A kit for early diagnosis of colon cancer and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kit for early diagnosis of colon cancer and application thereof. The kit comprises an enzyme label plate coated with a capture antibody, and a HRP-labeled antibody working solution; the capture antibody is a monoclonal antibody T1A-1, the heavy chain amino acid sequence of the monoclonal antibody T1A-1 is shown as SEQ ID NO. 1, and the light chain amino acid sequence is shown as SEQ ID NO. 2; the HRP-labeled antibody is a monoclonal antibody T1A-2, the heavy chain amino acid sequence of the monoclonal antibody T1A-2 is shown as SEQ ID NO. 3, and the light chain amino acid sequence is shown as SEQ ID NO. 4. The kit for early diagnosis of colon cancer provided in the application has high specificity, can be used as an auxiliary diagnostic means for colon cancer with high expression of STK32A protein, and has high diagnostic value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a reagent kit for the early diagnosis of colon cancer and its application. Background Technology

[0002] Colorectal cancer, also known as colon cancer, is one of the most common malignant tumors of the digestive tract originating from the mucosal epithelium of the colon or rectum. High-fat, low-fiber diets, genetic factors, inflammatory bowel disease, sedentary lifestyles, and aging are the main risk factors for colorectal cancer. Global cancer statistics show that colorectal cancer has become a major public health problem threatening human health, ranking among the top malignant tumors in terms of incidence and mortality. Most colorectal cancer patients have no obvious symptoms or only specific symptoms in the early stages, leading to a significant decrease in the five-year survival rate when diagnosed at an advanced stage. In contrast, patients diagnosed early have a five-year survival rate exceeding 90%.

[0003] Based on histological characteristics, colorectal cancer can be classified into several types, including adenocarcinoma, mucinous adenocarcinoma, and undifferentiated carcinoma, with adenocarcinoma being the most common. Early diagnosis of colorectal cancer is a key measure to reduce patient mortality and improve prognosis. Currently, early diagnosis of colorectal cancer mainly employs techniques such as colonoscopy, fecal occult blood test, imaging examinations, and tumor marker detection. Colonoscopy is considered the gold standard for diagnosis, but its invasiveness, pre-examination bowel preparation requirements, and high cost limit its widespread use in population screening. While the fecal occult blood test is simple and non-invasive, its sensitivity is low and easily affected by diet and medication. Imaging examinations are of significant value in tumor staging assessment, but their ability to detect early, small lesions is limited, and they are also costly.

[0004] In recent years, an increasing number of tumor markers have been applied to the auxiliary diagnosis and efficacy monitoring of colorectal cancer, but their clinical application effectiveness remains significantly insufficient. Existing colorectal cancer diagnostic markers, such as CEA, while possessing some reference value, suffer from limitations such as low specificity, insufficient sensitivity, and inability to accurately predict tumor stage and early lesions, making it difficult to meet the needs of large-scale population screening and early, precise diagnosis. Therefore, there is an urgent need to develop novel tumor markers with higher specificity and sensitivity to achieve early, non-invasive, and precise detection of colorectal cancer.

[0005] To achieve the above objectives, the present invention provides a kit for the early diagnosis of colorectal cancer and its application. Summary of the Invention

[0006] The primary objective of this invention is to provide a kit for the early diagnosis of colon cancer.

[0007] A second objective of this invention is to provide an application of a kit for the early diagnosis of colon cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A kit for the early diagnosis of colorectal cancer, the kit comprising an enzyme-labeled plate coated with capture antibodies and HRP-labeled antibody working solution; The capture antibody is a monoclonal antibody T1A-1 against STK32A protein. The heavy chain amino acid sequence of the monoclonal antibody T1A-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. The antibody in the HRP-labeled antibody working solution is a monoclonal antibody T1A-2 against STK32A protein. The heavy chain amino acid sequence of the monoclonal antibody T1A-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. The amino acid sequence of the STK32A protein is shown in SEQ ID NO.5.

[0009] Furthermore, the kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

[0010] Furthermore, the antigen standard is STK32A protein antigen; the washing solution is PBST; the colorimetric solution is 2 mg / L-4 mg / L TMB; and the stop solution is 2M-4M sulfuric acid.

[0011] The kit for early diagnosis of colorectal cancer described in any of the above-mentioned applications is used to detect the content of STK32A protein in blood or serum.

[0012] The detection method for the kit described above for the early diagnosis of colorectal cancer includes the following steps: (1) Take the sample to be tested, centrifuge it, and collect the supernatant; (2) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing buffer and spin dry, add the sample treated in step (1), incubate at room temperature, wash the plate with washing buffer and spin dry; (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature, wash the plate with washing solution and spin dry; (4) Add the colorimetric solution to the system after the reaction in step (3), develop the color at room temperature in the dark, and add the stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to perform dual-wavelength detection. Subtract the OD measurement value of 630nm from the OD measurement value of 450nm and calculate the result using a standard curve.

[0013] Furthermore, the sample to be tested is blood or serum.

[0014] Compared with the prior art, the main advantages of the present invention are as follows: This invention discloses an ELISA kit for detecting colorectal cancer biomarkers and its application. The kit detects the content of STK32A protein in the blood, has high specificity, is simple to operate, and can effectively determine the risk of colorectal cancer in the target population, improving the detection and diagnosis efficiency of colorectal cancer, thereby improving the survival rate of colorectal cancer patients, and has broad application prospects. Attached Figure Description

[0015] Figure 1 A standard curve for a kit to specifically detect STK32A protein; Figure 2 The diagnostic value of serum biomarker STK32A protein in colorectal cancer was analyzed using ROC curve analysis. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0017] The BALB / c mice used in the embodiments of this invention were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0018] Example 1 Preparation of monoclonal antibodies T1A-1 and T1A-2 (1) Animal immunization and titer detection: Six-week-old BALB / c mice were immunized with STK32A protein as the immunogen. The immunization procedure was as follows: each mouse was immunized 4 times, with an interval of 2 weeks, and the antigen dosage was 30 μg each time. For the first immunization, STK32A protein was emulsified with an equal volume of complete Freund's adjuvant; for the second to fourth immunizations, incomplete Freund's adjuvant was used for emulsification. After the last immunization, mouse serum was collected, and the antibody titer was detected by indirect ELISA. Mice with the highest titer were selected for subsequent fusion.

[0019] (2) Hybridoma cell preparation and positive clone screening: Under aseptic conditions, spleens from mice with the highest titers were collected, and spleen cell suspensions were prepared. Spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1, and fusion was completed at 37°C for 2 min using 50% polyethylene glycol (PEG) as a fusion agent. The medium was replaced with HAT selective medium on the second day after fusion, and culture was continued, with half the medium being replaced every 3-4 days. The hybridoma culture supernatant was detected by indirect ELISA to screen for positive clones; positive clones were subcloned using the limiting dilution method three times consecutively to finally obtain hybridoma cell lines that stably secrete highly sensitive and specific antibodies.

[0020] (3) Preparation and purification of monoclonal antibodies: The hybridoma cell lines obtained in step (2) were inoculated into the peritoneal cavity of BALB / c mice pretreated with paraffin oil. Ascites fluid was collected after the mice's abdomens became significantly distended. The ascites fluid was centrifuged at 6,000 rpm for 20 min to remove cellular components. After filtration through a 0.22 μm filter, the antibody was purified to obtain high-purity monoclonal antibodies.

[0021] (4) Antibody identification and sequence analysis: The monoclonal antibodies obtained in step (3) were subjected to activity assays, and two paired antibodies recognizing different antigenic epitopes of the STK32A protein were screened through epitope competition experiments and named T1A-1 and T1A-2, respectively. The variable region sequences were further determined: the amino acid sequence of the heavy chain variable region of T1A-1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of T1A-2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; the amino acid sequence of the STK32A protein is shown in SEQ ID NO.5.

[0022] Table 1 Sequence List Example 2 Preparation of ELISA kit (1) Preparation of ELISA plates coated with capture antibody T1A-1: The monoclonal antibody T1A-1 prepared in Example 1 was used as the capture antibody. Using an ELISA 96-well plate as the solid-phase carrier, T1A-1 was diluted to 2 μg / mL with antibody coating buffer (50 mM carbonate buffer, pH=9.6), and 100 μL / well was added to the 96-well ELISA plate. After sealing, the plate was placed at 4℃ and coated overnight. The next day, the coating buffer was discarded, the plate was washed 3 times with PBST washing buffer, 200 μL / well was added with 1% (w / v) BSA blocking buffer, and the plate was blocked at room temperature for 2 h. The blocking buffer was discarded, the plate was patted dry, sealed, and stored at 4℃ for later use.

[0023] (2) Preparation of working solution for HRP-labeled antibody T1A-2: The monoclonal antibody T1A-2 prepared in Example 1 was selected as the labeling antibody. 20 mg of HRP was weighed and dissolved in 1.2% glutaraldehyde solution, and the solution was allowed to stand overnight at room temperature to complete the activation. The activated HRP solution was then purified by Sephadex G-25 chromatography column, using physiological saline as the elution buffer at a flow rate of 1 mL / min. The brown eluent was collected and placed in a 25 mL beaker and stirred slowly. 12.5 mg of the monoclonal antibody T1A-2 to be labeled was weighed and diluted to 5 mL with physiological saline. The solution was added dropwise to the above HRP solution while stirring. Then, 0.25 mL of 1 M, pH 9.5 carbonate buffer was added, and the mixture was stirred for 3 h. Then, 0.25 mL of 0.2 M lysine was added, mixed well, and allowed to stand at room temperature for 2 h. Subsequently, an equal volume of saturated ammonium sulfate was added dropwise to the beaker with stirring, and the reaction was carried out at 4°C for 1 h. The reaction solution was centrifuged at 3,000 rpm for 40 min, and the supernatant was discarded. The precipitate was washed twice with a semi-saturated ammonium sulfate solution, and finally dissolved in a small amount of 0.15 M, pH 7.4 PBS. The above solution was placed in a dialysis bag and dialyzed against 0.15 M, pH 7.4 PBS buffer to remove ammonium ions. The precipitate was then removed by centrifugation at 12,000 rpm for 30 min. The final supernatant was the working solution for the HRP-labeled antibody.

[0024] (3) Standard curve determination of the kit: Remove the ELISA plate coated with capture antibody T1A-1, allow it to return to room temperature, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of serially diluted STK32A protein antigen (50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 600 pg / mL, 800 pg / mL, 900 pg / mL), incubate at room temperature for 1 h, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of HRP-labeled antibody T1A-2 working solution to the reaction wells, incubate at room temperature for 1 h, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of 3 mg / L chromogenic reagent TMB to the reaction wells, incubate at room temperature in the dark for 30 min, and add 100 μL of 3M sulfuric acid to terminate the reaction. A dual-wavelength assay was performed using an ELISA reader to measure the OD values ​​at a reference wavelength of 450 nm and 630 nm. The OD value at 450 nm was then subtracted from the OD value at 630 nm. A standard curve was plotted with the standard concentration on the x-axis (pg / mL) and OD 450 - OD 630 on the y-axis. The results are as follows: Figure 1 The figure shows the standard curve for the kit specifically detecting STK32A protein. The detection range of the STK32A protein kit of this invention is 100-900 pg / mL, and the standard curve is y = 0.0055x + 0.0830, R0.2 =0.9972.

[0025] (4) Preparation of ELISA kit: The prepared enzyme-labeled plate coated with capture antibody T1A-1, working solution of HRP-labeled antibody T1A-2, antigen standard STK32A protein, PBST washing solution, colorimetric solution TMB, and stop solution sulfuric acid are packaged separately to form a kit for early diagnosis of colorectal cancer according to the present invention.

[0026] Experimental Example 1 Serum sample testing Ten blood samples were collected from healthy individuals (numbered 1–10) and ten blood samples from patients with colon cancer (numbered 11–20). The samples were centrifuged at 6,000 rpm for 10 min, and the supernatant was collected. The ELISA plates coated with the capture antibody T1A-1 were removed, brought to room temperature, washed three times with PBST, and then dried. 100 μL of the treated sample was added to each well, and the plates were incubated at room temperature for 1 h. The plates were washed three times with PBST and then dried. Subsequently, 100 μL of HRP-labeled antibody T1A-2 working solution was added to each well, and the plates were incubated at room temperature for 1 h. The plates were washed three times with PBST and then dried. 100 μL of 2 mg / L TMB chromogenic reagent was added to each well, and the plates were incubated at room temperature in the dark for 30 min. The reaction was terminated by adding 100 μL of 3M sulfuric acid. The samples were detected using a dual-wavelength ELISA reader. The sample concentration was calculated using a standard curve based on the OD450-OD630 values. A concentration ≥200 pg / mL was considered positive (+), otherwise negative (-).

[0027] Table 2 Blood Sample Test Results The results are shown in Table 2. The kit of the present invention can accurately detect the STK32A protein content in blood samples from colorectal cancer patients, with a positive result. Trace amounts of STK32A protein are present in the blood of healthy individuals, but the detection concentration is outside the linear range of the kit, resulting in a negative result. This demonstrates that the kit of the present invention can use blood as a sample to detect colorectal cancer markers, meeting the needs of basic research and clinical diagnosis.

[0028] Experimental Example 2 ROC curve assessment diagnostic value Eighty samples were collected, including 40 serum samples from healthy individuals as the control group and 40 serum samples from individuals with early-stage colorectal cancer as the observation group. STK32A protein levels were detected using the procedure described in Experiment 1 to evaluate the diagnostic performance of the kit for early-stage colorectal cancer. ROC curves were plotted based on the results, as shown below. Figure 2 As shown.

[0029] The results are as follows Figure 2The figure shows the ROC curve for the serum biomarker STK32A in diagnosing colorectal cancer. The AUC value of serum STK32A is 0.921. A larger ROC curve area indicates greater diagnostic value, and an AUC above 0.7 indicates good model classification ability. These results demonstrate that the serum biomarker STK32A protein can specifically distinguish between healthy individuals and colorectal cancer patients, and colorectal cancer can be screened by detecting the level of serum STK32A protein.

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A kit for the early diagnosis of colon cancer, characterized in that, The kit includes an ELISA plate coated with capture antibodies and HRP-labeled antibody working solution; The capture antibody is a monoclonal antibody T1A-1 against STK32A protein. The heavy chain amino acid sequence of the monoclonal antibody T1A-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.

2. The antibody in the HRP-labeled antibody working solution is a monoclonal antibody T1A-2 against STK32A protein. The heavy chain amino acid sequence of the monoclonal antibody T1A-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4. The amino acid sequence of the STK32A protein is shown in SEQ ID NO.

5.

2. The kit for early diagnosis of colon cancer according to claim 1, characterized in that, The kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

3. The kit for early diagnosis of colon cancer according to claim 2, characterized in that, The antigen standard is STK32A protein antigen; the washing solution is PBST; the colorimetric solution is 2 mg / L-4 mg / L TMB; and the stop solution is 2M-4M sulfuric acid.

4. The application of the kit for early diagnosis of colorectal cancer according to any one of claims 1 to 3, characterized in that, Used to detect the level of STK32A protein in blood or serum.

5. The detection method of the kit for early diagnosis of colorectal cancer according to any one of claims 1 to 3, characterized in that, The testing steps are as follows: (1) Take the sample to be tested, centrifuge it, and collect the supernatant; (2) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing buffer and spin dry, add the sample treated in step (1), incubate at room temperature, wash the plate with washing buffer and spin dry; (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature, wash the plate with washing solution and spin dry; (4) Add the colorimetric solution to the system after the reaction in step (3), develop the color at room temperature in the dark, and add the stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to perform dual-wavelength detection. Subtract the OD measurement value of 630nm from the OD measurement value of 450nm and calculate the result using a standard curve.

6. The detection method of the kit for early diagnosis of colorectal cancer according to claim 5, characterized in that, The sample to be tested is blood or serum.

Images