A kit for early diagnosis of lung cancer and its application

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to a kit for early diagnosis of lung cancer and its application. The kit includes an enzyme-labeled plate coated with a capture antibody, a working solution of an HRP-labeled antibody, an antigen standard, a washing solution, a chromogenic solution, and a termination solution; the heavy-chain amino acid sequence of the monoclonal antibody F6C-1 against 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 against the MAPRE2 protein in the HRP-labeled antibody is as shown in SEQ ID NO.3, and the light-chain amino acid sequence is as shown in SEQ ID NO.4. The kit for early diagnosis of lung cancer provided by the present invention has strong specificity, can be used as an auxiliary diagnostic method for lung cancer with high expression of the MAPRE2 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 early diagnosis of lung cancer and its application. Background Technology

[0002] Lung cancer, also known as primary bronchogenic carcinoma, is the most common malignant tumor of the lung 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 has become one of the greatest threats to human health. Most lung cancers are completely asymptomatic in their early stages, and despite continuous improvements in diagnostic methods, most are diagnosed at an advanced stage, with a five-year survival rate of only 16%.

[0003] Based on the morphological characteristics of lung cancer cells under a microscope, lung cancer 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 type, accounting for 80% of all lung cancers, and is further divided into adenocarcinoma, cellular carcinoma, and large cell carcinoma. Early diagnosis of lung cancer is a crucial measure to reduce lung cancer mortality. Currently, early diagnosis of lung cancer mainly utilizes techniques such as low-dose spiral CT, bronchoscopy, and pathological examination. Low-dose spiral CT is primarily used for lung cancer screening in high-risk populations and has achieved some success; however, its widespread adoption 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, limited sensitivity, and cost restrict the widespread adoption 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 diagnostic markers such as CEA, CA125, and SCC, while possessing some clinical value, have limitations and cannot accurately predict lung cancer stage. Therefore, there is an urgent need to develop more specific and sensitive tumor markers.

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

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

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

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

[0009] A kit for early diagnosis of lung cancer, the kit comprising an enzyme-linked immunosorbent assay (ELISA) plate coated with capture antibody, HRP-labeled antibody working solution, antigen standard, washing solution, chromogenic solution and stop solution;

[0010] The capture antibody is a monoclonal antibody F6C-1 against the MAPRE2 protein. 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 a monoclonal antibody H3A-2 against MAPRE2 protein. 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 recombinant MAPRET2 protein antigen; the washing solution is PBST; the chromogenic solution is TMB; and the stop solution is sulfuric acid.

[0013] Furthermore, the amino acid sequence of the recombinant MAPRET2 protein antigen is shown in SEQ ID NO.5.

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

[0015] The above-described kits for early diagnosis of lung cancer are used to detect the level of MAPRE2 protein in the blood.

[0016] Furthermore, the detection steps are as follows:

[0017] (1) Take the sample to be tested, centrifuge it, and collect the supernatant;

[0018] (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;

[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) Add color developing 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 stop solution to terminate the reaction;

[0021] (5) Use an ELISA reader to perform dual-wavelength detection. Subtract the OD value of 630nm from the OD value of 450nm and calculate the result using a standard curve.

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

[0023] Compared with the prior art, the main advantages of the present invention are as follows:

[0024] This 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 high specificity, is simple to operate, and can effectively determine the risk of lung cancer in the tested population, greatly improving the detection and diagnosis efficiency of lung cancer and having high diagnostic value. Attached Figure Description

[0025] Figure 1 Standard curve for a kit to specifically detect recombinant MAPRE2 protein;

[0026] Figure 2 The image shows the results of specific identification of F6C-1 and H3A-2 monoclonal antibodies.

[0027] Figure 3 ROC curve analysis was performed to assess the diagnostic value of serum marker MAPRE2 protein in lung cancer. 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] The preparation of F6C-1 and H3A-2 monoclonal antibodies includes the following steps:

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

[0033] (2) Hybridoma cell fusion: Mice with the highest antibody titers were selected, and their spleens were removed in a clean bench after sacrifice to prepare a spleen cell suspension. Spleen cells and myeloma cells were mixed at a ratio of 5:1, using polyethylene glycol as the fusion agent, and incubated at 37°C for 2 minutes. Five days after fusion, the cells were cultured in HAT selective medium. The supernatant from the hybridoma cell culture wells was detected using an indirect ELISA method to screen for positive hybridoma cells. These positive hybridoma cells were then subcloned using a three-stage limiting dilution method, and the hybridoma cells with the highest sensitivity and specificity were selected for further expansion 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 mice's abdomens swelled, the ascites fluid was collected. The collected mouse ascites fluid was centrifuged at 6000 rpm for 30 min to remove cellular components. The ascites fluid was filtered through a 0.2 μm filter. The collected ascites fluid was purified by ion exchange chromatography to obtain the purified monoclonal antibody.

[0035] (4) Analysis of monoclonal antibody sequences: The activity of the purified monoclonal antibodies was accurately measured. The two monoclonal antibodies with the highest activity 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 List

[0037]

[0038]

[0039] Example 2

[0040] Preparation of ELISA kit

[0041] (1) Preparation of the capture antibody-coated ELISA plate: The monoclonal antibody F6C-1 prepared in Example 1 was used 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 antibody coating buffer (50 mM carbonate buffer, pH = 9.6), and 100 μL / well was added to each well. The plate was sealed with sealing film and incubated overnight at 4°C. The coating buffer was discarded, and the plate was washed three times with PBST washing buffer (PBST buffer containing 0.2% Tween 20). 200 μL / well of 1% (w / v) BSA blocking buffer was added, and the plate was blocked at room temperature for 2 h. The liquid in the 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 50mM carbonate buffer for 24h, with the buffer changed twice during the process, and the concentration was adjusted to 2mg / mL. 5mg of HRP dry powder was weighed and dissolved in 1mL ddH2O. 200μL of 0.1M NaIO4 solution was added, and the solution was stirred at room temperature in the dark for 30min. The aldehyde-modified HRP solution was placed in a dialysis bag and dialyzed overnight in 10mM sodium acetate buffer at 4℃ to remove unreacted NaIO4. The pH 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 4h, followed by the addition of 0.1mL of 4mg / L NaBH4. After mixing, the solution was placed at 4℃ for 2h to obtain a stable enzyme-labeled antibody. The above sample was placed in 0.15M PBS (pH 7.6) and dialyzed overnight at 4°C. The dialysate was centrifuged to remove the precipitate, and the supernatant was the working solution of HRP-labeled H3A-2 antibody.

[0043] (3) Standard curve determination of the kit: Remove the ELISA plate coated with capture antibody, allow it to return to room temperature, wash the plate three times with PBST buffer and agitate dry. Add 100 μL of recombinant MAPRE2 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 the plate three times with PBST 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 the plate three times with PBST buffer and agitate dry. Add 100 μL of 4 mg / L TMB chromogenic reagent 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 standard curve was then plotted based on the results, as follows: Figure 1 As shown. The detection range of the kit for early diagnosis of lung cancer in this invention is 200-800 pg / mL, and the standard curve is y = 0.0040x + 0.6729, R0. 2 =0.9997.

[0044] (4) Preparation of ELISA kit: The prepared enzyme-labeled plate coated with capture antibody, HRP-labeled antibody working solution, antigen standard recombinant MAPRET2 protein, PBST washing solution, colorimetric 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] Experimental Example 1

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

[0047] BSA protein and recombinant MAPRE2 protein were mixed with SDS-containing loading buffer at a volume ratio of 2:1. The samples were subjected to SDS-PAGE, and the proteins were transferred to nitrocellulose membranes using a semi-dry gel transfer instrument. The membranes were immersed in blocking buffer and blocked at 37°C for 1 h, followed by washing three times with PBST washing buffer. 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 distributed over the membrane surface and incubated at 37°C for 1 h. The membranes were washed three times with PBST washing buffer to remove unbound antibodies. Anti-mouse IgG secondary antibody labeled with horseradish peroxidase was added, and the membranes were incubated at 37°C for 1 h to further amplify the signal. The membranes were washed three times with PBST washing buffer, and then immersed in a mixture of 2 mg / L TMB chromogenic solution for colorimetric reaction. The reaction was terminated by adding 100 μL of 2M sulfuric acid. The experimental results were observed to determine whether the monoclonal antibodies had the ability to specifically recognize 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 clear band in lane 2 (recombinant MAPRE2 protein). The H3A-2 protein monoclonal antibody showed no band in lane 3 (BSA protein) but a clear band in lane 4 (recombinant MAPRE2 protein). This indicates that the monoclonal antibodies F6C-1 and H3A-2 specifically bind to the recombinant MAPRE2 protein, but do not show significant binding to the BSA protein, suggesting that the F6C-1 and H3A-2 protein monoclonal antibodies have good specificity.

[0049] Experimental Example 2

[0050] Blood sample testing

[0051] (1) Collect blood samples from 10 healthy individuals (numbered 1-10) and 10 lung cancer patients (numbered 11-20), and centrifuge at 6000 rpm for 10 min to collect the supernatant;

[0052] (2) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate 3 times with PBST washing buffer and spin dry, add 100 μL of the sample treated in step (1), incubate at room temperature for 1 h, wash 3 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 3 times with PBST washing solution and spin dry;

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

[0055] (5) Use an 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 through the standard curve;

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

[0057] Table 2 Blood Sample Test Results

[0058] serial number OD 450-OD 630 MAPRE2 protein content detection 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] The results are shown in Table 2. The kit of the present invention can accurately detect the MAPRE2 protein content in blood samples from lung cancer patients, with a positive result. Trace amounts of MAPRE2 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 lung cancer biomarkers, meeting the needs of basic research and clinical diagnosis.

[0060] Experimental Example 3

[0061] ROC curve assessment kit for the diagnostic value of lung cancer

[0062] Forty samples were collected, including 20 serum samples from healthy individuals as the control group and 20 serum samples from early-stage lung cancer as the observation group. The MAPRE2 protein level was detected using the procedure described in Experiment 1. The results were plotted as ROC curves, and the AUC value was calculated to evaluate its diagnostic value in lung cancer.

[0063] The results are as follows Figure 3 The figure shows the ROC curve for the serum biomarker MAPRE2 in diagnosing lung cancer. The AUC value of serum MAPRE2 is 0.9780. A larger ROC curve area indicates greater diagnostic value, and an AUC above 0.7 indicates a good classification ability of the model. These results demonstrate that the serum biomarker MAPRE2 protein can specifically distinguish between healthy individuals and lung cancer patients, and lung cancer screening can be achieved by detecting the level of serum MAPRE2 protein.

[0064] 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 early diagnosis of lung cancer, characterized by, The kit includes an ELISA plate coated with capture antibody, HRP-labeled antibody working solution, antigen standard, washing solution, colorimetric solution, and stop solution; The capture antibody is a monoclonal antibody F6C-1 against the MAPRE2 protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody F6C-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 antibody in the HRP-labeled antibody working solution is a monoclonal antibody H3A-2 against MAPRE2 protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody H3A-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.

2. The kit for early diagnosis of lung cancer according to claim 1, characterized in that, The antigen standard is recombinant MAPRET2 protein antigen; the washing solution is PBST; the chromogenic 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 concentration of the colorimetric reagent is 2 mg / L-4 mg / L; the concentration of the stop solution is 2 M-4 M.

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