Anti-k i-67 protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human Ki-67 antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-Ki-67 protein rabbit monoclonal antibody, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1;The amino acid sequence of the variable region of the light chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.The antibody has high specificity, sensitivity, and can specifically recognize the cell expressing Ki-67 protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to an anti-Ki-67 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Ki-67 is a nuclear protein encoded by MKI-67 gene, also known as MKI-67. The protein size of MKI67 is 350-400 kDa, which belongs to the member of chromosome mitosis-related proteins. Ki-67 is related to ribosomal RNA transcription, and the inactivation of Ki-67 can inhibit the synthesis of ribosomal RNA. MKI67 can be used as a marker of cell proliferation. Ki-67 is expressed in all phases of cell proliferation (G1, S, G2 and M), but not expressed in the cell quiescent phase G0. It has been found that Ki-67 gene plays an important role in the development of various tumors, and the high and low Ki-67 index in pathological reports is closely related to the differentiation degree, infiltration, metastasis and prognosis of many tumors.

[0003] Ki-67 protein is a marker antigen universally present in the nuclei of all human proliferating cells. Studies at home and abroad have shown that Ki-67 gene is not expressed in normal tissues, but is strongly expressed in cancerous tissues. There is weak expression in the early stage of malignancy, and the expression level is positively correlated with the degree of lesion. Ki-67 also plays a promoting role in tumor metastasis. It can reduce the adhesion between homotypic cells, promote tumor angiogenesis, and remodel extracellular matrix. In clinical practice, Ki-67 is often closely related to colon cancer, gastric cancer, bladder cancer, kidney cancer and many other cancers. At the same time, Ki-67 can be used as one of the indicators for the prognosis and treatment of related cancers. SUMMARY

[0004] The present application provides an anti-Ki-67 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0005] SEQ ID NO. 1:

[0006] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGIDLSTNSLTWVRQAPGKGLEYIGIIDRYGSTYYATWAKGRFT

[0007] ISKTSTTVDLKITSPTMEDTATYFCSDLKIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGT

[0008] LTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISR

[0009] TPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARG

[0010] QPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSV

[0011] MHEALHNHYTQKSISRSPGK

[0012] SEQ ID NO.2:

[0013] MDTRAPTQLLGLLLLWLPGAICDPVMTQTPSSTSAAVGGTVTINCQSSQNVYNSNFLVWFQQKPGQPPKRLIYSASTLASGVPSRF

[0014] KGSGSGTQFTLTISDLQCDDAAIYYCAGGFSGNIVGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTW

[0015] EVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0016] Further, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 4.

[0017] SEQ ID NO. 3:

[0018] ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCT

[0019] GGTCACGCCTGGGACACCGCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTACCAATTCATTGACCTGGGTCCGCCAGG

[0020] CTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGATAGATATGGCAGCACATACTACGCGACCTGGGCAAAAGGCCGATTCACC

[0021] ATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAATGGAGGACACGGCCACTTATTTCTGTAGTGATTTGAA

[0022] GATTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGG

[0023] ACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACC

[0024] CTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAG

[0025] CAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGC

[0026] CCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGC

[0027] ACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGT

[0028] GCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACT

[0029] GGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGG

[0030] CAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAA

[0031] CGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGG

[0032] ACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTG

[0033] ATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG

[0034] SEQ ID NO.4:

[0035] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCATATGTGACCCTGTGATGACCCAGAC

[0036] TCCATCTTCCACGTCTGCGGCTGTGGGAGGCACAGTCACCATCAACTGTCAGTCCAGTCAGAATGTTTATAATAGCAACTTCTTAG

[0037] TCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTC

[0038] AAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGCAGTGTGACGATGCTGCCATTTACTATTGTGCAGGCGG

[0039] TTTTAGTGGTAATATTGTTGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAGGGTGATCCAGTTGCACCTACTGTCCTCATCTTCC

[0040] CACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGG

[0041] GAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAG

[0042] CACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGA

[0043] GCTTCAATAGGGGTGACTGTTAG

[0044] Further, the monoclonal antibody specifically recognizes the Ki-67 protein.

[0045] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0046] Further, the rabbit monoclonal antibody has a clone number of 10G1.

[0047] The inventors also provide a method for preparing an anti-Ki-67 protein monoclonal antibody, which selects the amino acid sequence shown in SEQ ID NO. 5 in the Ki-67 protein, adds a cysteine at the carboxyl end, and then couples it with the carrier protein KLH as an immunogen.

[0048] SEQ ID NO.5:

[0049] CTPKEKAQALEDLAGFKELFQ

[0050] The inventors also provide a Ki-67 protein immunodetection reagent containing the above-mentioned anti-Ki-67 protein monoclonal antibody as an effective component.

[0051] Differing from the prior art, the application has the beneficial technical effects that the above-mentioned technical solution provides an anti-Ki-67 protein rabbit monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize cells expressing Ki-67 protein, and is suitable for immunological detection, in particular, immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Comparison chart of esophageal squamous cell carcinoma immunohistochemical staining results (left: rabbit monoclonal antibody Ki-67 of the application, right: commercially available Ki-67).

[0053] Figure 2 Comparison chart of intestinal adenoma immunohistochemical staining results (left: rabbit monoclonal antibody Ki-67 of the application, right: commercially available Ki-67). DETAILED DESCRIPTION

[0054] In order to describe the possible application scenarios, technical principles, specific implementation schemes, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described in this paper are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0055] In this paper, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0056] Unless otherwise defined, the meanings of the technical terms used in this paper are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms in this paper is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0057] In the description of the present application, the phrase "and / or" is a description of the logical relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " herein generally represents that the associated objects before and after are a "or" logical relationship.

[0058] In the present application, phrases such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary, or order relationship between the entities or operations.

[0059] In the present application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0060] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions "greater than", "less than", "exceed" and the like are understood as not including the number; the expressions "above", "below", "within" and the like are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0061] Example 1 Preparation of Ki-67 polypeptide

[0062] I. Selection of antigen polypeptide

[0063] The protein sequence of accession number P46013 in Uniprot was used for sequence and secondary structure analysis. The full-length Ki-67 protein has a molecular weight of about 35.8 kDa. According to the parameters of secondary structure and surface accessibility of the protein predicted by the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , the amino acid sequence CTPKEKAQALEDLAGFKELFQ at positions 1213-1232 was selected as the antigen for chemical synthesis (Jinsirui Biotechnology Co., Ltd.). For the convenience of coupling, a cysteine was added at the carboxy terminus of the polypeptide to provide a thiol group for coupling.

[0064] II. Coupling and purification of polypeptide

[0065] The maleimide-activated keyhole limpet hemocyanin kit of Thermo Scientific (catalog number: 77653) was selected, and the operation was carried out according to the procedure provided in the kit. For the polypeptide to be coupled, first detect the free thiol group in the polypeptide with Ellman reagent (Thermo Scientific, catalog number: 22582): add 100 μL of Ellman reagent stock solution and 10 μL of polypeptide solution to a 96-well plate, and measure the ultraviolet absorption value at λ = 412 nm with a Nano Drop spectrophotometer. If the OD value is > 0.15, proceed to the next step; if the OD value is < 0.15 and > 0.05, supplement the polypeptide until the requirement is met; if the OD value is < 0.05, return to the polypeptide synthesis step for quality control. When starting the coupling, add 200 μL of deionized water to each mcKLH package to prepare a 10 mg / mL KLH solution, dissolve 2 mg of hapten in 500 μL of Imject EDC coupling buffer, add 500 μL of polypeptide solution to 200 μL of carrier protein solution, add 1 mL of deionized water to a package of EDC (10 mg), and slowly shake until completely dissolved. Take 50 μL and add to the mcKLH polypeptide solution. After 2 hours of reaction, remove the uncoupled cross-linking agent and salts by desalting column treatment to obtain KLH-Ki-67.

[0066] Example 2 Production of rabbit anti-human Ki-67 monoclonal antibody by single B cell sorting

[0067] I. Immunization and ELISA detection

[0068] The KLH-Ki-67 obtained in Example 1 was emulsified with Freund's complete adjuvant, and 6 rabbits were selected for immunization at a dose of 0.7 mg / rabbit. One-time booster immunization was performed on the 7th, 19th, 40th, 61st, and 112th days, respectively, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 0.35 mg / rabbit. Impact immunization was performed on the 126th day, and the antigen was mixed with normal saline at a dose of 0.7 mg / rabbit. After the third, fourth, and fifth immunization, serum ELISA titer detection, endogenous WB detection, and IHC detection were performed, and according to the results (i.e., using a positive control photograph corresponding to the antibody for detection, and immunohistochemical staining can be observed on the corresponding detection site in the positive control photograph), one of the rabbits was selected for subsequent monoclonal antibody screening.

[0069] II. Spleen cell separation and B lymphocyte sorting

[0070] Monoclonal antibody preparation was performed on the selected rabbit. The spleen was removed 3 days after impact immunization, and the rabbit spleen was placed in RMPI basic medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, cut into pieces with a surgical blade, and then transferred to a 100-μm cell screen for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes, and after centrifugation at 400 g for 5 minutes, the supernatant was removed and the spleen cell clumps were retained. The spleen cell clumps were resuspended with a hypotonic solution and lysed red blood cells, and then centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI basic medium containing 100 U / ml penicillin and 100 μg / ml streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended with complete medium (RMPI basic medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin) for standby.

[0071] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells".

[0072] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the KLH-Ki-67 protein in Example 1 were preliminarily screened by ELISA. According to the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0073] III. B cell culture positive clone detection

[0074] The single B cell culture supernatant was verified by IHC using a multi-tumor tissue chip containing Ki-67 positive and negative and a normal tissue chip. The IHC results of 13 clone culture supernatants were good, and the LEM supernatant was prepared for IHC verification to determine the clone (10G1) with excellent sensitivity and specificity.

[0075] The sensitivity and specificity screening criteria are: using the positive control photo corresponding to the antibody for detection, and the corresponding detection site in the positive control photo can observe immunohistochemical staining, while no immunohistochemical staining is observed at the non-detection site. Compared with the control antibody, the staining intensity reaches or is even higher than that of the control antibody.

[0076] IV. Cloning of the gene encoding the rabbit monoclonal antibody (10G1) and construction of the rabbit monoclonal antibody expression plasmid

[0077] After the cell collection and lysis of the positive clone, RNA is extracted and reverse transcribed into cDNA. Using PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes are amplified from the cDNA corresponding to the positive clone, and sequenced. The above-mentioned naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences are used to construct the rabbit monoclonal antibody expression vector plasmid.

[0078] Example 2 was completed by Wuhan Aibotek Biotechnology Co., Ltd. commissioned by Fujian Maynew Biotechnology Development Co., Ltd., in which immunohistochemical screening was performed by Fujian Maynew Biotechnology Development Co., Ltd.

[0079] Example 3 Rabbit monoclonal antibody expression of Ki-67

[0080] I. Plasmid amplification and extraction

[0081] Take one tube (100 μl) of competent bacteria (DH5α), insert into ice, ice bath for 5-10 min; add 5 μl of plasmid, gently shake and place on ice for 30 min; gently shake and place in a 42°C water bath for 90 s for heat shock, then quickly place back on ice, stand for 5 min; add 800 μl of LB medium (note: without antibiotics) to the above-mentioned mixture in the clean bench, mix gently, and fix on a shaker at 37°C for 1 h; take 50-100 μl of the above-mentioned transformation mixture in the clean bench, and add to the labeled solid LB plate containing Amp, and evenly spread with a glass spreader (sterilized); first stand in a 37°C incubator for 30 min, so that the surface bacteria penetrate into the medium, then invert and place in a 37°C incubator for overnight culture. Use a gun head to pick a single colony into 4 ml of LB medium (containing 2 uL of 200 mg / ml Amp), and culture at 37°C for 16 h at 220 rpm.

[0082] Add 1 ml of bacteria to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp), and culture at 37°C for 16 h on a shaker; use SanPrep endotoxin-free DNA small amount extraction kit (Shenguo), and follow the instructions for plasmid extraction.

[0083] II. Transfection

[0084] Adjust the concentration of 293F cells to 2.5-3x10 6 viable cells / ml with Expi293F™ Expression Medium and incubate overnight.

[0085] 1) Count the viable cells with a hemocytometer, the viable cell concentration is about 4.5-5.5x10 6 viable cells / ml, and the viable cell number should meet the requirements of the expression system;

[0086] 2) Dilute the cell concentration to 3x10 6 viable cells / ml with Expi293F™ Expression Medium;

[0087] 3) Add the plasmid DNA into Opti-MEN™ I Reduced Serum Medium, gently blow, and mix well by inverting;

[0088] 4) Gently invert ExpiFectamine TM 293 Reagent 4-5 times, mix ExpiFectamine TM 293 Reagent and Opti-MEN™ I Reduced Serum Medium, gently blow and invert 2-3 times, and place at room temperature for 5 min;

[0089] 5) Mix the solutions of steps 3) and 4), gently blow and invert 2-3 times, and mix well;

[0090] 6) Place the solution of step 5) at room temperature for 10-20 min;

[0091] 7) Slowly suck the mixture into the cell culture solution, and gently shake the flask;

[0092] 8) Incubate at 37℃ with 8% CO2 for 5-7 days;

[0093] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer1 and ExpiFectamine TM 293 Transfection Enhancer2 (Note: mix in advance before use), and gently shake to mix well, and continue to culture.

[0094] III. Purification of Monoclonal Antibodies

[0095] The antibodies were purified from the supernatant by HiTrap rProtein A FF affinity chromatography according to the manufacturer's instructions. The purity was determined by SDS-PAGE and the concentration was determined by the Bradford method. The purified antibodies were stored at -20°C.

[0096] Example 4. Immunohistochemical tissue microarray staining and identification

[0097] I. Preparation of the microarray

[0098] Each sample was first stained by HE section to determine the tumor site. The tumor target site was circled and prepared for punching. When making the blank recipient wax block, a plastic frame was placed on the mold, melted wax (melting point at 55-58°C) was poured into the mold, and after cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was taken out of the mold. On the tissue sample machine, a sample needle with a diameter of 1 mm was selected to punch a hole in the recipient wax block, with a hole depth of 3-4 mm, and another punching needle with a diameter of 1 mm was used to punch a hole in the marked part of the wax block to collect the tissue core, with a length of about 0.1 mm shallower than the hole depth of the recipient wax block. The collected tissue core was directly inserted or carefully clamped with tweezers into the empty hole of the recipient wax block. This was repeated until all sample points were prepared. Finally, a glass slide was used to flatten all the tissue cores, making the tissue chip wax block flat and smooth. The prepared tissue chip wax block was placed in the wax block making mold and placed in a 60°C oven for 15 min to fuse the tissue core and the wax of the recipient wax block into one body, then the mold was gently taken out of the oven, and the semi-melted wax was cooled at room temperature for about 30 min, then placed in a -20°C refrigerator for 6 min, and the tissue chip wax block was taken out of the mold, sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm, and the continuous sections were floated in cold water to naturally expand, then the separated sections were transferred to warm water at 45°C for 30 seconds, and the sections were mounted on slides treated with polylysine, and the prepared tissue chip was placed in a 65°C oven for 2 hours, then taken out and cooled at room temperature, and stored in a -4°C refrigerator.

[0099] II. IHC staining and analysis

[0100] Routine dewaxing in xylene 3 times, 6 minutes each, hydration in 100%, 100%, 95%, 85% gradient ethanol, 3 minutes each, final tap water rinse. Antigen retrieval was performed, then the slides were placed in a humidified chamber and rinsed in PBS 3 x 3 minutes. Slides were incubated with 3% H2O2 for 10 minutes, rinsed in PBS 3 x 3 minutes. Slides were blotted dry, and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody) and incubated at room temperature (25°C) for 1 hour, rinsed in PBS 3 x 3 minutes, the secondary antibody was added and incubated for 15-30 minutes at room temperature, rinsed in PBS 3 x 3 minutes, blotted dry, and developed with freshly prepared DAB for 3-10 minutes. Slides were counterstained with hematoxylin for 25 seconds, blued in PBS for 30 seconds. Slides were dehydrated in an alcohol gradient of 85% (3 minutes) - 95% (3 minutes) - 100% (3 minutes) - 100% (3 minutes), cleared in xylene for 3 minutes, and mounted with neutral balsam.

[0101] The results of immunohistochemical staining were divided into: positive and negative. Positive expression must be at the site of cell and tissue-specific antigens to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results were further divided according to the difference in staining intensity, as follows:

[0102] 1. The sample is weakly positive; marked as "+";

[0103] 2. The sample is moderately positive; marked as "++";

[0104] 3. The sample is highly positive; marked as "+++".

[0105] 4. The sample is negative, marked as "-".

[0106] III. Data Statistics

[0107] 1. Tumor tissue chip test results:

[0108] The antibody Ki-67 (10G1) and commercially available antibody Ki-67 (rabbit polyclonal) were simultaneously detected in 25 cases of esophageal squamous cell carcinoma and the detection results were compared. The immunohistochemical results of Ki-67 were statistically analyzed. The whole test process adopted a double-blind design, and the statistical results are as follows:

[0109]

[0110] The results show that the rabbit monoclonal antibody Ki-67 (10G1) has accurate staining localization, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive intensity of 3 cases is higher than that of the commercially available antibody, indicating that the sensitivity of the rabbit monoclonal antibody Ki-67 (10G1) is higher than that of the commercially available antibody.

[0111] Figure 1Comparison chart of immunohistochemical staining results of esophageal squamous cell carcinoma (left: rabbit monoclonal antibody Ki-67 of the application, right: commercially available Ki-67).

[0112] 2. Normal tissue chip test results:

[0113] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0114] The rabbit monoclonal antibody Ki-67 (10G1) and the commercially available Ki-67 antibody are simultaneously detected on the normal tissue chip, and the positive and negative detection results are consistent, indicating that the specificity of the antibody in the normal tissue is equivalent to that of the commercially available antibody.

[0115] Figure 2 Comparison chart of immunohistochemical staining results of intestinal gland (left: rabbit monoclonal antibody Ki-67 of the application, right: commercially available Ki-67).

[0116] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-Ki-67 protein monoclonal antibody, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The coding DNA sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes Ki-67 protein.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is a rabbit monoclonal antibody.

5. A Ki-67 protein immunoassay reagent, characterized by, The immunodetection reagent contains the anti-Ki-67 protein monoclonal antibody of claim 1 as an effective component.

6. The immunoassay test reagent of claim 5, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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