Mouse monoclonal antibody against Cyfra21-1 and its preparation method and application

Abstract

The present invention discloses a murine monoclonal antibody against Cyfra21-1, designated 4C10 and 6F9, respectively. The murine monoclonal antibody 4C10 has a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO.2 and a light chain variable region with the amino acid sequence set forth in SEQ ID NO.4; the murine monoclonal antibody 6F9 has a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO.3 and a light chain variable region with the amino acid sequence set forth in SEQ ID NO.5. An ELISA method for detecting Cyfra21-1 using a double-antibody sandwich method was established using the murine monoclonal antibodies prepared in the present invention. Compared to existing techniques, the method has higher sensitivity and specificity, and is more highly correlated with the Roche Cyfra21-1 detection kit.

Description

Technical Field

[0001] The present invention relates to in vitro diagnostic technology, in particular to an anti-Cyfra21-1 mouse monoclonal antibody. The present invention also relates to a preparation method of the antibody and application of the antibody in preparing a kit for detecting Cyfra21-1. Background Art

[0002] Lung cancer is one of the leading causes of cancer death. Approximately 70% of lung cancer patients present with mid- or late-stage disease, missing the window for surgical rescue. There are many types of lung cancer, but they can be broadly categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC accounts for approximately 20% of all types, while NSCLC accounts for approximately 80%. Numerous tumor markers have been identified for lung cancer, among which Cyfra21-1 (cytokeratin fraction 19) has recently become recognized as one of the more sensitive tumor markers for diagnosing NSCLC.

[0003] Cyfra21-1 is a soluble fragment of cytokeratin 19 (CK19). CY and FRA are abbreviations for Cytokeratin and Fragent, respectively, while 21-1 represents two monoclonal antibodies used to detect CK19 fragments: CM19.21 and KS19.1. Cyfra21-1 is primarily present in the cytoplasm of epithelial tumor cells, such as lung and esophageal cancers. It is released into the serum due to cell necrosis or lysis. Its concentration in the serum of healthy individuals is correlated with age, gender, and smoking status.

[0004] Cyfra21-1 is highly valuable in lung cancer diagnosis and patient prognosis. If a patient has a vague annular shadow in the lungs and a serum Cyfra21-1 concentration >30 ng / ml, the likelihood of primary bronchogenic carcinoma is very high. The current positive detection rate for various types of non-small cell lung cancer is 70% to 85%. Because serum Cyfra21-1 levels are positively correlated with clinical tumor stage, it can serve as an effective marker for tracking early recurrence after lung cancer surgery, chemoradiotherapy, and chemotherapy. High serum Cyfra21-1 levels indicate advanced disease and a poor prognosis. Successful treatment is marked by a rapid decrease in serum Cyfra21-1 levels; a decrease indicates incomplete lesion clearance. A subsequent increase in serum levels after a decrease indicates recurrence. However, a negative Cyfra21-1 level does not rule out the possibility of lung cancer. The sensitivity of Cyfra21-1 for diagnosing various types of lung cancer is as follows: squamous cell carcinoma > adenocarcinoma > large cell carcinoma > small cell carcinoma. The latest research report suggests that the diagnostic sensitivity of Cyfra21-1 for squamous cell carcinoma of the lung is as high as 93.5%. Therefore, Cyfra21-1 has guiding significance in the clinical diagnosis of squamous cell carcinoma of the lung.

[0005] Currently, leading manufacturers of Cyfra21-1 detection kits, such as Abbott and Roche, utilize two mouse monoclonal antibodies, CM19.21 and KS19.1, in their sandwich ELISA kits. The CM19.21 and KS19.1 antibodies were generated using MCF-7 cells as immunogens and specifically recognize the 346-367aa and 311-335aa fragments of the Cytokeratin 19 protein, respectively. U.S. Patent No. 5,780,032 similarly discloses the purification of native cytokeratin antigens from MCF-7 cells. Immunization of Balb / c mice resulted in the identification of a monoclonal antibody that specifically recognizes cytokeratin 19 and has no cross-reactivity with cytokeratin 8 or cytokeratin 18. Meanwhile, Chinese patent document [CN105132382A] discloses the use of Cyfra21-1 antigen to immunize Balb / c mice, resulting in the identification of a pair of mouse monoclonal antibodies that specifically recognize Cyfra21-1. The double antibody sandwich assay detects Cyfra21-1 with a sensitivity of 1 ng / ml. U.S. patent document [US9255142B2] discloses the use of recombinantly expressed Cytokeration 19 protein in Escherichia coli to immunize Balb / c mice. A pair of paired mouse monoclonal antibodies, specifically recognizing the 325-350 aa fragment and the 375-400 aa fragment of Cytokeration 19, were identified using Cytokeration 19 protein peptide fragments. These antibodies, tested against 69 lung cancer patient sera, achieved a detection rate of 88.41%, exceeding the 52.17% achieved by the Roche Cyfra21-1 test kit. Summary of the Invention

[0006] Based on the above-mentioned prior art, the present invention provides a pair of novel mouse monoclonal antibodies that can specifically recognize the Cyfra21-1 protein; another object of the present invention is to provide a method for preparing mouse monoclonal antibodies against Cyfra21-1; and a third object of the present invention is to provide a test paper or kit for detecting Cyfra21-1.

[0007] To achieve the above purpose, the present invention can adopt the following technical solutions:

[0008] The anti-Cyfra21-1 mouse monoclonal antibodies of the present invention are named 4C10 and 6F9, respectively, wherein:

[0009] The murine monoclonal antibody 4C10 comprises the following CDRs:

[0010] Heavy chain CDR1 to CDR3, whose amino acid sequences are shown in SEQ ID NOs. 1-3, respectively;

[0011] Light chain CDR1 to CDR3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO. 4, the amino acid sequence of light chain CDR2 is GAT, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO. 5;

[0012] The murine monoclonal antibody 6F9 comprises the following CDRs:

[0013] Heavy chain CDR1 to CDR3, whose amino acid sequences are shown in SEQ ID NOs. 6 to 9, respectively;

[0014] Light chain CDR1 to CDR3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO.9, the amino acid sequence of light chain CDR2 is KVS, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO.10.

[0015] The mouse monoclonal antibody 4C10:

[0016] The amino acid sequence of SEQ ID NO.1 is GFTFSAHY;

[0017] The amino acid sequence of SEQ ID NO.2 is INDGGSST;

[0018] The amino acid sequence of SEQ ID NO.3 is ARSYGNYWFFDV;

[0019] The amino acid sequence of SEQ ID NO.4 is EDIYIR;

[0020] The amino acid sequence of SEQ ID NO.5 is QQYWSTPPT;

[0021] The mouse monoclonal antibody 6F9:

[0022] The amino acid sequence of SEQ ID NO.6 is VFTFNNDA;

[0023] The amino acid sequence of SEQ ID NO.7 is IRSKSNNYAT;

[0024] The amino acid sequence of SEQ ID NO.8 is VRAYGYNVMDY;

[0025] The amino acid sequence of SEQ ID NO.9 is QSLVHRDGNTY;

[0026] The amino acid sequence of SEQ ID NO.10 is SQSTHVPPT.

[0027] Furthermore, the murine monoclonal antibody 4C10 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.11 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.12; the murine monoclonal antibody 6F9 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.13 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.14.

[0028] Specifically, the amino acid sequence of SEQ ID NO.11 is as follows:

[0029] EVMLVESGGDLVKPGGSLKLSCAASGFTFSAHYMYWVRQTPEKRLEWVATINDGGSSTFFPDSVQGRFTVSRDNAKNNLYLHMSSLRSEDTAMYYCARSYGNYWFFDVWGAGTTVTVSS.

[0030] The amino acid sequence of SEQ ID NO.12 is as follows:

[0031] DIQMTQSSSSFSVSLGDRVITTCKASEDIYIRLAWYQQKPGNAPRLLLISGATNLETGVPSRFSGSGYGKDYTLRITSLQTEDIGTYYCQQYWSTPPTTFGGGTKLEIK.

[0032] The amino acid sequence of SEQ ID NO.13 is as follows:

[0033] EVKLVESGGGLVQPKGSLKLSCAASVFTFNNDAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMVYLQMNNLKTEDTAVYYCVRAYGYNVMDYWGQGTSVTVS.

[0034] The amino acid sequence of SEQ ID NO.14 is as follows:

[0035] DAVMTQTPLSLPVSLGDQASISCRSSQSLVHRDGNTYLHWFLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYFCSQSTHVPPTFGGGTKLELK.

[0036] Furthermore, the murine monoclonal antibody also includes a heavy chain constant region and a light chain constant region.

[0037] Furthermore, the heavy chain constant region and the light chain constant region are derived from mouse IgG.

[0038] The anti-Cyfra21-1 mouse monoclonal antibody described in the present invention is obtained by immunizing Balb / c mice with a recombinant protein containing all 400 amino acids of Cyfra21-1 expressed in HEK293F cells as an immunogen. The spleen is then fused with myeloma cells NS1, followed by screening and purification. The prepared anti-Cyfra21-1 mouse monoclonal antibody has an affinity for the recombinant Cyfra21-1 antigen expressed in HEK293 cells of greater than 1.0×10 -9 M.

[0039] The anti-Cyfra21-1 mouse monoclonal antibody prepared by the present invention is used to establish a double-antibody sandwich ELISA detection kit, in which the anti-Cyfra21-1 mouse monoclonal antibody 4C10 is coated on magnetic beads as a capture antibody, and the anti-Cyfra21-1 mouse monoclonal antibody 6F9 is labeled with HRP as a detection antibody. The double-antibody sandwich method is used to detect Cyfra21-1 protein in the serum of lung cancer patients. The kit has extremely high sensitivity and specificity and can be applied to the clinical detection of lung cancer.

[0040] Compared with the prior art, the advantages of the present invention are:

[0041] The present invention uses the Cyfra21-1 recombinant protein expressed in HEK293F cells as an antigen to prepare a new specific anti-Cyfra21-1 mouse monoclonal antibody. The mouse monoclonal antibody prepared by the present invention is used to establish an ELISA method for detecting Cyfra21-1 using a double antibody sandwich method. Compared with the existing technology, especially the mouse monoclonal antibody prepared in U.S. Patent document [US9255142B2], the ELISA method has higher sensitivity and specificity and is more highly correlated with the Roche Cyfra21-1 detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The enzyme digestion products were identified by 1% agarose gel electrophoresis.

[0043] Figure 2 The results of SDS-PAGE analysis of Cyfra21-1 recombinant protein.

[0044] Figure 3 This is the Western blot identification result of anti-Cyfra21-1 mouse monoclonal antibody.

[0045] Figure 4 The kit prepared for the present invention is associated with Roche. DETAILED DESCRIPTION

[0046] The present invention will be described in more detail below by way of specific examples for the convenience of understanding by those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all conventional reagents, and the instruments and methods employed are also conventional instruments and methods in the art.

[0047] Example 1 Preparation of Cyfra21-1 recombinant antigen

[0048] 1. Complete gene synthesis of Cyfra21-1 coding sequence

[0049] The mRNA sequence of Cyfra21-1 (NM_002276.5) was found from NCBI (National Center for Biotechnology Information). The CDS region sequence is (SEQ ID NO. 15):

[0050]

[0051] Shanghai Sangon Bioengineering Co., Ltd. was commissioned to carry out whole gene synthesis.

[0052] 2. Construction and identification of Cyfra21-1 recombinant plasmid

[0053] The full-length gene sequence of the Cyfar21-1 coding region and the pCMV3 vector were double-digested with Xbal I and Hind III restriction endonucleases for 4 hours, and the digestion products were run on 1% agarose gel electrophoresis (110V, 35min). The target fragment was recovered using a gel recovery kit. The target fragment of Cyfra21-1 was ligated to the linear vector using T4 DNA ligase and the ligation was incubated at 4°C overnight. After the ligation product was transformed into Escherichia coli Top10 competent cells, it was spread on a Luria-Bertani (LB) solid culture medium plate containing 100ug / ml of ampicillin and cultured at 37°C overnight. Positive clones were picked and shaken to extract the plasmid. The plasmid was identified by digestion with Xbal I and Hind III, and the digestion products were identified by 1% agarose gel electrophoresis. A specific band was visible at about 1200bp, as shown in Figure 2. Figure 1 As shown, it was shown that a fragment of about 1200 bp had been inserted into the pCMV3 vector.

[0054] The positive plasmids were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results showed that they were completely consistent with the Cyfra21-1 gene sequence reported in NCBI.

[0055] 3. Preparation of Cyfra21-1 recombinant protein

[0056] HEK293F cells were cultured in 500 ml cell culture flasks using SMM293-TII medium in a volume of 200 ml. On the day of transfection, the density of HEK293F cells was adjusted to 3 × 10 6 cells / mL, with a viability of no less than 90%. Mix 200 μg of plasmid and 1 ml of transfection reagent, incubate at room temperature for 10 minutes, then slowly add the solution to a shake flask and incubate in a 37°C, 5% CO2 incubator. Sample the cells every 24 hours after transfection to determine cell viability. When the cell viability falls below 40%, centrifuge at 2000 rpm to collect the culture supernatant.

[0057] The cell culture supernatant was dialyzed overnight against 20 mmol / ml PBS at pH 7.4. After equilibration with 20 mmol / ml PBS at pH 7.4, the dialyzed supernatant was slowly loaded onto the Ni column. Contaminants were eluted with 200 mmol / ml imidazole at pH 7.4 (20 mmol / ml), and the protein was dissociated using 500 mmol / ml imidazole at pH 7.4 (20 mmol / ml). The collected dissociation buffer was concentrated to a protein concentration of 1 mg / ml and stored at 4°C. Figure 2 The results show that the purified Cyfra21-1 recombinant protein is of high purity.

[0058] Example 2 Preparation of mouse monoclonal antibody against Cyfra21-1

[0059] 1. Mouse immunization

[0060] The Cyfra21-1 recombinant antigen, fully emulsified in Freund's complete adjuvant, was intraperitoneally immunized into 5-week-old female Balb / c mice at a primary dose of 100 μg / mouse. Second and third immunizations were administered 21 and 42 days after the first immunization, respectively, at a dose of 50 μg / mouse. Approximately 10 days after the third immunization, tail blood was collected, and serum titers were determined indirectly using a 96-well plate coated with Cyfra21-1.

[0061] 2. Hybridoma Cell Preparation

[0062] Select indirect method to detect serum titer greater than 10 4 Mice were given an intrasplenic booster immunization at a dose of 100 μg / mouse. Three days after the booster immunization, the spleens of the mice were harvested and fused with mouse myeloma cells NS1 at a ratio of 10:1 using PEG. The fused cells were then cultured in DMEM medium (Gibco) containing HAT.

[0063] About 6-7 days after fusion, the specific antibody content in the cell culture supernatant was indirectly detected using a 96-well plate coated with Cyfra21-1. Positive wells with an OD value of not less than 0.5 were selected and subcloned three times using the limiting dilution method. Finally, two hybridoma cell lines that can stably secrete anti-Cyfra21-1 were obtained and named 4C10 and 6F9, respectively.

[0064] 3. Purification of mouse monoclonal antibody against Cyfra21-1

[0065] The obtained mouse hybridoma cells capable of stably secreting anti-Cyfra21-1 were injected into the peritoneal cavity of mice, and the ascites was collected and purified by SPA to obtain anti-Cyfra21-1 mouse monoclonal antibodies with a purity of more than 90%.

[0066] 4. Anti-Cyfra21-1 monoclonal antibody titer detection

[0067] The Cyfra21-1 recombinant protein obtained in Example 1 was diluted to 1 μg / ml using 0.05 mmol / L CB buffer, pH 9.6. 50 μl was added to each well of a 96-well ELISA plate (Corning) and coated overnight at 4°C. The following day, the plate was washed three times with PBST and blocked with 100 μl / well of 1% Casein at 37°C for 2 hours. Purified monoclonal antibodies 4C10 and 6F9 (both at a concentration of 5 mg / ml) were serially diluted using 0.05 mmol / L CB buffer, pH 9.6, at ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and so on. The diluted antibodies were added to the Cyfra21-1 recombinant protein-coated ELISA plate. 50 μl / well of 0.05 mmol / L CB buffer, pH 9.6, was added to the negative control wells. After incubation at 37°C for 30 minutes, the plate was washed five times with PBST, patted dry, and 100 μl / well of 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA) was added. The plate was incubated at 37°C for 30 minutes. The plate was washed five times with PBST, patted dry, and then 100 μl / well of standard ELISA substrate was added. The plate was incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μl of 0.1 mol / L sulfuric acid, and the absorbance at 450 nm was measured. The results are shown in Table 1 below.

[0068] Table 1 Absorbance at different dilution factors

[0069]

[0070] As shown in Table 1, the average OD value of the negative control was 0.01, and the titers of the two monoclonal antibodies 4C10 and 6F9 were both above 1 / 256000, showing relatively high titers.

[0071] 5. Identification of anti-Cyfra21-1 mouse monoclonal antibodies

[0072] The recombinant protein obtained in Example 1 was diluted to 0.1 mg / ml with 0.05 mmol / L, pH=9.6 CB buffer, and 20 ul of the diluted recombinant protein was taken for 10% SDS-PAGE electrophoresis. After the electrophoresis, the gel was placed in the transfer buffer for equilibrium for 10 minutes, and the membrane was electrotransferred at 200 mA for 30 minutes. The membrane was placed in a blocking solution, blocked overnight at room temperature, the blocking solution was discarded, and purified 4C10 and 6F9 monoclonal antibodies were added at a concentration of 5 ug / ml, and incubated at 4°C for 2 hours. The membrane was washed 3 times with TBST, each time for 3 minutes. A 1 / 4000 diluted horseradish peroxidase-labeled goat anti-mouse IgG was added, incubated at 37°C for 2 hours, and the membrane was washed 4 times with TBST, each time for 3 minutes. A solution and a solution B of the DAB kit were added and mixed in equal proportions for color development. From Figure 3 The identification results shown show that clear bands are visible between the molecular weight of 35-45 kD.

[0073] 6. Monoclonal Antibody Sequencing

[0074] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0075] 4C-LF 5′-GACATTGTGATGACCCAGTCTCCT-3′;

[0076] 4C-LR 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′

[0077] 4C-HF 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′

[0078] 4C-HR 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′

[0079] 3×10 6Total RNA from hybridoma cells 4C10 and 6F9 was reverse transcribed into cDNA. PCR amplification of the heavy chain variable regions of monoclonal antibodies 4C10 and 6F9 was performed using primers 4C-HF and 4C-HR. PCR amplification of the light chain variable regions of monoclonal antibodies 4C10 and 6F9 was performed using primers 4C-LF and 4C-LR. Both PCR reactions used a hot start reaction condition: 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 55°C for 45 seconds, and 72°C for 30 seconds, and 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis, and the target fragments were recovered and purified. The fragments were cloned into the PM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB plates. White plaques were inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, and plasmids were extracted using QIAGEN's plasmid extraction kit and sequenced to determine the heavy chain and light chain variable region sequences of monoclonal antibodies 4C10 and 6F9.

[0080] The heavy chain variable region of the monoclonal antibody 4C10 has the amino acid sequence shown in SEQ ID NO.11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.12; the heavy chain variable region of the murine monoclonal antibody 6F9 has the amino acid sequence shown in SEQ ID NO.15, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.14.

[0081] Example 3 Establishment of an ELISA method for detecting Cyfra21-1 using a double antibody sandwich method

[0082] 1. Magnetic particle coating

[0083] 30 μl of the mixed magnetic microparticle stock solution was washed five times with 300 μl of PBS buffer. The magnetic particles were then activated with 10% glutaraldehyde for one hour and then washed twice with PBS buffer, pH 7-8. Anti-Cyfra21-1 mouse monoclonal antibody 4C10 was added to the magnetic beads at a concentration of 0.3 μg / dose and coated at 4°C for two hours. Finally, the beads were blocked with a blocking buffer containing BSA for two hours.

[0084] 2. HRP-labeled anti-Cyfra21-1 mouse monoclonal antibody 6F9

[0085] Anti-Cyfra21-1 mouse monoclonal antibody 6F9 was labeled with HRP (Roche) at a mass ratio of 1.5:1. The HRP-labeled anti-Cyfra21-1 mouse monoclonal antibody 6F9 was diluted to 1:4000 in diluent.

[0086] 3. Clinical sample testing

[0087] 504 lung cancer patient samples with Roche Cyfra21-1 (cobas e 411) values ​​were collected from a hospital and tested using the double antibody sandwich method of the present invention. The correlation between the results and those of the Roche kit was as follows: Figure 4 As shown, the overall correlation R between the kit of the present invention and the Roche kit 2 =0.99.

[0088] 4. Linear interval

[0089] Prepare a high-value clinical sample with a concentration close to approximately 130% of the upper limit of the expected linear range, labeled H. Prepare a low-value clinical sample with a concentration close to the minimum detection limit (LOB), labeled L. These two samples were mixed in varying proportions to prepare a series of sample concentrations. The linear range of the gradient samples was then assessed. The results are shown in Tables 2 and 3 below.

[0090] Table 2 Preparation of a series of linear samples using clinical high-value and low-value samples

[0091]

[0092] Table 3: Statistics of variation and total error

[0093]

[0094] Each sample was tested 4 times, and a linear regression (y=ax+b) was performed between the expected concentration and the average value of the detected concentration to calculate the linear coefficient R 2 , requiring R 2 ≥0.99, a value between 0.9-1.1, and a total error TE% less than 20%. The linear range of the sandwich assay for Cyfra21-1 established using the antibody of the present invention was determined to have a lower limit of 0.51 ng / mL and an upper limit of 512.67 ng / mL.

[0095] 5. Sensitivity

[0096] 5.1 LOB

[0097] Normal saline was used as the O value sample for testing. The measurement was repeated 20 times. The mean (M) and standard deviation (SD) of the RLU values ​​(relative luminescence value) of the 20 measurements were calculated to obtain M+2SD. The RLU value of M+2SD was substituted into the calibration curve equation of the calibrator to obtain the corresponding concentration value, which is the limit of blank (LOB). The sandwich method for detecting Cyfra21-1 using the antibody of the present invention was established and tested on an AutoLumo A2000 Plus. The results are shown in Table 4 below.

[0098] Table 4 LOB assessment results

[0099]

[0100] 5.2 LOD

[0101] Five clinical samples with concentrations ranging from 1 to 5 times the LOB were selected and tested three times daily for four consecutive days on the AutoLumo A2000 Plus detection system, yielding 60 data points. The lowest Cyfra21-1 concentration that the system could detect with a 95% probability of detection was calculated, and the LoD was calculated. The results are shown in Table 5.

[0102] Table 5 LOD assessment results

[0103]

[0104] From the above results, it can be calculated that the lowest concentration that can be detected when the detection probability reaches 95% is 0.45 ng / mL. The LoD of the sandwich method for detecting Cyfra21-1 established using the antibody of the present invention is determined to be 0.45 ng / mL.

[0105] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A mouse monoclonal antibody against Cyfra21-1, characterized in that: The murine monoclonal antibodies were designated as 4C10 and 6F9, respectively, wherein the murine monoclonal antibody 4C10 comprises the following CDRs: Heavy chain CDR1 to CDR3, whose amino acid sequences are shown in SEQ ID NOs. 1-3, respectively; Light chain CDR1 to CDR3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO. 4, the amino acid sequence of light chain CDR2 is GAT, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO. 5; The murine monoclonal antibody 6F9 comprises the following CDRs: Heavy chain CDR1 to CDR3, whose amino acid sequences are shown in SEQ ID NOs. 6 to 9, respectively; Light chain CDR1 to CDR3, the amino acid sequence of light chain CDR1 is shown in SEQ ID NO.9, the amino acid sequence of light chain CDR2 is KVS, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO.

10.

2. The anti-Cyfra21-1 mouse monoclonal antibody according to claim 1, characterized in that: The murine monoclonal antibody 4C10 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.11 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.12; the murine monoclonal antibody 6F9 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.13 and a light chain variable region with the amino acid sequence shown in SEQ ID NO.

14.

3. The anti-Cyfra21-1 mouse monoclonal antibody according to claim 1, characterized in that: The murine monoclonal antibody further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region and the light chain constant region are derived from murine IgG.

4. An isolated nucleic acid, characterized in that Encodes the anti-Cyfra21-1 mouse monoclonal antibody according to any one of claims 1 to 3.

5. A carrier, characterized in that It contains the nucleic acid according to claim 4.

6. A host cell, characterized in that Comprising the vector according to claim 5.

7. A method for preparing a mouse monoclonal antibody against Cyfra21-1, characterized in that: Cultivate the host cell according to claim 6 to obtain the anti-Cyfra21-1 mouse monoclonal antibody.

8. A test paper for detecting Cyfra21-1, characterized in that: The invention comprises the anti-Cyfra21-1 mouse monoclonal antibody according to any one of claims 1 to 3.

9. A kit for detecting Cyfra21-1, characterized in that: Comprising the anti-Cyfra21-1 mouse monoclonal antibody according to any one of claims 1 to 3 or the test paper according to claim 8.

10. Use of the anti-Cyfra21-1 mouse monoclonal antibody according to any one of claims 1 to 3 in the preparation of a kit for detecting Cyfra21-1.

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