A kit of snd1 protein and application thereof

Abstract

The application discloses a kit for SND1 protein and application thereof, and comprises an enzyme-labeled plate coated with a monoclonal capture antibody X, ALP-labeled monoclonal detection antibody Y, SND1 standard, a substrate, the monoclonal capture antibody X and the detection antibody Y respectively comprising a heavy chain variable region and a light chain variable region shown in amino acid sequences as shown in SEQ ID NO. 1-14 and SEQ ID NO. 29-42, the heavy chain variable region comprising a heavy chain FR1, FR2, FR3, FR4 and a heavy chain CDR1, CDR2, CDR3, and the light chain variable region comprising a light chain FR1, FR2, FR3, FR4 and a light chain CDR1, CDR2, CDR3. The application prepares the monoclonal capture antibody X and the monoclonal detection antibody Y more suitable for early diagnosis of human serum and plasma SND1 tumor, and develops an early diagnosis kit capable of effectively detecting human serum and plasma SND1.

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 applications thereof. Background Art

[0002] As a serious threat to human health, cancer not only brings a heavy burden to society, but also has an increasing incidence rate. Unfortunately, many patients are already in the middle or 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 status 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 to promoting early diagnosis and treatment of cancer.

[0003] While existing tumor markers have demonstrated clinical value, they are generally only suitable for identifying specific tumor types and sometimes require the combination of multiple indicators to reach an accurate conclusion, which to some extent limits their practicality and accuracy in early screening. SND1 (Staphylococcal Nuclease and Tudor Domain Containing 1) protein, due to its widespread high expression in various cancer cells and its unique property of being released into the blood via exosomes, is considered a potential new biomarker that may overcome the shortcomings of traditional methods and enable more comprehensive and accurate early cancer detection.

[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. Alternatively, the information provided by these test kits is false, or the tests are only conducted under laboratory conditions. There is a lack of clinical early tumor diagnosis kits that can truly detect SND1 protein in human serum and plasma. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a kit for detecting 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. The monoclonal capture antibody X comprises a heavy chain variable region and a light chain variable region with amino acid sequences as shown in SEQ ID NOs. 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.

[0008] Furthermore, the monoclonal capture antibody X and detection antibody Y can efficiently identify the human tumor-associated protein SND1 in serum and plasma. The brief process is as follows:

[0009] Based on an in-depth analysis of the spatial conformational characteristics of the SND1 antigen epitope, a 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 an 8-140aa fragment of the N-terminal SN1 domain was selected as the immunogen for preparing the monoclonal detection antibody Y.

[0010] Injecting the immunogen into mice successfully stimulated the production of B lymphocytes specific for a specific epitope of SND1. These B cells were isolated from the mice and fused with myeloma cells to form hybridoma cells capable of producing monoclonal antibodies.

[0011] The surviving hybridoma cells are screened in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned and cultured using the 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 and detection antibodies.

[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 the antibodies.

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

[0015] Furthermore, the amino acid sequences of the variable regions of the monoclonal capture antibody X are the heavy chain variable region and the light chain variable region shown in SEQ ID NOs. 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 sequences of the monoclonal capture antibody X are shown in SEQ ID NOs. 15-28.

[0017] Furthermore, the monoclonal detection antibody Y comprises a heavy chain variable region and a light chain variable region having amino acid sequences as shown in SEQ ID NOs. 29-42, 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. Furthermore, the variable region base sequences of the monoclonal capture antibody Y are shown in SEQ ID NOs. 43-56.

[0018] Furthermore, the SND1 standard is a standard of human tumor-associated protein SND1 prepared using a mammalian expression system. The brief process is as follows:

[0019] Using CHO eukaryotic cells as a tool, a His-tagged SND1 protein was expressed via a mammalian expression system. The protein sample was purified using an AKTA-nickel column system to remove impurities and improve the purity of the target protein.

[0020] The His tag was cleaved using enterokinase, and the size and purity of the SND1 standard were determined by Coomassie Brilliant Blue staining. Furthermore, the SND1 protein kit is divided into SND1-ELISA (enzyme-linked immunosorbent assay) kit and SND1-CLEIA (chemiluminescent enzyme immunoassay) kit:

[0021] The substrate in the SND1-ELISA kit is 4-nitrophenylphosphate disodium;

[0022] The substrate in the SND1-CLEIA kit is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane disodium salt (AMPPD).

[0023] The present invention also provides an application of a kit for detecting the content of SND1 protein, wherein the kit is used for detecting the content of SND1 protein in human serum and plasma.

[0024] Furthermore, the serum and plasma are serum and plasma from patients with rectal cancer, gastric cancer, colon cancer, and liver cancer.

[0025] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0026] (1) The present invention targets the novel tumor marker SND1. Based on its C-terminus (682-767aa) and N-terminus (8-140aa), a monoclonal capture antibody X and a monoclonal detection antibody Y are prepared that are more suitable for early diagnosis of SND1 tumors in human serum and plasma. An early diagnosis kit that can effectively detect SND1 in human serum and plasma is developed 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 translation into clinical applications. The kits will provide physicians with a rapid, efficient, and cost-effective means of tumor screening, diagnosis, and assessment.

[0028] (3) The present invention adopts a mammalian cell expression system and accurately removes the His tag using enterokinase technology, 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 1 is the immunogenic information and Western blot identification diagram of the monoclonal capture antibody X according to an embodiment of the present invention.

[0031] Figure 2 1 is the immunogenic information and Western blot identification diagram of the monoclonal detection antibody Y according to an embodiment of the present invention.

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

[0033] Figure 4 ELISA standard curve results of the embodiment of the present invention.

[0034] Figure 5 1 is a CLEIA standard curve result diagram of an embodiment of the present invention.

[0035] Figure 6 is the detection result of a clinical serum sample based on the ELISA kit of the embodiment of the present application.

[0036] Figure 7 is the detection result of a clinical serum sample based on the CLEIA kit of the embodiment of the present application. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the present application and not restrictive of the same. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

[0038] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within the range of the intermediate values and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of conflict between the content of this specification and any incorporated document, the content of this specification prevails.

[0039] Various modifications and changes can be made to the specific embodiments of the present application described in this specification without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0040] As used herein, "comprise", "comprising", "including", "include", "contain", "containing", "have", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0041] ① Compared with CN118818050A, based on 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 monoclonal antibodies prepared are different.

[0042] ②The invention CN118818050A only tested mouse serum samples and did not involve the detection of SND1 in clinical human serum.

[0043] 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 its native conformation. By using enterokinase technology to accurately remove the His tag, 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 ).

[0044] ④ The present invention uses ALP labeling, and the R 2 The value reached 0.9986, and the R 2 The value reached 0.9987.

[0045] Example 1 Preparation of Monoclonal Capture Antibody X and Detection Antibody Y that Can Efficiently Identify Human Tumor-Associated Protein SND1 in Serum Based on an in-depth analysis of the spatial conformational characteristics of the SND1 antigen epitope, a 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 as immunogens for preparing monoclonal detection antibody Y ( Figure 2 ).

[0046] Brief process:

[0047] After injecting these immunogens into mice, they successfully stimulated the mice 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] By screening the surviving hybridoma cells in HAT medium and cloning and culturing them using the limiting dilution method, we can 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 amino acid sequences, base sequences (Tables 1-4) and antibody subtypes of the variable regions 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 the 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 blot 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 knockout (KO).

[0054] Table 1 Amino acid sequence information of the variable region of monoclonal capture antibody X

[0055]

[0056]

[0057] Note: The sequence is shown as SEQ ID NO.1-14

[0058] Table 2: Variable region base sequence information of monoclonal capture antibody X

[0059]

[0060]

[0061] Note: The sequence is shown as SEQ ID NO.15-28

[0062] Table 3: Amino acid sequence information of the variable region of monoclonal detection antibody Y

[0063]

[0064] Note: The sequence is shown as SEQ ID NO.29-42

[0065] Table 4: Variable region base sequence information of monoclonal detection antibody Y

[0066]

[0067]

[0068] Note: The sequence is shown as SEQ ID NO.43-56

[0069] Example 2: Preparation of a Standard of the Human Tumor-Associated Protein SND1 with a Native Conformation Using a Mammalian Expression System. Mammalian cell expression systems can provide post-translational modifications that closely resemble those found in the human body, resulting in proteins with activity closer to that of the native protein. Therefore, a standard of the human tumor-associated protein SND1 was prepared using a mammalian expression system.

[0070] Brief process:

[0071] First, CHO eukaryotic cells were used as a tool to express the His-tagged SND1 protein through a mammalian expression system;

[0072] The protein sample was then purified using an AKTA-nickel column system to remove impurities and improve the purity of the target protein. The His tag was then cleaved using enterokinase, and the size and purity of the SND1 standard were determined by Coomassie Brilliant Blue staining. Figure 3 ); Finally, the standard curves of the target protein were drawn based on the developed ELISA and CLEIA kits ( Figure 4 、 Figure 5 Result description: Coomassie Brilliant Blue staining results showed that the human tumor-related protein SND1 obtained through mammalian cell expression and subsequent purification system was of correct size and position and had high purity ( Figure 3 The results of standard curve drawing showed that the standard curve can be successfully drawn using the human SND1 standard. The R 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 ).

[0073] Example 3 SND1-ELISA kit

[0074] 1. Preparation of capture and detection antibodies

[0075] Targeting the novel tumor marker SND1, immunogens for monoclonal capture antibody X and monoclonal capture antibody Y were obtained based on the C-terminal (682-767 aa) and N-terminal (8-140 aa) antigen recognition epitopes of SND1, respectively. After injecting these immunogens into mice, the mice successfully produced specific B lymphocytes against specific epitopes 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 culture by limiting dilution, positive hybridoma cells secreting the desired monoclonal antibodies were obtained. Sequencing the variable region of the antibodies encoded by the hybridoma cells determined the variable region amino acid sequences, base sequences (Tables 1-4) and antibody subtypes of the capture antibodies and detection antibodies. Based on this information, antibody expression plasmids were constructed using genetic engineering methods, and antibody expression and purification were performed using CHO eukaryotic cell lines. The specificity and sensitivity of the antibodies were determined by Western blotting technology. Figure 1 , Figure 2 ) Finally, monoclonal capture antibody X was coated in a 96-well plate and blocked; monoclonal detection antibody Y was ALP labeled.

[0076] 2. Sample preparation: Collect whole blood into a test tube without anticoagulant, and place it at room temperature for 1 h. After the whole blood is naturally coagulated, centrifuge at 1500 g at 4°C for 10 min, and take the yellow supernatant as the serum sample.

[0077] 3. SND1 standard: As shown in Figure 3 , human SND1 protein was obtained after purification. Prepare gradient concentrations of SND1 protein standards.

[0078] 4. Loading: Dilute the sample 5-10 times with the diluent. Add 100 μL of sample and standard per well into the corresponding wells coated with monoclonal capture antibody X as the test group and standard group, and cover with a plate sealing film.

[0079] 5. Add detection antibody Y: Incubate at 37°C for 45 min, discard the liquid, and dry on thick absorbent paper; add 300 μL of washing solution to wash the plate for 1 min x 3 times, and dry on thick absorbent paper each time. Add 100 μL of ALP-conjugated monoclonal detection antibody Y per well, cover with a plate sealing film, and incubate at 37°C in the dark for 45 min.

[0080] 6. Add substrate: Discard the liquid and dry on thick absorbent paper; add 300 μL of washing solution per well to wash the plate for 1 min x 3 times, and dry on thick absorbent paper each time. Add 100 μL of 4-nitrophenyl phosphate disodium color developing solution per well, and develop at room temperature in the dark for 10 min.

[0081] 7. Detection and analysis: Add 100 μL of stop solution and measure the A405 value with a microplate reader. 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.

[0082] Example 4 SND1-CLEIA kit

[0083] 1. Preparation of capture and detection antibodies

[0084] Targeting the novel tumor marker SND1, based on its C-terminal (682-767aa) and N-terminal (8-140aa) antigen recognition epitopes, immunogens for 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 surviving hybridoma cells in HAT medium and cloning and culturing them using the 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 variable region amino acid sequence, base sequence (Table 1-4) and antibody subtype of the capture antibody and detection antibody. Based on this information, antibody expression plasmids were constructed using genetic engineering methods, and the antibodies were expressed and purified using the CHO eukaryotic cell line. The specificity and sensitivity of antibody recognition were determined by Western blotting ( 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.

[0085] 2. Sample preparation: Collect whole blood into a test tube without anticoagulant, place it at room temperature for 1 hour, wait for the whole blood to naturally coagulate, centrifuge at approximately 1500g for 10 minutes at 4°C, and collect the yellow supernatant to obtain the serum sample.

[0086] 3. SND1 standard: Figure 3 As shown, purified SND1 protein was obtained and SND1 protein standards with gradient concentrations were prepared.

[0087] 4. Sample loading: Dilute the sample 5-10 times with diluent. Add 100 μL / well of sample and standard to the corresponding wells coated with monoclonal capture antibody X as the test group and standard group, respectively, and cover with sealing film.

[0088] 5. Add Detection Antibody Y: Incubate at 37°C for 45 minutes, discard the liquid, and pat dry on thick absorbent paper. Add 300 μL of rinse buffer and wash the plate three times for 1 minute, patting dry on thick absorbent paper each time. Add 100 μL / well of ALP-conjugated monoclonal detection antibody Y, cover with sealing film, and incubate at 37°C in the dark for 45 minutes.

[0089] 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 three times for 1 minute, patting dry on thick absorbent paper each time. Add AMPPD enzymatic chemiluminescent substrate to each well. The enzyme catalyzes the decomposition of the substrate, generating a light signal.

[0090] 7. Detection and analysis: Measure the chemiluminescence value with a chemiluminescence immunoassay. Use the concentration of the standard as the horizontal axis and the chemiluminescence detection value as the vertical axis to draw the SND1 standard curve ( 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.

[0091] Finally, it should be noted that:

[0092] 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 they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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 NOs. 1-14 and SEQ ID NOs. 29-42, 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.

2. The kit for producing the SND1 protein according to claim 1, wherein: 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 NOs. 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.

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 producing the SND1 protein according to claim 1, wherein: The monoclonal detection antibody Y includes a heavy chain variable region and a light chain variable region as shown in the amino acid sequence of SEQ ID NO.29-42, 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.

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

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

7. The kit for producing the SND1 protein according to any one of claims 1 to 6, wherein: The kit can detect the content of SND1 protein in human serum and plasma.

8. The kit for producing the SND1 protein according to claim 7, wherein: The human serum and plasma are the serum and plasma of patients with rectal cancer, gastric cancer, colon cancer, and liver cancer.

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