照组(p<0.01)。
图13显示ROC曲线用于评价抗双链DNA(dsDNA)抗体对先天性巨结肠的诊断效果。AUC值为0.8313，最佳界限对应灵敏性为96.88％，特异性为69.23％。由此可见，抗双链DNA(dsDNA)抗体可以有效诊断先天性巨结肠。
实施例6.先天性巨结肠病变血浆中抗SSA抗体检测以及ROC曲线分析
为了验证抗SSA抗体对先天性巨结肠的诊断效果，我们收集来源于巨结肠病人(HSCR，37例)、其它肠疾病对照(DC，包括肛门狭窄和肠狭窄造瘘儿童的血浆样品共18例)、健康儿童对照(HC，30例)的血浆，用酶联免疫(ELISA)检测血浆中抗SSA抗体的水平，检测试剂盒为人抗SSA抗体(SSA-Ab)酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。结果如图14显示巨结肠病人的血浆中抗SSA抗体显著高于健康儿童对照组(p<0.01)，与肠疾病对照组无差异，也高于肠疾病与健康儿童合并的对照组(p<0.01)。
图15显示ROC曲线用于评价抗SSA抗体对先天性巨结肠的诊断效果。AUC为0.7756；最佳界限对应灵敏性为62.50％，特异性为74.36％。由此可见，抗SSA抗体可以有效诊断先天性巨结肠。
实施例7.先天性巨结肠病变血浆中抗nRNP/Sm抗体检测以及ROC曲线分析
为了验证抗nRNP/Sm抗体对先天性巨结肠的诊断效果，我们收集来源于巨结肠病人(HSCR，37例)、其它肠疾病对照(DC，包括肛门狭窄和肠狭窄造瘘儿童的血浆样品共18例)、健康儿童对照(HC，30例)的血浆，用酶联免疫(ELISA)检测血浆中抗nRNP/Sm抗体的水平，检测试剂盒为人抗nRNP/Sm抗体(nRNP/Sm-Ab)酶联免疫(ELISA)试剂盒(上海臻科生物科技有限公司)。结果如图16显示巨结肠病人的血浆中抗nRNP/Sm抗体显著高于健康儿童对照组(p<0.01)，与肠疾病对照组无差异，也高于肠疾病与健康儿童合并的对照组(p<0.01)。
图17显示ROC曲线用于评价抗nRNP/Sm抗体对先天性巨结肠的诊断效果。AUC值为0.8165，最佳界限对应灵敏性为100％，特异性为64.10％由此可见，抗nRNP/Sm抗体可以有效诊断先天性巨结肠。
实施例8.联合ROC曲线分析
进一步检测抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的组 合，联合应用对先天性巨结肠的诊断效果。将前述收集的来源于巨结肠病人、其它肠疾病对照、健康儿童对照的血浆，用酶联免疫(ELISA)检测血浆中的抗体水平。
结果如图18显示用逻辑回归的方法评价联合NMDAR和ssDNA指标诊断先天性巨结肠的效果，结果显示NMDAR和ssDNA联合诊断的效果好于两个单独指标，产生了协同作用。AUC为0.9351；最佳界限对应灵敏性为82.05％，特异性为93.75％。由此可见，NMDAR和ssDNA抗体联合可以更好的诊断先天性巨结肠。此外，用同批数据和同样的逻辑回归的方法评价其他联合抗体指标诊断先天性巨结肠的效果，结果显示：AUC＞0.9167的组合还包括：1.抗单链DNA抗体、抗双链DNA抗体组合；2.抗单链DNA抗体、抗SSA抗体组合；3.抗单链DNA抗体、抗nRNP/Sm抗体组合；4.抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体组合；5.NMDAR抗体、抗单链DNA抗体、抗SSA抗体组合；6.NMDAR抗体、抗单链DNA抗体、抗nRNP/Sm抗体组合；7.抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体组合；8.抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体组合；9.抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；10.NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体组合；11.NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗nRNP/Sm抗体组合；12.NMDAR抗体、抗单链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；13.抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体组合；14.抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体的组合。其中，五个标志物组合的AUC也达到了0.93以上。可见，上述1～14的组合均显示出了高于它们单个指标中的最高AUC值(0.9167)的预料不到的协同作用。而其余组合(包括其余的选自抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的两个、三个、四个、五个指标的组合，例如，抗NMDAR抗体+抗dsDNA抗体；抗NMDAR抗体+抗SSA抗体；抗NMDAR抗体+抗nRNP/Sm抗体；抗dsDNA抗体+抗SSA抗体；抗dsDNA抗体+抗nRNP/Sm抗体；抗SSA抗体+抗nRNP/Sm抗体；抗NMDAR抗体+抗dsDNA抗体+抗SSA抗体；抗NMDAR抗体+抗dsDNA抗体+抗nRNP/Sm抗体；抗NMDAR抗体+抗SSA抗体+抗nRNP/Sm抗体；抗dsDNA抗体+抗SSA抗体+抗nRNP/Sm抗体；抗NMDAR抗体+抗dsDNA抗体+抗SSA抗体+抗nRNPSm抗体)的AUC也均达到了0.79-0.88，也均可有效地用于先天性巨结肠的诊断。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (11)
- 一种先天性巨结肠的诊断标志物组合，其特征在于，所述诊断标志物组合包含选自抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少两种、至少三种、至少四种或五种抗体；优选的，所述抗体为自身抗体。
- 根据权利要求1所述的诊断标志物组合，其特征在于，所述诊断标志物组合包含抗NMDAR抗体，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种、至少两种、至少三种抗体或四种抗体；优选的，所述抗体为自身抗体。
- 一种先天性巨结肠的诊断产品，所述产品包含检测剂，所述检测剂包含：抗NMDAR抗体、抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体、抗nRNP/Sm抗体中的至少一种、至少两种、至少三种、至少四种或五种抗体的检测剂；优选的，所述抗体为自身抗体。
- 根据权利要求3所述的诊断产品，其特征在于，所述检测剂包含抗NMDAR抗体的检测剂，以及抗单链DNA抗体、抗双链DNA抗体、抗SSA抗体和抗nRNP/Sm抗体中的至少一种、至少二种、至少三种或四种的检测剂；优选的，所述抗体为自身抗体。
- 根据权利要求3或4所述的诊断产品，其特征在于，所述抗NMDAR抗体是抗NMDAR的IgG抗体；所述抗单链DNA抗体为抗单链DNA的IgG抗体；所述抗双链DNA抗体为抗双链DNA的IgG抗体；所述抗SSA抗体为抗SSA的IgG抗体；所述抗nRNP/Sm抗体为抗nRNP/Sm的IgG抗体。
- 根据权利要求3或4所述的诊断产品，其特征在于，所述检测剂用于执行如下任一方法：放射免疫法、间接免疫荧光法、斑点免疫金渗滤法、生物质谱法、免疫印记法和酶联免疫吸附法。
- 根据权利要求3或4所述的诊断产品，其特征在于，所述检测剂包括抗原，其中，抗NMDAR抗体的抗原是NMDAR蛋白；抗单链DNA抗体的抗原是ssDNA；抗双链DNA抗体的抗原是dsDNA；抗SSA抗体的抗原是SSA蛋白；抗nRNP/Sm抗体的抗原是nRNP/Sm蛋白，所述nRNP/Sm蛋白选自nRNP、Sm蛋白、以及Sm蛋白和nRNP的复合物。
- 根据权利要求7所述的诊断产品，其特征在于，所述抗原缀合于固相支持物上；优选的，所述固相支持物选自试管、EP管、多孔板、微量反应板凹孔和微球。
- 根据权利要求3-8任一所述的诊断产品，其特征在于，所述检测剂还包括抗人Ig抗体；优选的，所述抗人Ig抗体为抗人IgG抗体；更进一步优选的，所述抗人Ig抗体缀合有信号物质。
- 根据权利要求3-9任一所述的诊断产品，其特征在于，所述检测剂所适用的检测样本为血液、血浆、血清、组织、细胞、组织或细胞裂解物样品中的至少一种；优选为血液、血浆、血清中的至少一种；或优选为肠道组织、肠道细胞或肠道细胞裂解物中的至少一种。
- 权利要求3-10所述的诊断产品在制备先天性巨结肠诊断试剂、试剂盒或试纸条中的应用。
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| CN202110336372.1A CN113092752B (zh) | 2021-03-29 | 2021-03-29 | 抗ssa自身抗体作为先天性巨结肠诊断标志物的应用 |
| CN202110336409.0 | 2021-03-29 | ||
| CN202110336372.1 | 2021-03-29 | ||
| CN202110336404.8A CN113075399B (zh) | 2021-03-29 | 2021-03-29 | 抗单链dna抗体作为先天性巨结肠诊断标志物的应用 |
| CN202110336410.3 | 2021-03-29 | ||
| CN202110336409.0A CN113092779B (zh) | 2021-03-29 | 2021-03-29 | 抗nmdar自身抗体作为先天性巨结肠诊断标志物的应用 |
| CN202110333807.7A CN113075410B (zh) | 2021-03-29 | 2021-03-29 | 抗nRNP/Sm抗体作为先天性巨结肠诊断标志物的应用 |
| CN202110333807.7 | 2021-03-29 | ||
| CN202110336404.8 | 2021-03-29 | ||
| CN202110336410.3A CN113075400B (zh) | 2021-03-29 | 2021-03-29 | 抗双链dna抗体作为先天性巨结肠诊断标志物的应用 |
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