Monoclonal antibody H2 specifically combined with lung cancer marker CYFRA21-1 and application

Abstract

The invention discloses a monoclonal antibody H2 specifically bound with a lung cancer marker CYFRA21-1 and application, and relates to the technical field of biological medicines. The method comprises the following steps: firstly, screening from a Tomlinson I + J phage library by utilizing a phage display technology to obtain a lung cancer marker CYFRA21-1 monoclonal antibody H2, and the antibody has a heavy chain complementary determining region and a light chain complementary determining region which are formed by novel amino acid sequences and can be specifically combined with CYFRA21-1 protein; the monoclonal antibody H2 of the lung cancer marker CYFRA21-1 has the characteristics of high affinity, high specificity, stability and the like, and the monoclonal antibody H2 of the lung cancer marker CYFRA21-1 not only can efficiently and accurately detect the concentration of CYFRA21-1 protein, but also can be used for immunostaining of lung cancer tumor cells and lung cancer tissues in pathological diagnosis, and has important significance for detecting the concentration of CYFRA21-1.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, in particular to a monoclonal antibody H2 specifically binding to a lung cancer marker CYFRA21-1 and its application. Background Art

[0002] Cytokeratin 19 fragment antigen (CYFRA21-1) is a soluble acidic protein, composed of soluble fragments of CK19 keratin. It is mainly present in the cytoplasm of tumor cells of epithelial origin such as lung cancer and esophageal cancer. Lung adenocarcinoma and lung squamous cell carcinoma both express CYFRA21-1, belonging to the cytokeratin family. CYFRA21-1 has a wide range of clinical applications. It is a non-organ-specific tumor marker that plays an important role in a variety of cell types and tumors. It is mainly used to detect lung cancer, especially non-small cell lung cancer (NSCLC), with a detection rate of 70% to 85%. When tumor cells dissolve and die, a large number of proteases are activated, which in turn leads to the degradation of CK19 keratin, generating CYFRA21-1 and releasing it into the blood circulation, resulting in an increase in the serum level of CYFRA21-1. Therefore, the serum level of CYFRA21-1 can effectively indicate the occurrence and development of tumors. The serum CYFRA21-1 level is positively correlated with the progression of the clinical stage of non-small cell lung cancer. The concentration of CYFRA21-1 decreases significantly after radical surgery for lung cancer. If it increases again, tumor progression and recurrence should be considered. Therefore, the detection of serum CYFRA21-1 levels can provide an important basis for the diagnosis, treatment, and prognosis of non-small cell lung cancer.

[0003] Theoretically, detecting the concentration of CYFRA21-1 in tumors can provide important basis for the diagnosis of tumor patients' condition, selection of treatment methods, observation of therapeutic effects, prediction of recurrence, etc. However, there are relatively few publicly available monoclonal antibodies specific for CYFRA21-1 protein. Therefore, the development of antibodies with high affinity and specificity for identifying CYFRA21-1 is of great significance for detecting the concentration of CYFRA21-1. Summary of the invention

[0004] To solve the above problems, the purpose of the present invention is to provide a monoclonal antibody H2 that specifically binds to the lung cancer marker CYFRA21-1 and its application.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0006] Lung cancer marker CYFRA21-1 antibody H2, wherein the lung cancer marker CYFRA21-1 antibody H2 has a human antibody sequence skeleton, and the amino acid sequences of the three complementary determining regions of the heavy chain and the three complementary determining regions of the light chain are artificially designed and synthesized amino acid sequences, and specifically bind to the CYFRA21-1 protein;

[0007] The three complementarity determining regions of the heavy chain are CDR-H1, CDR-H2 and CDR-H3;

[0008] The three complementarity determining regions of the light chain are CDR-L1, CDR-L2 and CDR-L3;

[0009] The amino acid sequence of the H2 heavy chain variable region (H2-VH) of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 1, the amino acid sequence of CDR-H1 is SEQ ID NO: 2, the amino acid sequence of CDR-H2 is SEQ ID NO: 3, and the amino acid sequence of CDR-H3 is SEQ ID NO: 4;

[0010] The amino acid sequence of the H2 light chain variable region (H2-VL) of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 5, the amino acid sequence of CDR-L1 is SEQ ID NO: 6, the amino acid sequence of CDR-L2 is SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is SEQ ID NO: 8.

[0011] H2-VH:

[0012] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSINTT GAYTKYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSDAFDYWGQG TLVTV(SEQ ID NO: 1);

[0013] CDR-H1 GFTFSSYA (SEQ ID NO: 2);

[0014] CDR-H2 NTTGAYT (SEQ ID NO: 3);

[0015] CDR-H3 AKSSDAFDY (SEQ ID NO: 4);

[0016] H2-VL:

[0017] DIQMTQSPSSSLSASVGDRVTITCRASQSISLNWYQQKPGKAPKLLIYSASNL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYAYPTTFGQGTKVEIKR (SEQ ID NO: 5);

[0018] CDR-L1 RASQSISSYLN (SEQ ID NO: 6);

[0019] CDR-L2 SASNLQS (SEQ ID NO: 7);

[0020] CDR-L3 QQSYAYPTT (SEQ ID NO: 8).

[0021] A diagnostic or assay kit, comprising the lung cancer marker CYFRA21-1 antibody H2 according to claim 1; the lung cancer marker CYFRA21-1 antibody H2 can be prepared with an antigen CYFRA21-1 recombinant protein, an enzyme-labeled anti-human antibody that can bind to H2, or an anti-His-tag antibody, an enzyme substrate, and a color developing solution to form a kit for diagnosing lung cancer tumor cells and lung cancer tissues, or a kit for measuring the concentration of CYFRA21-1 protein.

[0022] The present invention also includes the use of the above-mentioned lung cancer marker CYFRA21-1 antibody H2 in detecting the concentration of lung cancer marker CYFRA21-1.

[0023] The present invention also includes the use of the lung cancer marker CYFRA21-1 antibody H2 in immunostaining of lung cancer tumor cells and lung cancer tissues.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The present invention uses phage display technology to screen a lung cancer marker CYFRA21-1 monoclonal antibody H2 from the Tomlinson I+J phage library. The antibody has a heavy chain complementary determining region and a light chain complementary determining region composed of a novel amino acid sequence, and can specifically bind to the CYFRA21-1 protein. The binding has the characteristics of high affinity, high specificity and stability, and can become a powerful tool for studying the physiological effects of the CYFRA21-1 protein in cells.

[0026] The monoclonal antibody H2 of the lung cancer marker CYFRA21-1 of the present invention can not only detect the concentration of CYFRA21-1 protein efficiently and accurately, but also can be used for immunostaining of lung cancer tumor cells and lung cancer tissues in pathological diagnosis, which is of great significance for detecting the concentration of CYFRA21-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of the polyclonal antibody ELISA obtained by screening;

[0028] Figure 2 This is a diagram showing the screening results of monoclonal antibodies against CYFRA21-1;

[0029] Figure 3 This is the result of monoclonal antibody competitive enzyme-linked immunosorbent assay;

[0030] Figure 4 This is a dose-response curve of the lung cancer marker CYFRA21-1 antibody H2. DETAILED DESCRIPTION

[0031] The purpose of the present invention is to provide a monoclonal antibody that specifically binds to the lung cancer marker CYFRA21-1 and a preparation method and application thereof. The present invention is further described below in conjunction with specific examples.

[0032] Example 1

[0033] 1. Amplification of phage display antibody library

[0034] Add the E. coli stock solution containing Tomlinson I+J phage library to 25 mL of 2YT liquid medium (2YTAG) containing 100 μg / mL ampicillin (Amp) and 1% glucose (Glucose), and culture overnight at 37°C and 250 rpm. Take 5 mL of overnight bacteria and add them to 50 mL of 2YTAG, and culture them in a conical flask at 37°C and 250 rpm until the OD reaches 0. 600 =0.4, add 25 μL helper phage M13K07 (titer: 10 12 cfu / mL), after infection at 37°C for 30 minutes, the infected E. coli was dissolved in a high-speed centrifuge, centrifuged at 5000rpm for 30 minutes, the supernatant was discarded, the centrifugation was repeated twice, and the supernatant was completely removed. Use 100mL of 2YT liquid culture medium (2YTAGK) containing 100μg / mL Amp, 50μg / mL kanamycin and 0.1% glucose to resuspend the bacteria, and place the solution in a conical flask and culture at 30°C and 250rpm for 20 hours. The next day, centrifuge at 5000rpm for 30 minutes, take the supernatant into a new 50mL sterile centrifuge tube, add 20mL of PEG / NaCl (20% polyethylene glycol 6000, 2.5M NaCl) solution to the supernatant, mix well, let stand on ice for 1 hour, centrifuge at 5000rpm for 1 hour, discard the supernatant, repeat the centrifugation twice, and completely remove the supernatant. 4 mL of sterile PBS buffer was added to dissolve the precipitate, which was used as the phage display antibody library solution. The phage display antibody library was titrated using E. coli. The concentration of the prepared antibody library was 10 12 cfu / mL.

[0035] 2. Screening of phage display antibody library

[0036] Coat 50 μg / mL of CYFRA21-1 protein (antigen) diluted in PBS buffer (PBS used in this experiment has been sterilized) in a 96-well microplate, 100 μL / well, and incubate overnight at 4°C. The next day, pour out the antigen solution, add 300 μL of PBS solution containing 5% skim milk powder (MPBS), and stand at room temperature for 2 hours. Wash the microplate three times with PBST solution (PBS containing 0.1% Tween 20), mix Tomlinson I phage library and Tomlinson J phage 1:1, and dilute to 10 with MPBS. 10 cfu / mL, as phage library R0. Add 100 μL of MPBS solution containing phage library R0 to each well of the microplate, shake on a horizontal shaker at room temperature for 1 hour, and then let it stand at room temperature for 1 hour. Wash 10 times with PBST solution, add 100 μL of trypsin solution (1 mg / mL, dissolved in sterilized PBS) to each well, let it stand at room temperature for 10 minutes, and elute the phage bound to the CYFRA21-1 antigen. Add 100 μL of glycine-hydrochloric acid buffer (GlycinHCl, pH=2.2) to each well, let it stand at room temperature for 5 minutes, elute the phage bound to the CYFRA21-1 protein, and then add 50 μL Tris-HCl buffer (pH=7.4) to each well for neutralization.

[0037] Take 10 μL of Escherichia coli TG-1 strain and add it to 4 mL of 2YT liquid culture medium without antibiotics and glucose, shake at 37°C and 250 rpm until OD600 = 0.4, add 500 μL of eluted phage solution to 4 mL of bacterial solution, infect at 37°C for 30 min, centrifuge at 5000 rpm for 30 min, discard the supernatant, resuspend the bacteria in 2YT liquid culture medium (2YTAG) containing 100 μg / mL ampicillin and 1% glucose, and shake the bacteria overnight at 37°C and 250 rpm. The next day, add 40 μL of overnight bacteria to 4 mL of 2YTAG liquid culture medium, shake at 37°C and 250 rpm until OD600 = 0.2. Add 10 μL of helper phage M13K07 (titer: 5×10 11 cfu / mL), infected at 37°C for 30 min, centrifuged the infected E. coli solution at 25°C and 5000 rpm for 20 min using a high-speed centrifuge, poured out the supernatant, centrifuged again, and completely removed the supernatant. Resuspend the bacteria in 20 mL of 2YT liquid culture medium (2YTAGK) containing 100 μg / mL Amp, 50 μg / mL kanamycin, and 0.1% glucose, and place in a conical flask and culture at 30°C and 250 rpm for 20 h.

[0038] Centrifuge overnight at 5000rpm for 30min at 4℃, separate and recover the supernatant, add 4mL PEG / NaCl solution to the supernatant solution, mix well and place on ice for 1h, centrifuge at 5000rpm for 1h at 4℃, discard the supernatant, centrifuge again, and completely remove the supernatant. Add 200μL sterile PBS solution as the antibody library (R1) for the second round of panning; repeat the above steps three times to obtain phage R2 and R3 respectively. After the third panning, the binding specificity of the original antibody library R0 and the R1, R2, and R3 obtained by panning to the CYFRA21-1 protein was verified by enzyme-linked immunosorbent assay.

[0039] The titers of the four phage libraries were determined, and the titers of the four antibody libraries were calculated. 5 μg / mL bovine serum albumin (BSA) and CYFRA21-1 protein diluted with PBS buffer were coated in 96 microwell plates, 3 wells each, 100 μL per well, a total of 4 groups, and incubated overnight at 4°C. The next day, the solution in the microwell plate was poured out, 300 μL of PBS solution containing 5% skimmed milk powder (MPBS) was added, and the solution was blocked at room temperature for 2 hours. The microwell plate was washed 3 times with PBST solution, and 100 μL of 10% was added to each of the 4 groups of microwells. 10 cfu of phage library (R0, R1, R2, R3) in 5% MPBS solution, incubate at room temperature for 1 hour. Wash the microplate 6 times with PBST solution, then add 1:5000 diluted mouse anti-M13-HRP antibody, incubate at room temperature for 1 hour. Wash the microplate 12 times with PBST, add 100μL TMBZ to each well for color development, react at 37℃ for 5-10min, add 50μL 10% sulfuric acid solution to each well to terminate the reaction, and use iMarkTM microplate reader (Bio-Rad) to measure the absorbance at 450nm and 630nm, and draw a bar graph.

[0040] The results of the ELISA test were Figure 1 As shown in the figure, when comparing the binding abilities of the phage libraries R0, R1, R2, and R3 obtained by panning with CYFRA21-1 protein, it was found that with the increase in the number of panning times, the binding ability of phage solution R3 with CYFRA21-1 protein increased significantly, while the binding performance of the four groups of phage libraries to BSA was very weak and did not change, indicating that the antibodies against CYFRA21-1 protein in the constructed phage display antibody library were successfully enriched.

[0041] 3. Monoclonal antibody screening

[0042] The titer of R3 was measured. The next day, 96 single colonies were randomly selected in a 96-well microplate. 1.5 mL of 2YTAG liquid culture medium was added to each of the 96 wells. The 96-well plate was sealed with a sealing film to prevent the bacterial liquid from shaking out and causing cross contamination. The plate was placed in a shaker at 37°C and 200 rpm for overnight culture. The next day, 10 μL of overnight bacteria were taken from each well and added one by one to a new 96-well plate containing 90 μL of 2YTAG liquid culture medium. The plate was sealed with a sealing film and shaken at 37°C and 200 rpm until the OD 600 = 0.2, 75 μL of helper phage M13K07 (titer: 2.5×10 11 cfu / mL) was mixed with 5 mL of 2YTAG liquid medium, and 150 μL was added to each well of a 96-well plate, and the plate was sealed with a sealing film and infected at 37°C for 30 min. After the infection, 1.35 mL of 2YTAK (without glucose) was added to each well, the plate was sealed with a sealing film, and cultured in a shaking incubator at 30°C and 250 rpm for 20 h.

[0043] 100 μL of PBS solution containing BSA (5 μg / mL) and CYFRA21-1 protein (5 μg / mL) was added to the 96-well plate, one by one, and incubated at 4°C overnight. The next day, the solution in the microplate was poured out, 300 μL of 5% MPBS was added, and the plate was blocked at room temperature for 2 hours. The bacterial solution cultured for 20 hours was taken out into a 2mL centrifuge tube and centrifuged at 5000 rpm for 30 minutes at 4°C. During this period, the 96-well plate blocked with MPBS was washed 3 times with PBST, 80 μL of MPBS solution was added to each well, and then 20 μL of phage supernatant after centrifugation was added one by one, and the mixture was mixed by blowing gently, and the plate was incubated at room temperature for 1 hour. The microplate was washed 6 times with PBST solution, 100 μL of 1:5000 diluted mouse anti-M13-HRP antibody was added to each well, and the plate was incubated at room temperature for 1 hour. The microplate was washed 12 times with PBST, 100 μL TMBZ was added to each well for color development, and after reacting at 37°C for 5-10 min, 50 μL 10% sulfuric acid solution was added to each well to terminate the reaction. The absorbance at 450 nm and 630 nm was measured using an iMarkTM microplate reader (Bio-Rad), and a bar graph was drawn as shown in FIG. Figure 2 As shown. Antibodies with high binding activity to CYFRA21-1 were screened out, re-validated, and the positive clones were further identified.

[0044] 4. Antibody sequence comparison and analysis

[0045] According to the above experimental results, 8 positive clones were selected, plasmids were extracted and gene sequencing was performed, and an antibody strain was obtained and named H2. By comparing with the antibody sequence registered in the antibody gene library, no sequence identical to the antibody gene of the present invention was found, so the antibody is a new antibody. The amino acid sequence of the H2 antibody is described in detail as follows.

[0046] The lung cancer marker CYFRA21-1 antibody H2 has a human antibody sequence backbone. The amino acid sequences of the three complementarity determining regions of the heavy chain (CDR-H1, CDR-H2, CDR-H3) and the three complementarity determining regions of the light chain (CDR-L1, CDR-L2, CDR-L3) are artificially designed and synthesized amino acid sequences that specifically bind to CYFRA21-1.

[0047] The amino acid sequence of the H2 heavy chain variable region (H2-VH) of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 1, the amino acid sequence of CDR-H1 is SEQ ID NO: 2, the amino acid sequence of CDR-H2 is SEQ ID NO: 3, and the amino acid sequence of CDR-H3 is SEQ ID NO: 4;

[0048] The amino acid sequence of the H2 light chain variable region (H2-VL) of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 5, the amino acid sequence of CDR-L1 is SEQ ID NO: 6, the amino acid sequence of CDR-L2 is SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is SEQ ID NO: 8;

[0049] H2-VH:

[0050] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSINTT GAYTKYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSDAFDYWGQG TLVTV(SEQ ID NO: 1);

[0051] CDR-H1 GFTFSSYA (SEQ ID NO: 2);

[0052] CDR-H2 NTTGAYT (SEQ ID NO: 3);

[0053] CDR-H3 AKSSDAFDY (SEQ ID NO: 4);

[0054] H2-VL:

[0055] DIQMTQSPSSSLSASVGDRVTITCRASQSISLNWYQQKPGKAPKLLIYSASNL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYAYPTTFGQGTKVEIKR (SEQ ID NO: 5);

[0056] CDR-L1 RASQSISSYLN (SEQ ID NO: 6);

[0057] CDR-L2 SASNLQS (SEQ ID NO: 7);

[0058] CDR-L3 QQSYAYPTT (SEQ ID NO: 8).

[0059] 5. Antigen specificity of monoclonal antibodies

[0060] 2μg / mL BSA and CYFRA21-1 protein were coated in a 96-well microplate, with 3 wells coated with BSA and 6 wells coated with CYFRA21-1, 100μL per well, and incubated at 4°C overnight. The next day, the solution in the microplate was discarded, 300μL of PBS solution (MPBS) containing 5% skimmed milk powder was added, and the plate was blocked at room temperature for 2h. The microplate was washed 3 times with PBST solution, and 80μL of MPBS and 20μL of phage display antibody supernatant were added to each well. Among them, 3 wells coated with CYFRA21-1 protein were added with the above mixture and the final concentration of 10μg / mL CYFRA21-1 protein was added to compete, and the plate was incubated at room temperature for 1h. The microplate was washed 6 times with PBST solution, and then a 1:5000 diluted mouse anti-M13-HRP antibody was added, and the plate was incubated at room temperature for 1h. The microplate was washed 12 times with PBST, 100 μL of TMBZ was added to each well for color development, and after reacting at 37°C for 5-10 min, 50 μL of 10% sulfuric acid solution was added to each well to terminate the reaction. The absorbance at 450 nm and 630 nm was measured using an iMarkTM microplate reader (Bio-Rad) to draw a bar graph.

[0061] The results of competitive ELISA Figure 3 As shown, the H2 antibody specifically binds to the CYFRA21-1 protein and does not bind to the coated BSA. At the same time, when there is competition from free CYFRA21-1 protein, the binding ability of the H2 antibody to the CYFRA21-1 protein is significantly reduced, indicating that the H2 antibody is indeed a specific antibody for the CYFRA21-1 protein and has specific binding to the CYFRA21-1 protein.

[0062] 6. Detection of CYFRA21-1 protein concentration using competitive ELISA using monoclonal antibodies

[0063] 2 μg / mL BSA and CYFRA21-1 protein were coated in a 96-well microplate, 100 μL per well, and incubated overnight at 4°C. The next day, the solution in the microplate was discarded, and 300 μL of PBS solution (MPBS) containing 5% skim milk powder was added, and the plate was blocked at room temperature for 2 hours. The microplate was washed 3 times with PBST solution, and the phage display antibody solution (10 μg / mL) diluted with MPBS was added to the microwells coated with BSA. 10 cfu / mL), a series of phage-displayed antibody dilution solutions with a final concentration of CYFRA21-1 of 0, 0.1, 1, 10, 100 and 1000 μg / mL were added to the microwells coated with CYFRA21-1 protein, and each concentration was repeated three times and incubated at room temperature for 1 hour. Wash with PBST 8 times, and add HRP-labeled mouse anti-M13 antibody. Wash the microplate 6 times with PBST solution, and then add 1:5000 diluted mouse anti-M13-HRP antibody and incubate at room temperature for 1 hour. Wash the microplate 12 times with PBST, add 100 μL TMBZ to each well for color development, react at 37°C for 5 to 10 minutes, add 50 μL 10% sulfuric acid solution to each well to terminate the reaction, measure the absorbance at 450nm and 630nm, and draw a dose-response curve. The presence of free CYFRA21-1 in the solution was detected by competitive ELISA using phage-displayed antibodies ( Figure 4 ). When the concentration of free CYFRA21-1 in the solution is low, the phage-displayed antibody binds to the CYFRA21-1 protein coated on the microplate, resulting in a higher absorbance intensity. As the concentration of free CYFRA21-1 protein in the solution increases, the phage-displayed antibody competitively binds to the free CYFRA21-1 protein, resulting in a decrease in binding to the CYFRA21-1 protein coated on the microplate and a decrease in absorbance.

[0064] The half inhibitory concentration (IC50) of H2 antibody was determined to be 3.1 μg / mL through the dose-response curve, and the lowest detection limit (LOD) of CYFRA21-1 protein was calculated to be 1.75 ng / mL.

[0065] 7. In addition to detecting the concentration of lung cancer marker CYFRA21-1, the lung cancer marker CYFRA21-1 antibody H2 can also be used for immunostaining of lung cancer tumor cells and lung cancer tissues for pathological diagnosis.

[0066] (1) For immunohistochemical staining

[0067] For lung cancer tissue sections, the tissue is first fixed, embedded and sliced. The H2 antibody can specifically recognize the CYFRA21-1 antigen on the surface or in the lung cancer cell.

[0068] When the CYFRA21-1 antibody H2 is added to the tissue section, it will bind to CYFRA21-1 in the lung cancer cells. Then add the secondary antibody, which is usually an antibody against the primary antibody and carries a marker (such as fluorescein or enzyme). If a fluorescently labeled secondary antibody is used, lung cancer cells can be observed to show fluorescent signals under a fluorescence microscope; if an enzyme-labeled secondary antibody is used, lung cancer cells will show color changes after adding a substrate for color development, so that they can be observed under an optical microscope.

[0069] (2) Tumor cell localization: Through immunostaining, the distribution of CYFRA21-1 antibody H2 in lung cancer cells can be clearly seen, whether it is in the cell membrane, cytoplasm or nucleus, which helps to understand the biological characteristics of tumor cells.

[0070] (3) Tumor type identification: Different types of lung cancer (such as adenocarcinoma, squamous cell carcinoma, etc.) may differ in the expression level and distribution pattern of CYFRA21-1. Immunostaining using the CYFRA21-1 antibody H2 can assist pathologists in distinguishing different types of lung cancer. For example, squamous cell carcinoma of the lung usually has higher CYFRA21-1 expression than adenocarcinoma of the lung, and the intensity and range of immunostaining can provide clues for diagnosis.

[0071] (4) Tumor staging assessment: During the progression of the tumor, the expression of CYFRA21-1 may change. By immunostaining lung cancer tissue and observing the expression of CYFRA21-1, it can help determine the degree of tumor invasion and metastatic potential. For example, high expression of CYFRA21-1 in the tumor margin area accompanied by irregular cell morphology may indicate that the tumor is highly invasive.

[0072] 8. Lung cancer marker CYFRA21-1 antibody H2 is prepared into a kit for detecting CYFRA21-1 concentration

[0073] The lung cancer marker CYFRA21-1 antibody H2 of the present invention can be prepared into a kit for detecting CYFRA21-1 concentration with antigen CYFRA21-1 recombinant protein, enzyme-labeled anti-human antibody capable of binding to H2, or anti-His-tag antibody, enzyme substrate and color developing solution.

[0074] The specific materials of the kit are as follows:

[0075] (1) Antibody H2 for lung cancer marker CYFRA21-1: As a core reagent, it is used to specifically identify the lung cancer marker CYFRA21-1. It needs to undergo strict purification and quality testing to ensure its high affinity and specificity for CYFRA21-1. It is usually produced by hybridoma technology or recombinant DNA technology.

[0076] (2) Labeled secondary antibody: If an indirect detection method is used, a labeled secondary antibody is required. The label can be an enzyme (such as horseradish peroxidase, alkaline phosphatase), a fluorescein (such as fluorescein isothiocyanate, rhodamine) or biotin, etc. For example, a secondary antibody labeled with horseradish peroxidase can be combined with the H2 antibody for subsequent color reaction detection.

[0077] (3) Buffer and diluent

[0078] Phosphate buffered saline (PBS): used to dissolve antibodies, dilute samples, etc. PBS can maintain a stable pH environment (usually pH 7.2-7.4) to ensure the activity of antibodies and antigens.

[0079] (4) Blocking buffer: Commonly used are bovine serum albumin (BSA) solution or skimmed milk powder solution. Its function is to block nonspecific binding sites and reduce background interference.

[0080] (5) Sample diluent: used to dilute samples such as blood and tissue fluid to reach a suitable detection concentration. In addition to buffer, the sample diluent may also contain some additives, such as preservatives and protease inhibitors, to prevent the degradation of components in the sample.

[0081] (6) Substrate and color developer (for enzyme-labeled detection)

[0082] If using horseradish peroxidase as the labeling enzyme:

[0083] The substrate can be 3,3',5,5'-tetramethylbenzidine (TMB). TMB will undergo a color reaction under the catalysis of horseradish peroxidase, changing from colorless to blue, and then the reaction can be terminated with sulfuric acid to change the color to yellow. The depth of the color is related to the concentration of CYFRA21-1 in the sample.

[0084] (7) Standards and quality control products

[0085] Standard: A pure CYFRA21-1 with a known concentration or a sample containing a known concentration of CYFRA21-1. It is used to establish a standard curve so that the concentration of CYFRA21-1 in an unknown sample can be calculated based on the detection signal (such as absorbance). The concentration range of the standard should cover the linear range of the detection kit.

[0086] Quality control products: used to monitor the accuracy and repeatability of the test kit. It includes high, medium and low concentrations of CYFRA21-1 samples, which are tested together with unknown samples in each test to ensure the reliability of the test results.

[0087] (9) Other materials

[0088] Microplate (for ELISA testing): usually made of polystyrene, with a specially treated surface that can adsorb antibodies and antigens well. Its specifications can be selected according to the requirements of the test throughput, such as 96-well plates.

[0089] Packaging materials: such as the test kit shell, reagent bottle, dropper, instruction manual, etc. The test kit shell should be sturdy and easy to transport and store; the reagent bottle should be able to ensure the stability and sealing of the reagent; the dropper should be able to accurately absorb and distribute the reagent; the instruction manual should explain in detail the use of the test kit, the detection principle, the storage conditions, etc.

Claims

1. Antibody H2 for lung cancer marker CYFRA21-1, characterized by: The lung cancer marker CYFRA21-1 antibody H2 has a human antibody sequence skeleton, and the amino acid sequences of the three complementary determining regions of the heavy chain and the three complementary determining regions of the light chain are artificially designed and synthesized amino acid sequences, which specifically bind to the CYFRA21-1 protein; The three complementarity determining regions of the heavy chain are CDR-H1, CDR-H2 and CDR-H3; The three complementarity determining regions of the light chain are CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the H2 heavy chain variable region of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 1, the amino acid sequence of CDR-H1 is SEQ ID NO: 2, the amino acid sequence of CDR-H2 is SEQ ID NO: 3, and the amino acid sequence of CDR-H3 is SEQ ID NO: 4; The amino acid sequence of the H2 light chain variable region of the lung cancer marker CYFRA21-1 antibody is SEQ ID NO: 5, the amino acid sequence of CDR-L1 is SEQ ID NO: 6, the amino acid sequence of CDR-L2 is SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is SEQ ID NO:

8.

2. A diagnostic or assay kit, characterized in that: The invention comprises the lung cancer marker CYFRA21-1 antibody H2 according to claim 1.

3. The use of the lung cancer marker CYFRA21-1 antibody H2 according to claim 1, characterized in that: Application in detecting the concentration of lung cancer marker CYFRA21-1.

4. The use of the lung cancer marker CYFRA21-1 antibody H2 according to claim 1, characterized in that: Application in immunostaining of lung cancer tumor cells and lung cancer tissues.

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