Kit of SND1 protein and application thereof

Abstract

The invention discloses an SND1 protein kit and application thereof. The SND1 protein kit comprises an elisa plate coated with a monoclonal capture antibody X, an ALP-labeled monoclonal detection antibody Y, an SND1 standard substance and a substrate, the monoclonal capture antibody X and the detection antibody Y respectively comprise a heavy chain variable region and a light chain variable region, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID NO.1-14 and SEQ ID NO.29-42; the heavy chain variable region comprises heavy chains FR1, FR2, FR3 and FR4 and heavy chains CDR1, CDR2 and CDR3, and the light chain variable region comprises light chains FR1, FR2, FR3 and FR4 and light chains CDR1, CDR2 and CDR3. The monoclonal capture antibody X and the monoclonal detection antibody Y which are more suitable for early diagnosis of human serum and plasma SND1 tumors are prepared, and the early diagnosis kit capable of effectively detecting the human serum and plasma SND1 is developed.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a kit for preparing SND1 protein and application thereof. Background Art

[0002] As a serious threat to human health, cancer not only brings a heavy burden to society, but also its incidence rate is rising. Unfortunately, many patients are already in the middle and late stages when diagnosed, which highlights the importance of early detection and helps implement more effective treatment plans, thereby improving patients' chances of survival and quality of life. Tumor markers refer to a class of biomarkers that can characterize the characteristics and state of tumor cells. They can be detected in patients' body fluids or tissue samples. The use of these markers for detection is crucial to assist in the early identification of tumors, thereby supporting customized treatment strategies, enhancing treatment effects and improving prognosis. Given the advantages of tumor marker testing, such as simplicity and speed, easy sample collection, high cost-effectiveness, and convenience for continuous monitoring of disease changes, exploring efficient and reliable tumor markers is particularly critical for promoting early diagnosis and treatment of cancer.

[0003] Although existing tumor markers have shown certain value in clinical applications, they are usually only applicable to the identification of specific types of tumors, and sometimes multiple indicators must be combined to draw accurate conclusions, which to a certain extent restricts their practicality and accuracy in early screening. SND1 (Staphylococcal Nuclease and Tudor Domain Containing 1) protein is regarded as a potential new biomarker due to its widespread high expression in various cancer cells and its unique property of being released into the blood via exosomes. It is expected to overcome the shortcomings of traditional methods and achieve more comprehensive and accurate early detection of cancer.

[0004] Although there are currently products that display test kits for SND1 protein, they cannot actually be applied to the detection of human serum and plasma. Or the information provided by the test kits is untrue, or they are only tested under laboratory conditions. There is a lack of clinical tumor early diagnosis kits that can truly detect SND1 protein in human serum and plasma. Summary of the invention

[0005] The object of the present invention is to provide a kit for SND1 protein and its application, so as to solve the technical problem that there is a lack of a tumor early diagnosis kit for detecting SND1 protein in human serum and plasma in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a kit for SND1 protein, comprising an ELISA plate coated with a monoclonal capture antibody X, an ALP-labeled monoclonal detection antibody Y, an SND1 standard, and a substrate, wherein the monoclonal capture antibody X comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.1-14, wherein the heavy chain variable region comprises heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, and CDR3, and the light chain variable region comprises light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, and CDR3.

[0007] Furthermore, the monoclonal capture antibody X and detection antibody Y can efficiently identify the human tumor-related protein SND1 in serum and plasma. The brief process is as follows:

[0008] Based on an in-depth analysis of the spatial conformational characteristics of the SND1 antigen epitope, the 682-767aa (amino acid) fragment of the C-terminal Tudor domain of the SND1 protein was selected as the immunogen for preparing the monoclonal capture antibody X, and the 8-140aa fragment of the N-terminal SN1 domain was selected as the immunogen for preparing the monoclonal detection antibody Y.

[0009] After the immunogen was injected into mice, the mice were successfully stimulated to produce specific B lymphocytes targeting specific epitopes of SND1.

[0010] These B cells are isolated from mice and fused with myeloma cells to form hybridoma cells that can produce monoclonal antibodies.

[0011] The surviving hybridoma cells are selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned by limiting dilution method to ensure that positive hybridoma cells secreting the desired monoclonal antibody are obtained.

[0012] The variable regions of the antibodies encoded by the hybridoma cells were sequenced to determine the variable region base sequences, amino acid sequences and antibody subtypes of the capture antibody and detection antibody.

[0013] Based on this information, antibody expression plasmids were constructed using genetic engineering methods, and CHO eukaryotic cell lines were used to express and purify antibodies.

[0014] Finally, the specificity and sensitivity of antibody recognition were determined by Western blotting.

[0015] Furthermore, the amino acid sequence of the variable region of the monoclonal capture antibody X is a heavy chain variable region and a light chain variable region as shown in SEQ ID NO.1-14, wherein the heavy chain variable region includes heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, and CDR3, and the light chain variable region includes light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, and CDR3.

[0016] Furthermore, the variable region base sequence of the monoclonal capture antibody X is shown in SEQ ID NO.15-28.

[0017] Furthermore, the monoclonal detection antibody Y includes a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.29-42, the heavy chain variable region includes heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, CDR3, and the light chain variable region includes light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, CDR3.

[0018] Furthermore, the variable region base sequence of the monoclonal capture antibody Y is shown in SEQ ID NO.43-56.

[0019] Furthermore, the SND1 standard is a standard of human tumor-related protein SND1 prepared using a mammalian expression system. The brief process is as follows:

[0020] Using CHO eukaryotic cells as a tool, SND1 protein with a His tag was expressed through a mammalian expression system.

[0021] The protein samples were purified using the AKTA-nickel column system to remove impurities and improve the purity of the target protein.

[0022] The His tag was cleaved by enterokinase, and the size and purity of the SND1 standard were determined by Coomassie blue staining.

[0023] Furthermore, the kit for the SND1 protein is divided into a SND1-ELISA (enzyme-linked immunosorbent assay) kit and a SND1-CLEIA (chemiluminescent enzyme immunoassay) kit: The substrate in the SND1-ELISA kit is disodium 4-nitrophenylphosphate; The substrate in the SND1-CLEIA kit is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane disodium salt (AMPPD).

[0024] The present invention also provides an application of a kit for detecting the content of SND1 protein in detecting the content of SND1 protein. The kit is used for detecting the content of SND1 protein in human serum and plasma.

[0025] Furthermore, the serum and plasma are serum and plasma from patients with rectal cancer, gastric cancer, colon cancer, and liver cancer.

[0026] Based on the above technical solution, the embodiments of the present invention can at least produce the following technical effects: (1) The present invention targets the novel tumor marker SND1, and based on its C-terminus (682-767 aa) and N-terminus (8-140 aa), prepares a monoclonal capture antibody X and a monoclonal detection antibody Y that are more suitable for early diagnosis of human serum and plasma SND1 tumors, and develops an early diagnosis kit that can effectively detect human serum and plasma SND1 for use in early screening and diagnosis of tumors.

[0027] (2) The ELISA and CLEIA kits of the present invention will not only provide powerful tools for basic medical research under laboratory conditions, but more importantly, they have great potential for clinical application. The kits will provide doctors with a fast, efficient and cost-effective means of tumor screening, diagnosis and evaluation.

[0028] (3) The present invention adopts a mammalian cell expression system and uses enterokinase technology to accurately remove the His tag, so that the SND1 standard can more accurately simulate the tumor-related protein SND1 in human serum and plasma, which not only improves the linear detection range of the standard curve, but also significantly enhances the accuracy of detection.

[0029] (4) R of the ELISA kit of the present invention 2 The value reached 0.9986, and the R 2 The value reached 0.9987. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the immunogenic information and Western blot identification diagram of the monoclonal capture antibody X according to the embodiment of the present invention.

[0031] Figure 2 It is the immunogenic information and protein blotting identification diagram of the monoclonal detection antibody Y in the embodiment of the present invention.

[0032] Figure 3 This is the Coomassie brilliant blue staining result of the human tumor-related protein SND1 standard product according to the embodiment of the present invention.

[0033] Figure 4 It is the ELISA standard curve result diagram of the embodiment of the present invention.

[0034] Figure 5 It is a CLEIA standard curve result diagram of an embodiment of the present invention.

[0035] Figure 6 This is the clinical serum sample test result based on the ELISA kit in the embodiment of the present invention.

[0036] Figure 7 This is the clinical serum sample test result based on the CLEIA kit in the embodiment of the present invention. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] ① Compared with the invention of CN118818050A, based on an in-depth analysis of the spatial conformation of the SND1 antigen epitope, the 682-767 amino acid fragment of the C-terminal Tudor domain of the SND1 protein was selected as the immunogen for preparing the monoclonal capture antibody X, and the 8-140 amino acid fragment of the N-terminal SN1 domain was selected as the immunogen for preparing the monoclonal detection antibody Y, and the prepared monoclonal antibodies are different.

[0043] ②The invention CN118818050A only detected mouse serum samples and did not involve the detection of SND1 in clinical human serum.

[0044] ③ The invention of CN118818050A uses a bacterial culture system to produce a His-tagged SND1 protein standard. The present invention uses a mammalian expression system to prepare a human tumor-related protein SND1 standard that is closer to the natural conformation. The His tag is precisely removed by using enterokinase technology. This improvement enables the SND1 standard to more accurately simulate the SND1 protein in human serum and plasma. This optimization not only improves the linear detection range of the standard curve, but also significantly enhances the accuracy of the detection (see Figure 3 ).

[0045] ④ The present invention adopts ALP labeling, and the R of ELISA kit 2 The value reached 0.9986, and the R 2 The value reached 0.9987.

[0046] Example 1 Preparation of monoclonal capture antibody X and detection antibody Y capable of efficiently identifying human tumor-related protein SND1 in serum Based on an in-depth analysis of the spatial conformational characteristics of the SND1 antigen epitope, the 682-767aa (amino acid) fragment of the C-terminal Tudor domain of the SND1 protein was selected as the immunogen for preparing the monoclonal capture antibody X ( Figure 1 ), and the 8-140aa fragment of the N-terminal SN1 domain was used as the immunogen for the preparation of the monoclonal detection antibody Y ( Figure 2 ).

[0047] Brief process: After these immunogens were injected into mice, the mice were successfully stimulated to produce specific B lymphocytes targeting specific epitopes of SND1.

[0048] These B cells were then isolated from the mice and fused with myeloma cells to form hybridoma cells that can produce monoclonal antibodies.

[0049] The surviving hybridoma cells are screened in HAT medium and cloned using the limiting dilution method to ensure that positive hybridoma cells secreting the desired monoclonal antibody are obtained.

[0050] The variable regions of the antibodies encoded by the hybridoma cells were sequenced to determine the variable region amino acid sequences, base sequences (Tables 1-4) and antibody subtypes of the capture and detection antibodies.

[0051] Based on this information, antibody expression plasmids were constructed using genetic engineering methods, and CHO eukaryotic cell lines were used to express and purify antibodies.

[0052] Finally, the specificity and sensitivity of antibody recognition were determined by Western blotting ( Figure 1 , Figure 2 ).

[0053] Result description: Mouse monoclonal capture antibody X and detection antibody Y were successfully prepared. The corresponding variable region amino acid sequences are shown in Table 1 and Table 3, and the base sequences are shown in Table 2 and Table 4. The subtype of capture antibody X is IgG1, and the subtype of detection antibody Y is IgG2. Western blotting results ( Figure 1 , Figure 2 ) showed that both antibodies could specifically recognize WT (wild type) human melanoma cells A375 and human cervical cancer cells HeLa, but no obvious band signal was detected in the cell samples with SND1 knocked out (KO).

[0054] Table 1 Amino acid sequence information of the variable region of monoclonal capture antibody X Note: The sequence is shown in SEQ ID NO.1-14 Table 2: Variable region base sequence information of monoclonal capture antibody X Note: The sequence is shown in SEQ ID NO.15-28 Table 3: Variable region amino acid sequence information of monoclonal detection antibody Y Note: The sequence is shown in SEQ ID NO. 29-42 Table 4: Variable region base sequence information of monoclonal detection antibody Y Note: The sequence is shown in SEQ ID NO.43-56 Example 2 Preparation of human tumor-related protein SND1 standard product simulating native conformation using mammalian expression system The mammalian cell expression system can provide post-translational modifications that are very similar to the human body environment, so that the activity of the produced protein is closer to that of the natural protein. Therefore, the mammalian expression system was used to prepare the standard of the human tumor-related protein SND1.

[0055] Brief process: First, CHO eukaryotic cells were used as a tool to express the His-tagged SND1 protein through a mammalian expression system; The protein sample was then purified using an AKTA-nickel column system to remove impurities and improve the purity of the target protein; Enterokinase was then used to cleave the His tag, and Coomassie Brilliant Blue staining was used to determine the size and purity of the SND1 standard ( Figure 3 ); Finally, standard curves of the target protein were drawn based on the developed ELISA and CLEIA kits ( Figure 4 , Figure 5 ).

[0056] Result description: Coomassie Brilliant Blue staining results show that the human tumor-related protein SND1 obtained through mammalian cell expression and subsequent purification system has the correct size and position and high purity ( Figure 3 The standard curve plotting results showed that the standard curve can be successfully plotted using the human SND1 standard. 2 The value reaches 0.9986 ( Figure 4 ), R of CLEIA kit 2 The value reached 0.9987, and the latter had a wider detection linear range ( Figure 5 ).

[0057] Example 3 SND1-ELISA kit 1. Preparation of capture and detection antibodies Targeting the novel tumor marker SND1, based on the antigen recognition epitopes at its C-terminus (682-767aa) and N-terminus (8-140aa), the immunogens of monoclonal capture antibody X and monoclonal capture antibody Y were obtained respectively. After these immunogens were injected into mice, the mice were successfully stimulated to produce specific B lymphocytes targeting the specific epitope of SND1. Subsequently, these B cells were isolated from the mice and fused with myeloma cells to form hybridoma cells capable of producing monoclonal antibodies. By screening the surviving hybridoma cells in HAT medium and cloning and culturing them by limiting dilution method, it was ensured that positive hybridoma cells secreting the desired monoclonal antibodies were obtained. The variable regions of the antibodies encoded by the hybridoma cells were sequenced to determine the amino acid sequences, base sequences (Table 1-4) and antibody subtypes of the variable regions of the capture antibody and the detection antibody. Based on this information, antibody expression plasmids were constructed by genetic engineering, and the CHO eukaryotic cell line was used for antibody expression and purification. The specificity and sensitivity of antibody recognition were determined by Western blotting technology ( Figure 1 , Figure 2 Finally, the monoclonal capture antibody X was coated in a 96-well plate and blocked; the monoclonal detection antibody Y was labeled with ALP.

[0058] 2. Sample preparation: Collect whole blood into a test tube without anticoagulant, place at room temperature for 1 hour, wait for the whole blood to coagulate naturally, centrifuge at about 1500g for 10 minutes at 4℃, and take the yellow supernatant to obtain the serum sample.

[0059] 3. SND1 standard: Figure 3 As shown, the purified human SND1 protein was obtained and the SND1 protein standard with gradient concentrations was prepared.

[0060] 4. Sample loading: Dilute the sample 5-10 times with diluent. Add the sample and standard into the corresponding wells coated with monoclonal capture antibody X at 100 μL / well as the test group and standard group, respectively, and cover with sealing film.

[0061] 5. Add detection antibody Y: incubate at 37℃ for 45min, discard the liquid, and pat dry on thick absorbent paper; add 300μL rinsing solution to wash the plate for 1 min×3 times, and pat dry on thick absorbent paper each time. Add 100μL / well of monoclonal detection antibody Y conjugated to ALP to all wells, cover with sealing film, and incubate at 37℃ in the dark for 45min.

[0062] 6. Add substrate: discard the liquid and pat dry on thick absorbent paper; add 300μL of rinse solution to each well and wash the plate for 1min×3 times, placing it on thick absorbent paper and pat dry each time. Add 100μL of 4-nitrophenylphosphate disodium colorimetric solution to each well and color develop for 10min at room temperature away from light.

[0063] 7. Detection and analysis: Add 100 μL of stop solution and measure the A405 value with an ELISA instrument. Draw the SND1 standard curve with the standard concentration as the horizontal axis and the A405 value as the vertical axis ( Figure 4 ). The corresponding concentration of the sample is calculated by the absorbance value of the sample and the standard curve ( Figure 6 ), the results showed that compared with the normal control group, the SND1 concentration in the serum of tumor patients was higher, and the difference was statistically significant.

[0064] Example 4 SND1-CLEIA Kit 1. Preparation of capture and detection antibodies Targeting the novel tumor marker SND1, based on the antigen recognition epitopes at its C-terminus (682-767 aa) and N-terminus (8-140 aa), the immunogens of monoclonal capture antibody X and monoclonal capture antibody Y were obtained respectively. After these immunogens were injected into mice, the mice were successfully stimulated to produce specific B lymphocytes targeting the specific epitope of SND1. Subsequently, these B cells were isolated from the mice and fused with myeloma cells to form hybridoma cells capable of producing monoclonal antibodies. By screening the surviving hybridoma cells in HAT medium and cloning and culturing them by limiting dilution method, it was ensured that positive hybridoma cells secreting the desired monoclonal antibodies were obtained. The variable regions of the antibodies encoded by the hybridoma cells were sequenced, and the variable region amino acid sequences, base sequences (Table 1-4) and antibody subtypes of the capture antibody and detection antibody were determined. Based on this information, antibody expression plasmids were constructed by genetic engineering, and the CHO eukaryotic cell line was used for antibody expression and purification. The specificity and sensitivity of antibody recognition were determined by Western blotting technology ( Figure 1 , Figure 2 Finally, the monoclonal capture antibody X was coated in a 96-well plate and blocked; the monoclonal detection antibody Y was labeled with ALP.

[0065] 2. Sample preparation: Collect whole blood into a test tube without anticoagulant, place at room temperature for 1 hour, wait for the whole blood to coagulate naturally, centrifuge at about 1500g for 10 minutes at 4℃, and take the yellow supernatant to obtain the serum sample.

[0066] 3. SND1 standard: Figure 3 As shown, the purified SND1 protein was obtained. SND1 protein standards with gradient concentrations were prepared.

[0067] 4. Sample loading: Dilute the sample 5-10 times with diluent. Add the sample and standard into the corresponding wells coated with monoclonal capture antibody X at 100 μL / well as the test group and standard group, respectively, and cover with sealing film.

[0068] 5. Add detection antibody Y: incubate at 37℃ for 45min, discard the liquid, and pat dry on thick absorbent paper; add 300μL rinsing solution to wash the plate for 1 min×3 times, and pat dry on thick absorbent paper each time. Add 100μL / well of monoclonal detection antibody Y conjugated to ALP to all wells, cover with sealing film, and incubate at 37℃ in the dark for 45min.

[0069] 6. Add substrate: discard the liquid and pat dry on thick absorbent paper; add 300 μL of rinse solution to each well and wash the plate for 1 min × 3 times, placing it on thick absorbent paper and pat dry each time. Add AMPPD enzymatic chemiluminescent substrate to each well, and the enzyme catalyzes the decomposition of the substrate to generate a light signal.

[0070] 7. Detection and analysis: Measure the chemiluminescence value with a chemiluminescence immunoassay. Draw the SND1 standard curve with the standard concentration as the horizontal axis and the chemiluminescence detection value as the vertical axis ( Figure 5 ). The corresponding concentration of the sample is calculated by the chemiluminescence detection value of the sample and the standard curve ( Figure 7 ), the results showed that compared with the normal control group, the SND1 concentration in the serum of tumor patients was higher, and the difference was statistically significant.

[0071] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for SND1 protein, characterized in that: The invention comprises an enzyme labeling plate coated with a monoclonal capture antibody X, an ALP-labeled monoclonal detection antibody Y, an SND1 standard, and a substrate. The monoclonal capture antibody X and the detection antibody Y respectively comprise a heavy chain variable region and a light chain variable region with amino acid sequences as shown in SEQ ID NO.1-14 and SEQ ID NO.29-42, the heavy chain variable region comprises heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, and CDR3, and the light chain variable region comprises light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, and CDR3.

2. The kit for the SND1 protein according to claim 1, characterized in that: The monoclonal capture antibody X comprises a heavy chain variable region and a light chain variable region as shown in the amino acid sequences of SEQ ID NO.1-14, wherein the heavy chain variable region comprises heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, CDR3, and the light chain variable region comprises light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, CDR3.

3. The kit for the SND1 protein according to claim 1, characterized in that: The variable region base sequences of the monoclonal capture antibody X are shown in SEQ ID NOs. 15-28.

4. The kit for the SND1 protein according to claim 1, characterized in that: The monoclonal detection antibody Y includes a heavy chain variable region and a light chain variable region as shown in the amino acid sequences of SEQ ID NO.29-42, wherein the heavy chain variable region includes heavy chain FR1, FR2, FR3, FR4 and heavy chain CDR1, CDR2, CDR3, and the light chain variable region includes light chain FR1, FR2, FR3, FR4 and light chain CDR1, CDR2, CDR3.

5. The kit for the SND1 protein according to claim 1, characterized in that: The variable region base sequences of the monoclonal capture antibody Y are shown in SEQ ID NOs. 43-56.

6. The kit for the SND1 protein according to claim 1, characterized in that: The substrates include disodium 4-nitrophenyl phosphate and AMPPD enzymatic chemiluminescent substrate.

7. Use of the kit for detecting the content of SND1 protein according to any one of claims 1 to 6, characterized in that: The kit is used to detect the content of SND1 protein in human serum and plasma.

8. Use of the kit for detecting the content of SND1 protein according to claim 7, characterized in that: The human serum and plasma are serum and plasma from patients with rectal cancer, gastric cancer, colon cancer, and liver cancer.

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