Biomarker combination, kit and application for diagnosing Hirschsprung's disease

Abstract

The present invention provides a biomarker combination for diagnosing Hirschsprung's disease, which is any one of combination 1, combination 2, and 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; the combination 3 is a combination of SCGN and S100. The present invention also provides the application of the above biomarker combination in the preparation of a diagnostic reagent for Hirschsprung's disease. At the same time, an immunohistochemistry kit is provided. The present invention discovers that SCGN can effectively identify ganglion cells in rectal mucosal biopsy specimens / entire colon specimens of children with Hirschsprung's disease, and the diagnostic sensitivity and specificity are higher than the existing detection schemes after combining with PGP9.5 and / or S100; the present invention provides a new, accurate, and sensitive detection method for the diagnosis of Hirschsprung's disease.

Description

Technical Field

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

[0002] Hirschsprung's disease is one of the most common surgical diseases causing childhood constipation and belongs to congenital digestive tract malformations. The incidence varies greatly among different ethnic groups. As one of the regions with the highest incidence of neonatal HD, the incidence of live births in China is about 2.8 / 10,000.

[0003] Rectal mucosal biopsy is the most sensitive and specific diagnostic method before Hirschsprung's disease surgery, which is diagnosed by determining the number of submucosal ganglion cells and the thickness and shape of nerve fibers. However, during the implementation process, the smaller the tissue obtained due to less damage to the patient during mucosal biopsy, the higher the difficulty of pathological diagnosis. The development of acetylcholinesterase histochemical method has significantly promoted the diagnosis of rectal mucosal biopsy for Hirschsprung's disease. The basic histological feature of Hirschsprung's disease is the congenital absence of ganglion cells in the affected intestinal tract and the increased expression of acetylcholinesterase. Although the acetylcholinesterase histochemical method has high accuracy, there are still a certain proportion of false positive and false negative results. Different biopsy sites, immature enzyme systems and technical differences may all lead to incorrect acetylcholinesterase test results. In addition, the limitations of acetylcholinesterase staining also include: (1) it needs to be carried out on frozen sections, and the diagnostic accuracy is lower than that of paraffin sections; (2) there are immature nerve fibers in some Hirschsprung's disease specimens, 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 staining multiple indicators can diagnose the cause by locating the number of ganglion cells in the mucosa and observing the shape and distribution of nerve fibers, which improves the accuracy while reducing the diagnostic trauma. However, there are currently no mature and efficient diagnostic markers and diagnostic methods for identifying intestinal ganglion cells and nerve fibers on the market.

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

[0006] To solve the problem of the 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 Hirschsprung's disease, providing a new, accurate and sensitive detection method for the diagnosis of Hirschsprung's disease.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A biomarker combination for diagnosing Hirschsprung's disease, which is any one of combination 1, combination 2, and combination 3; wherein, the combination 1 is a combination of SCGN (secretin), PGP9.5 (brain-specific protein product 9.5), and S100 (S100 protein); the combination 2 is a combination of SCGN (secretin) and PGP9.5 (brain-specific protein product 9.5); the combination 3 is a combination of SCGN (secretin) and S100 (S100 protein).

[0009] Use of the above biomarker combination in the preparation of a diagnostic reagent for Hirschsprung's disease.

[0010] As one of the preferred embodiments of the present invention, the diagnostic reagent for Hirschsprung's disease is based on immunohistochemistry or immunofluorescence.

[0011] An immunohistochemistry kit, comprising a primary antibody reagent and a secondary antibody reagent; wherein, the primary antibody reagent is one of a combination of anti-human SCGN monoclonal antibody, anti-human PGP9.5 monoclonal antibody, and anti-human S100 monoclonal antibody, a combination of anti-human SCGN monoclonal antibody and anti-human PGP9.5 monoclonal antibody, or a combination of anti-human SCGN monoclonal antibody and anti-human S100 monoclonal antibody; the secondary antibody reagent is HRP-labeled goat anti-rabbit IgG.

[0012] As one of the preferred embodiments of the present invention, it further comprises a chromogenic agent and a counterstain.

[0013] As one of the preferred embodiments of the present invention, the chromogenic agent is a DAB chromogenic solution, and the counterstain is a hematoxylin counterstain solution.

[0014] As one of the preferred embodiments of the present invention, it further comprises an antigen retrieval solution, a blocking agent, and a blocking buffer.

[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 buffer is hydrogen peroxide.

[0016] Design principle:

[0017] There are two criteria for the diagnosis of Hirschsprung's disease (mucosal biopsy): (1) the absence of ganglion cells in the submucosa; (2) thick nerve fibers. The present invention uses SCGN to identify ganglion cells and simultaneously uses PGP9.5 and / or S100 to observe the thickness of nerve fibers, thereby improving the diagnostic accuracy.

[0018] The advantages of the present invention compared with the prior art are as follows:

[0019] The present invention discovers that SCGN can effectively identify ganglion cells in rectal mucosal biopsy specimens / entire colon specimens of children with Hirschsprung's disease, and the diagnostic sensitivity and specificity are higher than those of existing detection methods after combining with PGP9.5 and / or S100; the diagnostic method of the present invention has a diagnostic sensitivity of 98.99% (95%CI, 97.02% - 100.00%) for Hirschsprung's disease in 321 constipated children, 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%); the present invention provides a new, accurate, and sensitive detection method for the diagnosis of Hirschsprung's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a result diagram of proteomics screening for diagnostic markers of children with Hirschsprung's disease in Experimental Example 1;

[0021] Figure 2 It is the result of staining SCGN, PGP9.5, and S100 in rectal mucosal biopsy sections of the Hirschsprung's disease group and the control group in Experimental Example 2 (in the figure, Figure A is the SCGN staining result of the Hirschsprung's disease group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung's disease group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung's disease group, and Figure F is the S100 staining result of the control group; brown indicates positive staining);

[0022] Figure 3 It is the result of staining SCGN, PGP9.5, and S100 in entire colon biopsy pathological sections of the Hirschsprung's disease group and the control group in Experimental Example 2 (in the figure, Figure A is the SCGN staining result of the Hirschsprung's disease group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung's disease group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung's disease group, and Figure F is the S100 staining result of the control group; brown indicates positive staining). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. In the following embodiments:

[0024] Monoclonal antibody targeting human SCGN: Abclonal A19615 is 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: MXB biotechnologies KIT-9902 was used;

[0028] In addition, unless otherwise specified, the reagents used in the following examples 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 elaborated.

[0029] Example 1

[0030] An immunohistochemistry kit in this example includes primary antibody reagent, secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstain solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). Among them, 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 HRP-labeled goat anti-rabbit IgG.

[0031] The kit in this example is used for the diagnostic detection of Hirschsprung's disease, and during the detection, it is based on the biomarker combination of "SCGN + PGP9.5 + S100".

[0032] Example 2

[0033] An immunohistochemistry kit in this example includes primary antibody reagent, secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstain solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). Among them, the primary antibody reagent is 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 example is used for the diagnostic detection of Hirschsprung's disease, and during the detection, it is based on the biomarker combination of "SCGN + PGP9.5".

[0035] Example 3

[0036] An immunohistochemistry kit in this example includes primary antibody reagent, secondary antibody reagent, DAB chromogenic solution, hematoxylin counterstain solution, EDTA (antigen retrieval solution), hydrogen peroxide (blocking agent), and bovine serum albumin (blocking agent). Among them, the primary antibody reagent is 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 of this example is used for the diagnostic detection of Hirschsprung's disease, and when detecting, it is based on the biomarker combination of "SCGN + S100".

[0038] Example 4

[0039] An immunohistochemistry kit of this example is basically the same as that of Example 1, and the main difference is that: sodium citrate is used as the antigen retrieval solution.

[0040] Example 5

[0041] A method for diagnosing Hirschsprung's disease in this example is based on the immunohistochemistry kit of Example 1, and the operation is as follows:

[0042] (1) Deparaffinization, hydration and antigen retrieval of tissue sections

[0043] Take a pathological section of a clinical rectal mucosal biopsy or a full-thickness colon biopsy, put the section into xylene, soak for 15 min, 2 times. Subsequently, place it in absolute ethanol, 95% ethanol, 80% ethanol and 75% ethanol for 5 min in sequence. Rinse with RO water for 5 min. Place the section in 2% EDTA, perform antigen retrieval in a water bath at 100 °C for 20 min, and cool to room temperature naturally. Use 3% hydrogen peroxide to block the activity of endogenous peroxidase, and rinse with PBS solution 3 times, 5 min each time.

[0044] (2) Blocking

[0045] Drop 50 μL of bovine serum albumin onto the section, incubate at 37 °C for 20 min, discard the excess liquid, and do not wash.

[0046] (3) Incubating with the primary antibody

[0047] Incubate the section with a monoclonal antibody targeting human SCGN (diluted 1:200), a monoclonal antibody targeting human PGP9.5 (diluted 1:200), and a monoclonal antibody targeting human S100 (diluted 1:200) overnight at 4 °C or for 2 h at 37 °C. Rinse with PBS solution 3 times, 5 min each time.

[0048] (4) Incubating with the secondary antibody

[0049] Drop a total of 50 μL of an HRP-labeled goat anti-rabbit IgG antibody (ready-to-use, no dilution required) onto the tissue section, incubate at 37 °C for 30 min, and rinse with PBS solution 3 times, 5 min each time.

[0050] (5) Color development

[0051] Drop 50 μL of DAB staining solution onto the section, incubate at room temperature for 3 - 10 min, and rinse with PBS solution once for 5 min.

[0052] (6) Counterstaining

[0053] Counterstain the tissue sections with hematoxylin for 1 min, wash with water for 30 - 40 s, rinse with PBS for 30 s, add hydrochloric acid alcohol for differentiation for 10 s, and rinse with PBS solution for 1 min.

[0054] (7) Dehydration, clearing, and mounting with resin mounting medium

[0055] Place the sections successively in 75% ethanol, 80% ethanol, 95% ethanol, and absolute ethanol for 5 min, then soak in xylene for 5 min, twice. After air drying in a fume hood, mount the sections with resin mounting medium.

[0056] (8) Microscopic photographing and observation

[0057] Take microscopic photographs of the mounted sections to observe whether ganglion cells positively expressing SCGN and thick nerve fibers are visible.

[0058] If a large number of ganglion cells positively expressing SCGN and thin nerve fibers positively stained for PGP9.5 are visible in the sections, it is judged as normal rectum / colon.

[0059] If no ganglion cells significantly expressing SCGN are visible in the sections, and thick nerve fibers positively stained for PGP9.5 and S100 are visible, it is judged as Hirschsprung's disease.

[0060] Example 6

[0061] A method for diagnosing Hirschsprung's disease in this example, based on the immunohistochemistry kit of Example 2, is operated as follows:

[0062] (1) The same as Example 5.

[0063] (2) The same as Example 5.

[0064] (3) Incubate the sections with monoclonal antibody targeting human SCGN (diluted 1:200) and monoclonal antibody targeting human PGP9.5 (diluted 1:200) at 4°C overnight or at 37°C for 2 h. Rinse with PBS solution 3 times, 5 min each time.

[0065] (4) - (8) The same as Example 5.

[0066] If a large number of ganglion cells positively expressing SCGN and thin nerve fibers positively stained for PGP9.5 are visible in the sections, it is judged as normal rectum / colon.

[0067] If no ganglion cells expressing SCGN are observed in the section, and thick nerve fibers with PGP9.5 positive staining are seen at the same time, it is diagnosed as Hirschsprung's disease.

[0068] Example 7

[0069] A method for diagnosing Hirschsprung's disease in this example, based on the immunohistochemical kit of Example 3, is operated as follows:

[0070] (1) The same as Example 5.

[0071] (2) The same as Example 5.

[0072] (3) Incubate the section with the monoclonal antibody targeting human SCGN (diluted 1:200) and the monoclonal antibody targeting human S100 (diluted 1:200) overnight at 4°C or for 2 h at 37°C. Rinse with PBS solution 3 times, 5 min each time.

[0073] (4) - (8) The same as Example 5.

[0074] If a large number of ganglion cells with positive expression of SCGN are observed in the section, and fine nerve fibers with S100 positive staining are seen at the same time, it is diagnosed as normal rectum / colon.

[0075] If no ganglion cells expressing SCGN are observed in the section, and thick nerve fibers with S100 positive staining are seen at the same time, it is diagnosed as Hirschsprung's disease.

[0076] Example 8

[0077] A method for diagnosing Hirschsprung's disease in this example, based on the immunohistochemical kit of Example 4, is operated as follows:

[0078] (1) Take a pathological section of clinical rectal mucosal biopsy or full-thickness colon biopsy, put the section into xylene, soak for 15 min, 2 times. Subsequently, place it in absolute ethanol, 95% ethanol, 80% ethanol and 75% ethanol for 5 min in sequence. Rinse with RO water for 5 min. Place the section in 1% sodium citrate, perform antigen retrieval in a water bath at 100°C for 20 min, and cool to room temperature naturally. Use 3% hydrogen peroxide to block the activity of endogenous peroxidase, and rinse with PBS solution 3 times, 5 min each time.

[0079] (2) - (8) The same as Example 5.

[0080] If a large number of ganglion cells with positive expression of SCGN are observed in the section, and fine nerve fibers with PGP9.5 and S100 positive staining are seen at the same time, it is diagnosed as normal rectum / colon.

[0081] If there are no significantly SCGN-expressing ganglion cells in the section, while thick nerve fibers positive for PGP9.5 and S100 are seen, it is diagnosed as Hirschsprung's disease.

[0082] Experimental Example 1

[0083] This experimental example is used to reveal the process of proteomic screening for differential proteins in colon tissue samples of children with Hirschsprung's disease and other intestinal disease control groups:

[0084] The "colon tissue samples" were divided into the "group of children with Hirschsprung's disease (6 cases)" and the "other intestinal disease control group" (including colon tissues with anal stenosis and intestinal stenosis fistula, a total of 3 cases). Proteomic sequencing was performed by Shanghai GeneChem Co., Ltd.

[0085] The results of the differential gene analysis are as Figure 1 shown. It can be seen from it that the expression level of SCGN protein in the colon of children with Hirschsprung's disease is significantly lower than that of the control group (p = 0.008).

[0086] Experimental Example 2

[0087] This experimental example is used to verify the feasibility of diagnosing Hirschsprung's disease by combining SCGN with PGP9.5 and / or S100:

[0088] The samples were divided into the "group of children with Hirschsprung's disease" and the "control group (Hirschsprung's allied disease)". Referring to the method of Example 5, the corresponding rectal mucosal biopsy sections and full-thickness colon biopsy pathological sections were prepared respectively, and the sections were photographed under a microscope. The results are as Figure 2 、 3 shown.

[0089] Figure 2 Results of staining for SCGN, PGP9.5 and S100 in rectal mucosal biopsy sections of the Hirschsprung's disease group and the control group (in the figure, Figure A is the SCGN staining result of the Hirschsprung's disease group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung's disease group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung's disease group, and Figure F is the S100 staining result of the control group). Figure 3 Results of staining for SCGN, PGP9.5 and S100 in full-thickness colon biopsy pathological sections of the Hirschsprung's disease group and the control group (in the figure, Figure A is the SCGN staining result of the Hirschsprung's disease group, Figure B is the SCGN staining result of the control group, Figure C is the PGP9.5 staining result of the Hirschsprung's disease group, Figure D is the PGP9.5 staining result of the control group, Figure E is the S100 staining result of the Hirschsprung's disease group, and Figure F is the S100 staining result of the control group).

[0090] It can be seen from Figure 2 that: in the normal submucosa of the rectum, a large number of ganglion cells with positive expression of SCGN can be seen; in the submucosa of the rectum of patients with Hirschsprung's disease, no obvious expression can be seen. In the normal submucosa of the colon, fine nerve fibers with positive staining of PGP9.5 and S100 can be seen; in the submucosa of the rectum of patients with Hirschsprung's disease, thick nerve fibers (≥ 40 μm) with positive staining of PGP9.5 and S100 can be seen.

[0091] It can be seen from Figure 3 that: in the normal muscular layer of the colon, a large number of ganglion cells with positive expression of SCGN can be seen; in the muscular layer of the colon of patients with Hirschsprung's disease, no obvious expression can be seen. In the normal muscular layer of the colon, fine nerve fibers with positive staining of PGP9.5 and S100 can be seen; in the muscular layer of the colon of patients with Hirschsprung's disease, thick nerve fibers (≥ 40 μm) with positive staining of PGP9.5 and S100 can be seen.

[0092] Experimental Example 3

[0093] This experimental example is used to test the diagnostic efficacy of the SCGN combined with PGP9.5 and / or S100 of the present invention:

[0094] Among 321 children with constipation, different diagnostic marker combinations of the present invention were used respectively, and rectal mucosal biopsy specimens were stained by immunohistochemistry, and the diagnostic sensitivity and specificity were calculated.

[0095] Specifically, taking 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) combined with the corresponding diagnostic methods (refer to Examples 5 to 7) as examples, and at the same time using the existing detection scheme (calretinin+S100+PGP9.5) and the detection method based solely on the SCGN marker (using a monoclonal antibody targeting human SCGN alone as the primary antibody in the detection reagent, and the others are the same as in Example 1) as controls.

[0096] The results are shown in Tables 1-1 and 1-2.

[0097] Table 1-1 Diagnostic efficacy (sensitivity, specificity) under different diagnostic marker combinations

[0098]

[0099] In the table, CI represents the confidence interval.

[0100] Table 1-2 Diagnostic efficacy (positive, negative predictive values) under different diagnostic marker combinations

[0101]

[0102] In the table, CI represents the confidence interval.

[0103] As can be seen from Tables 1-1 and 1-2: Using SCGN combined with PGP9.5 and S100 immunohistochemical staining has the highest diagnostic efficacy for the diagnosis of Hirschsprung's disease in rectal mucosal biopsy specimens, and is superior to the existing detection methods.

[0104] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A biomarker combination for diagnosing Hirschsprung's disease, characterized in that: Any one of combination 1, combination 2 and 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; Wherein, the SCGN is used to identify and mark enteric ganglion cells, and PGP9.5 and / or S100 are used to mark enteric nerve fibers.

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 for diagnosing Hirschsprung's disease, 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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