CN120192419B - Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application - Google Patents

Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application

Info

Publication number
CN120192419B
CN120192419B CN202510432603.7A CN202510432603A CN120192419B CN 120192419 B CN120192419 B CN 120192419B CN 202510432603 A CN202510432603 A CN 202510432603A CN 120192419 B CN120192419 B CN 120192419B
Authority
CN
China
Prior art keywords
amino acid
acid sequence
monoclonal antibody
rabbit monoclonal
mab6
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
Application number
CN202510432603.7A
Other languages
Chinese (zh)
Other versions
CN120192419A (en
Inventor
扈晓敏
柳孟姣
吴娜
丁佳琦
任琪
W·付
张杨
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WUXI ORIGENE BIO-TECHNOLOGY CO LTD
Original Assignee
WUXI ORIGENE BIO-TECHNOLOGY CO LTD
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by WUXI ORIGENE BIO-TECHNOLOGY CO LTD filed Critical WUXI ORIGENE BIO-TECHNOLOGY CO LTD
Priority to CN202510432603.7A priority Critical patent/CN120192419B/en
Publication of CN120192419A publication Critical patent/CN120192419A/en
Application granted granted Critical
Publication of CN120192419B publication Critical patent/CN120192419B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Reproductive Health (AREA)
  • Dispersion Chemistry (AREA)
  • Plasma & Fusion (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The application belongs to the technical field of immunodetection, and discloses a rabbit monoclonal antibody mAb6 against a mouse immunoglobulin G2a subtype (IgG 2 a) and application thereof. The rabbit monoclonal antibody mAb6 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the LCDR1 comprises an amino acid sequence KSVYKNNY, the LCDR2 comprises an amino acid sequence GTN, the LCDR3 comprises an amino acid sequence AGGYID, the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence RFSLSNYR, the HCDR2 comprises an amino acid sequence IFTRGST, and the HCDR3 comprises an amino acid sequence ARGWNS. The rabbit monoclonal antibody can be combined with the high specificity of the mouse immunoglobulin G2a subtype (IgG 2 a), can be applied to immunodetection, and provides a basis for the preparation of the engineering antibody in the next step.

Description

Rabbit monoclonal antibody mAb6 against murine immunoglobulin G2a subtype (IgG 2 a) and uses thereof
Technical Field
The application relates to the technical field of immunodetection, in particular to a rabbit monoclonal antibody mAb6 of an anti-mouse immunoglobulin G2a subtype (mIgG 2 a) and application thereof.
Background
Affinity conjugates/antibodies are one of the most commonly used tools in life sciences for studying proteins and their functions in various biological pathways and diseases. The most widely applied antibody species in the scientific research field and the medicine field at present comprise human antibodies, murine antibodies and rabbit antibodies. These antibodies can specifically recognize the target of interest and then display a signal via an enzyme-labeled secondary antibody or a fluorescein-labeled secondary antibody. The existing anti-mouse IgG and anti-human IgG labeled antibodies are widely applied to the market, and can be applied to enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blot), immunohistochemical staining (IHC), flow Cytometry (FCM), co-immunoprecipitation and the like, and the most commonly used labeling methods comprise horseradish peroxidase (HRP), alkaline phosphatase, biotin, fluorescein and the like.
The main method for preparing the anti-mouse IgG antibody is to directly immunize animals such as rabbits, sheep, donkey and the like with IgG immunoglobulin to generate immune serum polyclonal antibody, and then use the immune serum polyclonal antibody after purification. However, the development of antibodies specific for different types of IgG heavy and light chains, polyclonal antibodies, is far from sufficient, and monoclonal antibodies, particularly directed against different subclasses of IgG, are required.
The heavy chains of the mouse IgG monoclonal antibodies comprise four subtypes of IgG1, igG2a, igG2b and IgG3, the light chains comprise two types of kappa chains and lambda chains, and the kappa chains are about 20:1. At present, polyclonal antibodies such as goat polyclonal antibodies, sheep polyclonal antibodies, donkey polyclonal antibodies and the like are mostly used in the market, and few monoclonal antibodies, particularly rabbit anti-mIgG 2a specific monoclonal antibodies are not reported.
To meet the demand for monoclonal antibodies specific to mIgG2a, development of monoclonal antibodies with high specificity and high sensitivity to mIgG2a is currently urgently required.
Disclosure of Invention
In order to overcome the technical problems, the application provides the anti-mIgG 2a rabbit monoclonal antibody mAb6 with high specificity and high sensitivity and the application thereof in immunodetection, including but not limited to chemiluminescence, fluorescence and chromogenic detection for primary antibodies, which are suitable for various applications, such as cell imaging, flow cytometry detection, western immunoblotting and immunohistochemistry, and also provide a foundation for the preparation of the next engineering antibody.
In one aspect, the application provides a rabbit monoclonal antibody mAb6 or antigen-binding fragment of the anti-murine immunoglobulin G2a subtype (IgG 2 a), said light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence of SEQ ID No.1, said LCDR2 comprising the amino acid sequence GTN, and said LCDR3 comprising the amino acid sequence of SEQ ID No. 3.
In certain embodiments, the light chain variable region is 106 amino acids in total, 26, 17, 36 and 10 in number of amino acids in the 4 domains of FR, 8, 3 and 6 in number of amino acids in the 3 domains of LCDR, and 27aa-34aa,52aa-54aa and 91aa-96aa in the regions of LCDR1, LCDR2 and LCDR3, respectively, with amino acid sequences KSVYKNNY (SEQ ID NO. 1), GTN (SEQ ID NO. 2) and AGGYID (SEQ ID NO. 3), respectively.
In certain embodiments, the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence of SEQ ID No.4, the HCDR2 comprising an amino acid sequence of SEQ ID No.5, and the HCDR3 comprising an amino acid sequence of SEQ ID No. 6.
In certain embodiments, the heavy chain variable region is 109 amino acids in full length, 24, 17, 36 and 11 in the number of amino acids in the 4 domains of FR, 8, 7 and 6 in the number of amino acids in the 3 domains of HCDR, 25aa-32aa,50aa-56aa and 93aa-98aa in the number of amino acids in the 3 domains of HCDR, and RFSLSNYR (SEQ ID NO. 4), IFTRGST (SEQ ID NO. 5), ARGWNS (SEQ ID NO. 6) in the amino acid sequence.
In certain embodiments, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO.7 or an amino acid sequence having at least 95% identity, preferably at least 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO.7, and the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8 or an amino acid sequence having at least 95% identity, preferably at least 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO. 8.
In certain embodiments, the antigen binding fragment is one of F (ab ') 2, fab', fab, fv, scFv, dsFv, a bispecific antibody, and an antibody minimal recognition unit, preferably, the species from which the remainder of the antibody sequences are derived includes one or more of rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.
In another aspect, the application also provides a biological material comprising a polynucleotide encoding the rabbit monoclonal antibody mAb6 or antigen binding fragment, a vector carrying the polynucleotide, or a cell carrying the polynucleotide, or containing the vector, or capable of expressing the rabbit monoclonal antibody mAb6 or antigen binding fragment.
In another aspect, the application also provides a method for preparing the rabbit monoclonal antibody mAb6 or antigen binding fragment, comprising culturing the cell, optionally, the cell is prepared by transforming a polynucleotide encoding the rabbit monoclonal antibody mAb6 or antigen binding fragment into the cell, the polynucleotide comprises a heavy chain expression plasmid and a light chain expression plasmid, and the transforming comprises co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cell.
In certain embodiments, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a 293 cell or CHO cell.
In another aspect, the application also provides the use of said rabbit monoclonal antibody mAb6 or antigen-binding fragment or said biological material in any of the following:
1) Non-diagnostic and therapeutic destinations detection of murine immunoglobulin G2a subtype (IgG 2 a);
2) Preparing a product for detecting the murine immunoglobulin G2a subtype (IgG 2 a);
3) For purifying the murine immunoglobulin G2a subtype (IgG 2 a);
4) A product was prepared for purification of the murine immunoglobulin G2a subtype (IgG 2 a).
In another aspect, the application also provides a detection reagent or detection kit comprising said rabbit monoclonal antibody mAb6 or antigen-binding fragment, or said biological material.
In certain embodiments, the detection kit is used for enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blot), immunohistochemical staining (IHC), flow Cytometry (FCM), immunoprecipitation, and the like.
Compared with the prior art, the mIgG2a rabbit monoclonal antibody can be combined with the mIgG2a in a high specificity way, has high affinity, and has an affinity constant Ka of 8 multiplied by 10 8 L/mol. The application also relates to the application of the anti-mouse immunoglobulin G2a subtype (IgG 2 a) specific rabbit monoclonal antibody in an immunodetection tool, including but not limited to chemiluminescence, fluorescence and chromogenic detection for primary antibodies, and is suitable for various applications, such as cell imaging, flow cytometry detection, western immunoblotting and immunohistochemistry, and also provides a basis for the preparation of the next engineering antibody.
Drawings
FIG. 1 is an electrophoretogram of full-length amplification products of heavy and light chains of a rabbit monoclonal antibody mAb6, M is a DNA molecular weight Marker.
FIG. 2 is a graph showing the Western blot detection results of specific recognition of the native mIgG2a antibody by rabbit monoclonal antibody mAb 6.
FIG. 3 is a graph showing ELISA detection results of specific recognition of mIgG2a by mAb6, a rabbit monoclonal antibody.
FIG. 4 is a graph showing the Western blot detection results of specific recognition of the natural mIgG2a antibody by horseradish peroxidase labeled rabbit monoclonal antibody mAb 6.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions, those described in the laboratory manual, or those suggested by the manufacturer.
EXAMPLE 1 preparation of murine IgG2a rabbit monoclonal antibodies
1) Preparation of immunogens
Third party mIgG2a antibodies were purchased as immunogens.
2) Immunization of animals
The purchased mIgG2a antibody is emulsified by complete Freund's adjuvant, about 2kg of New Zealand white rabbits are immunized by subcutaneous injection, the immunization dose is 500 mug/animal, the second immunization is carried out after two weeks interval, the incomplete Freund's adjuvant is used for emulsification, and the immunization dose is 250 mug/animal. And taking tail blood after two immunization, performing gradient dilution by ELISA method to determine serum titer, judging whether to collect PBMCs or continue immunization according to the result by taking OD450 when ELISA titer 128000 is larger than 1.0 as a standard, and selecting rabbits with highest antibody titers for collecting the PBMCs.
3) PBMCs separation, specific B cell separation, cloning recombination
Fixing the rabbit on the operating table in a supine manner, removing hair from the heart part, sterilizing skin with alcohol, selecting the most obvious part of heart beat, puncturing with a 50ml syringe, flushing blood into the syringe after the needle is punctured into the heart, rapidly pulling out the needle after the required blood volume is obtained, transferring whole blood in the syringe into a sterile 50ml tube, uniformly mixing with equal amount of PBS, slowly adding dropwise above lymphocyte separation liquid, centrifuging at room temperature of 400 Xg for 30min, separating the liquid level into four layers from top to bottom, namely a yellow plasma layer, a white film layer, a mononuclear cell layer, a separation liquid layer and a red blood cell layer, carefully sucking the mononuclear cell layer, and washing with PBS to remove platelets and lymphocyte separation liquid to obtain the rabbit PBMCs.
Antigen-specific B cells were further sorted from rabbit PBMCs for culture, and the supernatant of the cultured B cells was screened for positive clones using antigen-coated ELISA plates. The full-length sequence of the light chain and heavy chain of the naturally paired rabbit monoclonal antibody is amplified from cDNA of the corresponding positive clone, a rabbit monoclonal antibody expression vector is constructed by a cloning recombination method, and the sequence is determined by sequencing. The results of the amplified full length PCR products are shown in FIG. 1.
4) Preparation and purification of monoclonal antibodies
In order to obtain the rabbit monoclonal antibody for recognizing the human mIgG2a protein, the heavy chain and light chain genes of the rabbit monoclonal antibody are loaded on an expression vector, HEK293 cells are transfected with the plasmid, and the recombinant rabbit monoclonal antibody for recognizing the human mIgG2a protein is obtained from the culture supernatant after 120-144 hours of transfection. Collecting cell suspension, centrifuging to obtain supernatant, and performing antibody purification by affinity chromatography. The concentration of the purified monoclonal antibody was measured by BCA method, and then sub-packaged and lyophilized to give the purified antibody designated as murine IgG2a rabbit monoclonal antibody mAb6.
Example 2 specific identification of anti-mIgG 2a rabbit monoclonal antibody mAb6
1) Western blot identification of rabbit monoclonal antibody mAb6
Western Blot (WB) detection was used. 2 samples were selected for each subtype of mIgG1, mIgG2a, mIgG2b, and mIgG3, and 100ng of each protein was subjected to SDS-PAGE, and after transfer, WB detection was performed.
The results showed that rabbit monoclonal antibody mAb6 specifically recognized mIgG2a, but not mIgG1, mIgG2b, and mIgG3, as shown in FIG. 2.
2) ELISA identification of rabbit monoclonal antibody mAb6
The ELISA plate is coated by mIgG1, mIgG2a, mIgG2b and mIgG3 antibodies at 4 ℃ overnight, the ELISA plate is taken out the next day, PBST is washed once, 1% BSA solution is blocked for 2 hours at 37 ℃, PBST is washed 3 times, rabbit monoclonal antibody MAB6100 μl is added to each hole, the concentration is respectively 10, 3, 1, 0.3 and 0ng/ml, the incubation is carried out for 1 hour at 37 ℃, the ELISA plate is taken out after the incubation is completed, PBST is washed 3 times, HRP-labeled goat anti-rabbit secondary antibodies are respectively added as detection antibodies at 37 ℃ for 1 hour, the ELISA plate is taken out after the incubation is completed, PBST is washed 5 times, TMB substrate is added, and color development is carried out for 10 minutes at 37 ℃. After removal, stop solution was added and OD450 readings were measured on an microplate reader.
The results showed that rabbit monoclonal antibody mAb6 specifically recognized mIgG2a, but not mIgG1, mIgG2b, and mIgG3, as shown in FIG. 3.
Example 3 affinity identification of anti-mIgG 2a rabbit monoclonal antibody mAb6
Affinity constants (Ka) were determined by non-competitive ELISA.
Coating, namely diluting antigen with carbonate buffer solution to the concentration of 1, 0.5, 0.1 and 0.05 mug/mL, adding the antigen to a 96-well ELISA plate according to 100 mug/well, coating respectively, and incubating for 24 hours at 4 ℃;
blocking, namely, washing the plate 4 times by using PBST, adding BSA solution according to 200 mu L/hole, and incubating for 2 hours at 37 ℃;
adding the monoclonal antibody, namely washing the plate 4 times by using PBST, diluting the rabbit monoclonal antibody by a starting multiple ratio of 100 mu g/mL by using carbonate buffer solution, adding 100 mu L of the rabbit monoclonal antibody into each hole, and incubating for 2 hours at 37 ℃;
adding enzyme-labeled secondary antibody, namely washing the plate with PBST for 4 times, adding 100 mu L of goat anti-rabbit Ig secondary antibody which is diluted 1:10000 times and is marked by HRP enzyme into each hole, and standing for 30min at 37 ℃;
Color development and termination, namely, washing the plate for 4 times by using PBST, adding 100 mu L of a substrate color development solution into each hole, carrying out light-shielding reaction for 15min at 37 ℃, and adding 50 mu L of 1.0mol/L H 2SO4 termination solution into each hole to terminate the reaction;
detection the absorbance at a wavelength of 450nm (A450 nm) was measured.
The logarithmic scale of the antibody concentration is taken as the abscissa, the OD value is taken as the ordinate, an S-shaped curve is drawn, and the affinity constant Ka=8× 8 L/mol of the anti-mIgG 2a rabbit monoclonal antibody mAb6 is calculated.
Example 4 analysis of variable region genes and amino acid sequences of rabbit monoclonal antibody mAb6 recombinant plasmids of mAb6 antibodies were used as DNA templates, light chain variable region and heavy chain variable region sequencing primers were designed from the 5' end vector sequences of the light chain and heavy chain on the templates, and sequencing was performed using a sequencer ABI 3730. The nucleotide sequence of the variable region of the light chain and the heavy chain of the mAb6 of the rabbit monoclonal antibody is obtained through sequencing.
And (3) respectively analyzing the nucleotide sequences of the light chain variable region and the heavy chain variable region by using IMGT/V-QUEST analysis software on http:// www.imgt.org through utilizing the Internet to obtain the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody mAb6 shown as SEQ ID NO.7 and the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 8.
The total length of the light chain variable region is 106 amino acids, the number of FR 4 domain amino acids is 26, 17, 36 and 10, the number of LCDR3 domain amino acids is 8, 3 and 6, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-34aa,52aa-54aa and 91aa-96aa, respectively, with amino acid sequences KSVYKNNY (SEQ ID NO. 1), GTN (SEQ ID NO. 2) and AGGYID (SEQ ID NO. 3), respectively.
The total length of the heavy chain variable region is 109 amino acids, the number of 4 domain amino acids of FR is 24, 17, 36 and 11, the number of 3 domain amino acids of HCDR is 8, 7 and 6, HCDR1, HCDR2 and HCDR3 are 25aa-32aa,50aa-56aa and 93aa-98aa, respectively, and the amino acid sequences thereof are RFSLSNYR (SEQ ID NO. 4), IFTRGST (SEQ ID NO. 5), ARGWNS (SEQ ID NO. 6), respectively
EXAMPLE 5 identification of HRP-labeled Rabbit monoclonal antibody mAb6 as secondary antibody
(1) HRP-labeled rabbit monoclonal antibody mAb6
1. The rabbit monoclonal antibody mAb6 was dissolved in sodium bicarbonate solution at pH 9.6;
2. dissolving HRP with a certain mass in deionized water, adding sodium periodate for reaction for 30min, adding ethylene glycol for continuous reaction for 30min, and dialyzing overnight;
3. Weighing a certain amount of sodium borohydride, dissolving the sodium borohydride in deionized water, adding the sodium borohydride into the crosslinked antibody-HRP solution, reacting for 2 hours, and dialyzing overnight;
4. the HRP-labeled antibody obtained was stored at-20℃with the same amount of glycerol.
(2) HRP-labeled rabbit monoclonal antibody mAb6 Western blot identification
Western Blot (WB) detection was used. 100ng of mIgG2a antibody was subjected to SDS-PAGE, and after transfer, the HRP-labeled rabbit monoclonal antibody mAb6 was incubated for WB detection.
The results show that the HRP-labeled rabbit monoclonal antibody mAb6 can well recognize the mIgG2a Fc region, and the molecular weight is about 50KD, and the results are shown in FIG. 4.
(3) ELISA identification of HRP-labeled rabbit monoclonal antibody mAb6
Sheep anti-mouse IgG coats the ELISA plate at 4 ℃ overnight, the ELISA plate is taken out the next day, PBST is washed once, 1% BSA solution is blocked at 37 ℃ for 2 hours, PBST is washed 3 times, mIgG2a antibody is added into each hole, the concentration is 0.2ug/ml,37 ℃ is incubated for 1 hour, the ELISA plate is taken out after incubation is finished, PBST is washed 3 times, HRP-labeled mAb6 is added as a detection antibody, 1ug/ml is diluted by a factor of 7 gradients, 37 ℃ is incubated for 1 hour, the ELISA plate is taken out after incubation is finished, PBST is washed 5 times, TMB substrate is added, and color development is carried out at 37 ℃ for 10 minutes. After removal, stop solution was added and the OD450 reading was determined on an ELISA reader, the results of which are shown in Table 1.
TABLE 1 ELISA detection results of HRP-labeled rabbit anti-mIgG 2a mAb6 specifically recognizing mIgG2a antibody
The results show that HRP-labeled rabbit anti-mIgG 2a mAb6 as a secondary antibody can well detect mIgG2a antibody signals, superior to commercial goat anti-mouse IgG polyclonal antibodies (mIgG 1, mIgG2a, mIgG2b, mIgG3 can be detected simultaneously as a secondary antibody).
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (11)

1.抗鼠免疫球蛋白G2a亚型（IgG2a）的兔单克隆抗体mAb6或其抗原结合片段，其特征在于，包括轻链可变区和重链可变区，所述轻链可变区包括互补决定区LCDR1、LCDR2和LCDR3，所述LCDR1的氨基酸序列如SEQ ID NO.1所示，所述LCDR2的氨基酸序列为GTN，所述LCDR3的氨基酸序列如SEQ ID NO.3所示；所述重链可变区包括互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1的氨基酸序列如SEQ ID NO.4所示，所述HCDR2的氨基酸序列如SEQ ID NO.5所示，所述HCDR3的氨基酸序列如SEQ ID NO.6所示。1. A rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) or its antigen-binding fragment, characterized in that it comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises complementarity-determining regions LCDR1, LCDR2, and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO.1, the amino acid sequence of LCDR2 is GTN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.3; the heavy chain variable region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO.4, the amino acid sequence of HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.6. 2.根据权利要求1所述的兔单克隆抗体mAb6或其抗原结合片段，其特征在于，所述轻链可变区包括如SEQ ID NO.7所示的氨基酸序列或与SEQ ID NO:7所示的氨基酸序列具有至少95％同一性的氨基酸序列；所述重链可变区包括如SEQ ID NO:8所示的氨基酸序列或与SEQ ID NO:8所示的氨基酸序列具有至少95％同一性的氨基酸序列。2. The rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to claim 1, characterized in that the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:7; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:8. 3.根据权利要求1或2所述的兔单克隆抗体mAb6或其抗原结合片段，其特征在于，所述抗原结合片段为F(ab’)2、Fab’、Fab、Fv、scFv、dsFv和双特异抗体中的一种。3. The rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to claim 1 or 2, characterized in that the antigen-binding fragment is one of F(ab') 2 , Fab', Fab, Fv, scFv, dsFv and bispecific antibody. 4.生物材料，其特征在于，所述生物材料包括多核苷酸、载体或细胞，所述多核苷酸编码权利要求1-3任一项所述的兔单克隆抗体mAb6或其抗原结合片段；所述载体携带所述多核苷酸；所述细胞携带所述多核苷酸，或含有所述载体，或能够表达权利要求1-3任一项权利要求所述的兔单克隆抗体mAb6或其抗原结合片段。4. A biomaterial, characterized in that the biomaterial comprises a polynucleotide, a carrier, or a cell, wherein the polynucleotide encodes the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3; the carrier carries the polynucleotide; the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3. 5.权利要求1-3任一项所述的兔单克隆抗体mAb6或其抗原结合片段的制备方法，其特征在于，包括培养如权利要求4中所述的细胞；5. A method for preparing the rabbit monoclonal antibody mAb6 or its antigen-binding fragment according to any one of claims 1-3, characterized in that it includes culturing the cells as described in claim 4; 所述细胞为将编码包括所述兔单克隆抗体mAb6或其抗原结合片段的多核苷酸转化至细胞制得，所述多核苷酸包括重链表达质粒和轻链表达质粒，所述转化包括将所述重链表达质粒和所述轻链表达质粒共转化至细胞。The cells are prepared by converting cells with a polynucleotide encoding a rabbit monoclonal antibody mAb6 or its antigen-binding fragment, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells. 6.根据权利要求5所述的制备方法，其特征在于，所述细胞为真核细胞。6. The preparation method according to claim 5, wherein the cells are eukaryotic cells. 7.根据权利要求6所述的制备方法，其特征在于，所述细胞为哺乳动物细胞。7. The preparation method according to claim 6, wherein the cell is a mammalian cell. 8.根据权利要求7所述的制备方法，其特征在于，所述细胞为293细胞或CHO细胞。8. The preparation method according to claim 7, wherein the cells are 293 cells or CHO cells. 9.权利要求1-3任一项所述的兔单克隆抗体mAb6或其抗原结合片段或权利要求4所述的生物材料在以下任一中的应用：9. The use of the rabbit monoclonal antibody mAb6 according to any one of claims 1-3 or its antigen-binding fragment, or the biological material according to claim 4, in any of the following: 1）非诊断和治疗目的地检测鼠免疫球蛋白G2a亚型（IgG2a）；1) Non-diagnostic and therapeutic target detection of mouse immunoglobulin G2a subtype (IgG2a); 2）制备用于检测鼠免疫球蛋白G2a亚型（IgG2a）的产品；2) Prepare products for detecting mouse immunoglobulin G2a subtype (IgG2a); 3）用于纯化鼠免疫球蛋白G2a亚型（IgG2a）；3) Used for purifying mouse immunoglobulin G2a subtype (IgG2a); 4）制备用于纯化鼠免疫球蛋白G2a亚型（IgG2a）的产品。4) Prepare products for purifying mouse immunoglobulin G2a subtype (IgG2a). 10.检测试剂或检测试剂盒，其特征在于，所述检测试剂或检测试剂盒包含权利要求1-3任一项所述的兔单克隆抗体mAb6或其抗原结合片段、或权利要求4所述的生物材料。10. A detection reagent or detection kit, characterized in that the detection reagent or detection kit comprises the rabbit monoclonal antibody mAb6 or its antigen-binding fragment as described in any one of claims 1-3, or the biological material as described in claim 4. 11.根据权利要求10所述的检测试剂或检测试剂盒，其特征在于，所述检测试剂盒用于酶联免疫吸附（ELISA）、免疫印迹（Western blot）、免疫组织化学染色（IHC）、流式细胞术（FCM）及免疫共沉淀等。11. The detection reagent or detection kit according to claim 10, characterized in that the detection kit is used for enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation, etc.
CN202510432603.7A 2025-04-08 2025-04-08 Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application Active CN120192419B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202510432603.7A CN120192419B (en) 2025-04-08 2025-04-08 Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202510432603.7A CN120192419B (en) 2025-04-08 2025-04-08 Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application

Publications (2)

Publication Number Publication Date
CN120192419A CN120192419A (en) 2025-06-24
CN120192419B true CN120192419B (en) 2025-11-21

Family

ID=96073855

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202510432603.7A Active CN120192419B (en) 2025-04-08 2025-04-08 Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application

Country Status (1)

Country Link
CN (1) CN120192419B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116239697A (en) * 2023-01-30 2023-06-09 上海百英生物科技股份有限公司 Rabbit monoclonal antibody resisting mouse IgG and application thereof
CN116554337A (en) * 2022-05-31 2023-08-08 无锡傲锐东源生物科技有限公司 Rabbit monoclonal antibody against mouse immunoglobulin G3 subtype (IgG 3) and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115772224A (en) * 2022-07-11 2023-03-10 上海百英生物科技股份有限公司 Goat antibody for resisting mouse IgG2a and application thereof
CN119684465B (en) * 2024-12-25 2025-08-22 无锡傲锐东源生物科技有限公司 Human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and its application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116554337A (en) * 2022-05-31 2023-08-08 无锡傲锐东源生物科技有限公司 Rabbit monoclonal antibody against mouse immunoglobulin G3 subtype (IgG 3) and application thereof
CN116239697A (en) * 2023-01-30 2023-06-09 上海百英生物科技股份有限公司 Rabbit monoclonal antibody resisting mouse IgG and application thereof

Also Published As

Publication number Publication date
CN120192419A (en) 2025-06-24

Similar Documents

Publication Publication Date Title
CN112250763B (en) Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use
US12391745B2 (en) Adeno-associated virus antibodies and fragments thereof
CN118684770B (en) Monoclonal antibody against triiodothyronine, detection reagent based thereon and application thereof
CN116143931B (en) Anti-human IgM antibody and preparation method and application thereof
CN118496356B (en) Antibody or antigen-binding fragment thereof for detecting thyroxine, preparation method and application thereof
CN116284382A (en) Procalcitonin-resistant antibodies and uses thereof
CN115028713A (en) An antibody against coronavirus N protein and its application
CN119684465B (en) Human immunoglobulin G2 subtype (IgG2) rabbit monoclonal antibody and its application
CN117024595B (en) Monoclonal antibody against human ST2 and use thereof
CN102232087A (en) Antibodies to modified human IGF-1/E peptides
CN109336973B (en) Anti-transferrin antibody and its use
CN111269314A (en) anti-CK 18 monoclonal antibody and application thereof
CN119192368B (en) Monoclonal antibody or antigen-binding fragment thereof for detecting folic acid and its binding protein complex, preparation method and application thereof
KR101263913B1 (en) Bovine tuberculosis diagnosing kit and diagnosing method using the same
CN120192419B (en) Rabbit monoclonal antibody mAb6 against mouse immunoglobulin G2a subtype (IgG2a) and its application
WO2024146419A1 (en) Specific antibody for claudin-18.2 and use thereof
CN116554337A (en) Rabbit monoclonal antibody against mouse immunoglobulin G3 subtype (IgG 3) and application thereof
CN120192418A (en) Rabbit monoclonal antibody mAb8 against mouse immunoglobulin G2b subtype (IgG2b) and its application
EP3896082A1 (en) Pre-haptoglobin-2 monoclonal antibodies and uses thereof
CN120040588B (en) Anti-T3 antibody mAb68 or antigen-binding fragment, preparation method and application
CN120192412B (en) Anti-T3 antibody mAb32 or antigen-binding fragment, preparation method and application
CN119899269B (en) A monoclonal antibody and its detection application
CN118772269B (en) An antibody against parainfluenza virus type 3 NP protein and its application
CN115677851B (en) Immune blocking antibody or antigen binding fragment thereof and application thereof
KR101287602B1 (en) Antibody recognizing kidney type and respiratory type infectious bronchitis virus and use thereof

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