A monoclonal antibody H2 specifically binding to lung cancer marker CYFRA21-1 and its application

Abstract

The present invention discloses a monoclonal antibody H2 that specifically binds to the lung cancer marker CYFRA21-1 and its application, relating to the field of biomedicine technology. First, using phage display technology, a lung cancer marker CYFRA21-1 monoclonal antibody H2 is obtained by panning 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 novel amino acid sequences, and can specifically bind to the CYFRA21-1 protein. The binding has the characteristics of high affinity, high specificity and stability. The monoclonal antibody H2 for the lung cancer marker CYFRA21-1 of the present invention can not only 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, which is of great 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 primarily present in the cytoplasm of epithelial tumor cells, such as lung cancer and esophageal cancer. CYFRA21-1 is expressed in both lung adenocarcinoma and squamous cell carcinoma, and belongs to the cytokeratin family. CYFRA21-1 has extensive clinical applications as a non-organ-specific tumor marker, playing an important role in various cell types and tumors. It is primarily used to detect lung cancer, particularly 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, which is released into the blood circulation, resulting in elevated serum CYFRA21-1 levels. Therefore, serum CYFRA21-1 levels can effectively indicate the occurrence and progression of tumors. Serum CYFRA21-1 levels are positively correlated with the progression of clinical stages of non-small cell lung cancer. After radical resection of lung cancer, CYFRA21-1 concentrations significantly decrease. If they increase again, tumor progression and recurrence should be considered. Therefore, detecting serum CYFRA21-1 levels can provide important evidence 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 evidence for diagnosing the condition of tumor patients, selecting treatment methods, observing efficacy, and predicting recurrence. However, there are currently relatively few publicly available monoclonal antibodies specific for the 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 present invention aims to provide a monoclonal antibody H2 that specifically binds to the lung cancer marker CYFRA21-1 and its application.

[0005] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0006] Lung cancer marker CYFRA21-1 antibody H2, which has a human antibody sequence backbone, and the amino acid sequences of the three complementarity determining regions of the heavy chain and the three complementarity determining regions of the light chain are artificially designed and synthesized amino acid sequences, and specifically binds 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 the antigen CYFRA21-1 recombinant protein, an enzyme-labeled anti-human antibody capable of binding 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 the 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] This study utilizes phage display technology to screen a monoclonal antibody, H2, against the lung cancer marker CYFRA21-1, from the Tomlinson I+J phage library. This antibody, which features heavy and light chain complementary determining regions (CDRs) composed of novel amino acid sequences, specifically binds to the CYFRA21-1 protein. This binding exhibits high affinity, specificity, and stability, making it a powerful tool for studying the physiological effects of the CYFRA21-1 protein in cells.

[0026] The monoclonal antibody H2 against 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 enzyme-linked immunosorbent assay of polyclonal antibodies obtained by panning;

[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 the 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 with reference to specific examples.

[0032] Example 1

[0033] 1. Amplification of phage display antibody library

[0034] Add the E. coli stock containing the 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. Culture 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), and after infection at 37°C for 30 minutes, the infected E. coli was dissolved in a high-speed centrifuge and centrifuged at 5000rpm for 30 minutes. The supernatant was discarded and the centrifugation was repeated twice to completely remove the supernatant. The bacteria were resuspended in 100mL of 2YT liquid culture medium (2YTAGK) containing 100μg / mL Amp, 50μg / mL kanamycin and 0.1% glucose, and the solution was placed in a conical flask and cultured at 30°C and 250rpm for 20 hours. The next day, centrifuged at 5000rpm for 30 minutes, the supernatant was taken and placed in a new 50mL sterile centrifuge tube, and 20mL of PEG / NaCl (20% polyethylene glycol 6000, 2.5M NaCl) solution was added to the supernatant. After mixing evenly, the supernatant was allowed to stand on ice for 1 hour, centrifuged at 5000rpm for 1 hour, the supernatant was discarded, the centrifugation was repeated twice to 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 libraries

[0036] 50 μg / mL CYFRA21-1 protein (antigen) diluted in PBS buffer (PBS used in this experiment has been sterilized) was coated into a 96-well microplate, 100 μL / well, and incubated at 4°C overnight. The next day, the antigen solution was discarded and 300 μL of PBS solution containing 5% skim milk powder (MPBS) was added. The plate was blocked at room temperature for 2 hours. The microplate was washed three times with PBST solution (PBS containing 0.1% Tween 20). Tomlinson I phage library and Tomlinson J phage were mixed at a ratio of 1:1 and diluted 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 sterile PBS) to each well, and let it stand at room temperature for 10 minutes to elute phage bound to the CYFRA21-1 antigen. Add 100 μL of glycine-HCl buffer (pH = 2.2) to each well, let it stand at room temperature for 5 minutes to elute phage bound to the CYFRA21-1 protein, and then add 50 μL of Tris-HCl buffer (pH = 7.4) to each well for neutralization.

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

[0038] The supernatant was recovered by centrifugation at 5000 rpm for 30 minutes at 4°C overnight. 4 mL of PEG / NaCl solution was added to the supernatant, mixed thoroughly, and placed on ice for 1 hour. The supernatant was discarded and centrifuged again at 5000 rpm at 4°C for 1 hour. 200 μL of sterile PBS solution was added to serve as the second round of panning (R1). This step was repeated three times to obtain phage R2 and R3, respectively. After the third round of panning, the binding specificity of the original antibody library R0 and the panned R1, R2, and R3 to the CYFRA21-1 protein was verified using enzyme-linked immunosorbent assay.

[0039] The titer of the four groups of phage libraries was determined, and the titer of the four groups of antibody libraries was calculated. 5 μg / mL bovine serum albumin (BSA) and CYFRA21-1 protein diluted with PBS buffer were coated in 96 microwell plates, with 3 wells in each package, 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 discarded, 300 μL of PBS solution containing 5% skim milk powder (MPBS) was added, and the plate was blocked at room temperature for 2 hours. The microwell plate was washed 3 times with PBST solution, and 100 μL of 10% MPBS was added to each of the 4 groups of microwells. 10 Phage libraries (R0, R1, R2, and R3) containing 50 cfu of phage were added to a 5% MPBS solution and incubated at room temperature for 1 hour. The microplate was washed six times with PBST solution, and then a 1:5000 dilution of mouse anti-M13-HRP antibody was added and incubated at room temperature for 1 hour. The microplate was washed 12 times with PBST, and 100 μL of TMBZ was added to each well for color development. After incubation at 37°C for 5-10 minutes, 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 iMark™ microplate reader (Bio-Rad), and a histogram was plotted.

[0040] The results of enzyme-linked immunosorbent assay were Figure 1 As shown in the figure, when comparing the binding ability 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 determined. The next day, 96 single colonies were randomly picked and placed in a 96-well microplate. 1.5 mL of 2YTAG liquid 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 then placed in a shaker at 37°C and 200 rpm for overnight culture. The next day, 10 μL of overnight bacteria was taken from each well and added one by one to a new 96-well plate containing 90 μL of 2YTAG liquid 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. The plate was sealed with sealing film and infected at 37°C for 30 min. After infection, 1.35 mL of 2YTAK (without glucose) was added to each well, the plate was sealed with sealing film, and incubated at 30°C at 250 rpm in a shaking incubator for 20 h.

[0043] 100 μL of PBS solution containing BSA (5 μg / mL) and CYFRA21-1 protein (5 μg / mL) was added to each well of a 96-well plate, one for each well, and incubated overnight at 4°C. The next day, the solution in the microplate was discarded and 300 μL of 5% MPBS was added. The plate was blocked at room temperature for 2 hours. The 20-hour culture solution was removed from the microplate and transferred to a 2 mL centrifuge tube. The plate was centrifuged at 5000 rpm for 30 minutes at 4°C. During this time, the MPBS-blocked 96-well plate was washed three times with PBST. 80 μL of MPBS solution was added to each well. 20 μL of the centrifuged phage supernatant was then added to each well. The mixture was gently pipetted and incubated at room temperature for 1 hour. The microplate was washed six times with PBST solution. 100 μL of a 1:5000 dilution of mouse anti-M13-HRP antibody was added to each well and incubated at room temperature for 1 hour. The microplate was washed 12 times with PBST, and 100 μL of TMBZ was added to each well for color development. 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 iMark™ microplate reader (Bio-Rad), and a bar graph was drawn as shown in FIG. Figure 2 Antibodies with high binding activity to CYFRA21-1 were screened and re-validated to further identify positive clones.

[0044] 4. Antibody sequence comparison and analysis

[0045] Based on these experimental results, eight positive clones were selected, plasmids were extracted, and gene sequencing was performed. One antibody strain, designated H2, was obtained. Comparison with antibody sequences registered in an antibody gene library revealed no sequences identical to those of the antibody described herein, thus confirming that this antibody is novel. The amino acid sequence of the H2 antibody is detailed below.

[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] 2 μg / mL BSA and CYFRA21-1 protein were coated onto a 96-well microplate, with 3 wells coated with BSA and 6 wells coated with CYFRA21-1, with 100 μL per well incubated overnight at 4°C. The next day, the microplate contents were discarded and 300 μL of PBS containing 5% skim milk powder (MPBS) was added. The plate was blocked at room temperature for 2 hours. The microplate was washed three times with PBST solution, and a mixture of 80 μL of MPBS and 20 μL of phage-displayed antibody supernatant was added to each well. Three wells coated with CYFRA21-1 protein were added with this mixture and the competition protein was added at a final concentration of 10 μg / mL. The plate was incubated at room temperature for 1 hour. The microplate was washed six times with PBST solution, and a 1:5000 dilution of mouse anti-M13-HRP antibody was added and incubated at room temperature for 1 hour. The microplate was washed 12 times with PBST, and 100 μL of TMBZ was added to each well for color development. 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 iMark™ microplate reader (Bio-Rad), and a bar graph was plotted.

[0061] The results of competitive enzyme-linked immunosorbent assay were Figure 3 As shown in the results, 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 at 4°C overnight. The next day, the solution in the microplate was discarded, 300 μL of PBS solution containing 5% skim milk powder (MPBS) 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 10 cfu / mL), a series of phage display 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. Each concentration was repeated three times and incubated at room temperature for 1 hour. Washed 8 times with PBST, and HRP-labeled mouse anti-M13 antibody was added. Washed the microplate 6 times with PBST solution, and then added a 1:5000 diluted mouse anti-M13-HRP antibody and incubated at room temperature for 1 hour. Washed the microplate 12 times with PBST, and 100 μL TMBZ was added to each well for color development. After reacting at 37°C for 5 to 10 minutes, 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, and a dose-response curve was drawn. 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 limit of detection (LOD) of CYFRA21-1 protein was calculated to be 1.75 ng / mL.

[0065] 7. In addition to being used to detect the concentration of the 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 inside lung cancer cells.

[0068] When the CYFRA21-1 antibody H2 is added to the tissue section, it binds to CYFRA21-1 in the lung cancer cells. A secondary antibody is then added, which is usually an antibody against the primary antibody and carries a marker (such as fluorescein or an enzyme). If a fluorescently labeled secondary antibody is used, the lung cancer cells will show a fluorescent signal under a fluorescence microscope; if an enzyme-labeled secondary antibody is used, the lung cancer cells will show a color change after the addition of a substrate, allowing them to 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 have differences 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. The intensity and range of immunostaining can provide clues for diagnosis.

[0071] (4) Tumor staging assessment: The expression of CYFRA21-1 may change during the progression of the tumor. 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. Antibody H2 against lung cancer marker CYFRA21-1 was 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 the antigen CYFRA21-1 recombinant protein, enzyme-labeled anti-human antibody capable of binding to H2, or anti-His-tag antibody, enzyme substrate and color development solution.

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

[0075] (1) Antibody H2, a lung cancer marker, is a core reagent used to specifically identify the lung cancer marker CYFRA21-1. It undergoes rigorous purification and quality testing to ensure high affinity and specificity for CYFRA21-1. It is typically produced using 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. For example, a horseradish peroxidase-labeled secondary antibody can be combined with the H2 antibody for subsequent colorimetric 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), ensuring the activity of antibodies and antigens.

[0079] (4) Blocking buffer: Commonly used solutions include bovine serum albumin (BSA) solution or skim 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 achieve an appropriate detection concentration. In addition to buffer, the sample diluent may also contain additives such as preservatives and protease inhibitors to prevent degradation of sample components.

[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 reacts with horseradish peroxidase to produce a color change from colorless to blue. The reaction can then be terminated with sulfuric acid, resulting in a yellow color change. The intensity of the color is related to the concentration of CYFRA21-1 in the sample.

[0084] (7) Standards and quality control products

[0085] Standards are pure CYFRA21-1 or samples containing CYFRA21-1 of known concentration. They are used to establish a standard curve to calculate the CYFRA21-1 concentration in unknown samples based on the assay signal (e.g., absorbance). The concentration range of the standard should cover the linear range of the assay kit.

[0086] Quality Controls: Used to monitor the accuracy and reproducibility of the test kit. They include high, medium, and low concentrations of CYFRA21-1 samples, which are tested along with unknown samples during each test to ensure the reliability of the test results.

[0087] (9) Other materials

[0088] Microplates (used for ELISA testing): Typically made of polystyrene, the surface is specially treated to effectively adsorb antibodies and antigens. Specifications can be selected based on test throughput requirements, such as 96-well plates.

[0089] Packaging materials: such as the test kit housing, reagent bottles, droppers, and instructions. The test kit housing should be sturdy and easy to transport and store; the reagent bottles should ensure the stability and sealing of the reagents; the droppers should be able to accurately draw up and dispense the reagents; and the instructions should detail the kit's usage, detection principles, and storage conditions.

Claims

1. Antibody H2 for the lung cancer marker CYFRA21-1, characterized by: The lung cancer marker CYFRA21-1 antibody H2 has a human antibody sequence backbone, and the amino acid sequences of the three complementarity determining regions of the heavy chain and the three complementarity 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.

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