Anti-ck5&6 antibody and application thereof

Abstract

The application discloses an anti-CK5&6 antibody and application thereof, the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of CDR-H1 of the heavy chain variable region is shown as SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown as SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is shown as SEQ ID NO: 6; the amino acid sequence of CDR-L1 of the light chain variable region is shown as SEQ ID NO: 7, the amino acid sequence of CDR-L2 is shown as SEQ ID NO: 8, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO: 9. The anti-CK5&6 antibody is a rabbit monoclonal antibody, the obtained antibody has high titer, specific positioning is clear in immunohistochemical staining, and there is no non-specific background staining, the antibody has wide application, and can also be used in Western blot and flow cytometry experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody development, and specifically relates to an anti-CK5&6 antibody and its application. Background Art

[0002] CK5 & 6 refer to cytokeratin 5 and cytokeratin 6, members of the cytokeratin family. They primarily function in the cytoskeleton of epithelial cells, providing structural support and protecting cells from mechanical stress. In normal tissues, CK5 & 6 are primarily expressed in the basal cells of squamous and ductal epithelium, as well as in some squamous germinal cells, myoepithelial cells, and mesothelial cells, while they are typically negative in glandular epithelial cells.

[0003] Cytokeratins CK5 & 6 play a significant role in assisting diagnosis, disease progression, and prognosis in cancer. For patients with cancers such as breast and lung cancer, cytokeratin 5 / 6 expression may be elevated because tumor cells produce large amounts of cytokeratin and release it into surrounding tissues. Testing for cytokeratin CK5 & 6 can help determine the presence of cancerous lesions and further assess the patient's disease status. Furthermore, cytokeratin CK5 & 6 can be used to predict the survival time and prognosis of cancer patients. Studies have shown that patients with high expression of cytokeratin CK5 & 6 are generally more likely to experience metastasis and recurrence, and have a poorer prognosis, than those with low expression.

[0004] Immunohistochemical detection of cytokeratin CK5 and 6 has important clinical significance in cancer diagnosis and treatment. It can not only assist in the diagnosis of various diseases but also guide subsequent treatment decisions. Therefore, the development of anti-CK5 and 6 antibodies will help in the diagnosis of related diseases, including tumors. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-CK5&6 antibody and its application. To this end, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides an anti-CK5&6 antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 6; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 9.

[0007] In some embodiments of the present invention, the antibody comprises the following polypeptide sequences: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 using Kabat online database analysis.

[0008] (1) A CDR-H1 comprising at least 5 amino acid sequences; the sequence is NYAMG (SEQ ID NO: 4).

[0009] (2) A CDR-H2 comprising at least 16 amino acid sequences; the sequence is IIRNDRNTYYASWAKG (SEQ ID NO: 5).

[0010] (3) A CDR-H3 comprising at least 11 amino acid sequences; the sequence is GGCADCITANI (SEQ ID NO: 6).

[0011] (4) A CDR-L1 comprising at least 11 amino acid sequences; the sequence is QASQSISSYLN (SEQ ID NO: 7).

[0012] (5) A CDR-L2 comprising at least 7 amino acid sequences; the sequence is RASTLAS (SEQ ID NO: 8).

[0013] (6) A CDR-L3 comprising at least 12 amino acid sequences; the sequence is QQTYNYNNVDNL (SEQ ID NO: 9).

[0014] In some embodiments of the present invention, the antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 2, and a light chain having an amino acid sequence as shown in SEQ ID NO: 3.

[0015] In some embodiments of the present invention, the antibody is a rabbit anti-human monoclonal antibody, and the immunogen used to immunize the rabbit comprises the amino acid sequence shown in SEQ ID NO: 1.

[0016] In some embodiments of the present invention, a polypeptide fragment synthesized from aa231-aa245 DSIVDERGRLDSELR (SEQ ID NO: 1) in the amino acid sequence of human CK5 protein (Uniprot ID: P13647) is selected as the immunogen sequence for preparing the antibody. This sequence is consistent with the amino acid aa226-aa240 sequence in CK6A protein, Uniprot ID: P02538. This polypeptide sequence has a low molecular weight. To ensure the immune effect, one amino acid C, i.e., cysteine, is added to the left side of the polypeptide sequence for cross-linking the macromolecule KLH. The aforementioned polypeptide fragment cross-links the macromolecule KLH to form a complete antigen.

[0017] A nucleic acid encoding the antibody described in the first aspect. By isolating the nucleic acid encoding the antibody described in the first aspect, the antibody can be produced in a recombinant manner, the nucleic acid is isolated and inserted into a replicable vector, and then further cloned or further expressed. Based on this, the second aspect of the present invention provides an expression vector comprising the nucleic acid described above. The sequence of the nucleic acid encoding the heavy chain variable region and light chain variable region of the present invention can be changed, and such changes include the addition, deletion or non-conservative / conservative substitution of nucleotides. The DNA encoding the antibody can be easily isolated or synthesized using conventional procedures. A variety of vectors are available, and the vector components generally include but are not limited to one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0018] Preferably, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0019] A third aspect of the present invention provides a host cell comprising the expression vector described above. In some embodiments of the present invention, the expression vector is a prokaryotic expression vector and the host cell is a prokaryotic cell; in other embodiments, the expression vector is a eukaryotic expression vector and the host cell is a eukaryotic cell. In a specific embodiment of the present invention, the expression vector is an Escherichia coli expression vector and the host cell is a HEK293 cell.

[0020] The fourth aspect of the present invention provides use of the antibody of the first aspect in preparing an immunoassay tool for detecting and identifying CK5&6 proteins.

[0021] In some embodiments of the present invention, the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0022] In some embodiments of the present invention, the immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

[0023] A fifth aspect of the present invention provides the use of the antibody described in the first aspect in the preparation of a lung cancer diagnostic kit for squamous cell lung carcinoma and adenocarcinoma. Immunohistochemical staining revealed that the anti-CK5 & 6 rabbit monoclonal antibody was positively localized in the brown cytoplasm of tumor cells in squamous cell lung carcinoma tissue and negative in tumor cells in adenocarcinoma tissue.

[0024] The sixth aspect of the present invention provides a kit for detecting and identifying CK5&6 proteins, comprising the antibody described in the first aspect.

[0025] The seventh aspect of the present invention provides a method for obtaining the antibody of the first aspect, comprising:

[0026] (1) culturing the host cell described in the third aspect, and

[0027] (2) Isolating and purifying the antibody from the cultured cells.

[0028] Beneficial effects of the present invention

[0029] Compared to existing technologies, the present invention offers the following advantages: The anti-CK5 & 6 antibodies provided by the present invention have high titers, clear specific localization as verified by immunohistochemical staining, and no nonspecific background staining. These antibodies can be used not only in pathological immunohistochemical staining experiments but also in Western blot, immunoprecipitation, and flow cytometry experiments, demonstrating their wide application.

[0030] Anti-CK5&6 antibodies can also be used in the diagnosis of lung cancer types: to diagnose and distinguish between squamous cell carcinoma and adenocarcinoma. Immunohistochemical staining showed that anti-CK5&6 rabbit monoclonal antibodies were positively located in the brown cytoplasm of tumor cells in squamous cell carcinoma tumor tissue, but negative in tumor cells in adenocarcinoma tumor tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 ELISA binding curves of the antigens in Example 1 of the present invention are shown;

[0032] Figure 2 The figure shows the immunohistochemical staining results of anti-CK5&6 rabbit monoclonal antibodies in different tissue sections;

[0033] Figure 3 The figure shows the results of Western blot detection of anti-CK5&6 rabbit monoclonal antibodies; in the figure, 1 is a 1:500 dilution, 2 is a 1:1000 dilution, 3 is a 1:2000 dilution, and 4 is a 1:5000 dilution;

[0034] Figure 4 The figure shows the flow cytometry results of anti-CK5&6 rabbit monoclonal antibodies; the peak on the left indicates that the isotype control antibody does not recognize the CK5&6 protein on the A431 wild cell line, and the peak on the right indicates that the anti-CK5&6 rabbit monoclonal antibody can recognize the CK5&6 protein on the A431 wild cell line. DETAILED DESCRIPTION

[0035] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0037] The technical solution of this patent is further described in detail below in conjunction with specific embodiments. The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0038] Example 1 Anti-CK5&6 Monoclonal Antibodies and Their Preparation

[0039] A peptide fragment synthesized from the amino acid sequence aa231-aa245DSIVDERGRLDSELR (SEQ ID NO: 1) of the human CK5 protein (Uniprot ID: P13647) was selected as the immunogen sequence for the antibody preparation. This sequence is consistent with the amino acid sequence aa226-aa240 of the CK6A protein (Uniprot ID: P02538). This peptide sequence has a low molecular weight. To ensure the immune effect, an amino acid C, i.e., cysteine, was added to the left side of the peptide sequence for cross-linking of the macromolecule KLH. The aforementioned peptide fragment cross-links the macromolecule KLH to form a complete antigen.

[0040] The immunogen was used to immunize New Zealand white rabbits, and the steps were as follows:

[0041] First, the polypeptide immunogen was coupled to the KLH macromolecular protein and then dialyzed to immunize New Zealand white rabbits. The first immunization was performed using 1 mg / mL, 0.5 mL of which was mixed with an equal amount of complete Freund's adjuvant. Two weeks later, the second immunization was performed using 0.5 mg / mL, 0.5 mL of which was mixed with an equal amount of incomplete Freund's adjuvant. Subsequently, the third and fourth immunizations were performed every two weeks according to the second immunization method.

[0042] After 4 immunizations, blood was collected to test serum titers. B cells from qualified rabbits were cryopreserved after the titers met the criteria. B cells were obtained using the method described in our patent: CN201610724209.1. The cells were then lysed using the method described in our patent: CN201910004299.0, and mRNA was extracted for RT-PCR. After RT-PCR, the obtained cDNA was subjected to PCR to obtain the antibody's light and heavy chain DNA. The light and heavy chain DNAs were ligated to the pBV vector and transformed into competent cells, which were then plated and cultured for 12 hours. The cultured colonies were picked and shake-cultured. After 24 hours of culture, the heavy and light chain plasmids were extracted. The extracted light and heavy chain plasmids were transfected into HEK293 cells and cultured for 6 days.

[0043] After 6 days, the cell culture supernatant was harvested and purified to obtain rabbit monoclonal antibodies.

[0044] The purified rabbit monoclonal antibody was sequenced.

[0045] The amino acid sequence of the H chain (full-length heavy chain) of the CK5&6 rabbit monoclonal antibody (SEQ ID NO: 2) is as follows:

[0046] ETGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLNNYAMGWVRQAPGKGLEYIGIIRNDRNTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARG GCADCITANIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKT VAPSTCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPI EKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0047] The amino acid sequence of the L chain (full-length heavy chain) of the CK5&6 rabbit monoclonal antibody (SEQ ID NO: 3) is as follows:

[0048] DTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVECADAAAYYCQQTYNYNN VDNLFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0049] The obtained anti-CK5&6 antibodies were analyzed using the Kabat online database;

[0050] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are as follows:

[0051] (1) a CDR-H1 sequence of at least 5 amino acids; the sequence is NYAMG (SEQ ID NO: 4);

[0052] (2) a CDR-H2 sequence of at least 16 amino acids; the sequence is IIRNDRNTYYASWAKG (SEQ ID NO: 5);

[0053] (3) a CDR-H3 sequence of at least 11 amino acids; the sequence is GGCADCITANI (SEQ ID NO: 6);

[0054] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are as follows:

[0055] (1) a CDR-L1 sequence of at least 11 amino acids; the sequence is QASQSISSYLN (SEQ ID NO: 7);

[0056] (2) a CDR-L2 sequence of at least 7 amino acids; the sequence is RASTLAS (SEQ ID NO: 8);

[0057] (3) A CDR-L3 of at least 12 amino acid sequences; the sequence is QQTYNYNNVDNL (SEQ ID NO: 9).

[0058] Example 2 Enzyme-linked immunosorbent assay of anti-CK5&6 monoclonal antibodies

[0059] The antibody can specifically bind to the immunogen synthetic polypeptide sequence designed for human CK5&6.

[0060] The procedure was carried out according to the conventional iELISA method. The steps were as follows:

[0061] (1) Coating: Use a synthetic peptide designed to specifically bind to human MUM1 to coat the plate, 1µg / mL, 50µL / well, and coat overnight at 4°C.

[0062] (2) Blocking: The next day, wash the plate three times with PBST, then add 5% skim milk for blocking, 100 µL / well, and block at 30°C for 1 h.

[0063] (3) Primary antibody incubation: After washing the plate, the obtained anti-MUM1 rabbit monoclonal antibody was diluted 4-fold from 1 μg / mL, with a total of 7 gradients. The diluted antibody was added to the ELISA plate at 50 μL / well and incubated at 30°C for 1 h.

[0064] (4) Secondary antibody incubation: After washing the plate, add 1:30,000 diluted HRP-labeled goat anti-rabbit IgG (Jackson, Cat#111-035-144) to the ELISA plate at 50 µL / well and incubate at 30°C for 40-60 min.

[0065] Color development and termination: After washing, add TMB color development solution at 50µL / well and react at 30℃ in the dark for 15 minutes. After the reaction is completed, add 50µL 1M H2SO4 to each well to terminate the reaction and read the OD450 absorbance value on the microplate reader. The standard curve is prepared based on the reading. The ELISA binding curve is shown in Figure 1 .

[0066] The results showed that there were significant differences in the readings between the dilutions. The above antibodies could specifically bind to the immunogen synthetic peptide sequence designed for human CK5&6. The ELISA binding curve of the CK5&6 monoclonal antibody was as follows: Figure 1 As shown, the EC50 was 5.04 ng / mL.

[0067] Example 3 Application of anti-CK5&6 monoclonal antibodies in pathological immunohistochemistry

[0068] Tissue sections of normal tonsil tissue, normal esophageal tissue, lung squamous cell carcinoma tumor tissue, and lung adenocarcinoma tumor tissue were selected for pathological immunohistochemical staining. The tissue sections were subjected to the immunohistochemical staining experimental steps according to the conventional manual immunohistochemical operation process. After the immunohistochemical staining was completed, the sections were placed under a microscope for reading and photographing.

[0069] The immunohistochemical staining pictures taken under the microscope are shown in Figure 2 .

[0070] The results can be clearly seen from the pictures: anti-CK5&6 rabbit monoclonal antibodies have brown cytoplasm-positive localization in the squamous epithelial cells of normal tonsil tissue and normal esophageal tissue (indicated by arrows); brown cytoplasm-positive localization in tumor cells of lung squamous cell carcinoma tissue (indicated by arrows), and negative in tumor cells of lung adenocarcinoma tissue (indicated by arrows).

[0071] The results showed that the antibody can distinguish between lung squamous cell carcinoma and lung adenocarcinoma in the diagnosis of lung cancer, and the antibody expression spectrum meets the theoretical and practical immunohistochemistry application standards.

[0072] Example 4 Application of anti-CK5&6 monoclonal antibodies in Western blot

[0073] (1) A431 cell lysate was used for polyacrylamide gel electrophoresis. The loading amount of A431 lysate was 20 μg / lane. The electrophoresis conditions were: 80 V, 40 min; 120 V, 80 min.

[0074] (2) Transfer: Remove the gel from the glass plate after electrophoresis in step (1), soak it in transfer buffer for 30 minutes, and then perform the transfer experiment. The transfer conditions are: 0.4A constant current transfer for 20 minutes.

[0075] (3) After the transfer is completed, remove the PVDF membrane, rinse the PVDF membrane with ddH2O for 5 minutes, add 10mL of Ponceau red for staining, and wash the membrane with methanol for 5 minutes after staining. Dry it and set aside. At this time, the cell protein lysate on the PVDF membrane will appear a red area, indicating that the protein lysate on the cells has been transferred to the PVDF.

[0076] (4) Blocking: Take out the PVDF membrane prepared in step (3), activate it with methanol for 30 seconds, and then wash the membrane with ddH2O for 5 minutes. Then add 10 mL of 5% skim milk powder to block the nonspecific binding sites on the membrane and incubate at 60 rpm for 1 hour at room temperature.

[0077] (5) Primary antibody incubation: After step (4), add anti-CK5&6 monoclonal antibodies at dilutions of 1:500, 1:1000, 1:2000, and 1:5000, and incubate at room temperature and 60 rpm for 1 h.

[0078] (6) Secondary antibody incubation: After step (5), add 1:4000 diluted HRP-conjugated goat anti-rabbit IgG polyclonal antibody and incubate at room temperature at 60 rpm for 1 h.

[0079] (7) Development and photography: After step (6), add the prepared ECL developer to the membrane. Place the PVDF membrane in a UV exposure instrument and take a photo.

[0080] (8) The result of inverting the photograph is shown in the following figure. Figure 3 .

[0081] The results in the figure show that the anti-CK5&6 rabbit monoclonal antibody recognizes CK5&6 proteins in A431 cell lysates, with an apparent molecular weight of 60 kD. The target band is present at all dilutions, with no obvious secondary bands, consistent with theoretical and literature reports. This indicates that this antibody is suitable for Western blot applications.

[0082] Example 5 Application of anti-CK5&6 monoclonal antibodies in flow cytometry

[0083] (1) Use cultured A431 cells, wash with PBS, and adjust the cell density to 1M / mL.

[0084] (2) Divide the 1M / mL cells into 3 tubes, 1 tube for negative control experimental cells, 1 tube for isotype control experimental cells, and 1 tube for anti-CK5&6 antibody experimental cells.

[0085] (3) Cell fixation: Add 1 mL of 4% paraformaldehyde fixative to each of the three tubes of cells and fix the cells for 30 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0086] (4) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0087] (5) Cell membrane disruption: Add 1 mL of 0.1% Triton X-100 permeabilization reagent to each of the three tubes of cells and permeabilize the cells for 10 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0088] (6) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0089] (7) Primary Antibody Incubation: Add 100 µL of 1% BSA in PBS to one tube of cells, 100 µL of 1 µg / mL isotype control CK10 antibody to another tube, and 100 µL of 1 µg / mL anti-CK5&6 rabbit monoclonal antibody to the final tube. Resuspend the cells and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm for 5 min at room temperature.

[0090] (8) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0091] (9) Secondary antibody incubation: Add 100 µL of 1:2000 diluted goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) to each tube, resuspend the cells, and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm for 5 min at room temperature.

[0092] (10) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0093] (11) Flow cytometry detection: Using the isotype control well as the standard, adjust the voltage for flow cytometry detection. The detection results are as follows: Figure 4 shown.

[0094] The results showed that using anti-CK5&6 rabbit monoclonal antibodies in flow cytometry experiments on A431 wild-type tumor cells revealed that the isotype control antibody did not recognize CK5&6 proteins in the A431 wild-type cell line (left curve), while the anti-CK5&6 rabbit monoclonal antibody did recognize CK5&6 proteins in the A431 wild-type cell line (right curve). This antibody can be used in flow cytometry applications.

[0095] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. An anti-CK5&6 antibody, characterized in that: It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 6; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

9.

2. The antibody according to claim 1, characterized in that: The antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 2, and a light chain having an amino acid sequence as shown in SEQ ID NO:

3.

3. The antibody according to claim 1 or 2, characterized in that: The antibody is a rabbit anti-human monoclonal antibody, and the immunogen used to immunize rabbits includes the amino acid sequence shown in SEQ ID NO:

1.

4. An expression vector, characterized in that: Comprising a nucleic acid encoding the antibody according to claim 1 or 2.

5. A host cell, characterized in that: Comprising the expression vector according to claim 4.

6. Use of the antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting and identifying CK5&6 proteins.

7. The use according to claim 6, characterized in that: The immunoassay tool includes reagents, test kits, chips or test paper.

8. The use according to claim 6, characterized in that: The immunoassay tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

9. Use of the antibody according to any one of claims 1 to 3 in the preparation of a diagnostic kit for lung squamous cell carcinoma.

10. A kit for detecting and identifying CK5&6 proteins, characterized in that: Comprising the antibody of claim 3.

Images