CN113092752B - Application of anti-SSA autoantibody as congenital megacolon diagnosis marker - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to application of an anti-SSA autoantibody as a congenital megacolon diagnosis marker. The invention discovers that the anti-SSA autoantibody has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of congenital diseases of children, and fills the blank of the diagnosis of congenital megacolon plasma. The diagnostic sensitivity and specificity of the method are good, and the AUC is 0.7756; the optimal limit corresponds to sensitivity of 62.50% and specificity of 74.36%, and overcomes the defect of no plasma diagnosis in the prior art.

Description

Application of anti-SSA autoantibody as congenital megacolon diagnosis marker

Technical Field

The invention relates to the technical field of biological medicine, in particular to application of an anti-SSA autoantibody as a congenital megacolon diagnosis marker.

Background

Congenital megacolon (Hirschsprung disease, HSCR) is a birth defect disease of children with abnormal enteric neural development, the pathological mechanism is that enteric neural crest cells migrate and differentiate into enteric neurons to generate disorder, and the enteric neural deficiency is caused to generate persistent spasm, so that the congenital megacolon is one of common congenital intestinal diseases of children. The early stages of the congenital megacolon are marked by vomiting, abdominal distension, diarrhea and the like, which clinically cause complications such as neonatal death or repeated enteritis after operation, refractory constipation and the like, and seriously influence the growth and the quality of life of the infant.

Timely diagnosis and treatment of the congenital megacolon can reduce the occurrence risk of the congenital megacolon enteritis and obtain good prognosis. The diagnosis of the disease requires pathological sections of the postoperative lesion tissue. The preoperative diagnostic method is mainly barium enema, rectal biopsy and rectal pressure measurement to judge whether to implement 'megacolon radical operation'. At present, barium enema is the most important diagnosis method, and the principle is that the intestinal tract of a congenital megacolon infant has a stenosis without nerve segments and a proximal expansion, and the barium enema can be used for diagnosing the megacolon by visible expansion and stenosis. However, the method can only diagnose the infant with typical intestinal morphology change, the sensitivity needs to be improved, and the accuracy of diagnosis is about 80%. The rectal biopsy is to directly take out the intestinal tissue and detect whether ganglion cells are missing, so that the accuracy is high, but the sampling position has influence on the result. The method is invasive, and is very expensive, and is generally not obvious when applied to infants suffering from barium enema or inapplicable, such as when infants suffer from Necrotizing Enterocolitis (NEC), the barium enema can lead to intestinal perforation, and the method is not suitable for diagnosis by the barium enema, so that rectal biopsy is considered. Rectal manometry is a method for judging the abnormal innervation of intestinal nerves by detecting the lack of relaxation of the sphincter ani, and is only an auxiliary diagnosis method, and has more false positives and false negatives, and cannot be used for independent detection.

In view of this, the present invention has been made.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide an anti-SSA antibody of a congenital megacolon diagnosis marker and application thereof, and provides a new accurate and sensitive detection way for diagnosis of the congenital megacolon.

In order to achieve the above purpose, the present invention is realized by the following technical scheme.

The invention relates to an application of a quantitative detection agent of an anti-SSA autoantibody in preparing a congenital megacolon diagnosis reagent, a kit or a test strip.

Alternatively, the anti-SSA autoantibody is an anti-SSA autoantibody for use as described above.

Optionally, the use of the quantitative detection agent as described above is for performing any one of the following methods:

radioimmunoassay, indirect immunofluorescence, spot immunogold diafiltration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.

Alternatively, for use as described above, the quantitative detection agent is an SSA protein.

Alternatively, for use as described above, the SSA protein is conjugated to a solid support.

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, the quantitative detection agent further comprises an anti-human Ig antibody for use as described above.

Alternatively, the anti-human Ig antibody is an anti-human IgG antibody for use as described above.

Alternatively, the anti-human Ig antibodies are conjugated with a signal substance for use as described above.

Alternatively, for use as described above, the sample to be 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 discovers that the anti-SSA autoantibody has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of congenital diseases of children, and fills the blank of the diagnosis of congenital megacolon plasma. The diagnostic sensitivity and specificity of the method are good, and the AUC is 0.7756; the optimal limit corresponds to sensitivity of 62.50% and specificity of 74.36%, and overcomes the defect of no plasma diagnosis in the prior art.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a human self-antigen chip for screening diagnosis markers in blood plasma of congenital megacolon patients according to an embodiment of the invention;

FIG. 2 shows an enzyme-linked immunosorbent assay (ELISA) for detecting anti-SSA antibodies in congenital megacolon infants and other control groups, according to an embodiment of the invention;

FIG. 3 is a graph showing the diagnostic value of ROC curve analysis of anti-SSA antibodies in the congenital megacolon in one embodiment of the invention.

Detailed Description

Reference now will 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. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a 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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein 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-SSA autoantibody in preparing a congenital megacolon diagnosis reagent, a kit or a test strip.

The invention discovers that the anti-SSA autoantibody can be used as a diagnostic marker of congenital megacolon, has the advantages of simple operation, no intervention, high flux and low cost in the diagnosis of congenital diseases of children, and fills the blank of the diagnosis of congenital megacolon plasma. The diagnostic sensitivity and specificity of the method are good, and the AUC is 0.7756; the optimal limit corresponds to sensitivity of 62.50% and specificity of 74.36%, and overcomes the defect of no plasma diagnosis in the prior art.

The term "marker" or "biochemical marker" as used herein refers to a molecule to be used as a target for analysis of a patient experimental sample.

In some embodiments, the anti-SSA autoantibody is an anti-SSA autoantibody.

In the determination method according to the present invention, the expression analysis method of the autoantibody against the SSA protein is not particularly limited. For example, the relative amounts or concentrations may be determined not only by determining the absolute amounts or concentrations of these autoantibodies in a blood sample. More specifically, for example, the amount, concentration or activity of the above-mentioned autoantibody or the like in a blood sample can be measured. Such methods are well known in the art, and as an example, in some embodiments, the quantitative detection agent is used to perform any of the following methods:

radioimmunoassay, indirect immunofluorescence, spot immunogold diafiltration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.

Examples of the detection method include matrix assisted laser desorption ionization time-of-flight MASS spectrometry (MALDI-TOF-MASS), surface enhanced laser desorption ionization time-of-flight MASS spectrometry (SELDI-TOF-MASS), and the like time-of-flight MASS spectrometry (TOF-MASS). From the TOF-MASS images, the concentration or amount of autoantibodies can be grasped from the molecular weight peaks, other fragment peaks, their intensities, and the like. In addition, as in the case of ELISA, a secondary antibody having a labeling group and capable of binding to the respective antibodies may be used to detect an autoantibody selectively binding to the protein chip.

Among these methods, an immunoassay is preferable as a method for performing the above expression analysis. The immunoassay has high sensitivity and accuracy, and can detect even if the concentration of autoantibodies in blood slightly varies. When the expression analysis of the autoantibody according to the present invention is performed by enzyme-linked immunosorbent assay (ELISA), the diagnostic kit for congenital megacolon according to the present invention can be suitably used.

In some embodiments, the quantitative detection agent is SSA protein.

Autoantigen fragments 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 amino acids in length. The fragment may also be between 5, 10, 15, 20, 25, 50, 75, 100, 150, 200 or 250 and one amino acid less than the full length of the autoantigen. Typically, these epitopes are characterized in advance such that autoantibodies to a given autoantigen 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 an autoantigen disclosed herein, that is recognized by an autoantibody. Obviously, the quantitative detection agent can also be at least one peptide of fragment peptide, denaturation and modification of SSA protein. The denatured product of SSA protein is a denatured product that can specifically bind to the autoantibody obtained by performing physical treatment such as heating, freezing, and ultraviolet rays, or chemical treatment such as application of a surfactant or a denaturing agent. For example, a denatured product obtained by SDS or DTT treatment may be mentioned. The modified substance is a modified substance which is obtained by modifying 1 or more amino acids and which can specifically bind to the autoantibody. For example, a modified product obtained by treatment with glutaraldehyde is given. The above peptide may have a mutation, substitution, deletion and/or addition of 1 or several amino acid residues as long as it can specifically bind to the above autoantibody.

In some embodiments, the SSA protein is conjugated to a solid support.

In some embodiments, the solid support is selected from the group consisting of test tubes, EP tubes, multiwell plates, microplate wells, and microspheres.

The term "solid support" means a carrier material that is predominantly non-liquid in firmness, thereby allowing for accurate and traceable positioning 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 ELISA plate. It may contain holes of 16, 32, 48, 64, 96 or more.

In the present invention, the term "microsphere" may be a sphere, a spheroid, 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. Mu.m.

The microspheres have specific binding properties on their surface for the substance of interest (target or analyte) to be determined.

The microspheres are preferably magnetic beads, the components of which contain magnetic substances. The magnetic substance can be metal (metal simple substance or alloy), nonmetal, or a compound formed by metal and nonmetal. Metals such as iron, alnico metals, and the like; nonmetallic materials such as ferrite nonmetallic materials (preferably Fe ₂ O ₃ Or Fe (Fe) ₃ O ₄ Magnetic nanoparticles); composites of metals and non-metals such as neodymium iron boron rubber magnetic composites.

The surface of the microsphere is modified with one or more active functional groups, wherein the active functional groups comprise-OH, -COOH and-NH ₂ -CHO, and-SO ₃ H, one or more of H. In some embodiments, the coated antigen and antibody are conjugated or bound to the microsphere by physical adsorption 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 by a peptide, protein, antibody, linker, or 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 refer to antibodies to human Ig proteins, and anti-human IgG antibodies refer to antibodies to human IgG proteins.

In some embodiments, the anti-human Ig antibody is conjugated to a signaling substance.

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 to the anti-human Ig antibody labeled with a signal substance. The secondary antibody is allowed to act after washing and removing proteins or the like that bind non-specifically to peptides (SSA proteins) or the like with a buffer or the like. 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 a IgA, igD, igG, igE or IgM isotype or 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 one or more of the following components: phosphate buffer, naCl, EDTA, pluronic F-127, sodium azide, sorbitol, thiol-modified bovine serum, or any combination, variant or equivalent thereof.

In the present invention, the signal species is capable of providing a detected signal species, in some embodiments independently selected from any one or more of a chromophore, a digoxin-labeled probe, an electron dense species, colloidal gold, or an enzyme. These labels are listed in the following non-limiting section:

enzymes that produce detectable signals, such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.

Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds and dyes.

Groups having electron densities that can be detected by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltage measurement, and resistance.

A detectable group, such as a molecule of sufficient size to induce a modification detectable in its physical and/or chemical properties; such detection may be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic force spectroscopy and tunneling).

Electron dense matterSuch as radioactive molecules (e.g ³² P， ³⁵ S or ¹²⁵ I)。

In some embodiments, the signal species is an Acridinium Ester (AE).

Further, acridine chemiluminescent substances include acridine esters and acridine sulfonamides.

Further, the acridine chemiluminescent substance comprises 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 and the like.

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 detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cells, tissue, or cell lysate sample.

As used herein, "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., wherein the structural integrity of the tissue or cells has been compromised. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemicals to lyse, degrade or destroy the cell walls and cell membranes of such tissue or cells. The skilled artisan is well aware of suitable methods for obtaining lysates. This process is encompassed by the term "cleavage".

The diagnostic kit of the present invention preferably contains a normal control sample and a congenital megacolon control sample. If these samples are attached to the kit, the same experiment is performed on these samples, and the measurement value is compared with the result of the test sample, whereby the presence or absence of the congenital megacolon of the subject can be more objectively determined.

The concentration or amount of the autoantibody contained in the sample is indirectly obtained by the color development intensity or the like. The obtained measurement value can be converted into relative or absolute concentration, amount, activity, or the like by a calibration curve or the like.

The invention also relates to a method of diagnosing congenital megacolon comprising quantitatively detecting anti-SSA autoantibodies in a sample to be examined, wherein an elevated level of anti-SSA autoantibodies is indicative of congenital megacolon.

The term "indication" when used in the context of an autoantibody as used in embodiments of the invention includes that an autoantibody of which the embodiments of the invention determine the presence or absence is typically present in a subject having congenital megacolon. By "normally present" is meant that the autoantibody is often associated with the congenital megacolon. "frequently relevant" 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%.

An ideal scenario for diagnosis is one in which a single event or process can cause a variety of 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 will be appreciated by the skilled artisan, for a given multifactorial disease, diagnosis without biochemical markers is 100% specific and as sensitive as 100%. Conversely, biochemical markers can be used to assess, for example, the presence or absence or severity of a disease with some probability or predictive value. Thus, in routine clinical diagnosis, various clinical symptoms and biological markers are often taken into consideration in combination to diagnose, treat and manage underlying diseases.

In the method of the present invention, the presence or absence of the onset of congenital megacolon is then determined from the obtained results of the expression analysis. That is, the higher the onset of the congenital megacolon or the heavier the symptoms thereof, the higher the concentration or amount of the autoantibodies of the present invention in the blood. Thus, based on the results of the analysis of the expression of the autoantibody of the present invention, it can be judged positive if the amount of the autoantibody expressed is large, and it can be judged negative if the amount of the autoantibody expressed is small.

In fact, the boundary between positive and negative, i.e., cutoff value, can be changed according to the definition, severity, and the expression analysis method of the autoantibody according to the present invention of the congenital megacolon. Therefore, in a stage where there is no general standard, it is necessary for the practitioner of the method of the present invention to perform measurement after determining the expression analysis method and the cutoff value in advance by preliminary experiments or the like.

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, for example infants of 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 women and children medical centers, and all experimental tissues and blood samples are collected to obtain the ethical committee authorization and the patient consent of the Guangzhou women and children medical centers.

The results of the experiments of the invention were all statistically analyzed, the t-test was used to evaluate the differences between the two groups, p <0.05 was used to represent statistical significance, and all p-values were tested using two-sided. Statistical analysis was performed using R and graphpad8.0 software.

The congenital megacolon disease is also called Hirschman disease, is one of common congenital intestinal diseases of children because of continuous spasm of intestinal canal caused by lack of ganglion cells in colon, and is characterized by that the feces stagnates in proximal colon and the proximal colon becomes hypertrophic and dilated, wherein the congenital megacolon disease is clinically classified into short-segment type, common type, long-segment type and full-colon type according to increasing severity. The short-segment lesion is positioned at the near and middle sections of the rectum and is not more than 6.5cm away from the anal canal; the common lesions are located at the proximal end of the rectum or distal end of the sigmoid colon, about 9cm from the anal canal; the long-segment lesions extend to the sigmoid or descending colon; the lesions of the whole colon spread all over the colon and the terminal ileum, within 30cm of 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 SSA is a ribonucleoprotein in the cytoplasm of the RNase-resistant cell; the anti-SSA antibody is an autoantibody, preferably an IgG antibody, raised against SSA in the nucleus.

The "ROC curve" described herein 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 true positive to false positive rate that varies by greater than 1, away from 45 degrees.

The AUC in the invention refers to the area under the ROC curve, which is between 0.1 and 1, and is used for evaluating the quality of the classifier, and the classifier is better when the AUC is closer to 1.

The invention screens the blood plasma of congenital megacolon children by using a human self-antigen-free chip, and obtains the anti-SSA antibody with high expression of the blood plasma of congenital megacolon children by using the blood plasma of other intestinal diseases and healthy group children as a contrast. And further validated by enzyme-linked immunosorbent assay (ELISA) in a separate sample.

Example 1 plasma and tissue sample collection and grouping

Plasma samples were divided into congenital megacolon infant groups (37 cases), other intestinal disease control groups (18 cases), healthy children groups (30 cases), ages ranging from 3 months to 3 years, sexed males 3/4 were males, and disease and control groups were age and sex matched. All samples were from the Guangzhou women child medical center and healthy children group were blood samples remaining after physical examination. The blood sampling mode is anticoagulation blood sampling, centrifugal separation of blood plasma and freezing of the sample. The colon tissue samples were congenital megacolon infant group (36 cases) and were surgically excised lesion tissue. Other enteropathy control groups (11 cases of colon tissue including anal stenosis and intestinal stenosis fistulation).

Example 2 screening of human self-antigen chip and analysis of differentially expressed autoantibodies

5 cases of plasma from congenital megacolon (HSCR) infants, 5 cases from Healthy (HC) and other intestinal (DC) groups, were screened for human self-immune antigen chips provided by guangzhou Yijin biotechnology limited, lgG detection of more than 100 autoimmune antibodies, primary data, minus negative controls, normalized by RLM to obtain inter-group differential analysis of results using M statistics, fig. 1 shows a cluster analysis of differential autoantibodies, from which it can be seen that ribonucleoprotein antigen SSA was significantly higher in the plasma from congenital megacolon infants than in the control group (p=0.046).

Example 3 enzyme-linked immunosorbent assay (ELISA) detection of anti-SSA antibodies

To verify the diagnostic effect of anti-SSA antibodies on the congenital megacolon, we collected plasma from megacolon patients (37 cases), other intestinal disease controls (18 cases total of plasma samples including anal stenosis and enterostenosis fistulization children), healthy children controls (30 cases), and detected the level of anti-SSA antibodies in the plasma by enzyme-linked immunosorbent assay (ELISA), the detection kit was a human anti-SSA antibody (SSA-Ab) enzyme-linked immunosorbent assay (ELISA) kit (Shanghai zhen scientific biotechnology). The results as shown in figure 2 show that anti-SSA antibodies in the plasma of megacolon patients were significantly higher than in healthy pediatric controls (p < 0.01), no difference from intestinal disease controls, and higher than in controls where intestinal disease was combined with healthy children (p < 0.01).

EXAMPLE 4 ROC Curve analysis

Fig. 3 shows ROC curves for evaluating the diagnostic effect of anti-SSA antibodies on congenital megacolon. AUC is 0.7756; the optimum limit corresponds to a sensitivity of 62.50% and a specificity of 74.36%. Thus, it can be seen that anti-SSA antibodies can be effective in diagnosing congenital megacolon.

In conclusion, the anti-SSA antibody with high blood plasma expression of the congenital megacolon infant is obtained through the screening of the human self-immune antigen chip. And in independent samples, an enzyme-linked immunosorbent assay (ELISA) method proves that the anti-SSA antibody is obviously higher than other diseases and healthy control groups in a megacolon group (HSCR), and can effectively diagnose congenital megacolon children patients, and the AUC is 0.7756; the optimum limit corresponds to a sensitivity of 62.50% and a specificity of 74.36%. The anti-SSA antibody can be used as a congenital megacolon plasma diagnosis marker for diagnosing diseases, and fills the blank of congenital megacolon blood diagnosis.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. The application of the quantitative detection agent of the anti-SSA autoantibody in preparing a congenital megacolon diagnosis reagent, a kit or a test strip.

2. The use according to claim 1, wherein the anti-SSA autoantibody is an anti-SSA autoantibody.

3. The use according to claim 1, wherein the quantitative detection agent is used to perform any one of the following methods:

radioimmunoassay, indirect immunofluorescence, spot immunogold diafiltration, mass spectrometry, immunoblotting and enzyme-linked immunosorbent assay.

4. The use according to claim 1, wherein the quantitative detection agent is SSA protein.

5. The use according to claim 4, wherein the SSA protein 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, multiwell plates, wells of microplate and microspheres.

7. The use according to any one of claims 4 to 6, wherein the quantitative detection agent further comprises an anti-human Ig antibody.

8. The use according to claim 7, wherein the anti-human Ig antibody is an anti-human IgG antibody.

9. The use of claim 7, wherein the anti-human Ig antibody is conjugated to a signaling material.

10. The use according to any one of claims 1-6, 8, 9, wherein the sample to be detected by the quantitative detection agent is at least one of blood, plasma, serum, tissue, cells, a tissue lysate sample and a cell lysate sample.