CN113075400B - Application of anti-double-stranded DNA antibody as diagnosis marker of Hirschmannin - Google Patents
Application of anti-double-stranded DNA antibody as diagnosis marker of Hirschmannin Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to the technical field of biomedicine, in particular to application of an anti-double-chain DNA antibody as a diagnosis marker of Hirschsprung's disease. Screening samples of the infant congenital megacolon through a human self-immune antigen chip to obtain an anti-double-stranded DNA (dsDNA) antibody with high plasma expression of the infant congenital megacolon. And the effectiveness of anti-double stranded DNA (dsDNA) antibodies was verified in separate samples by enzyme-linked immunosorbent assay (ELISA). The double-stranded DNA (dsDNA) resistant antibody can effectively diagnose the infant with the congenital megacolon, and the AUC is 0.9167; the optimal limit corresponds to a sensitivity of 74.63% and a specificity of 96.88%. The anti-double-stranded DNA (dsDNA) antibody can be used as a diagnostic marker of the plasma of the Hirschsprung's disease for screening and diagnosing diseases, and fills the blank of diagnosing the plasma of the Hirschsprung's disease.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of an anti-double-chain DNA antibody as a diagnosis marker of Hirschsprung's disease.
Background
Hirschsprung disease (HSCR) is a birth defect disease of infantile enteric nerve dysplasia, and the pathological mechanism is that cells at the intestinal neural crest migrate and differentiate into enteric neurons to generate obstacle, so that the enteric nerve is deficient to generate persistent spasm, which is one of the common congenital intestinal tract diseases of infants. The early symptoms of the congenital megacolon include vomit, abdominal distension, diarrhea and the like, which can cause death of the newborn or complications such as repeated enteritis after operation, intractable constipation and the like clinically, and seriously affect the growth and the development and the life quality of the sick children.
The timely diagnosis and treatment of the congenital megacolon can reduce the risk of the congenital megacolon enteritis and obtain good prognosis. The diagnosis of the disease requires pathological sections of the diseased tissue after surgery. The preoperative diagnosis method mainly comprises barium enema, rectal biopsy and rectal manometry so as to judge whether 'megacolon radical operation' needs to be implemented. Currently, barium enema is the most important diagnostic method, and the principle is that no nerve segment stenosis and proximal dilatation exist in the intestinal tract of a child suffering from congenital megacolon, and the colon is diagnosed as megacolon by observing dilatation and stenosis after barium enema. However, the method can only diagnose children patients with typical intestinal tract shape changes, the sensitivity needs to be improved, and the diagnosis accuracy is about 80%. Rectal biopsy is to directly take the rectal tissue to detect whether the ganglion cells are deleted, the accuracy is high, but the sampling part has influence on the result. The method is invasive and very expensive, and is generally not readily applicable to infants who have a barium enema which is not obvious or is not suitable, for example, when Necrotizing Enterocolitis (NEC) is a possibility, the barium enema may cause intestinal perforation, and the barium enema is not suitable for diagnosis, and rectal biopsy is considered. Rectal manometry is to determine the innervation abnormality of the intestinal nerves by detecting the lack of relaxation of internal anal sphincter, is only an auxiliary diagnosis method, has more false positives and false negatives, and cannot be used for single detection.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an anti-double-stranded DNA (dsDNA) antibody of a diagnosis marker of the Hirschsprung's disease and an application thereof, and provides a new, accurate and sensitive detection way for the diagnosis of the Hirschsprung's disease.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention relates to an application of a quantitative detection agent of an anti-double-stranded DNA antibody in preparation of a congenital megacolon diagnostic reagent, a kit or a test strip.
Alternatively, for use as described above, the anti-double stranded DNA antibody is an anti-double stranded DNA IgG antibody.
Optionally, for use as described above, the quantitative detection agent is for performing any one of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
Alternatively, for use as described above, the quantitative detection agent is dsDNA.
Alternatively, the dsDNA is conjugated to a solid support, for use as described above.
Alternatively, the solid support is selected from the group consisting of test tubes, EP tubes, multiwell plates, microplate wells and microspheres, for use as described above.
Optionally, for use as described above, the quantitative detection agent further comprises an anti-human Ig antibody.
Alternatively, for use as described above, the anti-human Ig antibody is an anti-human IgG antibody.
Alternatively, the anti-human Ig antibody is conjugated with a signal agent for use as described above.
Optionally, in the above-mentioned application, the sample to be tested detected by the quantitative detection agent is at least one of a blood, plasma, serum, tissue, cell, tissue or cell lysate sample.
Compared with the prior art, the invention has the beneficial effects that:
the invention finds that dsDNA has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of children congenital diseases, and overcomes the defect of no plasma diagnosis in the prior art. The method has good diagnostic sensitivity and specificity, the AUC value is 0.8313, the optimal limit corresponds to the sensitivity of 96.88% and the specificity of 69.23%, and an effective method is provided for clinically diagnosing the congenital megacolon diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram of an example of the present invention of a human autoimmune antigen chip for screening diagnostic markers in the plasma of a child suffering from Hirschsprung's disease;
FIG. 2 is a graph showing enzyme-linked immuno sorbent assay (ELISA) detection of anti-double stranded DNA (dsDNA) antibodies in colon tissue of a child with congenital megacolon over other bowel diseases in one embodiment of the invention;
FIG. 3 is a graph showing the enzyme-linked immuno sorbent assay (ELISA) detection of anti-double stranded DNA (dsDNA) antibodies in one embodiment of the invention at a level in infants with congenital megacolon that is higher than in a control group of healthy children;
FIG. 4 is a graph showing the diagnostic value of ROC curve analysis of anti-double stranded DNA (dsDNA) antibodies in the native megacolon, in one embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention relates to an application of a quantitative detection agent of an anti-double-stranded DNA antibody in preparation of a congenital megacolon diagnostic reagent, a kit or a test strip.
The invention proves that the anti-double-stranded DNA (dsDNA) antibody can be used as a diagnostic marker of the Hirschsprung's disease and fills the blank in the field of plasma diagnosis of the Hirschsprung's disease. The technology has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of the congenital diseases of children, and overcomes the defect of no plasma diagnosis in the prior art. The method has good diagnostic sensitivity and specificity, the AUC value is 0.8313, the optimal limit corresponds to the sensitivity of 96.88% and the specificity of 69.23%, and an effective method is provided for clinically diagnosing the congenital megacolon diseases.
The term "marker" or "biochemical marker" as used herein refers to a molecule to be used as a target for analyzing a patient test sample.
In some embodiments, the anti-double stranded DNA antibody is an anti-double stranded DNA IgG antibody.
In the determination method according to the present invention, the method of analyzing the expression of the autoantibody against the double-stranded DNA is not particularly limited. For example, the absolute amount or concentration of these autoantibodies in a blood sample is not limited to measurement, and a relative amount or concentration may be measured. More specifically, for example, the amount, concentration or activity of the autoantibody or the like in a blood sample can be measured. Such methods are well known in the art, and by way of example, in some embodiments, the quantitative detection agent is used to perform any of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, biological mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
Examples of the above-mentioned detection method include a time-of-flight MASS spectrometry (TOF-MASS) 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). The concentration or amount of autoantibodies can be grasped from the TOF-MASS chart by the molecular weight peak, other fragment peaks, their intensities, and the like. In addition, as in the case of ELISA, autoantibodies that selectively bind to the protein chip can also be detected using a secondary antibody that has a labeling group and can bind to the respective antibodies.
Among these methods, immunoassay is preferable as a method for performing the expression analysis. The immunoassay method has high sensitivity and accuracy, and can detect a slight change in the concentration of autoantibodies in blood. When the expression of the autoantibody according to the present invention is analyzed by enzyme-linked immunosorbent assay (ELISA), the diagnostic kit for colons congenital megacolon according to the present invention can be suitably used.
In some embodiments, the quantitative detection agent is dsDNA.
An autoantigen fragment comprising an epitope recognized by an autoantibody can be at least 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, or 1000 nucleotides in length. The fragment can 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 dsDNA.
In some embodiments, the dsDNA is conjugated to a solid support.
In some embodiments, the solid support is selected from the group consisting of a test tube, an EP tube, a multi-well plate, a microplate well, and a microsphere.
The term "solid support" means that the carrier material is predominantly non-liquid-resistant, thereby allowing accurate and traceable localization of nucleic acids on the carrier material. The solid support can be selected from polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel, etc. The preferred solid support is an enzyme label plate. It may contain 16, 32, 48, 64, 96 or more holes.
In the present invention, the term "microsphere" may be a sphere, a nearly sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400nm to 10 μm.
The microspheres have specific binding properties for the substance of interest (target or analyte) to be assayed on their surface.
The microspheres are preferably magnetic beads, and the magnetic material is contained in the composition. The magnetic substance may be a metal (simple metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe)2O3Or Fe3O4Magnetic nanoparticles); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.
The surface of the microsphere is modified with one or moreActive functional group including-OH, -COOH, -NH2-CHO, and-SO3H. In some embodiments, the coated antigen and antibody are conjugated or bound to the microsphere by physisorption or direct chemical conjugation (e.g., bridging by a bridge). In particular, suitable techniques for constructing the bridge include, for example, covalent attachment, adsorption, non-covalent interactions, or combinations thereof. In some embodiments, direct bridging may be achieved by glutaraldehyde fixation, N-hydroxysuccinimide (NHS) chemistry, or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) NHS chemistry. Suitable means for indirect bridging include, for example, bridging via a peptide, protein, antibody, linker, or a combination thereof. In some embodiments, the indirect bridging to the solid support is via streptavidin and biotin.
In some embodiments, the quantitative detection agent further comprises an anti-human Ig antibody.
In some embodiments, the anti-human Ig antibody is an anti-human IgG antibody.
Anti-human Ig antibodies are antibodies directed against human Ig proteins, and anti-human IgG antibodies are antibodies directed against human IgG proteins.
In some embodiments, the anti-human Ig antibody is conjugated to a signal agent.
In other embodiments, the anti-human Ig antibody is not labeled with a signal substance, and the quantitative detection agent further comprises a second antibody against the anti-human Ig antibody labeled with a signal substance. After substances that bind non-specifically to double-stranded DNA or the like are washed and removed with a buffer solution or the like, the secondary antibody is allowed to act. The secondary antibody binds to the autoantibody or the like bound to the peptide or the like. The secondary antibody is detected by a method corresponding to the signal substance.
In the present invention, the antibody used as a quantitative detection agent may be in the form of an IgA, IgD, IgG, IgE or IgM isotype or a single domain, such as a single domain antibody from a camelid. In some embodiments, 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, variant or equivalent thereof.
In the present invention, the signal substance is a substance capable of providing a signal to be detected, and in some embodiments, the signal substance is independently selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme. The following non-limiting section lists these markers:
enzymes which produce a detectable signal, e.g.by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescent, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
Groups with electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltage measurement and resistance.
A detectable group, e.g., a molecule of sufficient size to induce a detectable modification in its physical and/or chemical properties; such detection can be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic spectroscopy and tunneling).
Electron-dense substances, e.g. radioactive molecules (e.g.32P，35S or125I)。
In some embodiments, the signal species is an Acridinium Ester (AE).
Further, acridine chemiluminescent substances include acridinium esters and acridinium sulfonamides.
Further, the acridine chemiluminescent substance includes acridine ester AE-NHS, acridine ester DMAE-NHS, acridine ester Me-DMAE-NHS, acridine ester NSP-DMAE-NHS, acridine salt NSP-SA-NHS, acridine hydrazide NSP-SA-ADH, etc.
Any biological sample containing autoantibodies may be used as the sample to be detected, including, but not limited to, serum, plasma, whole blood, saliva, urine, semen, sweat, tears, and body tissue. In some preferred embodiments, the sample to be tested for the quantitative detection agent is at least one sample of blood, plasma, serum, tissue, cells, tissue or cell lysate.
As used herein, "tissue or cell lysate" may also be used in common with the terms "lysate", "lysed sample", "tissue or cell extract", and the like, to denote a sample and/or biological sample material comprising lysed tissue or cells, i.e. where the structural integrity of the tissue or cells has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining lysates. This process is encompassed by the term "lysis".
The diagnostic kit of the present invention preferably contains a normal control sample and a colonscheim control sample. When these samples are attached to the kit, the presence or absence of the congenital megacolon of the subject can be determined more objectively by performing the same experiment on these samples and comparing the measurement values with the results of the test sample.
The concentration or amount of autoantibodies contained in the sample is indirectly obtained by the intensity of color development or the like. The obtained measurement values can be converted into relative or absolute concentrations, amounts, activities, and the like by a calibration curve or the like.
The invention also relates to a method for diagnosing Hirschsprung's disease, comprising the quantitative detection of anti-double-stranded DNA antibodies in a sample to be examined, wherein an elevated level of anti-double-stranded DNA antibodies is indicative for the Hirschsprung's disease.
The term "indicative" when used in the context of autoantibodies for use with embodiments of the present invention includes autoantibodies whose presence or absence is determined by embodiments of the present invention as typically present in subjects having Hirschsprung's disease. By "normally present" is meant that the autoantibody is often associated with the congenital megacolon. "frequently relevant" includes a probability of more than 50%, preferably more than 60%, more preferably more than 70%, even more preferably more than 80% and particularly preferably more than 90% or 95%.
An ideal scenario for diagnosis is a situation where a single event or process may cause 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. As the skilled artisan will appreciate, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Conversely, biochemical markers can be used to assess, for example, the presence or absence or severity of a disease with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.
In the method of the present invention, the presence or absence of the onset of Hirschsprung's disease is then determined from the results of the expression analysis obtained. That is, the more severe the onset of the congenital megacolon or the symptoms thereof, the higher the concentration or amount of the autoantibody according to the present invention in blood. Thus, based on the results of expression analysis of the autoantibody according to the present invention, a positive antibody can be determined when the expression level is high, and a negative antibody can be determined when the expression level is low.
In fact, the boundary between positive and negative, i.e., cutoff value, can be varied according to the definition and severity of the Hirschsprung's disease, the method of analysis of the expression of the autoantibodies to which the present invention relates. Therefore, at a stage where there is no general reference, it is necessary for the practitioner of the method of the present invention to determine the expression analysis method and the cutoff value in advance by preliminary experiments or the like and then perform the measurement.
In some embodiments, the subject is a patient under 18, e.g., 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year old. Or in infants, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months of age.
Embodiments of the present invention will be described in detail below with reference to examples.
The samples adopted by the invention are all from Guangzhou city woman child medical center, and all the experimental tissues and blood samples are collected by ethical committee authorization and patient consent of Guangzhou city woman child medical center.
The test results of the invention are all analyzed by statistics, t test is used for evaluating the difference between two groups, p is less than 0.05 for representing statistical significance, and two-sided test is used for all p values. Statistical analysis was performed using R and graphpad8.0 software.
The congenital megacolon disease is one of the common congenital intestinal diseases of children, wherein the congenital megacolon disease is clinically divided into a short segment type, a common type, a long segment type and a full colon type according to increasing severity degrees, and the congenital megacolon disease is characterized in that the colon is lack of ganglion cells to cause the continuous spasm of an intestinal canal, feces are stagnated in the proximal colon, and the proximal colon is thickened and dilated. The short-section lesions are positioned at the near and middle sections of the rectum and are not more than 6.5cm away from the anal canal; the common lesions were located at the proximal rectum or distal rectosigmoid, approximately 9cm from the anal canal; long segment lesions extend to the sigmoid or descending colon; the colon-wide lesion reaches the whole colon and the tail end of the ileum within 30cm from the ileocecal valve.
The autoantibodies of the present invention are antibodies that erroneously target and damage a specific tissue or organ of the body.
The dsDNA of the invention refers to double-stranded DNA in cell nucleus; anti-double stranded DNA (dsdna) antibodies refer to antibodies, preferably IgG antibodies, raised against double stranded DNA in the nucleus of a cell.
The "ROC curve" of the present invention is a curve of 1-specificity (false positive rate) and sensitivity (true positive rate) changes, reflecting the diagnostic capabilities of the classifier. A good classifier has a ratio of change of true positive rate to false positive rate of greater than 1, far from a 45 degree line.
The "AUC" refers to the area under the ROC curve, is between 0.1 and 1, and is used for evaluating the quality of the classifier, and the closer to 1, the better the classifier is.
The invention screens the plasma of the infant congenital megacolon through a human self-immune antigen chip, and uses the plasma of other intestinal diseases and healthy group infants as a contrast to obtain the anti-double-chain DNA (dsDNA) antibody with high expression of the plasma of the infant congenital megacolon. And the further effect is verified by an enzyme-linked immunosorbent assay (ELISA) method in an independent sample.
Example 1 plasma and tissue sample Collection and grouping
Plasma samples were divided into the hirschsprung child group (37 cases), the other enteropathy control group (18 cases), the healthy child group (30 cases), the age ranged from 3 months to 3 years, the gender male 3/4 was male, and the age and gender of the disease and control groups were matched. All samples were from the Guangzhou city women's Children medical center, and the healthy children group had blood samples left after physical examination. The blood sampling mode is anticoagulation blood sampling, centrifugal separation of blood plasma and freezing storage of samples. The colon tissue sample was a group of infant patients with congenital megacolon (36 cases), which was a lesion tissue excised by surgery. Other intestinal disease controls (total of 11 colonic tissues including anal stenosis and intestinal stenosis fistulization).
Example 2 screening of human Automation antigen chips and analysis of differentially expressed autoantibodies
Plasma of 5 children with megacolon congenitum (HSCR), 5 of each of the plasma of children with Healthy (HC) and other intestinal Diseases (DC), were screened against a human autoimmune antigen chip provided by cantonese biotechnology limited, guangzhou, which contained lgG detection of over 100 autoimmune antibodies, and the raw data, after subtraction of negative controls, were normalized by RLM method to obtain results for inter-group difference analysis using M statistics, and fig. 1 shows a cluster analysis graph of the different autoantibodies, from which it can be seen that double-stranded dna (dsdna) was significantly higher in plasma of children with megacolon congenitally than in the control group (p 0.033).
Example 3 anti-double stranded DNA (dsDNA) antibody detection in Hirschsprung's lesion tissue
Double-stranded dna (dsdna) is a neural tissue-specific expression protein whose autoantibodies are reported in encephalitis, but not in peripheral enteric nervous system disease. In order to verify that the anti-dsDNA (dsDNA) antibody is indeed derived from the enteric nerve tissue of the diseased section of the megacolon patient, the intestinal tissue of the diseased section of the megacolon patient (36 cases) and other intestinal diseased colon tissues (11 cases including colon tissue of anal stenosis and intestinal stenosis fistulization) were collected, and the level of the anti-dsDNA (dsDNA) antibody in the tissues was detected by enzyme-linked immunosorbent assay (ELISA), wherein the detection kit was a human anti-dsDNA (dsDNA) antibody enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Zheke science and technology, Inc.). The results shown in fig. 2 found that the level of anti-double-stranded dna (dsdna) antibodies in the colonscheit diseased tissue was significantly higher than in the colon tissue of other intestinal diseases (p <0.01), which demonstrated that there were indeed higher anti-double-stranded dna (dsdna) antibodies in the colonscheit tissue.
Example 4 enzyme-linked immunosorbent assay (ELISA) detection of anti-double-stranded DNA (dsDNA) antibodies in plasma
To verify the diagnostic effect of anti-dsDNA antibodies on hirschsprung, plasma from hiragana patients (37 cases), other intestinal disease controls (including 18 plasma samples from children with anal and intestinal stenosis fistulization), and healthy children controls (30 cases) were collected and the level of anti-dsDNA (dsDNA) antibodies in the plasma was measured by enzyme linked immunosorbent assay (ELISA), which was human anti-dsDNA (dsDNA) antibody (dsDNA-Ab) enzyme linked immunosorbent assay (ELISA) kit (shanghai zhencology co). The results are shown in figure 3, where anti-double stranded dna (dsdna) antibodies in the plasma of megacolon patients were significantly higher than the healthy children control group (p <0.01), not significantly different from the bowel disease control group, and also higher than the control group with bowel disease and healthy children combined (p < 0.01).
Example 5 ROC Curve analysis
FIG. 4 shows the ROC curve for evaluating the diagnostic effect of anti-double stranded DNA (dsDNA) antibodies on Hirschsprung's colon. AUC values were 0.8313, with the best limit corresponding to a sensitivity of 96.88% and a specificity of 69.23%. It follows that anti-double stranded dna (dsdna) antibodies are effective in diagnosing the congenital megacolon.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (9)
1. The application of the quantitative detection agent of the anti-double-stranded DNA autoantibody in the preparation of a diagnostic reagent, a kit or a test strip for Hirschsprung's disease.
2. The use of claim 1, wherein the anti-dsDNA autoantibody is an anti-dsDNA IgG antibody.
3. The use of claim 1, wherein the quantitative detection agent is used to perform any one of the following methods:
radioimmunoassay, indirect immunofluorescence, dot immunogold filtration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.
4. The use of claim 1, wherein the quantitative detection agent comprises dsDNA.
5. The use of claim 4, wherein the dsDNA is conjugated to a solid support.
6. The use according to claim 5, wherein the solid support is selected from the group consisting of test tubes, EP tubes, multi-well plates, wells of a microplate, and microspheres.
7. The use of any one of claims 4 to 6, wherein the quantitative detection agent further comprises an anti-human IgG antibody.
8. The use of claim 7, wherein said anti-human IgG antibody is conjugated to a signal agent.
9. The use of any one of claims 1 to 6 and 8, wherein the sample to be tested for the quantitative detection agent is at least one of blood, plasma, serum, tissue, cell, tissue lysate sample or cell lysate sample.
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