Double antibody sandwich elisa kit for detecting human cyfra 21-1 content

Abstract

The application belongs to the field of immunological detection, and provides a double-antibody sandwich ELISA kit for detecting human Cyfra21-1 content. The anti-human Cyfra21-1 monoclonal antibody provided by the application can specifically bind to human Cyfra21-1, has no cross reaction with other similar proteins, and has high stability; based on the antibody, the double-antibody sandwich ELISA method for detecting human Cyfra21-1 has high sensitivity, specificity and accuracy, and can accurately and high-throughput detect the content of Cyfra21-1 in human serum or other serum analogs containing human Cyfra21-1.

Description

Double antibody sandwich ELISA kit for detecting human Cyfra21-1 levels Technical Field

[0001] This invention belongs to the field of immunological detection, specifically, it relates to a double-antibody sandwich ELISA kit for detecting human Cyfra21-1 content. Background Technology

[0002] Cyfra21-1, a soluble fragment of cytokeratin 19, also known as cytokeratin, is a tumor-associated biomarker that has gained increasing attention in recent years. It is a fragment of cytokeratin 19 (CK19), produced during cancer cell differentiation. CK19 is a characteristic protein component of intermediate filaments in epithelial cells and is widely present in various normal epithelial tissues. When epithelial cells transform into tumors, its structure remains largely unchanged, but its expression level significantly increases. With the necrosis and lysis of tumor cells, the soluble fragment of CK19, Cyfra21-1, can be released into the bloodstream. Cyfra21-1 is present in the cytoplasm of epithelial-originating tumor cells such as those in lung cancer and esophageal cancer, thus it has some significance in the auxiliary detection of lung cancer and gastrointestinal tumors. Furthermore, Cyfra21-1 has also shown some correlation in diseases such as bladder cancer, head and neck tumors, breast cancer, and cervical cancer.

[0003] Currently, the main detection methods for Cyfra21-1 include electrochemical detection, radioimmunoassay, and luminescent oxygen channel immunoassay. Electrochemical detection relies on dedicated electrochemical sensors, analyzing changes in electrical signals caused by the target analyte, which places high demands on the performance of the detection instruments. Radioimmunoassay has a short iodine-labeled half-life, low sensitivity, and instability. Luminescent oxygen channel immunoassay typically requires biotinylation of antibodies and coupling them with streptavidin-labeled luminescent microspheres, resulting in a complex detection system and numerous operational steps. Furthermore, immunodiffusion has a long detection cycle, low sensitivity, and is easily affected by experimental conditions, making it only suitable for qualitative or semi-quantitative analysis. While high-performance liquid chromatography and capillary electrophoresis offer high separation capabilities, they require sophisticated equipment and skilled operators, resulting in high detection costs and hindering their widespread application in clinical or large-scale sample testing.

[0004] Therefore, there is an urgent need to develop a kit that can accurately, rapidly, and in large quantities detect the content of human Cyfra21-1 in samples. Summary of the Invention

[0005] The purpose of this invention is to provide a double-antibody sandwich ELISA kit for detecting human Cyfra21-1 content.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides an anti-human Cyfra21-1 monoclonal antibody 2H4, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody 2H4 are QSLLYSSNQRNY (SEQ ID NO:9), WAS, and QQYYSYPFT (SEQ ID NO:10), respectively; and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody 2H4 are GFTFSRYW (SEQ ID NO:11), IRLKSDNYAT (SEQ ID NO:12), and TGLYAMDY (SEQ ID NO:13), respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody 2H4 is as shown in SEQ ID NO:3, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:3;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2H4 is shown in SEQ ID NO:4, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:4.

[0009] Secondly, the present invention provides a nucleic acid molecule encoding the monoclonal antibody.

[0010] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0011] Fourthly, the present invention provides an antibody conjugate, which is obtained by conjugating the monoclonal antibody 2H4 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, etc.

[0012] Fifthly, the present invention provides a composition comprising the monoclonal antibody 2H4 and the anti-human Cyfra21-1 monoclonal antibody 6A4;

[0013] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody 6A4 are QGISNY (SEQ ID NO:14), YAS, and QQSNSWPHT (SEQ ID NO:15), respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody 6A4 are GFTFSNYW (SEQ ID NO:16), IRLKSNNYAT (SEQ ID NO:17), and PRYAMDY (SEQ ID NO:18), respectively.

[0014] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody 6A4 is as shown in SEQ ID NO:7, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:7;

[0015] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6A4 is shown in SEQ ID NO:8, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:8.

[0016] In a sixth aspect, the present invention provides the use of the monoclonal antibody or the nucleic acid molecule or the biological material or the antibody conjugate or the composition in the preparation of reagents, test strips or kits for detecting human Cyfra21-1.

[0017] In a seventh aspect, the present invention provides a double-antibody sandwich ELISA kit for detecting human Cyfra21-1 levels, using the monoclonal antibody 2H4 as the first antibody and the monoclonal antibody 6A4 as the second antibody; or,

[0018] The monoclonal antibody 6A4 was used as the first antibody, and the monoclonal antibody 2H4 was used as the second antibody.

[0019] In one specific embodiment of the present invention, the kit is prepared by immobilizing the monoclonal antibody 6A4 on a solid support, wherein the solid support is selected from enzyme-labeled plates, microspheres, nitrocellulose membranes, glass cellulose membranes, or nylon membranes, etc.

[0020] The labeling can be enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, or radioactive labeling, etc.

[0021] The enzyme labeled with the enzyme may be selected from any of the following: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc., with horseradish peroxidase being preferred.

[0022] Furthermore, the kit also includes at least one of a substrate chromogenic solution, a blocking solution, or a stop solution.

[0023] Commonly used substrates include, but are not limited to, o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS [2,2'-azino-di-(3-ethylbenziazobinesulfonate-6)], with TMB being preferred.

[0024] The sealing solution may be a 5% skim milk powder solution or a 1-2% BSA solution.

[0025] The terminating solution is such as a 1-2.5M (preferably 2M) sulfuric acid solution.

[0026] The principle of this invention, employing an enzyme-linked immunosorbent assay (ELISA) based on a double-antibody sandwich method for detecting human Cyfra21-1 content, is as follows: Anti-human Cyfra21-1 monoclonal antibody is coated onto an ELISA plate; serially diluted standards and pre-diluted samples are added, and the human Cyfra21-1 in the standards and samples binds fully to the coated antibody on the ELISA plate; after washing the plate, biotin-labeled anti-human Cyfra21-1 antibody is added, which specifically binds to the human Cyfra21-1 in the standards and samples captured by the coated antibody on the plate; finally… HRP-labeled streptavidin was added to the reaction wells. After washing the plate, the chromogenic substrate TMB was added. If different concentrations of human Cyfra21-1 were present in the sample, HRP would cause the colorless TMB to turn into different shades of blue (positively correlated). After adding the stop solution, the reaction wells would turn yellow. Finally, the absorbance (OD) of the sample in the reaction wells was measured at λmax = 450 nm (OD = 450 nm). The concentration of human Cyfra21-1 in the sample is directly proportional to the OD. The concentration of human Cyfra21-1 in the sample can be calculated using a standard curve. This method utilizes an enzyme chromogenic amplification system, has high detection sensitivity, and can detect samples with low concentrations.

[0027] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] The anti-human Cyfra21-1 monoclonal antibody provided by this invention can specifically bind to human Cyfra21-1 and has no cross-reactivity with other similar proteins (in the cross-reactivity experiment of similar proteins performed on the kit of this invention, the kit showed specificity and did not recognize human IgG, human IgA, human IgM, human IgE, mouse IgG, mouse IgG, human HSP27, or human albumin), and has high stability (in the accelerated stability test at 37°C performed on the kit of this invention, the kit showed no significant change within 13 days, indicating high stability); based on the antibody, the double-antibody sandwich ELISA method for detecting human Cyfra21-1 has high sensitivity, specificity, and accuracy, and can accurately and with high throughput detect the content of Cyfra21-1 in human serum or other serum analogs containing human Cyfra21-1. Attached Figure Description

[0029] Figure 1 shows the SDS-PAGE detection results of the purified antibody in a preferred embodiment of the present invention. M is the protein marker, 1 is monoclonal antibody 2H4, and 2 is monoclonal antibody 6A4.

[0030] Figure 2 is a standard curve for detecting human Cyfra21-1 content plotted based on the double-antibody sandwich ELISA method in a preferred embodiment of the present invention. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0032] Example 1: Preparation of anti-human Cyfra21-1 monoclonal antibody

[0033] 1. Animal immunization

[0034] Female Balb / c mice aged 6-8 weeks were selected and immunized with recombinant human Cyfra21-1 antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks, and serum titers were measured after four immunizations. A booster immunization was performed three days before fusion.

[0035] 2. Cell fusion

[0036] Mice were euthanized by cervical dislocation, and the spleen was aseptically removed. The spleen cells were then crushed and ground in a petri dish to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a specific ratio, and polyethylene glycol (PEG), a fusion promoter, was added. Under the action of PEG, various lymphocytes could fuse with myeloma cells to form hybridoma cells. The specific procedures are as follows:

[0037] Preparation of feeder cells:

[0038] Take an unimmunized Balb / c mouse, perform orbital bloodletting, and collect negative serum. Inject 2-3 mL of RPMI-1640 basal solution into the mouse's peritoneal cavity, pipette and transfer the solution to another centrifuge tube containing peritoneal macrophages. Centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells in HAT medium, and incubate at 37°C in a 5% CO2 incubator until use.

[0039] Preparation of immune spleen cells:

[0040] One Balb / c mouse that has been boosted with immunization was euthanized by exorbitant blood from the orbital cavity (serum was collected, which is the positive serum). The mouse was disinfected by soaking in 75% alcohol for 5-10 minutes. Then, it was fixed on a dissecting board for dissection. The spleen was removed, cut open, and placed in a sterile homogenizer. RPMI-1640 basal solution was added and the mixture was thoroughly homogenized. 10 mL of RPMI-1640 solution was added, and the mixture was allowed to stand for 2 minutes. The supernatant cell suspension was aspirated and placed in another centrifuge tube. 10 mL of RPMI-1640 solution was added again, and the homogenization was repeated twice. The cell suspension obtained above was centrifuged at 1000 r / min for 10 minutes, the supernatant was discarded, and the suspension was then resuspended in a small amount of basal RPMI-1640 solution for counting and later use.

[0041] Fusion:

[0042] 1×10 7 -2×10 7 SP2 / 0 and 1×10 8 Mix the immune cells in a 50 mL centrifuge tube and centrifuge at 1000 rpm for 8 min. Discard the supernatant and place the centrifuge tube containing the cell mixture in a 37°C water bath. Then add 0.8 mL of 50% PEG (Sigma) pre-warmed to 37°C, stir, and let stand for 30 s. After standing, add 10 mL of RPMI-1640 basal solution pre-warmed to 37°C. Mix well, centrifuge at 1000 rpm for 5 min, discard the supernatant, and incubate at 37°C for 5-8 min. Seed at 250 μL / well into 96-well culture plates pre-coated with feeder cells and incubate at 37°C in a 5% CO2 incubator. On day 4 after fusion, replace with HT medium and continue culturing. When the fused cell colonies have grown to 1 / 4 of the culture wells and the medium has turned slightly yellow, perform antibody detection.

[0043] 3. Screening of hybridoma-positive clones and cell cloning

[0044] The purpose of selective culture is to screen for fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells, lacking hypoxanthine-guanine-phosphoribotransferase, cannot synthesize DNA via the salvage pathway and die. Unfused lymphocytes, although possessing hypoxanthine-guanine-phosphoribotransferase, cannot survive long-term in vitro and gradually die. Only fused hybridoma cells, having acquired hypoxanthine-guanine-phosphoribotransferase from spleen cells and possessing the unlimited proliferation characteristic of myeloma cells, can survive and proliferate in HAT medium. The specific procedures are as follows:

[0045] The steps for screening positive hybridoma cells using indirect ELISA are as follows:

[0046] Coating with known antigens: Dilute the purified coating antigen to 1-10 μg / ml with coating buffer; add 100 μl to each well, gently shake, and incubate overnight at 4°C or for 1 h at 37°C; discard the liquid in the wells and pat dry on a thick stack of absorbent paper; wash 3 times, 1 minute each time.

[0047] To block the uncoated areas in the enzyme-labeled wells: Add 250 μl of blocking buffer (5% skim milk powder or 1% BSA) to each well, shake gently, and incubate at 37°C for 1 h; discard the liquid in the wells and pat dry on a thick stack of absorbent paper; fill each well with washing buffer, let stand for 1 min, discard the liquid in the wells, and pat dry. This method involves washing three times with washing buffer first.

[0048] Sample addition: Add 50 μl of supernatant from each well of the hybridoma to be tested to the enzyme-labeled wells in sequence, and gently shake to mix. Incubate at 37°C for 1 h, wash 3 times, and pat dry.

[0049] Add enzyme-labeled anti-antibody: First, dilute the enzyme-labeled secondary antibody to the appropriate working concentration according to the instructions using diluent. Add 100 μl to each well, shake gently, and incubate at 37°C for 1 hour. Then wash 3 times and pat dry.

[0050] Add colorimetric reagent: Add 100 μl of freshly prepared TMB colorimetric reagent to each well, shake gently, and incubate at 37°C in the dark for 10 min.

[0051] Termination of reaction: Add 50 μl of 1M sulfuric acid stop solution to each well.

[0052] Result: ELISA reader OD 450nm The reading is taken below; if it is greater than 3 times that of the negative well, it can be determined as positive.

[0053] Cloning of hybridoma cells (limiting dilution method):

[0054] Prepare mouse feeder cell layers before cloning; gently blow the hybridoma cells to be cloned from the culture wells and count the number of viable cells using a hemocytometer; dilute the cells with complete culture medium to 5, 10, and 30 cells / mL.

[0055] The three cell suspensions of the above concentrations were added to the prepared feeder cells in 96-well plates at 100 μl / well, so that each well contained 0.5, 1, and 3 cells, respectively. One drop of culture medium was added on day 4. The cell growth in each well was carefully observed and recorded on days 5 and 6.

[0056] Detection of specific antibodies: On days 7-9 after cloning, when the cell clones have grown to fill 1 / 3-1 / 2 of the field of view, they can be detected. Cells from positive wells can be transferred to 24-well culture plates. When the cells in the 24-well plates are growing well, mice can be inoculated intraperitoneally to collect ascites fluid.

[0057] 4. Sequencing of the variable regions of monoclonal antibodies 6A4 and 2H4

[0058] Collect more than 10 hybridoma cells 6 The subsequent construction and sequencing were commissioned to Sangon Biotech (Shanghai) Co., Ltd. The gene sequencing results were as follows: In the 2H4 cell line, the light chain variable region sequence is 339 bp long, encoding 113 amino acids; the DNA sequence is shown in SEQ ID NO:1, and the protein sequence is shown in SEQ ID NO:3. The heavy chain variable region sequence is 351 bp long, encoding 117 amino acids; the DNA sequence is shown in SEQ ID NO:2, and the protein sequence is shown in SEQ ID NO:4. In the 6A4 cell line, the light chain variable region sequence is 321 bp long, encoding 107 amino acids; the DNA sequence is shown in SEQ ID NO:5, and the protein sequence is shown in SEQ ID NO:7. The heavy chain variable region sequence is 348 bp long, encoding 116 amino acids; the DNA sequence is shown in SEQ ID NO:6, and the protein sequence is shown in SEQ ID NO:8.

[0059] 5. Large-scale preparation of monoclonal antibodies 2H4 and 6A4

[0060] After the hybridoma cells were established, they were injected into the peritoneal cavity of mice. Ascites fluid was collected after about 7 days. Antibodies were purified by rProtein G affinity chromatography and identified by SDS-PAGE (Figure 1).

[0061] Example 2: Construction of a double-antibody sandwich ELISA kit containing human Cyfra21-1 content

[0062] 1. Determination of relative affinity constant

[0063] Human Cyfra21-1 antigen was coated onto an ELISA plate and blocked. After washing with PBST, the monoclonal antibody was diluted to saturation concentration and added to the plate at 100 μL / well, and incubated at room temperature for 2 h. After washing with PBST, 60 μL / well of NaSCN solution at concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mol / L were added sequentially, and the plates were incubated at room temperature for 15 min. After washing with PBST, HRP-labeled goat anti-mouse IgG was added, and the plates were incubated at room temperature for 45 min for colorimetric detection. The sodium thiocyanate concentration corresponding to the decrease in OD value at 450 nm to 50% of the uneluted value after elution is the relative affinity constant of the antibody, expressed in mol / L. The results showed that the relative affinity constants of monoclonal antibodies 2H4 and 6A4 were both greater than 1.5 mol / L, indicating good affinity (Table 1).

[0064] Table 1

[0065]

[0066] 2. Biotin labeling of monoclonal antibody 2H4

[0067] Add antibody 2H4 to a dialysis bag and dialyze overnight at 4°C in 0.01M CB buffer. The next day, dissolve biotin-N-hydroxysuccinimide activated lipid (NHS-Biotin) in DMSO to prepare a concentration of 2.2 mg / ml. Then, add 100 μl to the extracted antibody and gently stir at room temperature in the dark for 4 hours. Remove the solution and place it in a dialysis bag for dialyzing in 0.01M PBS. Change the buffer every two hours and dialyze overnight at 4°C. Remove the labeled solution from the overnight dialyze, add an equal volume of glycerol, preservative, and BSA (final concentration 10 mg / ml), and store at -20°C.

[0068] 3. Preliminary pairing and screening of monoclonal antibody pairs

[0069] Monoclonal antibodies were diluted to 6 μg / ml using 0.05 M pH 9.6 carbonate coating buffer. 100 μl of each solution was added to the wells of a 96-well polystyrene reagent plate and incubated overnight at 4°C. The next day, the solution was discarded, and the plate was washed three times with washing buffer for 1 minute each time. After the above steps, 300 μl of the solution was used to block each well of the reaction plate, and the plate was incubated for 2 hours. The solution was discarded, the plate was washed three times, and the plate was dried. 100 μl of high-concentration (200 ng / ml) human Cyfra21-1 was added to each well (0 well). The plate was sealed and incubated at room temperature for 2 hours. The plate was washed four times and dried. 100 μl of biotin-labeled antibody working solution was added to the wells, the plate was sealed, and the plate was incubated at room temperature for 1 hour. The plate was washed four times and dried. Add 100 μl of HRP-SA working solution to the reaction wells, seal the plate, and incubate at 37°C for 30 min. Add 100 μl of chromogenic substrate TMB to the reaction wells and incubate at 37°C in the dark for 15 min. Add 50 μl of 1M sulfuric acid stop solution and immediately measure the OD value at 450 nm using a microplate reader. Table 2 shows that 6A4 is the best antibody pairing agent for coating. Based on the affinity assay results above, 6A4 was selected as the coating antibody, and 2H4 was selected as the antibody pair for detection.

[0070] Table 2

[0071]

[0072] 4. Preparation of monoclonal antibody 6A4 ELISA plate

[0073] Dilute monoclonal antibody 6A4 to 2 μg / ml with 0.05 M pH 9.6 carbonate coating buffer. Add 100 μl / well to a 96-well microplate and incubate overnight at 4°C. The next day, discard the solution in the wells and wash three times with washing buffer for 1 minute each time. After the above steps, block each well of the reaction plate with 300 μl of 2% BSA solution and incubate at room temperature for 2 hours. Discard the solution in the wells, dry in a desiccant chamber, and then vacuum-seal in an aluminum foil bag and store at 4°C.

[0074] 5. Establishment of the double-antibody sandwich ELISA method

[0075] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 6A4 and allow it to reach room temperature. Before adding samples, wash the plate three times and pat dry. Add 100 μl of human Cyfra21-1 standards at different dilutions: 60 ng / ml, 30 ng / ml, 15 ng / ml, 7.5 ng / ml, 3.75 ng / ml, 1.875 ng / ml, and 0.9375 ng / ml. A blank control was also included. After sealing the plate, incubate at 37°C for 90 minutes. Wash the plate four times and pat dry. Add 100 μl of biotin-labeled 2H4 antibody working solution to the reaction wells. After sealing the plate, incubate at 37°C for 60 minutes. Wash the plate four times and pat dry. Add 100 μl of HRP-SA working solution to the reaction wells, seal the plate, and incubate at 37℃ for 30 min. Add 100 μl of chromogenic substrate TMB to the reaction wells, and incubate at 37℃ in the dark for 15 min. Add 50 μl of 1M sulfuric acid stop solution, and immediately measure the OD value at 450 nm using a microplate reader. Plot a standard curve with different concentrations of human Cyfra21-1 standards on the x-axis and the corresponding OD values ​​on the y-axis to establish a regression equation. The results show that the detection range is 0.9735-60 ng / ml, the sensitivity is 0.87 ng / ml, and the R... 2 It is 0.9998 (Figure 2).

[0076] 6. Specificity detection of double-antibody sandwich ELISA

[0077] Thirty minutes before the experiment, remove the ELISA plate coated with monoclonal antibody 6A4, allow it to reach room temperature, wash it three times, and blot dry. Add 100 μl of human Cyfra21-1 standards at different dilutions and standards of indicators with high levels in serum, or structurally similar proteins of human Cyfra21-1 (human IgG, human IgA, human IgM, human IgE, mouse IgG, mouse IgG, human HSP27, human albumin). After sealing the plate, incubate it at 37°C for 90 minutes, wash it four times, and blot dry. Add 100 μl of biotin-labeled 2H4 working solution to the reaction wells, seal the plate, and incubate it at 37°C for 60 minutes, wash it four times, and blot dry. Add 100 μl of enzyme conjugate working solution to the reaction wells, seal the plate, and incubate at 37°C for 30 min. Add 100 μl of chromogenic substrate TMB to the reaction wells and incubate at 37°C in the dark for 15 min. Add 50 μl of stop solution and immediately measure the OD value at 450 nm using a microplate reader. The results showed that this antibody pair did not react with other similar proteins (Table 3).

[0078] Table 3

[0079]

[0080] 7. Stability testing of double-antibody sandwich ELISA

[0081] The ELISA plate coated with monoclonal antibody 6A4, biotin-labeled monoclonal antibody 2H4, and human Cyfra21-1 standard were placed at 37°C for accelerated stability testing for 12 days (approximately 18 months at 4°C). Afterward, the plate was removed for testing. The testing method involved washing the plate three times and patting it dry. 100 μl of human Cyfra21-1 standard at different dilutions (60 ng / ml, 30 ng / ml, 15 ng / ml, 7.5 ng / ml, 3.75 ng / ml, 1.875 ng / ml, and 0.9375 ng / ml) was added, along with a blank control. The plate was sealed and incubated at 37°C for 90 min, washed four times, and patted dry. 100 μl of Bio-2H4 working solution was added to the reaction wells, the plate was sealed, and incubated at 37°C for 60 min, washed four times, and patted dry. Add 100 μl of enzyme conjugate working solution to each well, seal the plate, and incubate at 37°C for 30 min. Add 100 μl of chromogenic substrate TMB to each well, and incubate at 37°C in the dark for 15 min. Add 50 μl of stop solution, and immediately measure the OD value at 450 nm using a microplate reader. Plot a standard curve with different concentrations of human Cyfra21-1 standard on the x-axis and the corresponding OD value on the y-axis to establish a regression equation. The results show that the kit has good stability (Table 4).

[0082] Table 4

[0083]

[0084] Example 3: Analysis of Serum Sample Measurement Results

[0085] Eleven randomly selected human serum samples were simultaneously measured (unit: ng / ml). A review of relevant literature indicated that the level of Cyfra21-1 in normal human serum should be less than 3.3 ng / ml, but can reach as high as 4.09 ng / ml with age. In cases of cancer, the level of Cyfra21-1 in serum can reach several hundred ng / ml (varying depending on age and individual differences). The results show that the level measured by this kit can effectively predict the development of cancer in the body (Table 5).

[0086] Table 5

[0087]

[0088] Example 4: Analysis of Cell Supernatant Sample Measurement Results

[0089] A549 (human non-small cell lung cancer cells), HT-29 (human colon cancer cells), and MCF-7 (human breast cancer cells) were selected, plated, and cultured in fresh medium for 3 days. Cell supernatants were then collected for analysis. The results (Table 6) showed that all three cell lines expressed Human Cyfra21-1 to varying degrees, consistent with previous reports.

[0090] Table 6

[0091]

[0092] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Anti-human Cyfra21-1 monoclonal antibody 2H4, characterized in that, The amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain variable region of the monoclonal antibody 2H4 are QSLLYSSNQRNY, WAS, and QQYYSYPFT, respectively; the amino acid sequences of the complementarity-determining regions (CDR1, CDR2, and CDR3) of the heavy chain variable region of the monoclonal antibody 2H4 are GFTFSRYW, IRLKSDNYAT, and TGLYAMDY, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody 2H4 is as shown in SEQ ID NO:3, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:3; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2H4 is as shown in SEQ ID NO:4, or an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:

4.

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. A biomaterial containing the nucleic acid molecule of claim 3, characterized in that, The biological material is an expression cassette, plasmid vector, viral vector, or engineered bacteria.

5. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the monoclonal antibody of claim 1 or 2 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

6. A composition, characterized in that, The composition comprises the monoclonal antibody of claim 1 or 2 and anti-human Cyfra21-1 monoclonal antibody 6A4; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody 6A4 are QGISNY, YAS, and QQSNSWPHT, respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody 6A4 are GFTFSNYW, IRLKSNNYAT, and PRYAMDY, respectively.

7. The use of the monoclonal antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the biological material of claim 4, the antibody conjugate of claim 5, or the composition of claim 6 in the preparation of reagents, test strips, or kits for the detection of human Cyfra21-1.

8. A double-antibody sandwich ELISA kit for detecting human Cyfra21-1 content, characterized in that, Monoclonal antibody 2H4 is used as the first antibody, and monoclonal antibody 6A4 is used as the second antibody; or, monoclonal antibody 6A4 is used as the first antibody, and monoclonal antibody 2H4 is used as the second antibody; wherein monoclonal antibody 2H4 is the same as the monoclonal antibody according to claim 1 or 2, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region of monoclonal antibody 6A4 are QGISNY, YAS, and QQSNSWPHT, respectively; and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of monoclonal antibody 6A4 are GFTFSNYW, IRLKSNNYAT, and PRYAMDY, respectively.

9. The reagent kit according to claim 8, characterized in that, The monoclonal antibody 6A4 is immobilized on a solid-phase support, which is selected from enzyme-labeled plates, microspheres, nitrocellulose membranes, glass cellulose membranes, or nylon membranes; the labeling is enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, or radioactive labeling; the enzyme labeling enzyme is selected from any one of the following: horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, and malate dehydrogenase.

10. The kit according to claim 8 or 9, characterized in that, The kit also includes at least one of a substrate chromogenic solution, a blocking solution, or a stop solution.

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