Anti-cd73 protein monoclonal antibodies, methods of making and uses thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human CD73 antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-CD73 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 CD73 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-CD73 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] CD73 is an ecto-5'-nucleotidase (eNT) composed of 523 amino acids encoded by the NT5E gene, with a relative molecular mass of 70 kDa, and is a kind of glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. CD73 protein is composed of 3 domains, the N-terminal domain contains 2 zinc ion binding sites and at least 1 N-glycosylation site at aspartic acid 311; the C-terminal domain contains a substrate binding site and is non-covalently bound to the plasma membrane through a GPI anchor; the N-terminal and C-terminal domains are connected by a short helix. CD73 exists in two conformations of "open" and "closed".

[0003] CD73 is widely distributed on the surface of human endothelial cells, lymphocytes and other tissue cells, has extracellular nucleotidase activity, and can also participate in the activation of T cells. Recent studies have found that CD73 is highly expressed in various tumors and is closely related to the occurrence and development of tumors, and plays an important role in tumor growth, apoptosis, invasion and metastasis, and is also a main molecule of vascular regeneration and immunosuppression in the occurrence and development of tumors. Studies have also shown that high expression of CD73 is related to HIF, IFN-α and ER. The hypoxic environment of tumor tissue induces HIF dimers, thereby promoting the expression of CD73. IFN-α promotes the expression of CD73 by indirectly inducing other regulatory mediators. ER can also down-regulate the expression of the CD73 gene. SUMMARY

[0004] The present application provides an anti-CD73 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] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSSNAMNWVRQAPGKGLEWIGIIGARGNTYYATWAKGRFSISRTSTTVDLKITGPTTEDTATYFCARMYAGSSHDNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0007] SEQ ID NO. 2:

[0008] MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGSTVTINCQASQSVYKNNYLAWFQQKSGQPPKRLIYSASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSADCHVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0009] 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.

[0010] SEQ ID NO. 3:

[0011]

[0012] SEQ ID NO. 4:

[0013] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTGCATCGCCCGTGTCTGCAGCTGTGGGAAGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAAGAACAACTACTTAGCCTGGTTTCAGCAGAAATCAGGGCAGCCTCCCAAGCGCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTC

[0014] AAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAG

[0015] TTATGATTGTAGTAGTGCTGATTGCCATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAGGGTGATCCAGTTGCACCTACTGTCC

[0016] TCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACC

[0017] GTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAA

[0018] CCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAG

[0019] TCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0020] Further, the monoclonal antibody specifically recognizes a CD73 protein.

[0021] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0022] Further, the clone number of the rabbit monoclonal antibody is 2C1.

[0023] The inventor also provides a preparation method of the anti-CD73 protein monoclonal antibody, and the CD73 protein antigen used for immunizing the rabbit is the amino acid sequence shown in SEQ ID NO. 5.

[0024] SEQ ID NO. 5:

[0025] MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQ

[0026] YQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVG

[0027] IVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEV

[0028] PAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIV

[0029] YLDGSSQSCRFRECNMGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGST

[0030] LKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELL

[0031] RHDSGDQDINVVSTYISKMKVIYPAVEGRIK

[0032] Further, the CD73 protein antigen is expressed by Expi293F cells.

[0033] Further, the CD73 protein antigen is transformed into Expi293F cells by plasmid vector pCDNA3.4.

[0034] The inventor also provides a CD73 protein immunodetection reagent containing the above-mentioned anti-CD73 protein monoclonal antibody as an effective component.

[0035] Different from the prior art, the beneficial technical effects of the present application are: the above-mentioned technical scheme provides an anti-CD73 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; 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, and can specifically recognize cells expressing CD73 protein, and is suitable for immunological detection, especially immunohistochemical detection.

[0036] Further, the immunodetection includes immunohistochemical method, immunoblotting method and enzyme-linked immunoassay. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a pCDNA3.4-CD73 protein purification result gel map.

[0038] Figure 2 It is a comparison chart of gastric cancer immunohistochemical staining results; the left is rabbit monoclonal antibody CD73 (2C1), and the right is commercially available CD73.

[0039] Figure 3 It is a comparison chart of tonsil immunohistochemical staining results; the left is rabbit monoclonal antibody CD73 (2C1), and the right is commercially available CD73. DETAILED DESCRIPTION

[0040] In order to detail the possible application scenarios, technical principles, specific schemes that can be implemented, purposes and effects that can be achieved, etc. of the present application, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described herein 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.

[0041] The term "embodiment" is mentioned herein means that the specific features, structures, or characteristics described in connection with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it 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.

[0042] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0043] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the 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.

[0044] In the present application, terms 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.

[0045] 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.

[0046] In the present application, "greater than", "less than", "exceeding" and the like are understood as not including the number; "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.

[0047] Example 1 Expression and purification of CD73 protein fragments

[0048] The CD73 adopts the protein sequence (1-547AA) with the accession number of NP_001191742.1 / NP_002517.1 in the NCBI database, which is the amino acid sequence shown in SEQ ID NO. 5; after being cloned into the plasmid vector pCDNA3.4, the Expi.293F cells are transformed by the plasmid vector pCDNA3.4, and the Expi.293F cells secrete a large amount of the target CD73 protein. The nickel column is used for affinity purification of the protein. After purification, the protein concentration is 3 mg / mL, the purity is 95%, and the immunization requirements are met.

[0049] Figure 1 The gel map of the pCDNA3.4-CD73 protein purification result is shown in the following table, and the final purified protein concentration is 0.754 mg / mL, which can be used for animal immunization and antibody screening and identification requirements.

[0050] Example 2: Single B cell sorting for producing rabbit anti-human CD73 monoclonal antibodies

[0051] I. Immunization and ELISA detection

[0052] The purified CD73 protein of Example 1 is emulsified with Freund's complete adjuvant, and 6 rabbits are selected for immunization, with a dose of 400 μg per rabbit. The rabbits are boosted once on the 7th, 21st, 42nd and 82nd days, and the antigen is emulsified with Freund's incomplete adjuvant, with a dose of 200 μg per rabbit. On the 91st day, the rabbits are given a boost, and the antigen is mixed with physiological saline, with a dose of 400 μg per rabbit. After the third and fourth immunizations, serum ELISA titer detection is performed, and the serum of 4 rabbits after the fourth immunization is subjected to IHC detection. According to the results (i.e., the positive control photo corresponding to the antibody is used for detection, and immunohistochemical staining can be observed at the corresponding detection site in the positive control photo), one rabbit (number: E17234) is selected for subsequent monoclonal antibody screening.

[0053] II. Spleen cell separation and B lymphocyte sorting

[0054] Single-antibody preparation was performed on selected rabbits. Three days after the booster immunization, the spleen was removed and placed in RMPI basal medium containing 100 U / ml penicillin and 100 ug / ml streptomycin. The spleen was cut into small pieces with a surgical blade and transferred to a 100-μm cell strainer for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes. 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 in a hypotonic solution and the red blood cells were lysed. After centrifugation at 400 g for 5 minutes, the spleen cells were retained. The spleen cells were resuspended in RMPI basal medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended in complete medium (RMPI basal medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 ug / ml streptomycin) for storage.

[0055] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".

[0056] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant CD73 protein in Example 1 were screened by ELISA. According to the ELISA data, 96 supernatants from high to low were selected for IHC verification.

[0057] III. Detection of positive clones of B cell culture

[0058] Single B cell culture supernatants were verified by IHC using a multi-tumor tissue chip and a normal tissue chip containing CD73 positive and negative. The IHC results of the supernatants of 14 clones were better, and the supernatant of LEM was prepared for IHC verification to determine the clone (2C1) with excellent sensitivity and specificity.

[0059] The screening criteria for sensitivity and specificity are as follows: the positive control photograph corresponding to the antibody is used for detection, and immunohistochemical staining can be observed on the corresponding detection site in the positive control photograph, while no immunohistochemical staining is observed on the non-detection site. Compared with the control antibody, the staining intensity is even higher than that of the control antibody.

[0060] IV. Cloning of rabbit monoclonal antibody gene (2C1) and construction of rabbit monoclonal antibody expression plasmid

[0061] After cell collection and lysis of the positive clone, RNA was extracted and reverse transcribed into cDNA. Using PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes were amplified and sequenced from the cDNA of the corresponding positive clone. The above naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences were used to construct rabbit monoclonal antibody expression vector plasmids.

[0062] Example 2 was completed by Wuhan Aibotek Biotechnology Co., Ltd. on behalf of Fujian Maynew Biotechnology Development Co., Ltd., in which immunohistochemical screening was performed by Fujian Maynew Biotechnology Development Co., Ltd.

[0063] Example 3 CD73 Rabbit Monoclonal Antibody Expression

[0064] I. Plasmid amplification and extraction

[0065] Take out a tube (100 μl) of competent bacteria (DH5a), 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 return to ice, stand for 5 min; add 800 μl of LB medium (note: without antibiotics) to the above-mentioned mixture in the clean bench, gently mix, 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, 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, allowing the surface bacteria to completely penetrate 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 on a shaker for 16 h at 220 rpm.

[0066] Add 1 ml of bacteria to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp), and culture at 37°C on a shaker for 16 h; use SanPrep Endotoxin-Free Plasmid DNA Mini Extraction Kit (Shenguo) to extract plasmid according to the instructions.

[0067] II. Transfection

[0068] Adjust the concentration of 293F cells to 2.5-3 x 10 6 viable cells / ml with Expi293FTM Expression Medium, and culture overnight.

[0069] 1) Count with a hemocytometer, the viable cell concentration is about 4.5-5.5 x 10 6 viable cells / ml, and the number of viable cells needs to meet the requirements of the expression system;

[0070] 2) Dilute the cell concentration to 3 x 10 6 viable cells / ml with Expi293FTM Expression Medium;

[0071] 3) Add plasmid DNA to Opti-MEM™ I Reduced Serum Medium, gently flick, invert to mix;

[0072] 4) Invert ExpiFectamine TM 293 Reagent 4-5 times, mix ExpiFectamine TM 293 Reagent and Opti-MEM™ I Reduced Serum Medium, gently flick and invert 2-3 times, let stand at room temperature for 5 min;

[0073] 5) Mix solutions from steps 3) and 4), gently flick and invert 2-3 times, mix well;

[0074] 6) Let solution from step 5) stand at room temperature for 10-20 min;

[0075] 7) Slowly pipette mixture into cell culture, gently shake flask;

[0076] 8) Incubate at 37°C, 8% CO2, 5-7 days;

[0077] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2 (Note: mix before use), gently shake to mix, continue incubation.

[0078] III. Purification of monoclonal antibodies

[0079] Purify antibodies from supernatant using HiTrap rCD73otein A FF affinity chromatography according to manufacturer's instructions. Determine purity by SDS-PAGE gel and concentration by Bradford method. Store purified antibodies at -20°C.

[0080] Example 4. Immunohistochemical tissue chip staining and identification

[0081] I. Chip preparation process

[0082] HE staining was performed on each sample 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, and melted wax (melting point at 55-58°C) was poured into the mold. After cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was removed from the mold. On the tissue sample machine, a 1 mm diameter sample needle was selected to punch a hole in the recipient wax block, with a hole depth of 3-4 mm. Another 1 mm diameter punching needle was used to punch a hole in the marked site of the wax block to collect a 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 core wax block flat and smooth. The prepared tissue core wax block was placed in the wax block making mold and placed in a 60°C oven for 15 min, so that the tissue core and the wax of the recipient wax block were fused together. Then the mold was gently taken out of the oven, and the semi-melted wax was allowed to cool at room temperature for about 30 min. Then the tissue core wax block was placed in a -20°C refrigerator for 6 min, and then removed from the mold. The tissue core wax block was sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm. The continuous sections were floated in cold water to naturally expand, and then transferred to warm water at 45°C for 30 seconds. The sections were mounted on slides treated with polylysine, and the prepared tissue core was placed in a 65°C oven for 2 hours. After cooling to room temperature, the tissue core was stored in a -4°C refrigerator.

[0083] II. IHC Staining and Analysis

[0084] Routine xylene de-waxing for 3 times, 6 minutes each time, hydration in 100%, 100%, 95%, 85% gradient ethanol for 3 minutes each time, and finally tap water rinse. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS for 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, and then rinsed with PBS for 3 x 3 minutes. The sections were spun dry, and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody). The sections were incubated at room temperature (25°C) for 1 hour, rinsed with PBS for 3 x 3 minutes, and then the secondary antibody was added and incubated at room temperature for 15-30 minutes. The sections were rinsed with PBS for 3 x 3 minutes, spun dry, and then fresh DAB developing solution was added for color development for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and then PBS was added for 30 seconds to return to blue. The sections were dehydrated in an alcohol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes), and finally xylene clearing for 3 minutes. The sections were mounted with neutral resin.

[0085] The immunohistochemical staining results are classified as positive and negative. Positive expression must be at the site of cell and tissue specific antigen to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further classified according to the difference in staining intensity, as follows:

[0086] 1. The sample is weakly positive, marked as "+".

[0087] 2. The sample is moderately positive, marked as "++".

[0088] 3. The sample is highly positive, marked as "+++".

[0089] 4. The sample is negative, marked as "-".

[0090] III. Data Statistics

[0091] 1. Tumor tissue chip detection results:

[0092] The present antibody CD73 (2C1) and commercially available antibody CD73 (rabbit polyclonal antibody) were synchronously detected in 32 cases of gastric cancer and the detection results were compared. The immunohistochemical results of CD73 were statistically analyzed. The whole test process adopted a double-blind design, and the statistical results are as follows:

[0093]

[0094] The results show that the rabbit monoclonal antibody CD73 (2C1) has accurate staining localization, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive rate is comparable to that of the commercially available antibody, but the positive intensity of 7 cases is higher than that of the commercially available antibody, indicating that it has higher sensitivity and effectively avoids false negative results.

[0095] Figure 2 Comparison chart of immunohistochemical staining results of gastric cancer (left: rabbit monoclonal antibody CD73 of the present application, right: commercially available CD73).

[0096] 2. Normal tissue chip detection results:

[0097] The normal tissue chip includes 30 kinds of normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens; each kind of tissue includes 3 different case samples. The 30 kinds of 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 cells.

[0098] The rabbit monoclonal antibody CD73 (2C1) and the commercial CD73 antibody were synchronously detected on a normal tissue chip, and the positive and negative detection results were consistent, indicating that the specificity of the antibody in normal tissues was equivalent to that of the commercial antibody.

[0099] Figure 3 A comparison chart of tonsil immunohistochemical staining results (left: rabbit monoclonal antibody CD73 of the application, right: commercial CD73).

[0100] 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-CD73 protein monoclonal antibody, characterized in that, 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; 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.

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 the nucleotide sequence shown in SEQ ID NO. 3; the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes CD73 protein.

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

5. A CD73 protein immunoassay test reagent, characterized by, The immunodetection reagent contains the anti-CD73 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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