A cancer early screening antigen, its detection kit and application

Abstract

The present invention discloses a monoclonal antibody that can specifically bind to both CYFRA21-1 protein and S100A10 protein. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4. The present invention also discloses a kit for detecting lung adenocarcinoma, which includes a calibrator, R1 reagent, and R2 reagent, wherein the R2 reagent is a monoclonal antibody conjugated with alkaline phosphatase. The monoclonal antibody of the present invention has good sensitivity and specificity. The kit of the present invention has a higher detection coincidence rate for lung adenocarcinoma than the CYFRA21-1 kit. Therefore, the kit of the present invention provides a good reference for the early diagnosis research of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a cancer early screening antigen, a detection kit thereof, and an application. Background Art

[0002] Lung cancer is the leading cause of cancer-related deaths globally, with an estimated 2.277 million new cases and 1.796 million deaths in 2020. The incidence and mortality of lung cancer are increasing year by year. Lung adenocarcinoma (LUAD) is the most common histological subtype, accounting for approximately 40% of all lung cancers. Each clinical stage of lung cancer differentiates the overall survival. The 60-month overall survival rate of lung cancer patients gradually decreases from 92% in stage IA1 to 0% in stage IVB, which reflects the importance of early diagnosis and treatment in improving the prognosis of lung cancer patients. Despite the improvement in early diagnosis and individualized treatment, the 5-year survival rate of patients remains approximately 23%, mainly because only 21% of cases are diagnosed at the early stage of lung cancer. Therefore, discovering new core molecular network characteristics and establishing new diagnostic and treatment models for early LUAD are crucial for reducing the damage caused by LUAD in the population.

[0003] CYFRA21-1 (amino acid sequence as shown in SEQ ID NO.1) is considered a tumor marker mainly used for the detection of lung cancer, and it has important value for the diagnosis of non-small cell lung cancer (NSCLC) in particular. If there are unclear circular shadows in the lungs and the serum CYFRA21-1 concentration > 3.3 ng / ml, the possibility of primary bronchogenic carcinoma is very high. The positive detection rate of various types of NSCLC is 70% - 85%. The serum concentration level of CYFRA21-1 is positively correlated with the clinical stage of the tumor, and it can also be used as an effective indicator for tracking early recurrence after lung cancer surgery and radiotherapy and chemotherapy. A high serum concentration level of CYFRA21-1 indicates that the disease is in the advanced stage and has a poor prognosis. The sign of successful treatment is a rapid decrease in the serum concentration of CYFRA21-1, otherwise it indicates that the lesion has not been completely cleared. If the serum level decreases and then increases, it indicates disease recurrence. However, a negative CYFRA21-1 does not exclude the possibility of having lung cancer. The sensitivity of CYFRA21-1 for the diagnosis of various types of lung cancer is in the order of: squamous cell carcinoma > adenocarcinoma > large cell carcinoma > small cell carcinoma. Therefore, for LUAD, the diagnostic positive rate of CYFRA21-1 needs to be improved. Therefore, on the basis of CYFRA21-1, it is necessary to screen for suitable early diagnostic antigens for LUAD and establish a combined diagnostic method for the early diagnosis of lung cancer. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a detection kit for the diagnosis of lung adenocarcinoma and its application.

[0005] Therefore, on the one hand, the present invention discloses a monoclonal antibody that can specifically bind to both CYFRA21-1 protein and S100A10 protein. The amino acid sequences of the heavy chain variable region and the light chain variable region of this monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0006] Preferably, the site where the monoclonal antibody of the present invention specifically binds to CYFRA21-1 protein is aa302-aa311, and its amino acid sequence is YRSLLEGQED.

[0007] Preferably, the site where the monoclonal antibody of the present invention specifically binds to S100A10 protein is aa39-aa48, and its amino acid sequence is FPGFLENQKD.

[0008] On the other hand, the present invention also discloses a kit for detecting lung adenocarcinoma, which includes a calibrator, R1 reagent and R2 reagent, wherein the R2 reagent is a monoclonal antibody conjugated with alkaline phosphatase.

[0009] Preferably, the calibrator of the present invention is CYFRA21-1 recombinant proteins with different concentrations, and their concentrations are 0 ng / ml, 5 ng / ml, 30 ng / ml, 100 ng / ml, 300 ng / ml, and 1000 ng / ml respectively.

[0010] Preferably, the R1 reagent of the present invention is magnetic particles conjugated with CYFRA21-1 monoclonal antibody and S100A10 monoclonal antibody.

[0011] Preferably, the Cut-off value of the kit of the present invention is 5 ng / ml. When the sample value > Cut-off value, it indicates that there may be lung adenocarcinoma. When the sample value ≤ Cut-off value, it indicates that the test result is normal.

[0012] On the other hand, the present invention also discloses the application of the monoclonal antibody in the preparation of a reagent for detecting lung adenocarcinoma.

[0013] Based on previous research, the present invention screened a monoclonal antibody that can specifically bind to both CYFRA21-1 protein and S100A10 protein through the analysis of CYFRA21-1 protein and S100A10 protein. This monoclonal antibody has good sensitivity and specificity.

[0014] Based on the CYFRA21-1 detection kit, a kit for the diagnosis of lung adenocarcinoma was established using the screened monoclonal antibodies of the present invention. This kit can detect the total content of CYFRA21-1 protein and S100A10 protein in a sample in a combined manner in one detection, and a new Cut-off value was established therefrom for the early diagnosis of lung adenocarcinoma by the kit. The kit of the present invention has a higher detection coincidence rate for lung adenocarcinoma than the CYFRA21-1 kit. Therefore, the kit of the present invention provides a good reference for the early diagnosis research of lung adenocarcinoma. Description of the Drawings

[0015] Figure 1 Western blot detection results of S100A10 protein in lung adenocarcinoma tissues.

[0016] Figure 2 Schematic diagram of monoclonal antibody preparation.

[0017] Figure 3 Western blot detection results of monoclonal antibodies against CYFRA21-1 recombinant protein and S100A10 recombinant protein respectively, where M is the marker. Figure 3 1 in A is CYFRA21-1 recombinant protein. Figure 3 1 in B is S100A10 recombinant protein. Detailed Description of the Invention

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of this invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0019] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0020] Example 1: Screening and Verification of Lung Adenocarcinoma Biomarkers

[0021] Researchers found that S100A10 (S100 calcium-binding protein A10, with the amino acid sequence shown in SEQ ID NO.2) was highly expressed in lung adenocarcinoma and was associated with poor prognosis [Wang Huijie, Sun Zhengui, Zhao Wenying, Geng Biao. S100A10 can promote the proliferation and invasion of lung adenocarcinoma cells: based on the activation of the Akt / mTOR signaling pathway [J]. Journal of Southern Medical University, 2023, 43(5): 733-740]. For this reason, the researchers in this study collected 3 pairs of lung adenocarcinoma tissues and adjacent tissues from a hospital in Guangdong, and used a monoclonal antibody against S100A10 (Wuhan Aimeijie Technology Co., Ltd., NDC-AMB-B9CX0C-100) to perform western blot detection on S100A10, and found that S100A10 was highly expressed in lung adenocarcinoma tissues, which was consistent with the literature reports, as specifically shown in Figure 1 shown. For this reason, the researchers in this study used this as a target for lung adenocarcinoma for subsequent research.

[0022] Example 2: Preparation and testing of monoclonal antibodies specifically binding to CYFRA21-1 protein and S100A10 protein

[0023] Dilute the CYFRA21-1 recombinant protein (Wuhan Aimeijie Technology Co., Ltd., MBS283658, with an N-terminal GST tag) and the S100A10 recombinant protein (Wuhan Aimeijie Technology Co., Ltd., PRO-384) to 0.5 mg / ml each, mix them in equal mass ratio for standby, and prepare monoclonal antibodies by the mouse hybridoma cell technology. The preparation schematic diagram is as shown in Figure 2 shown, and is briefly described as follows:

[0024] Immunize female BALB / c mice aged 6-8 weeks with the mixed protein respectively. For the first immunization, emulsify with an equal amount of Freund's complete adjuvant and inject intramuscularly in the leg, 0.2 ml per mouse; for the second immunization, emulsify with an equal amount of Freund's incomplete adjuvant and inject intramuscularly in the leg, 0.2 ml per mouse; for the third immunization, mix with an equal amount of physiological saline and inject intraperitoneally, 0.2 ml per mouse.

[0025] Collect blood 7 days after the third immunization, and use the CYFRA21-1 recombinant protein and the S100A10 recombinant protein respectively to measure the antibody titer by the indirect method to determine whether antibodies against the antigen are generated. Compare the titers of different mice, and finally select the mice with higher antibody titers against both proteins for cell fusion. Three days before fusion, boost the immunization once according to the method of the third immunization. Through detection, a total of 3 mice were screened, and their ELISA antibody titers against CYFRA21-1 protein and S100A10 protein were both ≥1:100000.

[0026] Take the spleens of the above 3 selected mice respectively, fuse spleen cells with mouse myeloma cells, and screen for hybridoma cells. After reverse screening and detection (i.e., indirectly measuring the antibody titer using CYFRA21-1 recombinant protein and S100A10 recombinant protein respectively), only one mouse screened out a good hybridoma cell strain, which was expanded and cultured and then stored in liquid nitrogen for later use.

[0027] Take 5 healthy female mice aged 6 - 8 weeks, and intraperitoneally inject 0.5 ml of liquid paraffin or other adjuvants into each of them to stimulate their immune responses. One week later, inject the screened hybridoma cell strain into the abdominal cavity of the mice, with about 800,000 to 1,000,000 cells injected into each mouse. About 7 days after injecting the cells, the abdomen of the mice will start to swell. At this time, daily observation is required to prevent sudden death of the mice. When the mice show symptoms such as difficulty in eating, inconvenient walking, and dull hair color, the mice should be sacrificed to extract ascites. After the mice are sacrificed by cervical dislocation, open the abdomen, and use a pipette to suck the effusion in the abdominal cavity into a centrifuge tube. Centrifuge at 4000 rpm at room temperature for 15 min, and the supernatant is the ascites, which is aliquoted and stored at -20°C for later use. Take the ascites after centrifugation treatment and purify it using a ProteinG column (the specific purification method is shown in Table 1). Measure the concentration of the purified antibody using the BCA method, aliquot it, and store it at -80°C for later use.

[0028] Table 1 Purification steps of monoclonal antibody from mouse ascites

[0029]

[0030] Use the prepared monoclonal antibody to perform western blot detection on CYFRA21-1 recombinant protein and S100A10 recombinant protein respectively. The results show that ( Figure 3 ), this monoclonal antibody can specifically bind to both CYFRA21-1 protein and S100A10 protein.

[0031] By testing and analyzing the sequence and antigen recognition site of this monoclonal antibody (entrusted to Beijing Bio-Tech Pack Technology Co., Ltd.), the amino acid sequences of the heavy chain variable region and light chain variable region of this monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4; among them, the site where this monoclonal antibody specifically binds to CYFRA21-1 protein (shown in SEQ ID NO.1) is aa302 - aa311, and its amino acid sequence is YRSLLEGQED; the site where this monoclonal antibody specifically binds to S100A10 protein (shown in SEQ ID NO.2) is aa39 - aa48, and its amino acid sequence is FPGFLENQKD.

[0032] Example 3: Indirect ELISA detection (reverse screening indirect ELISA detection)

[0033] Coating: Dilute the CYFRA21-1 recombinant protein and the S100A10 recombinant protein to 1 μg / ml respectively using the coating buffer (pH 9.6 CBS buffer), and add them to the enzyme-linked immunosorbent assay (ELISA) plates separately (i.e., the CYFRA21-1 recombinant protein and the S100A10 recombinant protein are coated separately), 0.1 ml per well, and coat overnight at 2-8°C. Washing: Aspirate the coating solution, add 0.3 ml of PBST buffer to each well, aspirate after shaking for 30 s, and repeat the washing 3 times and then aspirate the buffer. Blocking: Add 0.2 ml of PBST buffer containing 1% BSA to each well and incubate at 37°C for 2 h. Washing: Aspirate the blocking solution, add 0.3 ml of PBST buffer to each well, aspirate after shaking for 30 s, and repeat the washing 3 times and then aspirate the buffer. Adding samples: Dilute the mouse serum and the hybridoma cell culture supernatant 10-fold serially using the PBST buffer, mix well and then add them to the ELISA plates respectively, 0.1 ml per well, and incubate at 37°C for 1 h. Washing: Aspirate the samples, add 0.3 ml of PBST buffer to each well, aspirate after shaking for 30 s, and repeat the washing 3 times and then aspirate the buffer. Adding secondary antibody: Add 0.1 ml of HRP-labeled goat anti-mouse IgG secondary antibody (diluted 1:5000) to each well; incubate at 37°C for 0.5 h. Washing: Aspirate the secondary antibody, add 0.3 ml of PBST buffer to each well, aspirate after shaking for 30 s, and repeat the washing 3 times and then aspirate the buffer. Color development: Add 0.1 ml of TMB color development solution to each well and incubate at 37°C for 10 min. Termination: Add 0.05 ml of termination solution (2 M sulfuric acid) to each well, and immediately measure the OD450nm value after termination. Calculation: When the P / N value (sample OD450nm value / blank control OD value) > 2.1 and the OD450nm value > 0.1, it is judged as positive, otherwise it is negative.

[0034] Example 4: Preparation and detection of a lung adenocarcinoma detection kit

[0035] Preparation of calibrators: Dilute the CYFRA21-1 recombinant protein with 50 mM Tris-HCl pH = 8.0 to solutions with final concentrations of 0, 5, 30, 100, 300, 1000 (unit ng / ml) respectively, 0.5 ml per tube, and store at 2-8°C for standby.

[0036] Preparation of R1 reagent: Take 5 mg of carboxylated magnetic microparticles (purchased from thermo fisher, with a diameter of 2 μm), add 1 ml of 0.1 M MES buffer (pH 6.0), vortex and mix well. Let it stand on a magnetic rack for 5 min to separate the magnetic microparticles from the liquid. Discard the supernatant, add 1 ml of TBST buffer, wash 3 times, then add 0.5 ml of MES buffer with pH 6.0 and vortex. Add an equal proportion mixture of 0.1 mg of CYFRA21-1 monoclonal antibody (Wuhan AmyJet Scientific Co., Ltd., MBS1495558) and S100A10 monoclonal antibody (Wuhan AmyJet Scientific Co., Ltd., NDC-AMB-B9CX0C-100) (i.e., 0.05 mg CYFRA21-1 monoclonal antibody + 0.05 mg S100A10 monoclonal antibody), vortex, and incubate with rotation at room temperature for 20 min. Add 10 μl of EDC with a concentration of 10 mg / ml, vortex, and incubate with rotation at room temperature for 2 h. After centrifugation, discard the supernatant, add 1 ml of TBST buffer, and wash 3 times. Add 1 ml of Tris-HCl buffer with 1% BSA and pH = 8.0 for blocking, and block with rotation at room temperature for 2 h. After centrifugation, discard the supernatant, add 1 ml of TBST buffer, and wash 3 times. Add 0.5 ml of Tris-HCl buffer with 1% BSA and pH = 8.0 to resuspend the magnetic microparticles, dilute to 0.25 mg / ml with Tris-HCl buffer with 1% BSA and pH = 8.0, mix well after preparation, quantitatively dispense, and obtain R1 reagent. Store it in a dry place at 2 - 8°C.

[0037] Preparation of R2 reagent: Dissolve 2 mg of ALP (alkaline phosphatase) in 0.5 ml of monoclonal antibody solution (2 mg / ml), and load it into a dialysis bag. Dialyze overnight at 2 - 8°C with 10 mM PBS pH = 7.2, changing the solution three times during this period. Add 20 μl of 2.5% glutaraldehyde, react at room temperature for 2 h, and dialyze overnight at 2 - 8°C with 10 mM PBS pH = 7.2, changing the solution three times during this period. Change to 50 mM Tris-HCl buffer with pH = 8.0 for dialysis, dialyze overnight at 2 - 8°C, and change the solution three times during this period. Take out the labeled antibody, dilute it to 2 ml with Tris-HCl buffer with 1% BSA and pH = 8.0 to obtain the original solution of the AP-labeled substance. Dilute the original solution of the labeled substance 1000 times with Tris-HCl buffer with 1% BSA and pH = 8.0, quantitatively dispense, and obtain R2 reagent. Store it at 2 - 8°C for standby.

[0038] Kit detection: (1) Use a pipette to aspirate 20 μl of the sample to be tested into a dilution bottle (containing 180 μl of 50 mM Tris-HCl pH = 8.0 buffer), and mix well by inverting up and down. (2) Aspirate 50 μl of the sample in (1) and calibration products with different concentrations (the calibration products do not need to be diluted) and add them into reaction cups (containing 50 μl of R1 reagent and 50 μl of R2 reagent) respectively. Place the reaction cups in a 37 °C incubator and mix and incubate for 15 min (37 °C, 1000 rpm). (3) Wash 3 times with TBST buffer, 500 μl each time. (4) Add 200 μl of substrate solution (purchased commercially, such as Beckman or domestic manufacturers), incubate at 37 °C for 15 min (37 °C, 1000 rpm), and detect the RLU value with a chemiluminescence analyzer. (5) Fit a standard curve through the RLU value and the concentration of the calibration product for calculating the sample concentration. (6) Result determination: Cut-off value = 5 ng / ml; if the sample value > Cut-off value, it indicates that there may be lung adenocarcinoma; if the sample value ≤ Cut-off value, it indicates that the test result is normal.

[0039] Example 5: Detection of clinical samples

[0040] Collect 89 serum clinical samples (including 50 normal sera and 39 sera from lung adenocarcinoma patients), and use the kit prepared in Example 4, a commercially available CYFRA21-1 detection kit (produced by Beckman), and HumanProteinS100-A10, S100A10 ELISA KIT (Wuhan Aimeijie Technology Co., Ltd., JOTEK3801Hu) for detection respectively.

[0041] The results are shown in Table 2. The coincidence rate of the kit of the present invention is higher than that of the commercially available CYFRA21-1 kit and HumanProteinS100-A10, S100A10 ELISA KIT, indicating that the combined detection of CYFRA21-1 + S100A10 significantly improves the diagnostic coincidence rate of lung adenocarcinoma, which provides a good reference for the early diagnosis research of lung adenocarcinoma.

[0042] Table 2 Detection results of clinical samples

[0043]

[0044] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody can specifically bind to both CYFRA21-1 protein and S100A10 protein. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The monoclonal antibody according to claim 1, characterized in that The site where the monoclonal antibody specifically binds to the CYFRA21-1 protein is aa302-aa311, and its amino acid sequence is YRSLLEGQED.

3. The monoclonal antibody according to claim 1, characterized in that The site where the monoclonal antibody specifically binds to the S100A10 protein is aa39-aa48, and its amino acid sequence is FPGFLENQKD.

4. Use of the monoclonal antibody according to claim 1 in preparing a lung adenocarcinoma detection reagent.

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