Biomarker combination for diagnosing congenital megacolon, kit and application

Abstract

The invention provides a biomarker combination for diagnosing congenital megacolon. The biomarker combination is any one of a combination 1, a combination 2 and a combination 3, wherein the combination 1 is a combination of SCGN, PGP9.5 and S100; the combination 2 is a combination of SCGN and PGP9.5; and the combination 3 is a combination of SCGN and S100. The invention also provides application of the biomarker combination in preparation of a congenital megacolon diagnostic reagent. Meanwhile, the invention provides an immunohistochemical kit. The invention finds that the SCGN can effectively identify ganglion cells in a rectal mucosa biopsy specimen / colon full-thickness specimen of a child patient suffering from the congenital megacolon, and the diagnosis sensitivity and specificity are higher than those of an existing detection scheme after the SCGN is combined with PGP9.5 and / or S100; the invention provides a novel, accurate and sensitive detection way for the diagnosis of the congenital megacolon.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis, and in particular to a biomarker combination, a kit and an application thereof for diagnosing Hirschsprung's disease. Background Art

[0002] Hirschsprung's disease is the most common surgical disease that causes constipation in children. It is a congenital digestive tract malformation with a large difference in incidence among different races. As one of the regions with the highest incidence of neonatal HD, the incidence rate of live births in China is about 2.8 / 10,000.

[0003] Rectal mucosal biopsy is the most sensitive and specific preoperative diagnostic method for congenital megacolon. It is diagnosed by determining the number of submucosal ganglion cells and the thickness and shape of nerve fibers. However, in the implementation process, the less damage the mucosal biopsy causes to the patient, the smaller the tissue obtained, and the more difficult it is to make a pathological diagnosis. The development of acetylcholinesterase histochemical method has played a significant role in promoting the diagnosis of congenital megacolon by rectal mucosal biopsy. The basic histological characteristics of congenital megacolon are the congenital absence of ganglion cells in the affected intestine and the increased expression of acetylcholinesterase. Although the acetylcholinesterase histochemical method has a high accuracy, there is still a certain proportion of false positive and false negative results. Different biopsy sites, immature enzyme systems and technical differences may all lead to erroneous acetylcholinesterase test results. In addition, the limitations of acetylcholinesterase staining include: (1) it must be performed on frozen sections, and the diagnostic accuracy is not as good as that of paraffin sections; (2) some megacolon specimens contain immature nerve fibers, which may lead to false negatives; (3) acetylcholinesterase uses a quantitative diagnostic method, which is highly subjective and inevitably has certain inter-observer differences.

[0004] Using immunohistochemistry for multiple index staining, we can make a targeted diagnosis of the cause by locating the number of ganglion cells in the mucosa and observing the shape and distribution of nerve fibers, which reduces diagnostic trauma and improves accuracy. However, there are currently no mature and efficient diagnostic markers and methods for identifying intestinal ganglion cells and nerve fibers on the market.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the problem of lack of mature and efficient diagnostic markers for identifying intestinal ganglion cells and nerve fibers in the prior art, the present invention provides a biomarker combination, a kit and an application for diagnosing congenital megacolon, which provides a new, accurate and sensitive detection approach for the diagnosis of congenital megacolon.

[0007] The present invention adopts the following technical solutions to solve the above technical problems: A biomarker combination for diagnosing congenital megacolon, which is any one of combination 1, combination 2, and combination 3; wherein, combination 1 is a combination of SCGN (secretogen), PGP9.5 (brain-specific protein product 9.5), and S100 (S100 protein); combination 2 is a combination of SCGN (secretogen) and PGP9.5 (brain-specific protein product 9.5); and combination 3 is a combination of SCGN (secretogen) and S100 (S100 protein).

[0008] A use of the above biomarker combination in the preparation of a diagnostic reagent for congenital megacolon.

[0009] As one of the preferred embodiments of the present invention, the diagnostic reagent for Hirschsprung's disease is based on immunohistochemistry or immunofluorescence.

[0010] An immunohistochemistry kit comprises a primary antibody reagent and a secondary antibody reagent; wherein the primary antibody reagent is one of anti-human SCGN monoclonal antibody, anti-human PGP9.5 monoclonal antibody, anti-human S100 monoclonal antibody combination, anti-human SCGN monoclonal antibody, anti-human PGP9.5 monoclonal antibody combination, or anti-human SCGN monoclonal antibody, anti-human S100 monoclonal antibody combination; and the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.

[0011] As one of the preferred aspects of the present invention, it further comprises a developer and a counterstain.

[0012] As one of the preferred embodiments of the present invention, the color developer is DAB color developer, and the counterstain is hematoxylin counterstain.

[0013] As one of the preferred embodiments of the present invention, it also includes an antigen repair solution, a blocking agent and a sealing agent.

[0014] As one of the preferred embodiments of the present invention, the antigen repair solution is EDTA and / or sodium citrate, the blocking agent is bovine serum albumin, and the sealing agent is hydrogen peroxide.

[0015] Design principle: There are two criteria for the diagnosis of Hirschsprung's disease (mucosal biopsy): (1) lack of ganglion cells in the submucosal layer; (2) thick nerve fibers. The present invention uses SCGN to identify ganglion cells and uses PGP9.5 and / or S100 to observe the thickness of nerve fibers, thereby improving the accuracy of diagnosis.

[0016] The advantages of the present invention compared to the prior art are: The present invention finds that SCGN can effectively identify ganglion cells in rectal mucosal biopsy specimens / full-layer colon specimens of children with congenital megacolon, and the diagnostic sensitivity and specificity after combined with PGP9.5 and / or S100 are higher than those of existing detection schemes; the diagnostic sensitivity of the diagnostic scheme of the present invention for congenital megacolon in 321 children with constipation is 98.99% (95% CI, 97.02%~100.00%), the specificity is 97.75% (95% CI, 95.80%~99.70%), the positive predictive value is 95.15% (95% CI, 91.00%~99.30%), and the negative predictive value is 99.54% (95% CI, 98.64%~100.00%); the present invention provides a new, accurate and sensitive detection approach for the diagnosis of congenital megacolon. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the results of proteomics screening for diagnostic markers in children with Hirschsprung's disease in Experimental Example 1; Figure 2 are the results of staining of SCGN, PGP9.5 and S100 in the rectal mucosal biopsy sections of the Hirschsprung group and the control group in Experimental Example 2 (in the figure, Figure A is the SCGN staining result of the Hirschsprung group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung group, and Figure F is the S100 staining result of the control group; brown indicates positive staining); Figure 3 These are the results of staining for SCGN, PGP9.5 and S100 in the full-thickness colon biopsy pathological sections of the Hirschsprung's group and the control group in Experimental Example 2 (in the figures, Figure A is the SCGN staining result of the Hirschsprung's group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung's group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung's group, and Figure F is the S100 staining result of the control group; brown indicates positive staining). DETAILED DESCRIPTION

[0018] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following embodiment. In the following embodiment: Monoclonal antibody targeting human SCGN: Abclonal A19615 was used; Monoclonal antibody targeting human PGP9.5: Abclonal A23159; Monoclonal antibody targeting human S100: Abclonal A20380; HRP-labeled goat anti-rabbit IgG: MXB biotechnologies KIT-9902; In addition, unless otherwise specified, the reagents used in the following embodiments of the present invention are all commercially available products; the methods and equipment used are all conventional methods and equipment in the technical field and will not be described in detail.

[0019] Example 1 An immunohistochemistry kit of this embodiment includes a primary antibody reagent, a secondary antibody reagent, a DAB colorimetric solution, a hematoxylin counterstaining solution, EDTA (antigen repair solution), hydrogen peroxide (blocking agent) and bovine serum albumin (blocking agent). The primary antibody reagent is a monoclonal antibody targeting human SCGN, a monoclonal antibody targeting human PGP9.5 and a monoclonal antibody targeting human S100; the secondary antibody reagent is a goat anti-rabbit IgG labeled with HRP.

[0020] The kit of this embodiment is used for diagnosing and detecting Hirschsprung's disease, and the detection is based on the biomarker combination of "SCGN+PGP9.5+S100".

[0021] Example 2 An immunohistochemistry kit of this embodiment includes a primary antibody reagent, a secondary antibody reagent, a DAB colorimetric solution, a hematoxylin counterstaining solution, EDTA (antigen repair solution), hydrogen peroxide (blocking agent) and bovine serum albumin (blocking agent). The primary antibody reagent is a monoclonal antibody targeting human SCGN and a monoclonal antibody targeting human PGP9.5; the secondary antibody reagent is an HRP-labeled goat anti-rabbit IgG.

[0022] The kit of this embodiment is used for diagnosing and detecting Hirschsprung's disease, and the detection is based on the biomarker combination of "SCGN+PGP9.5".

[0023] Example 3 An immunohistochemistry kit of this embodiment includes a primary antibody reagent, a secondary antibody reagent, a DAB colorimetric solution, a hematoxylin counterstaining solution, EDTA (antigen repair solution), hydrogen peroxide (blocking agent) and bovine serum albumin (blocking agent). The primary antibody reagent is a monoclonal antibody targeting human SCGN and a monoclonal antibody targeting human S100; the secondary antibody reagent is a goat anti-rabbit IgG labeled with HRP.

[0024] The kit of this embodiment is used for diagnosing and detecting Hirschsprung's disease, and the detection is based on the biomarker combination of "SCGN+S100".

[0025] Example 4 The immunohistochemistry kit of this embodiment is basically the same as that of embodiment 1, with the main difference being that sodium citrate is used as the antigen repair solution.

[0026] Example 5 A method for diagnosing Hirschsprung's disease in this embodiment is based on the immunohistochemistry kit of Example 1, and the operation is as follows: (1) Tissue section dewaxing, hydration and antigen retrieval Take clinical rectal mucosal biopsy or full-thickness colon biopsy pathological sections, place the sections in xylene, and soak for 15 minutes, twice. Then, place them in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 minutes. Rinse with RO water for 5 minutes. Place the sections in 2% EDTA, perform antigen repair in a 100°C water bath for 20 minutes, and cool naturally to room temperature. Use 3% hydrogen peroxide to block endogenous peroxidase activity, and rinse with PBS solution 3 times, 5 minutes each time.

[0027] (2) Closed Add 50 μL of bovine serum albumin to the slices, incubate at 37°C for 20 min, shake off excess liquid, and do not wash.

[0028] (3) Incubation with primary antibody The sections were incubated with monoclonal antibodies targeting human SCGN (1:200 dilution), monoclonal antibodies targeting human PGP9.5 (1:200 dilution), and monoclonal antibodies targeting human S100 (1:200 dilution) at 4°C overnight or at 37°C for 2 hours. The sections were rinsed with PBS solution for 3 times, 5 minutes each time.

[0029] (4) Incubation with secondary antibody Add 50 μL of HRP-labeled goat anti-rabbit IgG antibody (ready-to-use, no dilution required) to the tissue sections, incubate at 37°C for 30 min, and rinse with PBS solution three times, 5 min each time.

[0030] (5) Color rendering Add 50 μL of DAB staining solution to the sections, incubate at room temperature for 3-10 min, and rinse once with PBS solution for 5 min.

[0031] (6) Re-staining The tissue sections were counterstained with hematoxylin for 1 min, washed with water for 30-40 s, rinsed with PBS for 30 s, differentiated with hydrochloric acid alcohol for 10 s, and rinsed with PBS solution for 1 min.

[0032] (7) Dehydration, transparency, and sealing with resin sealing medium The sections were placed in 75% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 5 min, and then immersed in xylene for 5 min, twice. After drying in a fume hood, the sections were sealed with a resin sealing agent.

[0033] (8) Microscope photography and observation The sections after sealing were photographed under a microscope to observe whether ganglion cells positively expressing SCGN and thick nerve fibers were visible.

[0034] If a large number of ganglion cells positively expressing SCGN are seen in the slice, and small nerve fibers positively stained with PGP9.5 and S100 are also seen, it is judged to be a normal rectum / colon.

[0035] If no ganglion cells expressing SCGN are clearly seen in the sections, but thick nerve fibers positively stained for PGP9.5 and S100 are seen, it is diagnosed as Hirschsprung's disease.

[0036] Example 6 A method for diagnosing Hirschsprung's disease in this embodiment is based on the immunohistochemistry kit of Example 2, and the operation is as follows: (1) Same as Example 5.

[0037] (2) Same as Example 5.

[0038] (3) Incubate the sections with monoclonal antibodies targeting human SCGN (1:200 dilution) or monoclonal antibodies targeting human PGP9.5 (1:200 dilution) at 4°C overnight or at 37°C for 2 h. Rinse with PBS solution three times, 5 min each time.

[0039] (4) to (8) are the same as in Example 5.

[0040] If a large number of ganglion cells positively expressing SCGN are seen in the slice, and small nerve fibers positively stained with PGP9.5 are also seen, it is judged to be a normal rectum / colon.

[0041] If no ganglion cells expressing SCGN are clearly seen in the slice, but thick nerve fibers positively stained with PGP9.5 are seen, it is diagnosed as Hirschsprung's disease.

[0042] Example 7 A method for diagnosing Hirschsprung's disease in this embodiment is based on the immunohistochemistry kit of Example 3, and the operation is as follows: (1) Same as Example 5.

[0043] (2) Same as Example 5.

[0044] (3) Incubate the sections with monoclonal antibodies targeting human SCGN (1:200 dilution) or monoclonal antibodies targeting human S100 (1:200 dilution) at 4°C overnight or at 37°C for 2 h. Rinse with PBS solution three times, 5 min each time.

[0045] (4) to (8) are the same as in Example 5.

[0046] If a large number of ganglion cells positively expressing SCGN are seen in the slice, and small nerve fibers positively stained with S100 are also seen, it is judged to be a normal rectum / colon.

[0047] If no ganglion cells expressing SCGN are clearly seen in the slice, but thick nerve fibers positively stained with S100 are seen, it is diagnosed as Hirschsprung's disease.

[0048] Example 8 A method for diagnosing Hirschsprung's disease in this embodiment is based on the immunohistochemistry kit of Example 4, and the operation is as follows: (1) Take a clinical rectal mucosal biopsy or a full-thickness colon biopsy pathological section, place the section in xylene, and soak for 15 minutes, twice. Then, place it in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 minutes. Rinse with RO water for 5 minutes. Place the section in 1% sodium citrate, perform antigen repair in a 100°C water bath for 20 minutes, and cool naturally to room temperature. Use 3% hydrogen peroxide to block endogenous peroxidase activity, and rinse with PBS solution 3 times, 5 minutes each time.

[0049] (2) to (8) are the same as in Example 5.

[0050] If a large number of ganglion cells positively expressing SCGN are seen in the slice, and small nerve fibers positively stained with PGP9.5 and S100 are also seen, it is judged to be a normal rectum / colon.

[0051] If no ganglion cells expressing SCGN are clearly seen in the sections, but thick nerve fibers positively stained for PGP9.5 and S100 are seen, it is diagnosed as Hirschsprung's disease.

[0052] Experimental Example 1 This experimental example is used to reveal the process of proteomics screening of differentially expressed proteins in colon tissue samples of children with Hirschsprung's disease and other intestinal disease control groups in the present invention: The "colon tissue samples" were divided into "Hercksprung's disease group (6 cases)" and "other intestinal disease control group" (including colon tissues of anal stenosis and intestinal stricture fistula, a total of 3 cases). Proteomic sequencing was performed by Shanghai GeneCare Gene.

[0053] The differentially expressed genes were analyzed as follows: Figure 1As shown, it can be seen that the expression level of SCGN protein in the colon of children with megacolon was significantly lower than that in the control group (p = 0.008).

[0054] Experimental Example 2 This experimental example is used to verify the feasibility of SCGN combined with PGP9.5 and / or S100 in diagnosing Hirschsprung's disease: The samples were divided into a "Hercksprung's disease group" and a "control group (Hercksprung's disease homologous to the disease)". The corresponding rectal mucosal biopsy sections and colon full-thickness biopsy pathological sections were prepared by referring to the method of Example 5. The sections were photographed under a microscope. The results are as follows: Figure 2 , 3 shown.

[0055] Figure 2 These are the results of staining for SCGN, PGP9.5, and S100 in the rectal mucosal biopsy sections of the Hirschsprung group and the control group (Figures, Panel A shows the SCGN staining result of the Hirschsprung group, Panel B shows the SCGN staining result of the control group, Panel C shows the PGP9.5 staining result of the Hirschsprung group, Panel D shows the PGP9.5 staining result of the control group, Panel E shows the S100 staining result of the Hirschsprung group, and Panel F shows the S100 staining result of the control group). Figure 3 These are the results of staining for SCGN, PGP9.5 and S100 in the full-thickness colon biopsy pathological sections of the Hirschsprung group and the control group (Figures, Panel A shows the SCGN staining result of the Hirschsprung group, Panel B shows the SCGN staining result of the control group, Panel C shows the PGP9.5 staining result of the Hirschsprung group, Panel D shows the PGP9.5 staining result of the control group, Panel E shows the S100 staining result of the Hirschsprung group, and Panel F shows the S100 staining result of the control group).

[0056] Depend on Figure 2 It can be seen that: in the normal rectal submucosa, a large number of ganglion cells positively expressing SCGN can be seen; no obvious expression is seen in the rectal submucosa of patients with congenital megacolon. In the normal colonic submucosa, small nerve fibers positively stained with PGP9.5 and S100 can be seen; in the rectal submucosa of patients with congenital megacolon, thick nerve fibers (≥ 40μm) positively stained with PGP9.5 and S100 can be seen.

[0057] Depend on Figure 3 It can be seen that: in the normal colonic muscular layer, a large number of ganglion cells positively expressing SCGN can be seen; no obvious expression is seen in the colonic muscular layer of patients with congenital megacolon. In the normal colonic muscular layer, small nerve fibers positively stained with PGP9.5 and S100 can be seen; in the colonic muscular layer of patients with congenital megacolon, thick nerve fibers (≥ 40 μm) positively stained with PGP9.5 and S100 can be seen.

[0058] Experimental Example 3 This experimental example is used to test the diagnostic efficacy of SCGN combined with PGP9.5 and / or S100 of the present invention: In 321 children with constipation, different diagnostic marker combinations of the present invention were used to stain rectal mucosal biopsy specimens by immunohistochemistry, and the diagnostic sensitivity and specificity were calculated.

[0059] Specifically, the kits of Example 1 (based on the SCGN+PGP9.5+S100 marker combination), Example 2 (based on the SCGN+PGP9.5 marker combination), and Example 3 (based on the SCGN+S100 marker combination) are combined with the corresponding diagnostic methods (refer to Examples 5 to 7) as examples, and the existing detection scheme (calretinin+S100+PGP9.5) and the detection method based on the SCGN marker alone (the detection reagent uses a single monoclonal antibody targeting human SCGN as the primary antibody, and the rest is the same as Example 1) are used as controls.

[0060] The results are shown in Table 1-1 and Table 1-2.

[0061] Table 1-1 Diagnostic efficacy (sensitivity, specificity) of different diagnostic marker combinations

[0062] In the table, CI stands for confidence interval.

[0063] Table 1-2 Diagnostic efficacy (positive and negative predictive values) of different diagnostic marker combinations

[0064] In the table, CI stands for confidence interval.

[0065] As shown in Tables 1-1 and 1-2, the use of SCGN combined with PGP9.5 and S100 immunohistochemical staining for the diagnosis of Hirschsprung's disease in rectal mucosal biopsy specimens has the highest diagnostic efficacy and is superior to existing detection schemes.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A biomarker combination for diagnosing Hirschsprung's disease, characterized in that: It is any one of combination 1, combination 2 and combination 3; wherein, combination 1 is a combination of SCGN, PGP9.5 and S100; combination 2 is a combination of SCGN and PGP9.5; and combination 3 is a combination of SCGN and S100.

2. Use of the biomarker combination as claimed in claim 1 in the preparation of a diagnostic reagent for congenital megacolon.

3. The use according to claim 2, characterized in that: The Hirschsprung's disease diagnostic reagent is based on immunohistochemistry or immunofluorescence.

4. An immunohistochemistry kit, characterized in that: It comprises a primary antibody reagent and a secondary antibody reagent; wherein the primary antibody reagent is one of anti-human SCGN monoclonal antibody, anti-human PGP9.5 monoclonal antibody, anti-human S100 monoclonal antibody combination, anti-human SCGN monoclonal antibody, anti-human PGP9.5 monoclonal antibody combination, or anti-human SCGN monoclonal antibody, anti-human S100 monoclonal antibody combination; and the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.

5. The immunohistochemistry kit according to claim 4, characterized in that: Also includes a developer and counterstain.

6. The immunohistochemistry kit according to claim 5, characterized in that The color developer is DAB color developer, and the counterstain is hematoxylin counterstain.

7. The immunohistochemistry kit according to claim 4, characterized in that: It also includes antigen retrieval solution, blocking agents and sealing agents.

8. The immunohistochemistry kit according to claim 7, characterized in that: The antigen repair solution is EDTA and / or sodium citrate, the blocking agent is bovine serum albumin, and the sealing agent is hydrogen peroxide.

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