Biomarker combination and kit for diagnosing hirschsprung's disease, and use
Patent Information
- Application Number
- PCT/CN2025/105851
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-27
- Filing Date
- 2025-06-30
- Publication Date
- 2026-10-01
Smart Images
Figure CN2025105851_01102026_PF_FP_ABST
Abstract
Description
Biomarker combinations, kits, and applications for the diagnosis of congenital megacolon Technical Field
[0001] This invention relates to the field of disease diagnosis, and more particularly to a combination of biomarkers, a kit, and applications for diagnosing congenital megacolon. Background Technology
[0002] Congenital megacolon is the most common surgical cause of constipation in children. It is a congenital malformation of the digestive tract, and its incidence varies greatly among different ethnic groups. As one of the regions with the highest incidence of neonatal Hip Hop, China has an incidence of approximately 2.8 / 10,000 live births.
[0003] Rectal mucosal biopsy is the most sensitive and specific diagnostic method for congenital megacolon preoperatively, determining the number of submucosal ganglion cells and the thickness and shape of nerve fibers. However, during the procedure, the less damage to the patient the mucosal biopsy causes, the smaller the tissue obtained, and the more difficult the pathological diagnosis becomes. The development of acetylcholinesterase histochemistry has significantly promoted the diagnosis of congenital megacolon through rectal mucosal biopsy. The basic histological features of congenital megacolon are the congenital absence of ganglion cells in the affected intestinal segment and increased expression of acetylcholinesterase. Although acetylcholinesterase histochemistry has high accuracy, a certain percentage of false positives and false negatives still exist. Different biopsy sites, immature enzyme systems, and technical differences can all lead to erroneous acetylcholinesterase test results. In addition, the limitations of acetylcholinesterase staining include: (1) it needs to 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 is diagnosed quantitatively, which is highly subjective and inevitably leads to some inter-observer differences.
[0004] Immunohistochemistry, using multiple staining markers, can enable targeted diagnosis by locating the number of ganglion cells in the mucosa and observing the shape and distribution of nerve fibers, thus reducing diagnostic trauma and improving accuracy. However, there are currently no mature and efficient diagnostic markers or methods available on the market for identifying intestinal ganglion cells and nerve fibers.
[0005] In view of this, the present invention is hereby proposed. Summary of the Invention
[0006] To address the lack of mature and efficient diagnostic markers for identifying intestinal ganglion cells and nerve fibers in existing technologies, this invention provides a combination of biomarkers, a kit, and applications for diagnosing congenital megacolon, offering a new, accurate, and sensitive detection method for the diagnosis of congenital megacolon.
[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:
[0008] A combination of biomarkers for diagnosing congenital megacolon, comprising any one of combination 1, combination 2, and combination 3; wherein combination 1 is a combination of SCGN (secretin), PGP9.5 (brain-specific protein product 9.5), and S100 (S100 protein); combination 2 is a combination of SCGN (secretin) and PGP9.5 (brain-specific protein product 9.5); and combination 3 is a combination of SCGN (secretin) and S100 (S100 protein).
[0009] Application of a combination of the above-mentioned biomarkers in the preparation of diagnostic reagents for congenital megacolon.
[0010] As one of the preferred embodiments of the present invention, the diagnostic reagent for congenital megacolon is based on immunohistochemistry or immunofluorescence.
[0011] An immunohistochemical kit includes a primary antibody reagent and a secondary antibody reagent; wherein the primary antibody reagent is one of the following: 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.
[0012] As one of the preferred embodiments of the present invention, it also includes a color developer and a re-dyeing agent.
[0013] As one of the preferred embodiments of the present invention, the color developer is DAB color developer solution and the counterstaining agent is hematoxylin counterstaining solution.
[0014] As one of the preferred embodiments of the present invention, it also includes an antigen retrieval solution, an inhibitor, and a blocking agent.
[0015] As one of the preferred embodiments of the present invention, the antigen retrieval solution is EDTA and / or sodium citrate, the blocking agent is bovine serum albumin, and the blocking agent is hydrogen peroxide.
[0016] Design principles:
[0017] The diagnosis of congenital megacolon (mucosal biopsy) has two criteria: (1) lack of ganglion cells in the submucosa; and (2) large nerve fibers. This invention utilizes SCGN to identify ganglion cells and simultaneously uses PGP9.5 and / or S100 to observe the thickness of nerve fibers, thereby improving diagnostic accuracy.
[0018] The advantages of this invention compared to the prior art are:
[0019] This invention discovers that SCGN can effectively identify ganglion cells in rectal mucosal biopsy specimens / full-thickness colonic specimens from children with congenital megacolon. Furthermore, when combined with PGP9.5 and / or S100, the diagnostic sensitivity and specificity are higher than existing detection methods. In 321 children with constipation, the diagnostic method of this invention showed a sensitivity of 98.99% (95% CI, 97.02%–100.00%), a specificity of 97.75% (95% CI, 95.80%–99.70%), a positive predictive value of 95.15% (95% CI, 91.00%–99.30%), and a negative predictive value of 99.54% (95% CI, 98.64%–100.00%) for the diagnosis of congenital megacolon. This invention provides a new, accurate, and sensitive detection method for the diagnosis of congenital megacolon. Attached Figure Description
[0020] Figure 1 shows the results of proteomics screening for diagnostic markers in children with congenital megacolon in Experiment Example 1.
[0021] Figure 2 shows the staining results of SCGN, PGP9.5, and S100 in rectal mucosal biopsy sections from the congenital megacolon group and the control group in Experimental Example 2 (Figure A shows the SCGN staining results of the congenital megacolon group, Figure B shows the SCGN staining results of the control group, Figure C shows the PGP9.5 staining results of the congenital megacolon group, Figure D shows the PGP9.5 staining results of the control group, Figure E shows the S100 staining results of the congenital megacolon group, and Figure F shows the S100 staining results of the control group; brown indicates positive staining).
[0022] Figure 3 shows the staining results of SCGN, PGP9.5, and S100 in the full-thickness colon biopsy pathological sections of the congenital megacolon group and the control group in Experimental Example 2 (Figure A shows the SCGN staining results of the congenital megacolon group, Figure B shows the SCGN staining results of the control group, Figure C shows the PGP9.5 staining results of the congenital megacolon group, Figure D shows the PGP9.5 staining results of the control group, Figure E shows the S100 staining results of the congenital megacolon group, and Figure F shows the S100 staining results of the control group; brown indicates positive staining). Detailed Implementation
[0023] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments. In the following embodiments:
[0024] Monoclonal antibody targeting human SCGN: Abclonal A19615 was used;
[0025] Monoclonal antibody targeting human PGP9.5: Abclonal A23159 was used;
[0026] Monoclonal antibody targeting human S100: Abclonal A20380 was used;
[0027] HRP-labeled goat anti-rabbit IgG: using MXB biotechnologies KIT-9902;
[0028] 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 this technical field, and will not be described in detail again.
[0029] Example 1
[0030] An immunohistochemical kit according to this embodiment includes a primary antibody reagent, a secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstaining solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). The primary antibody reagent consists of 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 HRP-labeled goat anti-rabbit IgG.
[0031] The kit in this embodiment is used to diagnose and detect congenital megacolon, and the detection is based on the combination of biomarkers “SCGN+PGP9.5+S100”.
[0032] Example 2
[0033] An immunohistochemical kit according to this embodiment includes a primary antibody reagent, a secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstaining solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). The primary antibody reagent consists of a monoclonal antibody targeting human SCGN and a monoclonal antibody targeting human PGP9.5; the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.
[0034] The kit in this embodiment is used to diagnose and detect congenital megacolon, and the detection is based on the combination of biomarkers “SCGN+PGP9.5”.
[0035] Example 3
[0036] An immunohistochemical kit according to this embodiment includes a primary antibody reagent, a secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstaining solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). The primary antibody reagent consists of a monoclonal antibody targeting human SCGN and a monoclonal antibody targeting human S100; the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.
[0037] The kit in this embodiment is used to diagnose and detect congenital megacolon, and the detection is based on the combination of biomarkers “SCGN+S100”.
[0038] Example 4
[0039] The immunohistochemistry kit in this embodiment is basically the same as that in Example 1, except that sodium citrate is used as the antigen retrieval solution.
[0040] Example 5
[0041] This embodiment provides a method for diagnosing congenital megacolon, based on the immunohistochemical kit of Example 1, and the procedure is as follows:
[0042] (1) Dewaxing, hydration and antigen retrieval of tissue sections
[0043] Clinical rectal mucosal biopsy or full-thickness colonic biopsy pathological sections were obtained. The sections were immersed in xylene for 15 minutes, twice. Subsequently, they were placed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 minutes each. They were then rinsed with RO water for 5 minutes. The sections were then placed in 2% EDTA and incubated in a 100°C water bath for 20 minutes for antigen retrieval, followed by natural cooling to room temperature. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, and the sections were rinsed three times with PBS solution for 5 minutes each time.
[0044] (2) Closed
[0045] Add 50 μL of bovine serum albumin to the slice, incubate at 37°C for 20 min, remove excess liquid, and do not wash.
[0046] (3) Incubation of primary antibody
[0047] The slides were incubated overnight at 4°C or for 2 hours at 37°C with monoclonal antibodies targeting human SCGN (1:200 dilution), human PGP9.5 (1:200 dilution), and human S100 (1:200 dilution). The slides were then washed three times with PBS solution for 5 minutes each time.
[0048] (4) Incubation of secondary antibodies
[0049] Add 50 μL of HRP-labeled goat anti-rabbit IgG antibody (ready-to-use, no dilution required) to the tissue section, incubate at 37°C for 30 min, and wash three times with PBS solution for 5 min each time.
[0050] (5) Color development
[0051] Add 50 μL of DAB staining solution to the slide and incubate at room temperature for 3–10 min. Rinse once with PBS solution for 5 min.
[0052] (6) Re-staining
[0053] Counterstain the tissue sections with hematoxylin for 1 min, wash with water for 30-40 s, rinse with PBS for 30 s, differentiate with hydrochloric acid alcohol for 10 s, and rinse with PBS solution for 1 min.
[0054] (7) Dehydration, clearing, and sealing with resin sealing tablets.
[0055] The sections were sequentially immersed in 75% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol for 5 minutes each, followed by immersion in xylene for 5 minutes, repeated twice. After air-drying in a fume hood, the sections were mounted with resin mounting medium.
[0056] (8) Microscopic photography and observation
[0057] The slides were photographed under a microscope after mounting to observe whether ganglion cells expressing positive SCGN and thick nerve fibers were visible.
[0058] If a large number of ganglion cells expressing SCGN are visible in the section, along with fine nerve fibers stained positive for PGP9.5 and S100, it can be determined to be a normal rectum / colon.
[0059] If no ganglion cells clearly expressing SCGN are seen in the section, but thick nerve fibers positively stained with PGP9.5 and S100 are also seen, it is determined to be congenital megacolon.
[0060] Example 6
[0061] This embodiment provides a method for diagnosing congenital megacolon, based on the immunohistochemical kit from Example 2, and the procedure is as follows:
[0062] (1) Same as Example 5.
[0063] (2) Same as Example 5.
[0064] (3) Incubate the slides with monoclonal antibodies targeting human SCGN (1:200 dilution) and monoclonal antibodies targeting human PGP9.5 (1:200 dilution) at 4°C overnight or at 37°C for 2 hours. Rinse three times with PBS solution for 5 minutes each time.
[0065] (4)~(8) are the same as in Example 5.
[0066] If a large number of ganglion cells expressing SCGN are visible in the section, along with fine nerve fibers stained with PGP9.5, it can be determined to be a normal rectum / colon.
[0067] If no ganglion cells clearly expressing SCGN are seen in the section, but large nerve fibers with positive PGP9.5 staining are also seen, it is determined to be congenital megacolon.
[0068] Example 7
[0069] This embodiment provides a method for diagnosing congenital megacolon, based on the immunohistochemical kit from Example 3, and the procedure is as follows:
[0070] (1) Same as Example 5.
[0071] (2) Same as Example 5.
[0072] (3) Incubate the slides with monoclonal antibodies targeting human SCGN (1:200 dilution) and monoclonal antibodies targeting human S100 (1:200 dilution) at 4°C overnight or at 37°C for 2 hours. Rinse three times with PBS solution for 5 minutes each time.
[0073] (4)~(8) are the same as in Example 5.
[0074] If a large number of ganglion cells expressing SCGN are visible in the section, along with fine nerve fibers stained with S100, it can be determined to be a normal rectum / colon.
[0075] If no ganglion cells clearly expressing SCGN are seen in the section, but thick nerve fibers with positive S100 staining are seen, it is determined to be congenital megacolon.
[0076] Example 8
[0077] This embodiment provides a method for diagnosing congenital megacolon, based on the immunohistochemical kit from Example 4, and the procedure is as follows:
[0078] (1) Obtain pathological sections from clinical rectal mucosal biopsies or full-thickness colonic biopsies. Immerse the sections in xylene for 15 min, twice. Subsequently, place them in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 5 min each. Rinse with RO water for 5 min. Place the sections in 1% sodium citrate and incubate at 100°C for 20 min for antigen retrieval, then allow to cool naturally to room temperature. Block endogenous peroxidase activity with 3% hydrogen peroxide, and rinse three times with PBS solution for 5 min each time.
[0079] (2)~(8) are the same as in Example 5.
[0080] If a large number of ganglion cells expressing SCGN are visible in the section, along with fine nerve fibers stained positive for PGP9.5 and S100, it can be determined to be a normal rectum / colon.
[0081] If no ganglion cells clearly expressing SCGN are seen in the section, but thick nerve fibers positively stained with PGP9.5 and S100 are also seen, it is determined to be congenital megacolon.
[0082] Experimental Example 1
[0083] This experimental example illustrates the process of screening differentially expressed proteins using proteomics in colon tissue samples from children with congenital megacolon and other control groups with intestinal diseases, as described in this invention.
[0084] The colon tissue samples were divided into a "congenital megacolon group (6 cases)" and a "control group of other intestinal diseases" (including colon tissue samples from anal stenosis and intestinal stenosis stomas, totaling 3 cases). Proteomics sequencing was performed by Shanghai Genomics.
[0085] The differential gene results obtained from the analysis are shown in Figure 1. 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).
[0086] Experimental Example 2
[0087] This experimental case was used to verify the feasibility of using SCGN in combination with PGP9.5 and / or S100 to diagnose congenital megacolon.
[0088] The samples were divided into a "congenital megacolon group" and a "control group (megacolon-related disease)". Following the method in Example 5, corresponding rectal mucosal biopsy sections and colonic full-thickness biopsy pathological sections were prepared. The sections were photographed under a microscope, and the results are shown in Figures 2 and 3.
[0089] Figure 2 shows the staining results of SCGN, PGP9.5, and S100 in rectal mucosal biopsy sections from the congenital megacolon group and the control group (Figure A shows the SCGN staining result in the congenital megacolon group, Figure B shows the SCGN staining result in the control group, Figure C shows the PGP9.5 staining result in the congenital megacolon group, Figure D shows the PGP9.5 staining result in the control group, Figure E shows the S100 staining result in the congenital megacolon group, and Figure F shows the S100 staining result in the control group). Figure 3 shows the staining results of SCGN, PGP9.5, and S100 in full-thickness colonic biopsy pathological sections from the congenital megacolon group and the control group (Figure A shows the SCGN staining result in the congenital megacolon group, Figure B shows the SCGN staining result in the control group, Figure C shows the PGP9.5 staining result in the congenital megacolon group, Figure D shows the PGP9.5 staining result in the control group, Figure E shows the S100 staining result in the congenital megacolon group, and Figure F shows the S100 staining result in the control group).
[0090] As shown in Figure 2, a large number of ganglion cells expressing SCGN are visible in the normal rectal submucosa; however, no significant expression is observed in the rectal submucosa of patients with congenital megacolon. In the normal colonic submucosa, fine nerve fibers stained positive for PGP9.5 and S100 are visible; in the rectal submucosa of patients with congenital megacolon, large nerve fibers (≥40 μm) stained positive for PGP9.5 and S100 are visible.
[0091] As shown in Figure 3, a large number of ganglion cells expressing SCGN are visible in the normal colonic muscle layer; however, no significant expression is observed in the colonic muscle layer of patients with congenital megacolon. Fine nerve fibers stained positive for PGP9.5 and S100 are visible in the normal colonic muscle layer; while large nerve fibers (≥40 μm) stained positive for PGP9.5 and S100 are visible in the colonic muscle layer of patients with congenital megacolon.
[0092] Experimental Example 3
[0093] This experimental example is used to test the diagnostic efficacy of the present invention SCGN combined with PGP9.5 and / or S100:
[0094] In 321 children with constipation, different combinations of diagnostic markers of the present invention were used to stain rectal mucosal biopsy specimens by immunohistochemistry, and the diagnostic sensitivity and specificity were calculated.
[0095] Specifically, the kits of Example 1 (based on the combination of SCGN+PGP9.5+S100 markers), Example 2 (based on the combination of SCGN+PGP9.5 markers), and Example 3 (based on the combination of SCGN+S100 markers) were combined with the corresponding diagnostic methods (refer to Examples 5-7) as examples. At the same time, the existing detection scheme (calciretinal protein + S100 + PGP9.5) and the detection method based solely on SCGN markers (the detection reagent uses a single monoclonal antibody targeting human SCGN as the primary antibody, and the rest is the same as in Example 1) were used as controls.
[0096] The results are shown in Tables 1-1 and 1-2.
[0097] Table 1-1 Diagnostic efficacy (sensitivity, specificity) of different combinations of diagnostic markers
[0098]
[0099] In the table, CI represents the confidence interval.
[0100] Table 1-2 Diagnostic efficacy (positive and negative predictive values) of different combinations of diagnostic markers
[0101]
[0102] In the table, CI represents the confidence interval.
[0103] As shown in Tables 1-1 and 1-2, the use of SCGN combined with PGP9.5 and S100 immunohistochemical staining has the highest diagnostic efficacy for the diagnosis of congenital megacolon in rectal mucosal biopsy specimens, and is superior to existing detection methods.
[0104] The above description is merely 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 within the protection scope of the present invention.
Claims
1. A combination of biomarkers for diagnosing congenital megacolon, characterized in that, It can be any one of combination 1, combination 2, or combination 3; combination 1 is a combination of SCGN, PGP9.5, and S100; combination 2 is a combination of SCGN and PGP9.5; combination 3 is a combination of SCGN and S100. The SCGN is used to identify and label enteric ganglion cells, and PGP9.5 and / or S100 are used to label enteric nerve fibers.
2. The application of the biomarker combination as described in claim 1 in the preparation of a diagnostic reagent for congenital megacolon.
3. The application according to claim 2, characterized in that, The diagnostic reagents for congenital megacolon are based on immunohistochemistry or immunofluorescence.
4. An immunohistochemical kit for diagnosing congenital megacolon, characterized in that, It includes a primary antibody reagent and a secondary antibody reagent; wherein, the primary antibody reagent is one of the following: 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; the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.
5. The immunohistochemistry kit according to claim 4, characterized in that, It also includes color developers and counterstains.
6. The immunohistochemical reagent kit according to claim 5, characterized in that, The color developer is DAB color developer solution, and the counterstaining agent is hematoxylin counterstaining solution.
7. The immunohistochemical reagent kit according to claim 4, characterized in that, It also includes antigen retrieval solutions, blocking agents, and sealing agents.
8. The immunohistochemical reagent kit according to claim 7, characterized in that, The antigen retrieval solution is EDTA and / or sodium citrate, the blocking agent is bovine serum albumin, and the blocking agent is hydrogen peroxide.