A kit for diagnosing cervical cancer and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kit for diagnosing cervical cancer and application thereof. The kit comprises an enzyme-labeled plate coated with a capture antibody E3G-1, a working solution of an HRP-labeled antibody K6T-2, an antigen standard, a washing solution, a color developing solution and a termination solution. The heavy chain amino acid sequence of the capture antibody E3G-1 is shown as SEQ ID NO. 1, and the light chain amino acid sequence is shown as SEQ ID NO. 2. The heavy chain amino acid sequence of the HRP-labeled antibody K6T-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 diagnosing cervical cancer can specifically detect the content of BEND3 protein in serum, and can be used as an auxiliary detection means for cervical cancer with high expression of BEND3 protein. The kit has high specificity, good stability and wide application prospect.

Description

Technical Field

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

[0002] Cervical cancer is one of the most common malignant tumors among women worldwide, and its incidence rate ranks second among malignant tumors unique to my country, second only to breast cancer. Persistent infection with high-risk human papillomavirus (HPV) is the main pathogenic factor for cervical cancer. Although most HPV infections can be cleared by the immune system, persistent infection may lead to cervical intraepithelial neoplasia, which can then develop into cervical cancer.

[0003] Currently, standard treatments for cervical cancer mainly include surgical resection and radiotherapy / chemotherapy; however, these methods have significant side effects. Furthermore, metastatic and recurrent cervical cancer is difficult to cure completely and is a major risk factor for patient mortality. Most patients with early-stage cervical cancer can be successfully cured, but the survival rate for patients diagnosed at a late stage is only 15%. Therefore, early detection and treatment can significantly reduce the mortality rate of cervical cancer patients, and early detection of cervical cancer has become a hot topic in clinical research.

[0004] Currently, early screening for cervical cancer mainly includes the following methods: 1) Cytological examination: observing morphological changes in exfoliated cervical cells, but this is highly subjective and prone to missed diagnoses; 2) HPV DNA testing: detecting high-risk HPV nucleic acids, with high sensitivity but low specificity, easily leading to overtreatment; 3) Methylation testing: detecting gene methylation, with strong objectivity, but not yet widely adopted; 4) Protein biomarker testing: common biomarkers for cervical cancer include SCC, CA12-5, and urinary gonadotropin fragments. Among them, CA12-5 is currently the most widely used biomarker in clinical practice, but its specificity is not high and it cannot completely solve the problem. The most critical issue in the early clinical diagnosis and treatment of cervical cancer is the lack of sensitive protein biomarkers to identify tumor development in the early stages of the disease.

[0005] To achieve the above objectives, the present invention provides a cervical cancer diagnostic kit and its application. Summary of the Invention

[0006] The primary objective of this invention is to provide a diagnostic kit for cervical cancer.

[0007] The second objective of this invention is to provide an application of a reagent kit for cervical cancer diagnosis.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A diagnostic kit for cervical cancer, the kit comprising an ELISA plate coated with capture antibody E3G-1, working solution of HRP-labeled antibody K6T-2, antigen standard, washing solution, chromogenic solution and stop solution;

[0010] The capture antibody E3G-1 is a monoclonal antibody against BEND3 protein, and its heavy chain amino acid sequence is shown in SEQ ID NO.1, and its light chain amino acid sequence is shown in SEQ ID NO.2; the HRP-labeled antibody K6T-2 is a monoclonal antibody against BEND3 protein, and its heavy chain amino acid sequence is shown in SEQ ID NO.3, and its light chain amino acid sequence is shown in SEQ ID NO.4.

[0011] Furthermore, the amino acid sequence of the BEND3 protein is shown in SEQ ID NO.5.

[0012] Furthermore, the preparation process of the monoclonal antibody against BEND3 protein is as follows:

[0013] (1) Recombinant BEND3 protein was used as an antigen to immunize mice. The spleen cells of the immunized mice were fused with myeloma cells to form hybridoma cells.

[0014] (2) ELISA was used to screen hybridoma cells, and positive cell lines were subcloned and expanded. Then, they were inoculated into mice, and ascites fluid was collected and purified to obtain monoclonal antibodies against BEND3 protein.

[0015] Furthermore, the antigen standard is recombinant BEND3 protein antigen; the washing solution is PBST; the chromogenic solution is TMB; and the stop solution is sulfuric acid.

[0016] Furthermore, the concentration of the colorimetric solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2 M-3 M.

[0017] Furthermore, the detection method of the kit is as follows:

[0018] (1) Take out the ELISA plate coated with capture antibody E3G-1, wash the plate with washing solution, add the sample to be tested and the serially diluted antigen standard, incubate at room temperature for 1-3 hours, wash with washing solution and spin dry;

[0019] (2) Add the HRP-labeled antibody K6T-2 working solution to the system in step (1), incubate at room temperature for 1-3 hours, wash with washing solution and spin dry;

[0020] (3) Add colorimetric solution to the system after the reaction in step (2) and develop color at room temperature in the dark for 5-10 minutes;

[0021] (4) Add a stop solution to the system after step (3) to terminate the reaction;

[0022] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at a wavelength of 450 nm and calculate the result based on the standard curve.

[0023] The above-described cervical cancer diagnostic kit is used to detect the content of BEND3 protein in serum.

[0024] Compared with the prior art, the main advantages of the present invention are as follows:

[0025] This invention provides a diagnostic kit for cervical cancer that can specifically detect the level of BEND3 protein in serum. As an auxiliary detection method for cervical cancer with high expression of BEND3 protein, it has high specificity and good stability, greatly improving the diagnostic efficiency of cervical cancer and has broad application prospects. Attached Figure Description

[0026] Figure 1 The serum titer of mice;

[0027] Figure 2 A standard curve for a kit to specifically detect serum BEND3 protein levels. Detailed Implementation

[0028] 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.

[0029] Example 1

[0030] Preparation of monoclonal antibodies:

[0031] (1) Immunization of experimental animals: Recombinant BEND3 protein was used as an antigen to immunize mice. Balb / c female mice were immunized by subcutaneous injection at multiple sites in the abdomen for about 8 weeks, and a total of 8 mice were injected. The antigen was diluted to 2 mg / mL. For the first immunization, recombinant BEND3 protein was mixed with an equal volume of complete Freund's adjuvant. For subsequent immunizations, recombinant BEND3 protein was mixed with incomplete Freund's adjuvant. The entire immunization process was carried out three times, with 200 μg injected each time. Starting from 7 days after the second immunization, blood was collected 7 days after each booster immunization to detect the titer. Blood was collected from the eye sockets of mice, allowed to stand at room temperature for 2 hours, and then centrifuged to collect serum. The antibody titer of mouse serum was detected by indirect ELISA. Mice with the highest titer were selected for hybridoma cell fusion. The ELISA method was performed as follows: 100 μL of 10 μg / mL BEND3 recombinant protein was coated onto a plate and incubated overnight at 4°C. Block with 5% BSA at 37℃ for 1 h. After washing with PBST, add 100 μL / well of mouse serum diluted with PBST (dilution gradients: 1:1000, 1:2000, 1:4000, 1:6000, 1:8000, 1:10000, 1:15000, 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000). Use unimmunized mouse serum as a negative control. Incubate at 37℃ for 1 h. After washing, add 100 μL of HRP-labeled goat anti-mouse antibody to each well. Incubate at 37℃ for 30 min, wash with PBST, develop with TMB for 10 min, and stop the reaction with 2M sulfuric acid. Read the OD value at 450 nm using a microplate reader. Results are as follows. Figure 1 As shown, the antibody titer in mouse 4 serum was around 40,000, indicating the best effect.

[0032] (2) Hybridoma cell fusion and screening: The highest titer mouse No. 4 was selected and euthanized by cervical dislocation. The mice were disinfected by immersing in 75% alcohol for 5 minutes. The abdominal skin was cut open with sterile scissors, and the spleen was completely removed and transferred to a cell culture dish with incomplete culture medium. The surface of the spleen was punctured with a sterile syringe needle and gently squeezed and ground into a cell suspension. The cell suspension was filtered through a 70 μm filter, then centrifuged and the supernatant was discarded. The pellet was resuspended in HAT medium, and the cells were counted and distributed into 96-well plates. Spleen cells and myeloma cells were mixed at a ratio of 10:1 and fused using PEG. The effect of PEG was then terminated with incomplete culture medium. The fused cells were resuspended in HAT medium and plated in 96-well plates and cultured in an incubator at 37°C and 5% CO2. After 10 days of screening culture in HAT selection medium, the supernatant was collected for ELISA detection. The mother clone with high OD value and good growth status was selected for subcloning. Subcloning was repeated 3 times, and the single clone with the strongest positive reaction was selected for expansion culture.

[0033] (3) Cell fusion and screening: 1×10 7 Selected subclonal cells were injected intraperitoneally into Balb / c female mice. The abdominal condition of the mice was observed, and ascites fluid was collected when the abdomen became noticeably distended. The collected ascites fluid from different mice was purified using immunochromatography to obtain purified monoclonal antibodies.

[0034] (4) Analysis of monoclonal antibody sequences: The activity of the purified monoclonal antibodies was accurately measured, and the two monoclonal antibodies with the highest activity were named E3G-1 and K6T-2, respectively. Their variable region sequences were determined. Monoclonal antibodies E3G-1 and K6T-2 consist of heavy and light chains, respectively. The heavy chain amino acid sequence of monoclonal antibody E3G-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. The heavy chain amino acid sequence of monoclonal antibody K6T-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0035] Table 1 Sequence List

[0036]

[0037] Example 2

[0038] Preparation of ELISA kit:

[0039] (1) Preparation of ELISA plates coated with capture antibodies:

[0040] The monoclonal antibody E3G-1 prepared in Example 1 was selected as the capture antibody. Using a 96-well ELISA plate as the solid-phase carrier, the capture antibody was diluted to 2 μg / mL with coating buffer (50 mM carbonate buffer) to obtain the coating solution. 100 μL of the coating solution was added to the ELISA plate, and the plate was sealed and incubated overnight at 4°C. The coating solution was discarded, the plate was washed three times with washing buffer, and 300 μL of blocking buffer was added per well. The plate was blocked at room temperature for 4 hours, the liquid in the plate was discarded, and the plate was dried and stored at 4°C.

[0041] (2) Preparation of HRP-labeled antibody working solution:

[0042] The monoclonal antibody K6T-2 prepared in Example 1 was selected as the labeling antibody. The monoclonal antibody K6T-2 was dialyzed in 50 mM carbonate buffer for 24 h to adjust its concentration to 2 mg / mL. 5 mg of HRP was weighed and dissolved in 1 mL of distilled water. 10.5 mg of NaIO4 was weighed and dissolved in 1 mL of distilled water to prepare a 0.1 M NaIO4 solution. 0.2 mL of the NaIO4 solution was mixed with 1 mL of the HRP solution, stirred at room temperature in the dark for 40 min, and dialyzed overnight in 10 mM NaAc buffer at 4 °C. Carbonate buffer was added to the above aldehyde-modified HRP solution to raise its pH to 9.0-9.5, and then 5 mL of 2 mg / mL monoclonal antibody K6T-2 was added. The solution was gently stirred at room temperature in the dark for 2 hours. Measure 0.1 mL of 4 mg / L NaBH4 and add it all to the reaction solution from the previous step. Mix well and react at 4°C for 2 hours. Place the sample in a dialysis bag and dialyze overnight at 4°C. Centrifuge at 10000 r / min for 30 min to remove the precipitate. The supernatant is the enzyme conjugate. Add an equal volume of 60% glycerol and aliquot. Store at 4°C in the dark.

[0043] (3) Determination of standard curve of reagent kit:

[0044] Remove the ELISA plate coated with the capture antibody, allow it to return to room temperature, wash it three times with washing buffer, and agitate dry. Add 100 μL of recombinant BEND3 protein antigen at different dilutions (50 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL), incubate at room temperature for 1 h, wash three times with PBST washing buffer, and agitate dry. Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 1 h, wash three times with PBST washing buffer, and agitate dry. Add 100 μL of 2 mg / L TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 30 min, and stop the reaction by adding 100 μL of 2M sulfuric acid stop solution. The OD values ​​at a reference wavelength of 450 nm were measured using a microplate reader. A standard curve was plotted with the standard concentration on the x-axis (pg / mL) and OD450 on the y-axis. The standard curve was then plotted based on the results. Figure 2 As shown. The standard curve for the kit used in this application for the detection of serum BEND3 protein content is y = 0.0097x + 0.4714, R0 2 = 0.9952, the detection range of this kit is 100-700 pg / mL.

[0045] (4) The ELISA kit of the present invention includes an enzyme-labeled plate coated with capture antibody E3G-1, working solution of HRP-labeled antibody K6T-2, recombinant BEND3 protein antigen, washing solution (PBST), chromogenic solution (TMB) and stop solution (sulfuric acid).

[0046] The protein standard is a recombinant BEND3 protein antigen, and the amino acid sequence of the BEND3 protein is shown in SEQ ID NO.5.

[0047] SEQ ID NO.5:

[0048] MNSTEFTEDVEEVLKSITVKVETEAEDAALDCSVNSRTSEKHSVDSVLTALQDSSKRK 。

[0049] Test example 1

[0050] Serum sample test:

[0051] (1) Serum samples from 15 healthy individuals (numbered 1-15) and 15 cervical cancer patients (numbered 16-30) were collected. The purified serum samples were added to an ELISA plate coated with capture antibody E3G-1 and incubated at room temperature for 1 h. The plate was washed three times with PBST washing buffer and then dried. 100 μL of HRP-labeled detection antibody K6T-2 working solution was added to the reaction wells and incubated at room temperature for 1 h. The plate was washed three times with PBST washing buffer and then dried. 100 μL of 2 mg / L TMB chromogenic solution was added to the reaction wells and incubated at room temperature in the dark for 10 min. 100 μL of 2 M sulfuric acid was added to terminate the reaction. The OD value at 450 nm was measured using an ELISA reader, and the average value was taken after three repetitions. The content of BEND3 protein in the serum to be tested was calculated according to the standard curve. The results are shown in Table 2.

[0052] Table 2 Clinical Sample Test Results

[0053]

[0054] The results are shown in Table 2. The kit of the present invention can accurately detect the BEND3 protein content in serum samples from cervical cancer patients, with positive results. The BEND3 protein content in the serum of cervical cancer patients was 500-700 pg / mL. Serum from healthy individuals contained trace amounts of BEND3 protein, but the detection concentration was outside the linear range of the kit, resulting in negative results. This demonstrates that the kit of the present invention can use serum as a sample to detect cervical cancer markers, meeting the needs of basic research and clinical diagnosis.

[0055] Experimental Example 2

[0056] Reagent kit stability testing:

[0057] Serum samples from volunteers 17, 22, and 28 in Experiment Example 1 were taken as samples, and 10 biological replicates were performed for each sample to test the stability of the kit. The results are shown in Table 3.

[0058] Table 3 Clinical Sample Test Results

[0059]

[0060] The results are shown in Table 3. The kit of the present invention can quantitatively detect the content of human serum BEND3 protein and has good stability, which can meet the needs of basic research and clinical diagnosis.

[0061] 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 diagnostic kit for cervical cancer, characterized in that, The kit includes an ELISA plate coated with capture antibody E3G-1, working solution of HRP-labeled antibody K6T-2, antigen standards, washing solution, chromogenic solution, and stop solution; The capture antibody E3G-1 is a monoclonal antibody against BEND3 protein, and its amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and its amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the HRP-labeled antibody K6T-2 is a monoclonal antibody against BEND3 protein, and its amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3, and its amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. The cervical cancer diagnostic kit according to claim 1, characterized in that, The amino acid sequence of the BEND3 protein is shown in SEQ ID NO.

5.

3. The cervical cancer diagnostic kit according to claim 1, characterized in that, The preparation process of the monoclonal antibody against BEND3 protein is as follows: (1) Recombinant BEND3 protein was used as an antigen to immunize mice. The spleen cells of the immunized mice were fused with myeloma cells to form hybridoma cells. (2) ELISA was used to screen hybridoma cells, and positive cell lines were subcloned and expanded. Then, they were inoculated into mice, and ascites fluid was collected and purified to obtain monoclonal antibodies against BEND3 protein.

4. The cervical cancer diagnostic kit according to claim 1, characterized in that, The antigen standard is recombinant BEND3 protein antigen; the washing solution is PBST; the chromogenic solution is TMB; and the stop solution is sulfuric acid.

5. A cervical cancer diagnostic kit according to claim 4, characterized in that, The concentration of the colorimetric reagent is 2 mg / L-4 mg / L; the concentration of the stop solution is 2 M-3 M.

6. A cervical cancer diagnostic kit according to claim 1, characterized in that, The detection method of the kit is as follows: (1) Take out the ELISA plate coated with capture antibody E3G-1, wash the plate with washing solution, add the sample to be tested and the serially diluted antigen standard, incubate at room temperature for 1-3 hours, wash with washing solution and spin dry; (2) Add the HRP-labeled antibody K6T-2 working solution to the system in step (1), incubate at room temperature for 1-3 hours, wash with washing solution and spin dry; (3) Add colorimetric solution to the system after the reaction in step (2) and develop color at room temperature in the dark for 5-10 minutes; (4) Add a stop solution to the system after step (3) to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at a wavelength of 450 nm and calculate the result based on the standard curve.

7. A cervical cancer diagnostic kit according to claim 1, characterized in that, The kit is used to detect the content of BEND3 protein in serum.

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