Kit for early diagnosis of lung cancer and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a kit for early diagnosis of lung cancer and application of the kit. The kit comprises an elisa plate coated with a captured antibody, an HRP labeled antibody working solution, an antigen standard substance, a washing solution, a developing solution and a stop solution, the heavy chain amino acid sequence of the monoclonal antibody F6C-1 for resisting the MAPRE2 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 H3A-2 of the anti-MAPRE2 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 H3A-2 is as shown in SEQ ID NO.4. The kit for early diagnosis of lung cancer provided by the invention has strong specificity, can be used as an auxiliary diagnosis means for lung cancer with high expression of MAPRE2 protein, and has high diagnostic value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a kit for early diagnosis of lung cancer and application thereof. Background Art

[0002] Lung cancer, also known as primary bronchogenic carcinoma, is the most common lung malignancy originating from the trachea, bronchial mucosa, or glands. Smoking, environmental pollution, respiratory diseases, and genetic factors are the main causes of lung cancer. Global cancer statistics show that lung cancer is one of the greatest threats to human health. Most lung cancers are completely asymptomatic in the early stages. Despite advances in diagnostic methods, the majority are diagnosed in the late stages, with a five-year survival rate of only 16%.

[0003] Based on the morphological characteristics of lung cancer cells under a microscope, they can be preliminarily divided into two types: small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer is the most representative cancer and accounts for 80% of lung cancers. It is divided into adenocarcinoma, cell carcinoma, and large cell carcinoma. Early diagnosis of lung cancer is an important measure to reduce lung cancer deaths. At present, early diagnosis of lung cancer mainly uses low-dose spiral CT, bronchoscopy, pathological examination and other technologies. Low-dose spiral CT is mainly used for lung cancer screening in high-risk groups and has achieved success to a certain extent, but its popularization is limited by high costs and increased false positive rates. Bronchoscopy and pathological examination are irreplaceable in the diagnosis of lung cancer, but their invasiveness, sensitivity limitations and cost issues restrict the popularization of early diagnosis.

[0004] In recent years, an increasing number of disease markers have been used for clinical disease screening and diagnosis, but their effectiveness remains significantly limited. In lung cancer diagnosis, existing lung cancer markers such as CEA, CA125, and SCC, while valuable, have limitations and cannot accurately predict lung cancer stages. Therefore, there is an urgent need to develop more specific and sensitive tumor markers.

[0005] Based on the above purpose, the present invention provides a kit for early diagnosis of lung cancer and its application. Summary of the Invention

[0006] The first object of the present invention is to provide a kit for early diagnosis of lung cancer.

[0007] The second object of the present invention is to provide an application of a kit for early diagnosis of lung cancer.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A kit for early diagnosis of lung cancer, comprising an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody working solution, an antigen standard, a washing solution, a color developing solution, and a stop solution;

[0010] The capture antibody is the anti-MAPRE2 monoclonal antibody F6C-1, the heavy chain amino acid sequence of the monoclonal antibody F6C-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2;

[0011] The antibody in the HRP-labeled antibody working solution is the anti-MAPRE2 protein monoclonal antibody H3A-2. The heavy chain amino acid sequence of the monoclonal antibody H3A-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0012] Furthermore, the antigen standard is a recombinant MAPRE2 protein antigen; the washing solution is PBST; the color developing solution is TMB; and the stop solution is sulfuric acid.

[0013] Furthermore, the amino acid sequence of the recombinant MAPRE2 protein antigen is shown in SEQ ID NO.5.

[0014] Furthermore, the concentration of the developing solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2M-4M.

[0015] The use of any of the above-mentioned kits for early diagnosis of lung cancer is used to detect the content of MAPRE2 protein in blood.

[0016] Furthermore, the detection steps are:

[0017] (1) Centrifuge the sample to be tested and take the supernatant;

[0018] (2) removing the enzyme-labeled plate coated with the capture antibody, returning it to room temperature, washing the plate with a washing solution and drying it, adding the sample treated in step (1), incubating at room temperature, washing the plate with a washing solution and drying it;

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

[0020] (4) adding a color developing solution to the system after the reaction in step (3), developing the color for a period of time at room temperature in the dark, and then adding a stop solution to terminate the reaction;

[0021] (5) Use a microplate reader for dual-wavelength detection, subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the results using a standard curve.

[0022] Furthermore, the sample to be tested is blood.

[0023] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0024] The present invention discloses a kit for early diagnosis of lung cancer and its application. The kit can detect the MAPRE2 protein content in the blood, has strong specificity and is simple to operate, can effectively judge the risk of lung cancer in the tested population, greatly improves the detection and diagnosis efficiency of lung cancer, and has high diagnostic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the standard curve of the kit for specific detection of recombinant MAPRE2 protein;

[0026] Figure 2 This is the result of specific identification of F6C-1 and H3A-2 monoclonal antibodies;

[0027] Figure 3 ROC curve was used to analyze the diagnostic value of serum marker MAPRE2 protein in lung cancer. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0029] Example 1

[0030] Preparation of monoclonal antibodies

[0031] Preparation of F6C-1 and H3A-2 monoclonal antibodies, comprising the following steps:

[0032] (1) Immunization of mice: Recombinant MAPRE2 protein was used as the immunogen. The amino acid sequence of the recombinant MAPRE2 protein is shown in SEQ ID NO.5. The recombinant MAPRE2 protein was mixed with an equal volume of complete Freund's adjuvant and fully emulsified. Healthy female mice were selected as the experimental group and immunized by multiple subcutaneous injections in the abdomen. The first immunization dose was 100 μg / mouse. Three weeks after the first immunization, the second immunization was performed. The recombinant MAPRE2 protein was mixed with an equal volume of incomplete Freund's adjuvant, emulsified, and injected. The immunization dose was 50 μg / mouse. Four weeks after the second immunization, the third immunization was performed. The immunization dose was 50 μg / mouse. After the third immunization injection, the mouse serum was collected and the mouse serum antibody titer was analyzed using ELISA.

[0033] (2) Fusion of hybridoma cells: Select mice with the highest antibody titer, kill them, remove the spleen in a clean bench, and prepare a spleen cell suspension. Mix spleen cells and myeloma cells at a ratio of 5:1, use polyethylene glycol as a fusion agent, and incubate in a 37°C water bath for 2 minutes. Five days after fusion, replace the HAT selective medium and continue culturing. Use the indirect ELISA method to detect the supernatant of the hybridoma cell culture wells, screen out positive hybridoma cells, perform three limiting dilution subcloning on the screened positive hybridoma cells, and select the hybridoma cells with the strongest sensitivity and specificity for expanded culture.

[0034] (3) Preparation and purification of monoclonal antibodies: The hybridoma cells obtained in step (2) were inoculated into the peritoneal cavity of female mice sensitized with paraffin oil. After the abdomen of the mouse swelled, ascites was collected. The collected mouse ascites was centrifuged at 6000 rpm for 30 minutes to remove cellular components. The ascites was filtered through a 0.2 μm filter and the collected ascites was purified by ion exchange chromatography to obtain purified monoclonal antibodies.

[0035] (4) Analysis of Monoclonal Antibody Sequences: The activities of the purified monoclonal antibodies were accurately measured. The two most active monoclonal antibodies were named F6C-1 and H3A-2, and their variable region sequences were determined, as shown in Table 1. The heavy chain amino acid sequence of monoclonal antibody F6C-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 H3A-2 is shown in SEQ ID NO. 3, and the light chain amino acid sequence is shown in SEQ ID NO. 4.

[0036] Table 1 Sequence Listing

[0037]

[0038]

[0039] Example 2

[0040] Preparation of ELISA kit

[0041] (1) Preparation of a capture antibody-coated enzyme-labeled plate: The monoclonal antibody F6C-1 prepared in Example 1 was used as the capture antibody. A 96-well enzyme-labeled plate was used as a solid phase carrier. The capture antibody was diluted to 2 μg / mL with an antibody coating solution (50 mM carbonate buffer, pH = 9.6). 100 μL / well was added to the microwells of the enzyme-labeled plate. The plate was sealed with a sealing film and then placed at 4°C for overnight coating. The coating solution was discarded, and the plate was washed three times with PBST washing solution (PBST buffer containing 0.2% Tween 20). 200 μL / well of 1% (w / v) BSA blocking solution was added and the plate was placed at room temperature for blocking for 2 hours. The liquid in the plate wells was discarded, the plate was patted dry, sealed, and stored at 4°C.

[0042] (2) Preparation of HRP-labeled antibody working solution: The monoclonal antibody H3A-2 prepared in Example 1 was used as the HRP-labeled antibody. The monoclonal antibody H3A-2 was dialyzed in 50 mM carbonate buffer for 24 h, during which the solution was changed twice to adjust the concentration to 2 mg / mL. 5 mg of HRP dry powder was weighed and dissolved in 1 mL of ddH2O. 200 μL of 0.1 M NaIO4 solution was added and stirred at room temperature in the dark for 30 min. The aldehyde-treated HRP solution was placed in a dialysis bag and dialyzed overnight in 10 mM sodium acetate buffer at 4°C to remove unreacted NaIO4. The pH value of the dialyzed HRP solution was adjusted to 9.0, and then an equal volume of monoclonal antibody H3A-2 was immediately added. The solution was stirred at room temperature in the dark for 4 h, followed by the addition of 0.1 mL of 4 mg / L NaBH4. After mixing, the solution was allowed to react at 4°C for 2 h to obtain a stable enzyme-labeled antibody. The sample was dialyzed into 0.15 M PBS at pH 7.6 overnight at 4° C. The dialyzate was centrifuged to remove the precipitate, and the supernatant was the HRP-labeled H3A-2 antibody working solution.

[0043] (3) Kit standard curve determination: Remove the enzyme-labeled plate coated with the capture antibody, return it to room temperature, wash the plate three times with PBST washing solution and spin dry, add 100 μL of recombinant MAPRE2 protein antigen with 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 hour, wash the plate three times with PBST washing solution and spin dry. Add 100 μL of HRP-labeled antibody working solution to the reaction well, incubate at room temperature for 1 hour, wash the plate three times with PBST washing solution and spin dry. Add 100 μL of 4 mg / L color development solution TMB to the reaction well, develop color at room temperature in the dark for 30 minutes, and add 100 μL of 3 M sulfuric acid to terminate the reaction. Use a microplate reader for dual wavelength detection, measure the OD value at a reference wavelength of 450nm and the OD value at a reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. Draw a standard curve with the concentration of the standard as the horizontal axis (pg / mL) and OD 450-OD 630 as the vertical axis. Draw a standard curve based on the results, such as Figure 1 The detection range of the kit for early diagnosis of lung cancer of the present invention is 200-800 pg / mL, the standard curve y=0.0040x+0.6729, R 2 =0.9997.

[0044] (4) Preparation of ELISA kit: The prepared enzyme labeling plate coated with capture antibody, HRP labeled antibody working solution, antigen standard recombinant MAPRE2 protein, PBST washing solution, color development solution TMB, and stop solution sulfuric acid are packaged separately to form the kit for early rapid screening of lung cancer of the present invention.

[0045] Test Example 1

[0046] Specificity identification of F6C-1 and H3A-2 monoclonal antibodies:

[0047] BSA protein and recombinant MAPRE2 protein were mixed with a loading buffer containing SDS at a volume ratio of 2:1, and the samples were subjected to SDS-PAGE. The proteins were transferred to a nitrocellulose membrane using a semi-dry gel transfer apparatus. The membrane was immersed in blocking solution and blocked at 37°C for 1 hour. The membrane was washed three times with PBST washing solution. The F6C-1 and H3A-2 monoclonal antibodies prepared in Example 1 were diluted to a concentration of 1 μg / mL. The diluted monoclonal antibodies F6C-1 and H3A-2 were evenly coated on the membrane surface and incubated at 37°C for 1 hour. Unbound antibodies were washed three times with PBST washing solution to remove them. An anti-mouse IgG secondary antibody labeled with horseradish peroxidase was added and incubated at 37°C for 1 hour to further amplify the signal. The membrane was washed three times with PBST washing solution, and the membrane was immersed in a mixed 2 mg / L TMB color development solution for color development. The reaction was terminated by adding 100 μL of 2M sulfuric acid. The experimental results were observed to determine whether the monoclonal antibody had the ability to specifically recognize the MAPRE2 protein.

[0048] The results are as follows Figure 2 As shown, the F6C-1 protein monoclonal antibody showed no band in lane 1 (BSA protein) but a distinct band in lane 2 (recombinant MAPRE2 protein). The H3A-2 protein monoclonal antibody showed no band in lane 3 (BSA protein) but a distinct band in lane 4 (recombinant MAPRE2 protein). This indicates that the F6C-1 and H3A-2 monoclonal antibodies specifically bind to recombinant MAPRE2 protein, but have no significant binding to BSA protein, demonstrating that the F6C-1 and H3A-2 protein monoclonal antibodies have good specificity.

[0049] Test Example 2

[0050] Blood sample testing

[0051] (1) Blood samples from 10 healthy individuals (numbered 1-10) and 10 lung cancer patients (numbered 11-20) were collected and centrifuged at 6000 rpm for 10 min to obtain the supernatant;

[0052] (2) Remove the ELISA plate coated with the capture antibody, return it to room temperature, wash the plate three times with PBST washing solution and spin dry, add 100 μL of the sample treated in step (1), incubate at room temperature for 1 hour, wash it three times with PBST buffer and spin dry;

[0053] (3) Add 100 μL of HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for 1 h, wash the plate three times with PBST washing solution and spin dry;

[0054] (4) Add 100 μL of TMB colorimetric solution to the reaction system after step (3), develop the color at room temperature in the dark for 30 min, and then add 100 μL of 3 M sulfuric acid stop solution to terminate the reaction;

[0055] (5) Dual-wavelength detection was performed using a microplate reader. The OD value at 450 nm was subtracted from the OD value at 630 nm, and the results were calculated using a standard curve.

[0056] (6) Determine whether the blood sample is positive based on the data results. If the detected concentration is within the test kit detection range of 200-800 pg / mL, it is positive (+), otherwise it is negative (-).

[0057] Table 2 Blood sample test results

[0058] serial number OD 450-OD 630 MAPRE2 protein content test results (pg / mL) result 1 0.3672 Not detected Negative 2 0.3537 Not detected Negative 3 0.3856 Not detected Negative 4 0.4319 Not detected Negative 5 0.4207 Not detected Negative 6 0.4183 Not detected Negative 7 0.3908 Not detected Negative 8 0.3889 Not detected Negative 9 0.3974 Not detected Negative 10 0.3998 Not detected Negative 11 2.8039 532.76 Positive 12 2.6429 492.50 Positive 13 2.6451 493.05 Positive 14 2.8630 547.52 Positive 15 2.8010 532.02 Positive 16 2.5697 474.21 Positive 17 2.3244 412.88 Positive 18 2.4869 453.51 Positive 19 2.5419 467.26 Positive 20 2.6079 483.76 Positive

[0059] As shown in Table 2, the kit of the present invention accurately detected the MAPRE2 protein content in blood samples from lung cancer patients, resulting in a positive result. However, the blood of healthy individuals contained trace amounts of MAPRE2 protein, and the detection concentration was outside the linear range of the kit, resulting in a negative result. This demonstrates that the kit of the present invention can detect lung cancer markers using blood as a sample, meeting the needs of basic research and clinical diagnosis.

[0060] Test Example 3

[0061] ROC curve evaluation of the diagnostic value of the kit in lung cancer

[0062] 40 samples were collected, including 20 healthy serum samples as a control group and 20 early lung cancer serum samples as an observation group. The MAPRE2 protein level was detected using the operating steps of Experimental Example 1. The ROC curve was plotted for the test results, and the AUC value was calculated to evaluate the diagnostic value in lung cancer.

[0063] The results are as follows Figure 3 The figure below shows the receiver operating characteristic (ROC) curve for the serum marker MAPRE2 in diagnosing lung cancer. The area under the ROC curve (AUC) for the serum marker MAPRE2 is 0.9780. A larger ROC curve area indicates greater diagnostic value for the disease. An AUC above 0.7 indicates strong classification capability. These results demonstrate that the serum marker MAPRE2 protein can specifically distinguish healthy individuals from lung cancer patients. Detecting serum MAPRE2 protein levels can be used for lung cancer screening.

[0064] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A kit for early diagnosis of lung cancer, characterized in that: The kit includes an enzyme-labeled plate coated with a capture antibody, an HRP-labeled antibody working solution, an antigen standard, a washing solution, a color developing solution, and a stop solution; The capture antibody is the anti-MAPRE2 monoclonal antibody F6C-1, the heavy chain amino acid sequence of the monoclonal antibody F6C-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 the anti-MAPRE2 protein monoclonal antibody H3A-2. The heavy chain amino acid sequence of the monoclonal antibody H3A-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. The kit for early diagnosis of lung cancer according to claim 1, characterized in that The antigen standard is a recombinant MAPRE2 protein antigen; the washing solution is PBST; the color developing solution is TMB; and the stop solution is sulfuric acid.

3. The kit for early diagnosis of lung cancer according to claim 2, characterized in that The amino acid sequence of the recombinant MAPRE2 protein antigen is shown in SEQ ID NO.

5.

4. The kit for early diagnosis of lung cancer according to claim 2, characterized in that The concentration of the color developing solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2M-4M.

5. Use of the kit for early diagnosis of lung cancer according to any one of claims 1 to 4, characterized in that: Used to detect the level of MAPRE2 protein in the blood.

6. Use of the kit for early diagnosis of lung cancer according to claim 5, characterized in that: The detection steps are: (1) Centrifuge the sample to be tested and take the supernatant; (2) removing the enzyme-labeled plate coated with the capture antibody, returning it to room temperature, washing the plate with a washing solution and drying it, adding the sample treated in step (1), incubating at room temperature, washing the plate with a washing solution and drying it; (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for a period of time, wash the plate with washing solution and spin dry; (4) adding a color developing solution to the system after the reaction in step (3), developing the color for a period of time at room temperature in the dark, and then adding a stop solution to terminate the reaction; (5) Use a microplate reader for dual-wavelength detection, subtract the OD value at 630 nm from the OD value at 450 nm, and calculate the results using a standard curve.

7. Use of the kit for early diagnosis of lung cancer according to claim 6, characterized in that: The sample to be detected is blood.

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