An anti-c-MET antibody and its preparation and application

Abstract

The present application discloses an anti-c-MET antibody and its preparation and application, belonging to the field of biotechnology. The anti-c-MET antibody is obtained by immunizing New Zealand white rabbits with c-MET polypeptide as an immunogen and then undergoing cell sorting and other steps, including heavy and light chains. The anti-c-MET antibody provided in this application can be applied to detection and screening fields such as immunohistochemistry, indirect ELISA, and flow cytometry, and can specifically identify and detect the expression of c-MET protein on tumor tissue, which is conducive to obtaining accurate evaluation and detection results.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an anti-c-MET antibody and its preparation and application. Background Art

[0002] At present, the main technologies for developing monoclonal antibodies include hybridoma technology, phage display technology, and B cell technology. Traditional hybridoma technology relies on myeloma cells, and the rabbit myeloma cells used to prepare rabbit monoclonal antibodies are subject to patent technology restrictions. Currently, only companies that own this patented technology use this technology to prepare rabbit monoclonal antibodies. Phage display antibody libraries use E. coli as the expression host and adopt non-natural antibody molecule forms such as single-chain antibodies (scFv), single-domain antibodies (VHH) or Fab antibodies. The structure and function of the antibodies are far inferior to those of antibody products expressed by mammalian cells. In addition, after the antibodies screened by phage display antibody libraries are converted into IgG molecules, even if they are expressed and produced by mammalian cells, some clones will show reduced activity and loss. Natural antibody genes are expressed using B cell technology, and the expressed antibodies have high specificity and high affinity.

[0003] Cellular-mesenchymal epithelial transition factor (c-MET), a member of the receptor tyrosine kinase family and a multifunctional transmembrane tyrosine kinase that acts as a receptor for hepatocyte growth factor (HGF), is lowly or absent in normal tissues. However, c-MET overexpression has been observed in tissues such as lung cancer, liver cancer, pancreatic cancer, and thyroid cancer. Immunohistochemistry (IHC) can be used to detect c-MET protein overexpression. Measuring c-MET protein expression levels and mutations can help physicians better diagnose and predict the progression and prognosis of certain diseases. Mutations in the c-MET gene are crucial for drug resistance in many tumors. As a cell membrane target, c-MET offers opportunities for the development of antibody-based therapeutics.

[0004] Therefore, developing an anti-c-MET antibody with high affinity and strong specificity is of great significance for specifically identifying and detecting the expression of c-MET protein on tumor tissues and obtaining accurate evaluation and detection results. Summary of the Invention

[0005] 1. Purpose of the Invention

[0006] The present application aims to provide an anti-c-MET antibody and its preparation and use. The anti-c-MET antibody is obtained by immunizing New Zealand white rabbits with the intracellular polypeptide of the c-MET protein as an immunogen and then performing cell sorting and other steps. The antibody can specifically recognize the c-MET protein. Immunohistochemistry (IHC) testing of various tissues has shown that the anti-c-MET antibody can effectively detect the expression of the c-MET protein on cells. The antibody can be applied to immunohistochemistry, indirect ELISA, immunofluorescence, flow cytometry, and other techniques to detect and screen for the expression of the c-MET protein.

[0007] 2. Technical solution

[0008] In order to achieve the above objectives, the technical solutions adopted in this application are as follows:

[0009] In a first aspect, the present application provides an anti-c-MET antibody, which can specifically bind to the c-MET protein and includes a heavy chain and a light chain.

[0010] Specifically, the heavy chain includes the amino acid sequence shown in SEQ ID NO.4.

[0011] Specifically, the above light chain includes the amino acid sequence shown in SEQ ID NO.5.

[0012] In a second aspect, the present application also provides a nucleic acid encoding the above-mentioned anti-c-MET antibody.

[0013] Specifically, the nucleic acid encoding the anti-c-MET antibody comprises:

[0014] The nucleotide sequence shown in SEQ ID NO.2 is used to encode the heavy chain of the anti-c-MET antibody including the amino acid sequence shown in SEQ ID NO.4;

[0015] The nucleotide sequence shown in SEQ ID NO.3 is used to encode the light chain of the above-mentioned anti-c-MET antibody comprising the amino acid sequence shown in SEQ ID NO.5.

[0016] In a third aspect, the present application further provides a recombinant expression vector, which contains the above-mentioned nucleic acid encoding the anti-c-MET antibody and can express the anti-c-MET antibody.

[0017] Specifically, the above-mentioned recombinant expression vector includes: a pcDNA3.1 plasmid containing the above-mentioned nucleic acid encoding the anti-c-MET antibody and capable of expressing the anti-c-MET recombinant monoclonal antibody.

[0018] In a fourth aspect, the present application further provides a recombinant expression cell, which comprises the above-mentioned recombinant expression vector or the above-mentioned nucleic acid encoding the anti-c-MET antibody and can express the anti-c-MET antibody.

[0019] Specifically, the above-mentioned recombinant expression cells include: HEK293 cell line.

[0020] In a fifth aspect, the present application also provides the use of the above-mentioned nucleic acid, recombinant expression vector, and recombinant expression cell in the preparation of anti-c-MET antibodies.

[0021] In a sixth aspect, the present application also provides a method for preparing the above-mentioned anti-c-MET antibody, comprising: using the above-mentioned recombinant expression vector to transfect cells to obtain recombinant expression cells; culturing the recombinant expression cells; collecting and purifying the supernatant to obtain the anti-c-MET antibody.

[0022] In a seventh aspect, the present application also provides the use of the above-mentioned anti-c-MET antibodies, nucleic acids, recombinant expression vectors, recombinant expression cells or methods for preparing anti-c-MET antibodies in detecting c-MET protein or preparing devices for detecting c-MET protein.

[0023] Specifically, the above-mentioned detection of c-MET protein includes any one or more of immunohistochemistry, ELISA, immunofluorescence, and flow cytometry.

[0024] Specifically, the above-mentioned c-MET protein detection device includes any one or more of a kit, an antibody coupling, an antibody chip, and the like.

[0025] In an eighth aspect, the present application further provides a kit for detecting c-MET protein, wherein the kit comprises the above-mentioned anti-c-MET antibody, with the anti-c-MET antibody as the primary antibody.

[0026] Specifically, the above-mentioned kit is a flow cytometry detection kit, which includes the above-mentioned anti-c-MET antibody and a flow cytometry detection reagent.

[0027] Furthermore, the above-mentioned flow cytometry detection kit also includes: fluorescently labeled secondary antibodies, PBS, bovine serum albumin, human IgG, 7-AAD and other reagents.

[0028] Specifically, the above-mentioned kit is an immunohistochemical detection kit comprising: the above-mentioned anti-c-MET antibody and an immunohistochemical detection reagent.

[0029] Furthermore, the above-mentioned immunohistochemistry detection kit includes: HRP enzyme-labeled secondary antibody, EDTA repair solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, blueing solution, etc.

[0030] In a ninth aspect, the present application also provides the use of the above-mentioned kit for detecting c-MET protein in detecting c-MET protein.

[0031] Specifically, the application of the above-mentioned flow cytometry detection kit includes: cell recovery, FC receptor blocking, primary antibody incubation, secondary antibody incubation, 7-AAD activity staining, data collection on the machine, etc.

[0032] Specifically, the immunohistochemistry detection kit includes the following steps: dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, sealing, and microscopic examination.

[0033] 3. Beneficial effects

[0034] Compared with the prior art, the present application has the following advantages:

[0035] (1) The present application provides an anti-c-MET antibody and its preparation and use. The anti-c-MET antibody is obtained by immunizing New Zealand white rabbits with the intracellular polypeptide of the c-MET protein as an immunogen and then undergoing cell sorting and other steps. The anti-c-MET antibody comprises a heavy chain with the amino acid sequence shown in SEQ ID NO. 4 and a light chain with the amino acid sequence shown in SEQ ID NO. 5. The anti-c-MET antibody can specifically recognize and detect the expression of c-MET protein in tumors or cancer tissues such as lung adenocarcinoma, thyroid cancer, liver cancer, and pancreatic cancer, thereby facilitating accurate evaluation and detection results.

[0036] (2) The anti-c-MET antibody provided in this application and its preparation and application have a shorter cycle and are not restricted by myeloma cells compared with the traditional hybridoma method; compared with phage display technology, natural antibodies have higher affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the immunohistochemical result of purified serum after immunization.

[0038] Figure 2 This is a cell image after specific B cell proliferation.

[0039] Figure 3 This is a diagram showing the immunohistochemical results of specific B cell supernatant validation on human pancreatic cancer tissue, intestinal cancer tissue, and lung cancer tissue.

[0040] Figure 4 This is the immunohistochemistry result diagram of the specific validation of the antibody in human lung adenocarcinoma tissue, human thyroid cancer tissue, human liver cancer tissue, and human pancreatic cancer tissue. DETAILED DESCRIPTION

[0041] The present application is further described below with reference to specific embodiments.

[0042] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0044] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0045] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0046] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0047] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0048] Example 1

[0049] This example provides the design and synthesis of c-MET polypeptide immunogens, specifically including:

[0050] Using c-MET (Uniprot ID: P08581) as the target antigen, the c-MET protein sequence was analyzed. The structure, linearity, length, and hydrophilicity of the c-MET protein sequence were predicted. Finally, the regional peptide aa997-aa1007 was selected as the immunogen for preparing anti-c-MET antibodies. This peptide segment belongs to the intracellular segment and is more suitable for the development of immunohistochemistry applications. Its specific sequence is: HNESVDYRATF (SEQ ID NO.1).

[0051] At the same time, a cysteine ​​was added to the C-terminus of the peptide aa997-aa1007 to cross-link KLH, named polypeptide A, for animal immunization; a biotin was added to the N-terminus of the peptide aa997-aa1007 to cross-link avidin magnetic beads, named polypeptide B.

[0052] Both peptides were synthesized by Anhui Guoping Pharmaceutical Co., Ltd.

[0053] Example 2

[0054] This example provides the preparation and screening of anti-c-MET antibodies, which specifically includes the following steps:

[0055] (1) Animal immunization and serum testing

[0056] The polypeptide A synthesized in Example 1 was conjugated with KLH as an immunogen. 500 μg of the immunogen was mixed with 0.5 mL of complete Freund's adjuvant and emulsified. Two New Zealand white rabbits, numbered K0021 and K0022, were immunized subcutaneously at multiple sites on the back. Fifteen days later, 0.25 mL of incomplete Freund's adjuvant and 250 μg of the immunogen were mixed and emulsified for a second immunization. Fifteen days later, a third immunization was performed using the same immunization method as the second. Fifteen days later, a fourth immunization was performed using the same immunization method as the second. Ten days after the final immunization, blood was collected from the rabbit's ear vein and centrifuged at 3000 rpm for 20 minutes to obtain rabbit antiserum.

[0057] c-MET polypeptide A was diluted to 0.5 μg / mL in carbonate buffer (50 mM, pH 9.6). 100 μL / well of polypeptide A was coated on an ELISA plate overnight at 4°C. The next day, the plate was blocked with 2‰ gelatin at 37°C for 1 hour. A serial dilution of rabbit antiserum was used as the primary antibody. After incubation at 37°C for 1 hour, a 1:10,000 goat anti-rabbit secondary antibody was added and incubated at 37°C for 1 hour. The plate was then developed with a colorimetric solution.

[0058] The test results are shown in Table 1. The titer of the rabbit anti-c-MET specific polyclonal antibody serum numbered K0021 was OD 450The value was higher than 0.5; and the immunohistochemical detection was performed using the polyclonal antibody purified from the rabbit serum, and the results showed that there was membrane staining in human breast cancer tumor tissue, but there was nonspecific ( Figure 1 The spleen of a New Zealand white rabbit numbered K0021 was obtained and ground to obtain lymphocytes.

[0059] Table 1 Titers of K0021 rabbit anti-c-MET specific polyclonal antibody at different dilutions

[0060] New Zealand White Rabbit Number K0021 K0022 1:250 3.391 3.408 1:1000 3.25 3.315 1:4000 3.226 3.05 1:16000 2.781 2.261 1:64000 1.56 0.969 1:256000 0.502 0.285 0 value 0.041 0.057

[0061] (2) Specific B cell enrichment and proliferation

[0062] The streptavidin magnetic beads used in this application were purchased from Suzhou Weidu Biotechnology Co., Ltd., with the product number CMP1001SB. 25 μL of magnetic beads were washed with PBS and then directly cross-linked with 100 μg of polypeptide B to obtain a magnetic bead mixture; the lymphocytes obtained from the spleen were counted and placed in a 5×10 6 Place living cells in a 9 cm culture dish and incubate in a 37 °C incubator on a shaker for 2 h. Then, aspirate the cell and magnetic bead mixture into a 15 mL centrifuge tube and place it in a ThermoDynaMag TM Place the cells on a -15 magnetic stand (Cat. No. 12301D). Discard the supernatant, resuspend in fresh 2% FBS1640 medium, and continue washing until no cells are visible in the supernatant. Resuspend the magnetically isolated specific B cells in culture medium and count the cells. After counting, dilute the cells, plate them in a 96-well plate, and culture them in a 5% CO2 incubator at 37°C for 7 days to allow for proliferation.

[0063] (3) Clone screening

[0064] After proliferation, a small population of B cells that can efficiently secrete monoclonal antibodies is obtained in the 96-well plate, such as Figure 2 As shown, antigen-specific clones were obtained by indirect ELISA. The experimental method is as follows:

[0065] 1) Remove polypeptide A from the freezer and coat with carbonate coating buffer at a concentration of 0.5 μg / mL. Add 50 μL / well of the prepared coating buffer to a labeled 96-well ELISA plate. Gently shake the plate to cover the bottom of the wells. Apply a self-adhesive sticker to the top 96-well ELISA plate and place in a refrigerator at 2-8°C overnight. Shake the coating buffer to dry the next day.

[0066] 2) Blocking: Add 60 μL / well of 1% BSA to each well and incubate at room temperature for 45 minutes;

[0067] 3) Washing: Discard the blocking solution and wash once with 200 μL / well of 1×PBST.

[0068] 4) Sample addition: Add 50 μL of supernatant to each well using a pipette;

[0069] 5) Positive and negative controls: K0021 quadruple immunization rabbit serum, diluted 1:1000 with 1% BSA, mixed, and added to two wells in the last column of the plate. For the negative control, no primary antibody was added to the last two wells.

[0070] 6) Washing: Remove the primary antibody and wash the plate twice with 200 μL 1× PBST.

[0071] 7) Add secondary antibody: Fc-HRP 1:10000 dilution, 50 μL / well, incubate at room temperature for 40 min;

[0072] 8) Washing: Discard the secondary antibody, wash twice with 200 μL 1× PBST, and pat dry.

[0073] 9) Color development: Add 50 μL of TMB substrate to each well and develop color for 5 min at room temperature in the dark;

[0074] 10) Termination: Add 50 μL of 4.9% phosphoric acid to each well in the order of color development to terminate the reaction;

[0075] 11) Reading: Microplate reader OD 450 Read the plate within 15 minutes after termination.

[0076] The supernatant of ELISA antigen-specific positive clones was selected for immunohistochemical verification. The results were as follows: Figure 3 As shown, a B cell clone that can specifically recognize c-MET protein in cancer tissues was obtained.

[0077] (4) Construction and expression of recombinant antibody vectors

[0078] B cells that specifically recognize c-MET protein in cancer tissues, as detected by immunohistochemistry, are lysed and mRNA is harvested. cDNA encoding the heavy and light chain sequences of these specific B cells is then generated by RT-PCR. The resulting cDNA is then constructed into the expression vector pCDNA3.1 via homologous recombination, and sequencing analysis is performed to obtain the heavy and light chain gene sequences. The heavy and light chain gene sequences are then paired, and the constructed eukaryotic expression vectors are transfected into HEK293 cells using a transfection reagent, and the cell supernatant is collected.

[0079] (5) Antibody sequence

[0080] The collected HEK293 cell supernatants were verified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, confirming that the antibodies expressed by the plasmids containing the following nucleic acids had a good immune response to the antigen:

[0081] The nucleic acid sequence encoding the heavy chain is:

[0082]

[0083] The nucleic acid sequence encoding the light chain is:

[0084] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTCTGGGAGGCACAGTCACCATCAAT TGCCAGTCTAGTCGGAGCCTATGCAGCCAGAACAACATAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGGTCTTGATCTtGCCGGTATCGAATCTGCCATCTGGGGTCCCATCACGGTTCAGCGGCA GTGGATCTGGGACACAGTTCAGTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATACAAGTGGTGTTTATGGTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAG GTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCAC CACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG (SEQ ID NO. 3).

[0085] Based on the above nucleic acid sequence, the amino acid sequences of the corresponding antibody heavy and light chains were obtained:

[0086] Heavy chain amino acid sequence:

[0087] METGLRWLLLVAVFKGVQCQEQLKETGGGLVQPGGSLTLSCKASGYELSSNDMCWVRQAPGEGLEWIGELRTSGGLWYATWVNGRFSISRENTQNTVSLQLNSLTAADTATYFCA RGGWDTDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVA PSTCSKPMCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKT ISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.4);

[0088] Light chain amino acid sequence:

[0089] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAALGGTVTINCQSSRSLCSQNNIAWYQQKPGQPPKVLILPVSNLPSGVPSRFSGSGSGTQFSLTISDLECDDAATYYCAGGYTSGV YGFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.5).

[0090] In the examples, a large number of cells were transfected with a confirmed positive expression vector, and after 3-5 days of culture, the cell suspension was collected, centrifuged, and the supernatant was purified by affinity chromatography on a Protein A column. The purification steps were as follows:

[0091] 1) Supernatant filtration: The cell culture supernatant was filtered through a 0.45 μm filter before incubation;

[0092] 2) Sample loading: Calculate the volume of Protein A filler required for incubation or loading based on the supernatant volume. Add the filler to a 50 mL centrifuge tube and place it on a rotary shaker. Incubate at room temperature for 1 hour. Then transfer the filler to a 6 mL empty chromatography column. After the incubation, let it stand at room temperature for 10 minutes to allow the filler to settle. After loading, there is no need to let it stand and subsequent washing can be carried out directly.

[0093] 3) Washing: Wash the packing with 15 column volumes of Wash Buffer A and 1× PBS, respectively. Measure the protein concentration of the post-column effluent. Elution can be performed when OD280 is less than 0.01.

[0094] 4) Elution: Use Elute Buffer B for elution. Collect the effluent until the OD280 is greater than 0.1 and continue collecting until the OD280 is less than 0.1. Add 10× PBS to the neutralized eluate to reconstitute the eluate in 1× PBS. Determine the IgG concentration using a protein concentration meter (the rabbit IgG absorbance coefficient must be manually set to 1.37). Record the data and then perform ultrafiltration and concentration.

[0095] After the above steps, a rabbit anti-human c-MET antibody with a purity greater than 95% can be obtained.

[0096] Example 3

[0097] This example provides the use of the anti-c-MET antibodies screened in this application in detecting c-MET protein. The detection method is an immunohistochemical method, using the anti-c-MET antibody as the primary antibody and PolyHRP-GAR as the secondary antibody, specifically including:

[0098] (1) The cancer tissue (mc) chip was sliced ​​to a thickness of 4 μm, spread at 42°C, and baked in a drying oven at 63-65°C for approximately 1 h.

[0099] (2) Dewaxing and hydration: Sections were placed in a) tissue clearing agent twice, each for 15 minutes; b) 100% alcohol twice, each for 5 minutes; c) 95% alcohol twice, each for 5 minutes; d) 85% alcohol once, each for 5 minutes; e) 75% alcohol once, each for 5 minutes. After dewaxing and hydration, sections were removed and rinsed with distilled water three times, each for 5 minutes. (All operations were performed in a fume hood.)

[0100] (3) Antigen repair: Place the sections in a staining box containing sodium citrate buffer (prepared with 200 mL of deionized water, i.e., add 4 mL of 50× repair solution) or EDTA repair solution. Add 2 L of distilled water to the antigen repair pot, and place the staining box in a pressure cooker for high-temperature and high-pressure repair. After the pressure cooker begins to release gas, close the outlet valve and continue heating for 20 minutes. Then, end the heating, open the lid, take out the inner pot, cool it naturally, rinse it with distilled water 3 times, and place it on a shaker for 5 minutes each time.

[0101] (4) Blocking endogenous peroxidase: After washing three times, circle the slices on the slide with an immunohistochemical pen. When drawing circles, make sure that the circles are roughly the same size, spaced apart, and the slice is placed roughly in the middle of the circle. Block nonspecific antigens with 10% goat serum (prepared in PBS) for 30 minutes at room temperature.

[0102] (5) Addition of primary antibody: After blocking, remove the blocking solution from the slide and add 80-100 μL of diluted primary antibody (anti-c-MET antibody) to each well. Adjust the volume appropriately according to the size of the circle. Place the slide in a humidified chamber and then slowly place it in a refrigerator at 4°C overnight.

[0103] (6) Addition of secondary antibody working solution: The next morning, remove the wet box and let it stand at room temperature for 10 minutes; rinse with 1× PBST three times, 5 minutes each time; add secondary antibody, incubate at room temperature for 30 minutes, and wash with PBST three times, 5 minutes each time;

[0104] (7) DAB color development: According to the calculation of 100 μL per well, 6 mL is required, that is, 6 drops of color development solution are added to 6 mL of color development buffer; freshly prepared color development solution is added to each slide, and the color is developed for 1 to 2 minutes. It can be observed with the naked eye and the color is stopped when brownish yellow appears. When the time is up, rinse with tap water to stop the color development; restain with hematoxylin for 2 to 5 minutes; rinse with distilled water for 5 minutes, decolorize in PBS for 30 seconds, and rinse with distilled water for 5 minutes;

[0105] (8) Dehydration of sections: Dehydrate and dry in graded alcohols: a) 50% alcohol, 5 min; b) 75% alcohol, 5 min; c) 90% alcohol, 5 min; d) 100% alcohol 3 times, 5 min each; e) xylene 2 times, 5 min each;

[0106] (9) Sealing: Add 50-100 μL of neutral gum to each slide, and then slowly add a coverslip;

[0107] (10) Scanning: After sealing, the slides were dried overnight. The next day, they were observed under a microscope and scanned with a tissue slicer to obtain the immunohistochemical results.

[0108] The results are as follows Figure 4As shown, the anti-c-MET antibody of the present application can detect the expression of c-MET protein in lung adenocarcinoma, thyroid cancer, liver cancer, and pancreatic cancer tissues. Tumor cells are uniformly stained by the antibody, and the cell membrane shows strong staining, while staining is almost undetectable in normal cells.

Claims

1. An anti-c-MET antibody, characterized in that The anti-c-MET antibody includes a heavy chain and a light chain, the heavy chain has an amino acid sequence shown in SEQ ID NO.4, and the light chain has an amino acid sequence shown in SEQ ID NO.

5.

2. A nucleic acid, characterized in that The nucleic acid encodes the heavy chain and light chain of the anti-c-MET antibody of claim 1.

3. A nucleic acid according to claim 2, characterized in that The nucleic acid includes the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to claim 2 or 3.

5. A recombinant expression cell, characterized in that The recombinant expression cell comprises the recombinant expression vector according to claim 4, or the nucleic acid according to claim 2 or 3.

6. Use of the nucleic acid according to claim 2 or 3, or the recombinant expression vector according to claim 4, or the recombinant expression cell according to claim 5 in preparing an anti-c-MET antibody.

7. The method for preparing the anti-c-MET antibody according to claim 1, wherein The method comprises: The recombinant expression vector according to claim 4 is transfected into cells to obtain recombinant expression cells; the recombinant expression cells are cultured; and the supernatant is collected and purified to obtain an anti-c-MET antibody.

8. Use of the anti-c-MET antibody of claim 1, or the nucleic acid of claim 2 or 3, or the recombinant expression vector of claim 4, or the recombinant expression cell of claim 5, or the method for preparing the anti-c-MET antibody of claim 7 in preparing a device for detecting c-MET protein.

9. The use according to claim 8, characterized in that The method used by the c-MET protein detection device includes any one or more of immunohistochemistry, ELISA, immunofluorescence, and flow cytometry.

10. A kit for detecting c-MET protein, characterized in that: The kit comprises the anti-c-MET antibody according to claim 1.

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