CN114516916B - anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application - Google Patents
anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application Download PDFInfo
- Publication number
- CN114516916B CN114516916B CN202011309809.4A CN202011309809A CN114516916B CN 114516916 B CN114516916 B CN 114516916B CN 202011309809 A CN202011309809 A CN 202011309809A CN 114516916 B CN114516916 B CN 114516916B
- Authority
- CN
- China
- Prior art keywords
- gpr65
- monoclonal antibody
- gpr
- hybridoma cell
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention particularly relates to an anti-monoclonal antibody GPR65-9G7, a hybridoma cell secreting the antibody and application thereof. GPR65 is expressed in a variety of immune cells and has a correlation with a variety of diseases such as inflammation, multiple sclerosis, thrombocytopenia, and tumors. Because of the high difficulty of GPR65 expression and purification, and the high homology of GPR65 proteins of human and murine origin, there is currently no efficient GPR65 monoclonal antibody expression tool. The invention provides a hybridoma cell GPR65-9G7 secreting GPR65 monoclonal antibody, which can secrete GPR65 monoclonal antibody of an IgG1 subtype and has good prospects as a research tool and an immune preparation.
Description
Technical Field
The invention belongs to the technical field of preparation of monoclonal antibody hybridoma cells, and particularly relates to a monoclonal antibody GPR65-9G7, a cell strain GPR65-9G7 secreting the monoclonal antibody and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
G-Protein coupled receptors (G proteins-Coupled Receptors, GPCRs) are composed of a series of large superfamily Protein receptors with seven transmembrane structures that undergo conformational changes under the influence of various stimulators, which in turn bind intracellular signaling G proteins and other signaling proteins, triggering intracellular signaling cascades. GPCRs can sense various endogenous or exogenous signals, such as peptides, lipids, neurotransmitters, odors, etc., thereby eliciting corresponding cellular response responses. These receptors are involved in and play an important role in a variety of physiological and pathological processes in the human body, including metabolism, endocrine balance, cell proliferation and differentiation, apoptosis, and immune response. G-protein-coupled receptor 65 (GPR 65), which is a member of the GPCRs family, encodes a gene located in chromosome 14, region q 31.3. Initially, significant increases in gene expression were found during the entry of T lymphocytes into programmed death, and thus are also referred to as T-cell death-associated gene 8 (tdag 8). Northern blot experiments show that GPR65 gene is mainly expressed in immune cells and tissues rich in white blood cells, including white blood cells in peripheral blood, spleen, thymus and tonsils. GPR65 is a proton susceptor activated only under acidic conditions at pH <7.4 and activity is maximized between ph=6.2-6.8. It regulates intracellular response by sensing changes in extracellular pH. GPR65 is expressed in a variety of immune cells, such as T lymphocytes, B lymphocytes, neutrophils, and is particularly highly expressed in eosinophils and mast cells. Activation of GPR65 under acidic conditions correlates with increased survival of eosinophils and lymphoma cells. It has been found that GPR65 is capable of effectively resisting acidosis-induced apoptosis through the Bcl-2 pathway and acting as an inflammatory inhibitor, that the expression of GPR65 and various inflammatory factors is significantly increased in inflammatory bowel disease patients, that there is a more severe inflammatory response in GPR65 knockout model mice, and that the lack of GPR 65-expressing T cells rather enhances paracrine secretion of pro-inflammatory factors, exacerbating inflammatory lesions. Several genome-wide association studies have shown that Single Nucleotide Polymorphisms (SNPs) of GPR65 are associated with increased susceptibility to inflammatory bowel disease. The single nucleotide polymorphism of GPR65 is associated with exacerbation of bacterial-induced colitis, since it leads to impaired GPR65 signaling pathway and thus to impaired lysosomal function in the epithelial cells. The hypothesis that the GPR65 gene may play an important role in regulating the immune response of the organism has been demonstrated in immune diseases such as multiple sclerosis (multiple sclerosis, MS) and heparin-induced thrombocytopenia (HIT). In addition, GPR65 has been found to play a role in tumor cell proliferation, apoptosis, metastasis, angiogenesis, and immunity based on acidosis in the "Warburg effect" tumor microenvironment, and thus, functional studies of GPR65 protein are currently becoming hot spots for immunological and oncologic studies.
Antibodies, particularly monoclonal antibodies, serve a significant immunological tool in both basic and applied research in biology and medicine. Development of anti-GPR 65 monoclonal antibodies also necessarily provides a powerful immunological tool for development of GPR 65-related studies. Since GPR65 is a membrane protein having a 7-pass transmembrane structure, there are great technical difficulties in expressing and purifying GPR65 protein, and in addition, GPR65 proteins of human and murine origin have high homology, and the currently commercially available anti-GPR 65 antibodies are mainly rabbit-derived polyclonal antibodies based on the above 2 points. Although polyclonal antibodies play an important role in research, they do not specifically recognize the target protein, which severely limits the field and scope of application. The monoclonal antibody can singly recognize target protein, perfectly make up the defect of the polyclonal antibody, and ensure that the properties of the monoclonal antibodies derived from the same 1 cell strain have good consistency. This feature is an incomparable advantage of polyclonal antibodies.
Disclosure of Invention
Against the background of the above study, the present invention provides a cell line GPR65-9G7 capable of secreting an anti-GPR 65 monoclonal antibody, and the corresponding monoclonal antibody GPR65-9G7 is obtained. The study of the invention provides an effective tool for the study of the biological activity related to GPR 65. Secondly, the immune coupling polypeptide selected by the invention stimulates animals to produce a monoclonal antibody of an IgG1 subtype, and the immune coupling polypeptide is applied to immunotherapy and hopefully obtains better immune effect.
Based on the technical scheme, the invention provides the following technical scheme:
in a first aspect of the invention, there is provided an anti-GPR 65 monoclonal antibody GPR65-9G7, wherein the antigen binding fragment of said monoclonal antibody GPR65-9G7 is shown as SEQ ID NO. 1 or the antigen binding fragment thereof comprises the sequence shown as SEQ ID NO. 1.
According to the invention, the polypeptide shown in SEQ ID NO. 1 is selected as an antigen for animal immune acquisition, and through verification, the monoclonal antibody is an antibody of an IgG1 subtype, and as known in the art, igG1 is the subtype with the highest potential in tumor immunotherapy, can be effectively combined with Fc gamma receptor, and has longer half-life in vivo. In terms of industrialization, the expression content of the IgG1 subtype in the hybridoma is higher, and meanwhile, the purification difficulty is low, the stability is good, and the method has ideal industrialization performance.
In addition, the research of the invention also proves that the polypeptide shown in SEQ ID NO. 1 has good affinity with the monoclonal antibody GPR65-9G7. On the one hand, the affinity is favorable for ensuring the stability of the industrial expansion production of the monoclonal antibody, and on the other hand, the antigen polypeptide and the monoclonal antibody are hopefully applied to detection means related to antigen and antibody binding effect, and the detection accuracy is improved.
In addition, the antibodies obtained by carrying out chemical modification and genetic modification on the monoclonal antibodies based on the conventional research thought are also within the scope of the application protection of the invention.
In a second aspect of the invention, there is provided a nucleic acid molecule encoding the monoclonal antibody GPR65-9G7 or an antigen binding fragment as set forth in SEQ ID NO. 1.
In a third aspect of the present invention, there is provided a hybridoma cell GPR65-9G7 which secretes an anti-GPR 65 monoclonal antibody, which has been deposited at the institute of microbiology, national academy of sciences, 11/04, at the following address: the biological preservation number of the Beijing city, the Chaoyang district, north Chen Xili No. 1, no. 3 is: CGMCC No.21007.
According to a fourth aspect of the present invention, there is provided a method for producing the anti-GPR 65 monoclonal antibody GPR65-9G7 of the first aspect, wherein the monoclonal antibody GPR65-9G7 is produced by the monoclonal antibody hybridoma cell GPR65-9G7 of the third aspect.
In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a GPR65-9G7 monoclonal antibody against GPR65 of the first aspect, a polypeptide as set forth in SEQ ID No. 1 and/or a hybridoma cell GPR65-9G7 secreting a GPR65 monoclonal antibody of the third aspect.
Preferably, the pharmaceutical composition further comprises pharmaceutically necessary auxiliary materials.
In a sixth aspect of the invention, there is provided a detection kit comprising the anti-GPR 65 monoclonal antibody GPR65-9G7 of the first aspect, the polypeptide shown in SEQ ID NO. 1 and/or the anti-GPR 65 monoclonal antibody secreting hybridoma cell GPR65-9G7 of the third aspect.
The beneficial effects of the above technical scheme are:
the invention provides a monoclonal antibody GPR65-9G7 for resisting GPR65, which perfectly overcomes the defects of application detection and the like of a polyclonal antibody, and the monoclonal antibody is of an IgG1 subtype and has good application prospect as a scientific research tool or applied to industrial production.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing genotyping results of GPR65-KO mice described in example 1.
FIG. 2 is a graph showing the affinity results of the monoclonal antibody GPR65-9G7 of example 1 for an antigen.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. 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 invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As described in the background, the existing studies indicate that GPR65 has relevance to a variety of diseases, and monoclonal antibodies thereof are also important immunological and biological research tools. Direct expression and purification is difficult due to the complex structure of GPR 65. In order to solve the technical problems, the invention firstly provides a monoclonal antibody GPR65-9G7 of anti-GPR 65, a hybridoma cell GPR65-9G7 secreting the monoclonal antibody, and application of the monoclonal antibody as a medicine and application of a detection reagent.
In a first aspect of the invention, there is provided an anti-GPR 65 monoclonal antibody GPR65-9G7, wherein the antigen binding fragment of said monoclonal antibody GPR65-9G7 is shown as SEQ ID NO. 1 or the antigen binding fragment thereof comprises the sequence shown as SEQ ID NO. 1.
Preferably, monoclonal antibody GPR65-9G7 also includes modified substances thereof, including, but not limited to, polyethylene glycol (PEG), avidin, streptavidin, various molecules such as biotin, radioisotopes, fluorescers, enzymes, cytotoxic substances, anti-tumor agents, and secondary antibodies modified therewith. The specific modification can be carried out in a manner known per se.
Examples of such radioisotopes include 18 F， 15 O， 13 N， 11 C， 82 Rb， 68 Ga， 198 Au， 199 Au， 32 P， 33 P， 125 I， 131 I， 123 I， 90 Y， 186 Re， 188 Re， 62 Cu， 64 Cu， 67 Cu， 47 Sc， 103 Pb， 109 Pb， 212 Pb， 71 Ge， 77 As， 105 Rh， 113 Ag， 119 Sb， 131 Cs， 143 Pr， 161 Tb， 177 Lu， 191 Os， 193 Pt， 197 Hg, etc. The radioisotope may be passed directly or indirectly through the chelating agent or the likeKnown methods are ligation or modification.
As the chelating agent, for example, DTPA (diethylenetriamine pentaacetic acid), DOTA (1, 4,7, 10-tetraazacyclotetradecane-1, 4,7, 10-tetraacetic acid), DFO (deferoxamine) and the like can be used.
Examples of the fluorescent agent include FITC (fluorescein isothiocyanate), rhodamine, phycoerythrin, phycocyanin, allophycocyanin, OPA (o-phthaldehyde) and fluoroamine.
Examples of such enzymes include horseradish peroxidase, beta-galactosidase, luciferase, and alkaline phosphatase.
Examples of such cytotoxic substances include diphtheria A chain, pseudomonas aeruginosa exotoxin A, pertussis toxin, ricin A chain, modesin toxin, alpha-sarcosine, diandion, curcin, crotin, gelonin or mitogellin. Examples thereof include clindamycin, phenylmycin, nematodycin, trichothecene, trichosanthin, cytochalasin B, dihydroxyanthracene dione, mitoxantrone, emetine, colchicine and saporin.
The antitumor agent, and examples thereof include alkylating agents, antimetabolites, antitumor antibiotics, antitumor plant components, BRMs (biological response modifiers), angiogenesis inhibitors, cell adhesion inhibitors, matrix metalloproteinase inhibitors, and the like.
Preferably, the monoclonal antibody against GPR65, GPR65-9G7, is of the IgG1 subtype.
In a second aspect of the invention, there is provided a nucleic acid molecule encoding the monoclonal antibody GPR65-9G7 or an antigen binding fragment as set forth in SEQ ID NO. 1.
In a third aspect of the present invention, there is provided a hybridoma cell GPR65-9G7 which secretes an anti-GPR 65 monoclonal antibody, which has been deposited at the institute of microbiology, national academy of sciences, 11/04, at the following address: the biological preservation number of the Beijing city, the Chaoyang district, north Chen Xili No. 1, no. 3 is: CGMCC No.21007.
In a fourth aspect of the present invention, there is provided a method for producing the anti-GPR 65 monoclonal antibody GPR65-9G7 of the first aspect, wherein the monoclonal antibody GPR65-9G7 is produced by the hybridoma cell GPR65-9G7 of the third aspect.
Preferably, in the preparation method, hybridoma cells GPR65-9G7 are placed in a culture system, and the monoclonal antibody GPR65-9G7 against GPR65 is obtained by purifying the culture supernatant of the cells.
Preferably, the preparation method comprises the following steps: injecting the hybridoma cell GPR65-9G7 of the third aspect into the abdominal cavity of an animal for cell proliferation, obtaining ascites, and purifying to obtain the monoclonal antibody GPR65-9G7.
Preferably, the monoclonal antibody hybridoma cell GPR65-9G7 is prepared by the following method: constructing a GPR65 protein knockout animal, injecting an antigen and an adjuvant into the animal for immunization, screening an immune positive animal, obtaining an organ of the animal for producing an antibody for cell fusion, and screening positive hybridoma cells to obtain the monoclonal antibody hybridoma cell GPR65-9G7.
Further, the immunized animal includes monkey, rabbit, dog, guinea pig, mouse, rat, sheep and goat, preferably mouse is used.
Further, the antigen is a polypeptide modification shown in SEQ ID NO. 1, and the modification comprises a coupling mode by adopting carrier proteins, wherein the carrier proteins comprise, but are not limited to KLH, BSA or OVA.
Further, the adjuvant includes Freund's complete adjuvant and Freund's incomplete adjuvant.
Further, the antibody-producing organs include, but are not limited to, spleen or lymph nodes.
In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a GPR65-9G7 monoclonal antibody against GPR65 of the first aspect, a polypeptide as set forth in SEQ ID No. 1 and/or a GPR65 monoclonal antibody secreting hybridoma cell GPR65-9G7 of the third aspect.
Preferably, the pharmaceutical composition further comprises pharmaceutically necessary auxiliary materials.
In a sixth aspect of the invention, there is provided a detection kit comprising the anti-GPR 65 monoclonal antibody GPR65-9G7 of the first aspect, the polypeptide shown in SEQ ID NO. 1 and/or the GPR65 monoclonal antibody secreting hybridoma GPR65-9G7 of the third aspect.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Example 1
1. Selection of immunized animals
Since the preparation technology of the murine monoclonal antibody is very mature, the patent ownership problem is not involved, the operation is simple, and the preparation cost is low, so the monoclonal antibody for preparing the murine GPR65 protein is selected in the embodiment.
The murine GPR65 protein and the human GPR65 protein have higher homology, in particular to the LOOP2 region of the functional region of GPRshttps://www.uniprot.org/align/A20201028E5A08BB0B2D1C45B0C7BC3B55FD265560019 CC9) This presents great difficulty in preparing monoclonal antibodies using wild-type mice. Therefore, this example used GPR65 protein full knockout mice (GPR 65-KO) constructed in the context of C57/B6 to conduct experiments. In constructing GPR65-KO mice, eGFP protein was inserted. GPR65-KO was first identified with the aid of a test.
(1) Tissue DNA of mice was extracted using the tissue lysis kit. Cutting ear of rice grain size mouse, placing into 1.5mL centrifuge tube, adding tissue lysate A (45 μL/tube), performing lysis at 95deg.C for 30min, strongly oscillating for more than 30s, cooling, adding tissue lysate B (5 μL/tube), and strongly oscillating for more than 30 s. Preserving at-20 ℃ and carrying out subsequent PCR for later use.
(2) And amplifying the target gene by PCR. Primers for identifying wild type mice were:
WT-F:Forward Primer(5’CCCGGTGGTCTACATAATTGTGTTT 3’)；
WT-R:Reverse Primer(5’GCTTGCAGAAAGGATACGCATAA 3’)；
primers for identifying GPR65-KO mice were:
eGFP reaction Forward primer(5’CGTCGTCCTTGAAGAAGATGGT 3’)；
eGFP reaction Reverse Primer(5’CACATGAAGCAGCACGACTT 3’)。
the reaction system and the reaction conditions were as follows.
PCR system(total 25μL)
PCR conditions
(3) Detection of the size of the destination strip. Detecting the size of the target band by adopting a DNA gel running method, and further judging the genotype of the mice. Wild-type mice (WT mice) only amplified bands for WT primers, but not for eGFP primers; GPR65-KO mice had only bands amplified against the eGFP primer, but no bands amplified against the WT primer. Run DNA strip: 4 mu L of PCR product was taken, run with 2.5% gel, set at 120V for 50min, and the DNA mark was selected for 50bp DNA ladder, and the identification result was shown in FIG. 1.
2. Selection of antigens
2.1 immunization experiments in mice
On day 1, mice were cheek bled and serum was kept ready (titer to be determined) for use, CFA was mixed with synthetic KLH-conjugated polypeptide (KLH-KSNFTLCYDKYPLEK, GPR-225-239-KLH, 50 μg) in equal volumes, C57BL/B6 GPR65-KO mice were intraperitoneally injected, 200 μl each, and a total of 7 mice were immunized; day 14 and day 28 mice were immunized intraperitoneally with 200 μl of an equal volume of IFA and synthetic polypeptide (25 μg), respectively; on day 35, cheek blood was taken and serum titers were determined by indirect ELISA: with synthetic polypeptide (KK-KSNFTLCYDKYPLEK) as antigen, post-immunization (AI) serum was equally diluted 2000, 4000, 8000, 16000, 32000 and 64000 fold, pre-immunization (BI) serum was diluted 1000 fold as negative control, PBS as blank control, rabbit anti-mouse IgG (L+H) -HRP as secondary antibody (50. Mu.L/well, 1:3000, A9044-2ML, sigma), TMB single-component display solution (50. Mu.L/well, PR1200, solarbio) developed and incubated in the absence of light at 37℃for 15min,1M H 2 SO 4 After termination (50. Mu.L/well), the absorbance at 450nm was measured within 1min (experimental junctionSee table 1 for results).
TABLE 1 serum titers of immunized mice
5755-2#,5755-3# and 5755-4# mice were selected for subsequent fusion experiments according to the experimental results. 3 days prior to cell fusion, mice were boosted with 50 μg of synthetic KLH conjugated polypeptide tail vein; three days later, spleens of mice were taken and subjected to fusion experiments with SP2/0 cells. A total of 36 96 well plates were fused.
2.2 selection of hybridoma cells and subcloning of Positive hybridoma cells
On day 10 after cell fusion, the fused hybridoma cells were screened by indirect ELISA. 18 positive hybridoma cell wells were obtained by preliminary screening (see table 2) and subcloned against positive cloning wells.
TABLE 2 Positive hybridoma cell wells
The indirect ELISA method used was as follows: synthetic polypeptide (KK-KSNFTLCYDKYPLEK) was coated at a concentration of 1. Mu.g/m L (50. Mu.L/well), fused cell supernatant (50. Mu.L/well) as primary antibody, cell culture medium as negative control, rabbit anti-mouse IgG (L+H) -HRP as secondary antibody (50. Mu.L/well, 1:3000, A9044-2ML, sigma), TMB single-component display solution (50. Mu.L/well, PR1200, solarbio) was developed, incubated at 37℃for 15min,1M H in the absence of light 2 SO 4 After termination (50. Mu.L/well), absorbance was measured at 450nm within 1 min. In P/N>2.1 positive (P is sample well reading, N is blank well reading). Wherein 1 positive cloning well still shows better positive result after 3 times of subcloning, the monoclonal antibody secreted by the cloning well is named monoclonal antibody GPR65-9G7, and the screening result is shown in Table 3.
TABLE 3 Primary and subclone screening of monoclonal antibody GPR65-9G7
2.3 in vitro amplification of monoclonal antibody GPR65-9G7
(1) Preparing ascites. Pristane (Sigma) was intraperitoneally immunized in 5 female 8-week Balb/c mice, each injected 200. Mu.L, and after 7 days, each injected intraperitoneally with 2X 10 6 Monoclonal antibody hybridoma cells GPR65-9G7. After 10 days, the mice were sacrificed for ascites, and a total of 20mL was collected. The ascites is centrifuged at 5000rpm for 10min, then filtered by a quick filter paper at 4 ℃ and the filtrate is collected and stored at-20 ℃.
(2) Purifying ascites to prepare monoclonal antibody. And purifying the ascites by using protein G to obtain the monoclonal antibody. Melting ascites preserved at-20deg.C on ice, centrifuging at 13000rpm at 4deg.C for 10min, collecting supernatant, and removing precipitate. Supernatant is first passed through sepinose TM 4B agarose (Protect; C600014-0100), filtrate was passed directly through Protein G pre-loaded chromatography column (Protect; C600993-0105), and the specific experimental procedure was as described. Protein G filtrate was collected and dialyzed against PBS buffer at 4 ℃ for 2 days. Short-term storage at-20deg.C.
2.4 subtype identification of monoclonal antibody GPR65-9G7
The subtype of monoclonal antibody GPR65-9G7 was detected by indirect ELISA using a Mouse monoclonal antibody subtype identification kit (SEK 003-100,Sino biological), while PBS was used as a negative control, and a commercial antibody mAbcam 8226 (Mouse monoclonal [ mAbcam 8226]to beta Actin,1:2000,Abcam,Inc ]) was used as a positive control, and the specific experimental procedure was performed according to the instructions. Experimental results indicate that monoclonal antibody GPR65-9G7 is IgG1 (see Table 4).
TABLE 4 subtype identification of monoclonal antibody GPR65-9G7
2.5 affinity determination of monoclonal antibody GPR65-9G7 with antigen
The affinity of monoclonal antibody GPR65-9G7 to antigen KLH-KSNFTLCYDKYPLEK was determined by competition ELISA.
The experimental procedure was as follows: (1) KLH conjugated polypeptide was plated at a concentration of 1. Mu.g/mL, 50. Mu.L per well, and incubated overnight at 4 ℃. (2) incubation with 3% MPBS at room temperature for 2h. Washed with PBS. (3) Into a 1.5mL EP tube, an antigen-containing KLH conjugate polypeptide (initial concentration of 1X 10) -6 M, comparative gradient dilution to a concentration of 6.1X10 -11 M) and monoclonal antibody GPR65-9G7 (56 nM) in PBS in a total volume of 100. Mu.L. (4) After incubation at room temperature for 30min, 90. Mu.L of the reaction mixture was added to the antigen-coated microwells, and 30. Mu.L of 30% MPBS was added in advance to the microwells and incubated for 9min. (5) After the incubation was completed, the reaction mixture was removed and washed with 0.5% PBS-Tween 20. Rabbit anti-mouse IgG (L+H) -HRP was then added as secondary antibody (100. Mu.l/well, 1:2000, A9044-2ML, sigma) and incubated for 50min at room temperature. (6) after incubation, washing with 0.5% PBS-Tween 20. Adding TMB single-component display solution (50. Mu.L/well, PR1200, solarbio) for color development, incubating at room temperature in the dark for 7min,1M H 2 SO 4 After termination (50. Mu.L/well), the absorbance was measured at 450nm in 1 min.
Measurement results: kd=39.1 nM. The results are shown in Table 5 and FIG. 1.
TABLE 5 affinity of monoclonal antibody GPR65-9G7 for antigen
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A monoclonal antibody GPR65-9G7 against GPR65, wherein the monoclonal antibody GPR65-9G7 against GPR65 is of the IgG1 subtype;
the antibody is secreted by hybridoma cell GPR65-9G7, which has been deposited at the institute of microorganisms, national academy of sciences, at 11.04, 2020: the biological preservation number of the Beijing city, the Chaoyang district, north Chen Xili No. 1, no. 3 is: CGMCC No.21007.
2. The monoclonal antibody GPR65-9G7 against GPR65 of claim 1, wherein the monoclonal antibody GPR65-9G7 is further modified, and wherein the modified substance is selected from the group consisting of polyethylene glycol, avidin, streptavidin, biotin, a radioisotope, a fluorescent agent, an enzyme, a cytotoxic substance, and an anti-tumor agent.
3. The monoclonal antibody GPR65-9G7 against GPR65 of claim 2, wherein the radioisotope is selected from the group consisting of 18 F， 15 O， 13 N， 11 C， 82 Rb， 68 Ga， 198 Au， 199 Au， 32 P， 33 P， 125 I， 131 I， 123 I， 90 Y， 186 Re ， 188 Re， 62 Cu， 64 Cu， 67 Cu， 47 Sc， 103 Pb， 109 Pb， 212 Pb， 71 Ge， 77 As， 105 Rh， 113 Ag， 119 Sb， 131 Cs， 143 Pr， 161 Tb， 177 Lu， 191 Os， 193 Pt， 197 Hg；
The radioisotope may be directly or indirectly linked or modified by a chelator by known methods;
the chelating agent is selected from DTPA, DOTA, DFO;
the fluorescent agent is selected from FITC, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, OPA or fluoramine;
the enzyme is selected from horseradish peroxidase, beta-galactosidase, luciferase or alkaline phosphatase;
the cytotoxic substance is selected from diphtheria A chain, pseudomonas aeruginosa exotoxin A, pertussis toxin, ricin A chain, modesin toxin, alpha-sarcosine, diandianan, curcin, crotin, gelonin, mitogellin;
the antineoplastic agent is selected from alkylating agent, antimetabolite, antitumor antibiotic, antitumor plant component, BRM, angiogenesis inhibitor, cell adhesion inhibitor, matrix metalloproteinase inhibitor.
4. A hybridoma cell GPR65-9G7 which secretes an anti-GPR 65 monoclonal antibody, wherein the cell has been deposited at the institute of microbiology, national academy of sciences, at 11/04, 2020 at: the biological preservation number of the Beijing city, the Chaoyang district, north Chen Xili No. 1, no. 3 is: CGMCC No.21007.
5. A method for the preparation of the anti-GPR 65 monoclonal antibody GPR65-9G7 according to any one of claims 1 to 3, wherein the monoclonal antibody GPR65-9G7 is prepared by the hybridoma cell GPR65-9G7 according to claim 4.
6. The method for producing anti-GPR 65 monoclonal antibody GPR65-9G7 according to claim 5, wherein the hybridoma cells GPR65-9G7 are placed in a culture system, and the anti-GPR 65 monoclonal antibody GPR65-9G7 is obtained by purifying the culture supernatant of the cells.
7. The method for producing anti-GPR 65 monoclonal antibody GPR65-9G7 according to claim 5, comprising the steps of: injecting the hybridoma cell GPR65-9G7 of claim 4 into the abdominal cavity of an animal for cell proliferation, obtaining ascites, and purifying to obtain the monoclonal antibody GPR65-9G7.
8. A test kit comprising the anti-GPR 65 monoclonal antibody GPR65-9G7 of any one of claims 1-3 or the anti-GPR 65 monoclonal antibody secreting hybridoma cell GPR65-9G7 of claim 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011309809.4A CN114516916B (en) | 2020-11-20 | 2020-11-20 | anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011309809.4A CN114516916B (en) | 2020-11-20 | 2020-11-20 | anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114516916A CN114516916A (en) | 2022-05-20 |
| CN114516916B true CN114516916B (en) | 2024-03-05 |
Family
ID=81594372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011309809.4A Active CN114516916B (en) | 2020-11-20 | 2020-11-20 | anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114516916B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406792A (en) * | 2018-09-14 | 2019-03-01 | 中国人民解放军第二军医大学第二附属医院 | GPR65 albumen is preparing application and appraisal procedure in Gliblastoma prognosis evaluation reagent kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061087A2 (en) * | 2000-12-19 | 2002-08-08 | Lifespan Biosciences, Inc. | Antigenic peptides, such as for g protein-coupled receptors (gpcrs), antibodies thereto, and systems for identifying such antigenic peptides |
-
2020
- 2020-11-20 CN CN202011309809.4A patent/CN114516916B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406792A (en) * | 2018-09-14 | 2019-03-01 | 中国人民解放军第二军医大学第二附属医院 | GPR65 albumen is preparing application and appraisal procedure in Gliblastoma prognosis evaluation reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| Expression of proton-sensing G-protein-coupled receptors in selected skin tumors;Anaïs Nassios等;Exp Dermatol;第28卷(第1期);66-71 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114516916A (en) | 2022-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2758394B2 (en) | Monoclonal antibodies against human tumor necrosis factor | |
| AU600439B2 (en) | Ras oncogene peptides and antibodies | |
| CN101315382A (en) | Immunocolloidal gold test strip for detecting kanamycin residue and preparation method thereof | |
| JP2011136978A (en) | Process for production of hybridoma and antibody obtained therefrom, able to recognize more than one vitamin d metabolite | |
| EP0163141B1 (en) | Monoclonal anti-human igg antibody and process for preparing the same | |
| JP7013051B2 (en) | Antibodies that specifically bind to bovine pregnancy-related glycoprotein 1 and their uses | |
| CN114133452B (en) | Heparin binding protein antibody, kit and application thereof | |
| CN102124100A (en) | A monoclonal antibody specifically binding to VEGF and the hybridoma secreting same and uses thereof | |
| JP2949467B2 (en) | Determination of human promatrix metalloprotease 7 by immunoassay | |
| CN114516916B (en) | anti-GPR 65 monoclonal antibody, hybridoma cell strain secreting same and application | |
| CN115286715B (en) | anti-CD3 nano antibody or antigen binding portion thereof and preparation method thereof | |
| CN116903750A (en) | anti-PIP 4K2A splice variant polyclonal antibody and application thereof in liver cancer diagnosis | |
| EP4261228A1 (en) | Antibody specifically binding to strep-tag ii tag and use thereof | |
| JP2001122900A (en) | ANTI-DNASE gamma ANTIBODY AND ITS PREPARATION AND USE | |
| JP4663831B2 (en) | Monoclonal antibodies, cell lines, and methods for measuring N1, N12-diacetylspermine | |
| CN110049996B (en) | Immunogenic fragment peptides of EN2 protein or antibody compositions specifically recognizing same | |
| CN117949656B (en) | Cytochrome P4502D6 metabolic enzyme detection kit and application thereof | |
| CN118530365B (en) | Monoclonal antibody for specifically detecting MMP12 protein and application thereof | |
| JP5840274B2 (en) | Monoclonal antibody that specifically reacts with strome lysin 1 | |
| JP5749948B2 (en) | Method and kit for measuring feline-derived β2 microglobulin, and antibody and antibody-producing cell line therefor | |
| JP4664340B2 (en) | Monoclonal antibodies, cell lines, and methods for measuring N1, N12-diacetylspermine | |
| JP3522877B2 (en) | Anti-tyrosinase monoclonal antibody F (ab ') 2 fragment | |
| JP2006265138A (en) | Monoclonal antibody against feline trypsinogen and/or feline trypsin | |
| KR100330311B1 (en) | Monoclonal Antibodies, Hybridoma Cell Lines against Human S-100A-6 Proteins and Methods of Making the Same | |
| CN116836274A (en) | Anti-serum amyloid A antibodies, reagents and kits for detecting serum amyloid A |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |