Kit for detecting cervical cancer and application thereof

Abstract

The invention belongs to the field of biological detection, and discloses a kit for detecting cervical cancer and application of the kit. The kit comprises an elisa plate coated with a captured antibody, an HRP labeled antibody, an antigen standard substance, a washing solution, a developing solution and a stop solution, the heavy chain amino acid sequence of the monoclonal antibody CPT1A-1 of the anti-CPT1A protein in the capture antibody is as shown in SEQ ID NO. 1, and the light chain amino acid sequence is as shown in SEQ ID NO. 2; the heavy chain amino acid sequence of the monoclonal antibody CPT1A-2 of the anti-CPT1A protein in the HRP labeled antibody is as shown in SEQ ID NO.3, and the light chain amino acid sequence of the monoclonal antibody CPT1A-2 of the anti-CPT1A protein in the HRP labeled antibody is as shown in SEQ ID NO.4. The kit for diagnosis of cervical cancer provided by the invention has strong specificity, can specifically detect the content of CPT1A protein in serum, has good stability, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a kit for cervical cancer detection and its application. Background Art

[0002] Cervical cancer is one of the most common malignant tumors of the reproductive system in women worldwide, ranking second among female malignant tumors globally, and its incidence and mortality rate are second only to breast cancer. The physiological process of cervical cancer usually begins with the dysplasia of cervical epithelial cells, and these abnormal cells gradually invade the surrounding tissues to form tumors. The incidence age of cervical cancer is mostly between 40 and 50 years old, and there has been a trend of younger age in recent years. Most early-stage cervical cancer patients can be successfully cured, but the survival rate of cervical cancer patients diagnosed at an advanced stage is only 15%. Therefore, early diagnosis and treatment can significantly reduce the mortality rate of cervical cancer patients, and the early diagnosis of cervical cancer has become one of the hotspots in clinical research.

[0003] Currently, the diagnostic techniques for cervical cancer are as follows: 1) Cervical smear cytology examination: It is easily affected by subjective factors, has a certain false negative rate, and is prone to missed diagnosis; 2) Imaging techniques: MRI examination, B-ultrasound examination, CT examination, but they are not sensitive enough to small tumors and cannot make a definite diagnosis; 3) Colposcopy examination: Whether there is canceration cannot be observed with the naked eye, and it is not applicable to patients with vaginal stenosis, cervical surface necrosis, or bleeding; 4) Cervical biopsy: This method is relatively complex to operate and has a relatively heavy economic burden. Common biomarkers for cervical cancer include SCC, CA12-5, and urinary gonadotropin fragments. Among them, CA12-5 is the most widely used biomarker in clinical practice at present, but its specificity is not high and it cannot completely solve the problem.

[0004] Therefore, it is urgent to find a new biomarker for cervical cancer and a stable and reliable detection method for cervical cancer to solve the current international problems, improve the early diagnosis rate of cervical cancer, improve the treatment effect of cervical cancer, and reduce the mortality rate of cervical cancer, which has very important clinical significance. Summary of the Invention

[0005] The first object of the present invention is to provide a kit for cervical cancer detection, which includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a coloring solution, and a termination solution.

[0006] The second object of the present invention is to provide the application of the kit for cervical cancer detection.

[0007] The first object of the present invention is achieved by adopting the following technical solution: A kit for cervical cancer detection, which includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a coloring solution, and a termination solution; The heavy-chain amino acid sequence of the monoclonal antibody CPT1A-1 against the CPT1A protein in the capture antibody 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 the monoclonal antibody CPT1A-2 against the CPT1A protein in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light-chain amino acid sequence is shown in SEQ ID NO.4.

[0008] Further, the antigen standard is recombinant CPT1A protein antigen; the washing solution is PBST; the chromogenic solution is TMB; the termination solution is sulfuric acid.

[0009] Further, the preparation process of the monoclonal antibody against the CPT1A protein is as follows: (1) Immunize mice with recombinant CPT1A protein as an antigen, fuse the spleen cells of the immunized mice with myeloma cells to form hybridoma cells; (2) Screen the hybridoma cells by ELISA, subclone and expand the positive cell line, then inoculate them into mice, collect ascites and purify to obtain the monoclonal antibody against the CPT1A protein.

[0010] Further, the preparation steps of the enzyme-labeled plate coated with the capture antibody are as follows: Dilute the monoclonal antibody CPT1A-1 with the antibody coating solution and add it to the micro-wells of the enzyme-labeled plate, seal the plate with a sealing film and coat it overnight, add the blocking solution and block it at room temperature for a period of time, discard the liquid in the wells of the enzyme-labeled plate, dry it and store it sealed.

[0011] Further, the preparation steps of the HRP-labeled antibody are as follows: Add NaIO4 solution to the HRP solution to oxidize the glycosyl group of HRP into aldehyde group, then mix the monoclonal antibody CPT1A-2 with the aldehydeized HRP solution, react at room temperature in the dark, continue to add NaBH4 solution and react at 4°C to obtain a stable enzyme-labeled antibody, and finally dialyze it in PBS overnight to obtain the HRP-labeled antibody.

[0012] The second object of the present invention is achieved by the following technical solution: The application of the above-mentioned kit for cervical cancer detection is used to detect the content of CPT1A protein in blood.

[0013] Further, the detection steps are as follows: (1) Take the sample to be detected, centrifuge it and take the supernatant; (2) Take out the enzyme-labeled plate coated with the capture antibody, restore it to room temperature, wash the plate with the washing solution and centrifuge it dry, add the sample processed in step (1), incubate it at room temperature, then wash the plate with the washing solution and centrifuge it dry; (3) Add the HRP-labeled antibody to the system in step (2), incubate it at room temperature for a period of time, wash the plate with the washing solution and centrifuge it dry; (4) Add a chromogenic solution to the system after the reaction in step (3), allow it to develop color in the dark at room temperature for a period of time, and then add a stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader for dual-wavelength detection. Subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the result through a standard curve.

[0014] Compared with the prior art, the beneficial effects of the present invention mainly lie in that the present invention provides an ELISA kit for detecting cervical cancer markers, which can specifically detect the content of CPT1A protein in serum. As an auxiliary diagnostic method for cervical cancer with high expression of CPT1A protein, it has strong specificity and good stability, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the standard curve of the kit for specifically detecting recombinant CPT1A protein; Figure 2 It is the specific identification result diagram of monoclonal antibodies CPT1A-1 and CPT1A-2. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following further describes the technical solutions of the present invention in combination with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0017] Example 1: Preparation of monoclonal antibody against CPT1A protein, including the following steps: S1. Animal immunization: Use recombinant CPT1A protein as the immunogen, mix it with Freund's complete adjuvant in equal volume, and emulsify it for later use. Select healthy female mice for multiple subcutaneous injections in the abdomen. The first immunization dose is 100 μg / mouse.

[0018] S2. Booster immunization: Three weeks after the first immunization, perform the second immunization. Mix recombinant CPT1A protein with Freund's incomplete adjuvant in equal volume, emulsify it and then inject. The immunization dose is 100 μg / mouse. Four weeks after the second immunization, perform the third immunization to ensure that high-titer antibodies are produced in the mice.

[0019] S3. Hybridoma cell fusion: Sacrifice the mouse with the highest antibody titer obtained, take the spleen of the mouse to make a spleen cell suspension. Mix the spleen cells with myeloma cells SP2 / 0 at a ratio of 5:1, use 50% polyethylene glycol as the fusogen, and the fusion time is 2 minutes.

[0020] S4. Screening of positive hybridoma cells and subcloning: Five days after fusion, the HAT selective medium was replaced for continuous culture. On the 7th day, the supernatant of the cell culture medium was collected, and positive hybridoma cells were screened by indirect ELISA. The selected positive hybridoma cells were subcloned three times by the limited dilution method, and the monoclonal cells with the strongest positive reaction were selected for expansion culture.

[0021] S5. Preparation of monoclonal antibody: 0.5 mL of liquid paraffin was injected into healthy female mice. Seven days later, 8×10 5 hybridoma cells producing the CPT1A protein monoclonal antibody were injected intraperitoneally. After the abdomen of the mice bulged, ascites was collected. The ascites was purified by the caprylic acid-ammonium sulfate method. After dialysis with PBS, the protein concentration was measured by the BCA method and diluted to 10 mg / mL with PBS buffer. The purified antibody was stored at -20 °C.

[0022] S6. Analysis of monoclonal antibody sequences: The activities of the purified monoclonal antibodies were accurately measured. The two monoclonal antibodies with the highest activities were named CPT1A-1 and CPT1A-2, and their variable region sequences were determined. Monoclonal antibody CPT1A-1 and monoclonal antibody CPT1A-2 each included a light chain and a heavy chain; among them, the amino acid sequence of the heavy chain of monoclonal antibody CPT1A-1 was as shown in SEQ ID NO.1, and the amino acid sequence of the light chain was as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain of monoclonal antibody CPT1A-2 was as shown in SEQ ID NO.3, and the amino acid sequence of the light chain was as shown in SEQ ID NO.4.

[0023] Table 1 Sequence Listing: ;

[0024] Example 2: Preparation of ELISA kit: (1) Preparation of enzyme-labeled plate coated with capture antibody: Monoclonal antibody CPT1A-1 prepared in Example 1 was selected as the capture antibody. An enzyme-labeled 96-well plate was used as the solid-phase carrier, and the capture antibody was diluted to a certain concentration with carbonate buffer to obtain the coating solution. 100 μL of the coating solution was added to the enzyme-labeled plate, and after sealing the plate, it was placed at 4 °C for overnight coating. The coating solution was discarded, and the plate was washed 3 times with the washing solution. 200 μL / well of 1% BSA blocking solution was added, and it was blocked at room temperature for 2 h. The liquid in the plate wells was discarded, patted dry, sealed, and stored at 4 °C.

[0025] (2)Preparation of HRP-labeled antibody working solution: The monoclonal antibody CPT1A-2 prepared in Example 1 was dialyzed against 50 mM carbonate buffer for 24 h, with the buffer changed twice during this period. After dialysis, its concentration was adjusted to 2 mg / mL. Weigh 5 mg of HRP and dissolve it in 1 mL of distilled water, add 200 μL of freshly prepared 20 mg / mL NaIO4 solution, and stir it in the dark at room temperature for 30 min to oxidize the glycosyl group of HRP into aldehyde group. Load the above solution into a dialysis bag and dialyze it against 1 mM NaAc buffer at pH 4.4 overnight at 4°C. Add an appropriate amount of 0.2 M carbonate buffer at pH 9.5 to adjust the pH value to 9.0, then immediately add an equal volume of the monoclonal antibody CPT1A-2, and gently stir it in the dark at room temperature for 4 hours. Then add 20 μL of freshly prepared 20 mg / mL NaIO4 solution, mix well and react at 4°C for 3 hours to obtain a stable enzyme-labeled antibody. Then dialyze it against 0.15 M PBS at pH 7.4 overnight at 4°C, centrifuge at 10000 r / min for 30 min to remove the precipitate, and the supernatant is the enzyme conjugate. Add an equal volume of 60% glycerol, dispense it in aliquots, and store it in the dark at 4°C.

[0026] (3)Determination of the standard curve of the kit: Take out the enzyme-labeled plate coated with the capture antibody, let it return to room temperature, wash the plate 3 times with the washing solution and shake it dry. Add 100 μL of recombinant CPT1A protein antigens with different dilution factors (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 3 times with PBST washing solution and shake it dry. Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 1 h, wash 3 times with PBST washing solution and shake it dry. Add 100 μL of TMB chromogenic solution to the reaction wells, develop color in the dark at room temperature for 30 min, and add 100 μL of 2 M sulfuric acid to terminate the reaction. Use an enzyme-labeled instrument for double-wavelength detection, measure the OD value at the reference wavelength of 450 nm and the OD value at the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value measured at 450 nm. Plot the standard curve with the standard product concentration as the abscissa (pg / mL) and OD 450 - OD 630 as the ordinate. Draw the standard curve according to the results, as Figure 1 shown.

[0027] (4)Preparation of the ELISA kit: The enzyme-labeled plate coated with the capture antibody, HRP-labeled antibody working solution, antigen standard CPT1A protein, PBST washing solution, chromogenic solution TMB, and termination solution sulfuric acid prepared were respectively packaged to form the ELISA kit of the present invention.

[0028] Test Example 1: Specific Identification of Monoclonal Antibodies CPT1A-1 and CPT1A-2: Mix BSA protein and recombinant CPT1A protein with loading buffer containing SDS at a volume ratio of 2:1. Perform SDS-PAGE on the samples and transfer the proteins to a nitrocellulose membrane using a semi-dry transfer apparatus. Block the membrane with the blocking solution at 37°C for 1 h and wash it 3 times with PBST washing solution. Dilute the monoclonal antibodies CPT1A-1 and CPT1A-2 prepared in Example 1 to a concentration of 1 μg / mL, add the monoclonal antibodies CPT1A-1 and CPT1A-2 prepared in Example 1 respectively, and incubate at 37°C for 1 h. Wash the membrane to remove unbound antibodies. Add anti-mouse IgG secondary antibody labeled with alkaline phosphatase and incubate at 37°C for 1 h to further amplify the signal. Wash the membrane 3 times with PBST washing solution, immerse the membrane in the mixed TMB chromogenic solution at 2 mg / L for color reaction, and add 100 μL of 2M sulfuric acid to terminate the reaction. Observe the experimental results to determine whether the monoclonal antibodies have the ability to specifically recognize CPT1A protein.

[0029] The results are as Figure 2 shown. There is no band in lane 1 (BSA protein) for the monoclonal antibody of CPT1A-1 protein, and an obvious band appears in lane 2 (recombinant CPT1A protein). There is no band in lane 3 (BSA protein) for the monoclonal antibody of CPT1A-2 protein, and an obvious band appears in lane 4 (recombinant CPT1A protein). It shows that the monoclonal antibodies CPT1A-1 and CPT1A-2 can specifically bind to the recombinant CPT1A protein, and have no obvious binding characteristics with BSA protein, indicating that the monoclonal antibodies of CPT1A-1 and CPT1A-2 proteins have good specificity.

[0030] Test Example 2: Detection of Blood Samples: (1) Collect 10 blood samples from healthy people (numbered 1-10) and 10 blood samples from cervical cancer patients (numbered 11-20), centrifuge at 6000 rpm for 10 min to obtain the supernatant; (2) Take out the enzyme-labeled plate coated with the capture antibody, restore it to room temperature, wash the plate 3 times with PBST washing solution and drain it, add the samples processed in step (1), incubate at room temperature for 1 h, and wash 3 times with PBST buffer solution and drain it.

[0031] (3) Add the HRP-labeled antibody to the system in step (2), incubate at room temperature for 1 h, wash the plate 3 times with PBST washing solution and drain it; (4) Add 100 μL of TMB chromogenic solution to the system after the reaction in step (3), develop color in the dark at room temperature for 30 min, and add 100 μL of 2M sulfuric acid termination solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader for dual-wavelength detection. Subtract the OD value measured at 630 nm from the OD value measured at 450 nm, and calculate the result through the standard curve; (6) Determine whether the blood sample is positive based on the data results.

[0032] Table 2 Detection results of blood samples: ; As shown in Table 2, the kit of the present invention can accurately detect the content of CPT1A protein in the blood samples of cervical cancer patients, and the result is positive. There is a trace amount of CPT1A protein in the blood of healthy people, and the detected concentration is not within the linear range of the kit of the present invention, and the result is negative. It shows that the kit of the present invention can use blood as a sample to complete the detection of cervical cancer markers and can meet the needs of basic research and clinical diagnosis.

[0033] Test Example 3: Stability of the ELISA kit: Take recombinant CPT1A protein standards (200 pg / mL, 400 pg / mL, 600 pg / mL) as the samples to be tested. Place the enzyme-linked immunosorbent assay (ELISA) plate coated with CPT1A-1 antibody and the working solution of HRP-labeled CPT1A-2 antibody at 37°C to test their stability for 30 days, and perform the detection of the content of the samples to be tested every 15 days. Place the enzyme-linked immunosorbent assay (ELISA) plate coated with CPT1A-1 antibody and the working solution of HRP-labeled CPT1A-2 antibody in a 4°C refrigerator for storage for 2 years, and take them out every 12 months to perform the detection of the content of the samples to be tested. The detection results are shown in Table 3.

[0034] Table 3 Detection results of standards: ; As shown in Table 3, within 30 days of exposure at 37°C and 2 years of low-temperature storage of the kit, the OD value gradient of the standard curve is good, and the detected concentration of the recombinant CPT1A protein standard is also relatively stable, indicating that the stability of the kit is good.

[0035] 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 them. The basic principles and main features of the present invention have been described in the above with specific implementation schemes. On the basis of the present invention, some modifications or replacements can be made, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the present invention claimed.

Claims

1. A kit for cervical cancer detection, characterized in that, The kit includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody, an antigen standard, a washing solution, a chromogenic solution, and a termination solution; In the capture antibody, the heavy chain amino acid sequence of the anti-CPT1A protein monoclonal antibody CPT1A-1 is as shown in SEQ ID NO.1, and the light chain amino acid sequence is as shown in SEQ ID NO.2; in the HRP-labeled antibody, the heavy chain amino acid sequence of the anti-CPT1A protein monoclonal antibody CPT1A-2 is as shown in SEQ ID NO.3, and the light chain amino acid sequence is as shown in SEQ ID NO.

4.

2. The kit for cervical cancer detection according to claim 1, characterized in that, The antigen standard is a recombinant CPT1A protein antigen; the washing solution is PBST; the chromogenic solution is TMB; the termination solution is sulfuric acid.

3. The kit for cervical cancer detection according to claim 1, wherein The preparation process of the anti-CPT1A protein monoclonal antibody is as follows: (1) Use the recombinant CPT1A protein as an antigen to immunize mice, fuse the spleen cells of the immunized mice with myeloma cells to form hybridoma cells; (2) Screen the hybridoma cells by ELISA, subclone and expand the positive cell line, then inoculate it into mice, collect ascites and purify to obtain the anti-CPT1A protein monoclonal antibody.

4. The kit for cervical cancer detection according to claim 1, wherein The preparation steps of the enzyme-labeled plate coated with the capture antibody are as follows: Dilute the monoclonal antibody CPT1A-1 with an antibody coating solution and add it to the microwells of the enzyme-labeled plate, seal the plate with a sealing film and coat it overnight, add a blocking solution and block it at room temperature for a period of time, discard the liquid in the wells of the enzyme-labeled plate, dry it and store it sealed.

5. The kit for cervical cancer detection according to claim 1, wherein The preparation steps of the HRP-labeled antibody are as follows: Add a NaIO4 solution to the HRP solution to oxidize the glycosyl group of HRP into an aldehyde group, then mix the monoclonal antibody CPT1A-2 with the aldehydeized HRP solution, react at room temperature in the dark, continue to add a NaBH4 solution and react at 4°C to obtain a stable enzyme-labeled antibody, and finally dialyze it in PBS overnight to obtain the HRP-labeled antibody.

6. Use of the kit for cervical cancer detection according to any one of claims 1 to 5, characterized in that, It is used to detect the content of CPT1A protein in blood.

7. Use of the kit for cervical cancer detection according to claim 6, characterized in that, The detection steps are as follows: (1) Centrifuge the sample to be detected and take the supernatant; (2) Take out the enzyme-labeled plate coated with the capture antibody, let it return to room temperature, wash the plate with the washing solution and spin dry, add the sample processed in step (1), incubate at room temperature, then wash the plate with the washing solution and spin dry; (3) Add the HRP-labeled antibody to the system in step (2), incubate at room temperature for a period of time, wash the plate with the washing solution and spin dry; (4) Add the chromogenic solution to the system after the reaction in step (3), develop color at room temperature in the dark for a period of time, and add the termination solution to terminate the reaction; (5) Use an enzyme-labeled instrument for dual-wavelength detection, subtract the OD value at 630nm from the OD value at 450nm, and calculate the result through the standard curve.

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