CN116715763B - Mouse IL-6 rabbit monoclonal antibody pair and preparation method and application thereof - Google Patents

Mouse IL-6 rabbit monoclonal antibody pair and preparation method and application thereof Download PDF

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CN116715763B
CN116715763B CN202211469596.0A CN202211469596A CN116715763B CN 116715763 B CN116715763 B CN 116715763B CN 202211469596 A CN202211469596 A CN 202211469596A CN 116715763 B CN116715763 B CN 116715763B
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monoclonal antibody
rabbit monoclonal
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rabbit
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CN116715763A (en
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吴飞鸽
盛飞
刘政泽
张莹
景攀
程扬
汤欢欢
吴海
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Wuhan Abclonal Inc
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    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a Mouse IL-6 rabbit monoclonal antibody pair, a preparation method and application thereof, and belongs to the technical field of biological detection. The invention provides a Mouse IL-6 rabbit monoclonal antibody pair 12E1 and 2G9, and develops a double-antibody sandwich method ELISA detection method specific to Mouse IL-6. The rabbit monoclonal antibody pair 12E1 and 2G9 can specifically bind to the mouse IL-6 protein, wherein the capture antibody is the rabbit monoclonal antibody 12E1, and the detection antibody is the biotin-labeled rabbit monoclonal antibody 2G9, so that the method has the advantages of good specificity, good thermal stability, high detection sensitivity and the like when used for detecting MouseIL-6.

Description

Mouse IL-6 rabbit monoclonal antibody pair and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a Mouse IL-6 rabbit monoclonal antibody pair, and a preparation method and application thereof.
Background
Interleukin 6 (IL-6), a family of interleukins, is a cytokine that is produced mainly in fibroblasts, monocytes/macrophages, T lymphocytes, B lymphocytes and a variety of tumor cells. IL-6 plays an important role in immune responses, hematopoiesis, inflammation, and stress responses.
Mouse IL-6 is a potent inducer in stress, and rapid production of Mouse IL-6 contributes to host defense during infection and tissue damage, but excessive Mouse IL-6 synthesis is involved in the pathological processes of the disease. In addition, mouse IL-6 is in CD4 + T cell subsets play an important role in differentiation. Mouse IL-6 binds to IL-6Rα and by this binding IL-6 can induce gp130 homodimerization, leading to triggering of the Jak/Stat cascade and the SHP-2/ErkMAP kinase cascade.
Clinically, the content of IL-6 in blood is closely related to diseases such as inflammation, autoimmune diseases, viral bacterial infection and the like of the body. Generally, the level of IL-6 in normal healthy adult blood is less than 7pg/mL; when the IL-6 content is 7-150 pg/mL, the existence of slight inflammation or infection symptoms is indicated; when the content of IL-6 is 150-250 pg/mL, the existence of systemic inflammation or general infection symptoms is indicated; when the IL-6 content was >250pg/mL, sepsis was indicated. IL-6 has extremely low content in normal human serum, so that the research of the IL-6 detection method with high sensitivity has important significance.
Currently, there are patents disclosing antibody development with respect to IL-6, the immunogen employed is mainly a recombinant protein of IL-6 of human or canine origin, but there is no rabbit monoclonal antibody developed with a recombinant protein of murine origin; and most of the monoclonal antibodies disclosed in the prior art have low specificity for detection of binding of the monoclonal antibody or polyclonal antibody to the monoclonal antibody.
Disclosure of Invention
Based on this, there is a need to provide new Mouse IL-6 rabbit monoclonal antibody pairs, methods of making and uses thereof. The Mouse IL-6 rabbit monoclonal antibody pair provided by the invention can be used for establishing an ELISA detection method of Mouse IL-6 with good specificity, good thermal stability and high sensitivity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a Mouse IL-6 rabbit monoclonal antibody pair, which is rabbit monoclonal antibodies 12E1 and 2G9.
The protein sequences of the light chain full length, the heavy chain full length, the light chain variable region, the heavy chain variable region, the light chain and the heavy chain complementarity determining region of the rabbit monoclonal antibody 12E1 are respectively shown as SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18.
The protein sequences of the light chain full length, the heavy chain full length, the light chain variable region, the heavy chain variable region, the light chain and the heavy chain complementarity determining region of the rabbit monoclonal antibody 2G9 are respectively shown as SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28.
The invention also provides application of the Mouse IL-6 rabbit monoclonal antibody to preparation of a reagent or a kit for detecting the Mouse IL-6. The reagent or the kit is used for enzyme-linked immunosorbent assay of Mouse IL-6, wherein the rabbit monoclonal antibody 12E1 is used as a capture antibody, and the rabbit monoclonal antibody 2G9 is used as a labeled antibody.
The invention also provides a reagent or a kit for detecting the Mouse IL-6, which comprises the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9. The Mouse IL-6 is at least one of recombinant Mouse IL-6 and Mouse IL-6 secreted by cells.
The invention also provides a preparation method of the Mouse IL-6 rabbit monoclonal antibody pair, which comprises the following steps: the heavy chain gene and the light chain gene of the Mouse IL-6 rabbit monoclonal antibody pair are respectively loaded on an expression vector, and 293F cells are transfected; culturing to obtain a supernatant containing the Mouse IL-6 rabbit monoclonal antibody pair; purifying to obtain the final product.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a Mouse IL-6 protein rabbit monoclonal antibody pair: 12E1 and 2G9, and according to the method for establishing double-antibody sandwich method ELISA detection of Mouse IL-6, the established method is used for detecting Mouse IL-6 protein, and has high specificity, high sensitivity and high stability.
Drawings
FIG. 1 is a graph showing the measurement of Serum titer (Serum titer) of a rabbit immunized with recombinant Mouse interleukin IL-6 protein of example 1.
FIG. 2 is a schematic diagram of the construction of an expression vector containing the heavy chain constant region of a rabbit monoclonal antibody in example 1.
FIG. 3 is a schematic diagram of the construction of an expression vector containing the light chain constant region of a rabbit monoclonal antibody according to example 1.
FIG. 4 is a graph of K assay data for example 1, rabbit monoclonal antibody pair 12E 1.
FIG. 5 is a graph of K assay data for 2G9 in the rabbit monoclonal antibody pair of example 1.
FIG. 6 is a graph of EP assay data for rabbit monoclonal antibodies 12E1 and 2G9 of example 1
FIG. 7 is a VL sequence alignment of rabbit monoclonal antibodies 12E1 and 2G9 of example 1.
FIG. 8 is a VH sequence alignment of rabbit monoclonal antibodies 12E1 and 2G9 of example 1.
FIG. 9 is a standard graph of example 2 for a sandwich ELISA based on rabbit monoclonal antibodies 12E1 and 2G9.
FIG. 10 is a graph showing cross-protein recognition data of sandwich ELISA based on rabbit monoclonal antibodies 12E1 and 2G9 of example 3.
FIG. 11 is a statistical chart of the results of the thermal stability test based on the rabbit monoclonal antibodies 12E1 and 2G9 of example 3.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention. In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Description of partial terms: "double antibody sandwich ELISA", "double antibody sandwich ELISA" and "double antibody sandwich ELISA" are used interchangeably. "Mouse interleukin IL-6", "Mouse interleukin IL-6 protein", "recombinant Mouse interleukin IL-6 protein", "Mouse IL-6" mean the same concept, and are used interchangeably.
Example 1
The embodiment provides a preparation method of a monoclonal antibody pair of anti-mouse interleukin IL-6, which specifically comprises the following steps:
1.1, immunogen preparation:
the immunogen used was Mouse recombinant Mouse IL-6 protein (commercially available from ablonal): a high quality recombinant murine IL-6 mature protein with biological activity was expressed in CHO cells using the Mouse IL-6NP_112445.1 (Uniprot ID: P08505) protein sequence corresponding to NM-031168.2 constructed to the pYURK-Chis vector.
1.2, animal immunization: taking recombinant MouseIL-6 protein as an immunogen to immunize 4 New Zealand white rabbits; each white rabbit was immunized with 200 μg of immunogen, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma Co.) to prepare an emulsifier prior to the first immunization, and injected subcutaneously in the abdomen and back of the rabbits at multiple points; 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare an emulsifier, which was subcutaneously injected at the abdomen and back of rabbits at multiple points to boost the immunization twice. After three immunizations, rabbit serum samples were collected, titers against MouseIL-6 were determined by ELISA, serum was diluted 1:243K and titers were determined by ELISA, OD was obtained 450nm The serum titer test for rabbits exceeding 0.2 is shown in FIG. 1, and the spleens were taken three days later by boosting with 200. Mu.g of immunogen at multiple subcutaneous injections.
1.3, spleen cells were isolated: taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40 mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by using the tail end of the pressed part of the grinding rod. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company), gently blowing off cell clusters by using a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
1.4, B lymphocyte sorting: the method of paragraphs [0030] to [0044] in the specification is adopted in Chinese patent CN110016462B (patent name: method for efficiently isolating single antigen-specific B lymphocytes from spleen cells).
1.5 cloning of Gene encoding Rabbit monoclonal antibody
The cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cells of positive clones were collected and lysed, and RNA was extracted using Quick-RNATMMicroPrep kit (available from ZYMO Co.) and reverse transcribed into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of a naturally paired rabbit monoclonal antibody from the cDNA of the corresponding positive clone, and a plurality of clones are selected for sequencing, and the sequencing work is completed by Jin Kairui biotechnology limited company.
Wherein, the PCR reaction system is as follows: 4. Mu.L cDNA, 1. Mu.L forward primer (10 mM), 1. Mu.L reverse primer (10 mM), 12.5. Mu.L 2 XGloriaHiFi, 6.5. Mu. L H 2 O. The 2 XGloriaHiFi reagent is available from Wuhan Aibolag Biotechnology Inc.
Wherein, the light chain variable region primer pair is:
VL-Primer-F：
5'-tgaattcgagctcggtacccatggacacgagggcccccac-3'(SEQ ID NO:1)；
VL-Primer-R：
5'-cacacacacgatggtgactgttccagttgccacctgatcag-3'(SEQ ID NO:2)；
the heavy chain variable region primer pair is:
VH-Primer-F：
5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3'(SEQ ID NO:3)；
VH-Primer-R：
5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'(SEQ ID NO:4)。
PCR amplification procedure: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.
1.6 preparation and purification of monoclonal antibodies
1.6.1 in order to obtain a plurality of rabbit monoclonal antibodies recognizing MouseIL-6 protein, the heavy chain genes and the light chain genes of a plurality of rabbit monoclonal antibodies selected in the step 1.5 are respectively loaded on an expression vector, and a mammal expression vector pBR322 is used, wherein the mammal expression vector pBR322 is shown in figures 2 and 3. In FIGS. 2 and 3, pBR322origin and f1origin are replication promoters in E.coli (E.Coli), ampcilin is a plasmid resistance gene, CMV immearly promotor is a promoter in eukaryotes, SV40PA terminator is a tailing signal, heavy chain constant in FIG. 2 is a nucleotide sequence of a heavy chain constant region of a rabbit monoclonal antibody, and Light chain constant in FIG. 3 is a nucleotide sequence of a light chain constant region of a rabbit monoclonal antibody.
1.6.2 mammalian cell expression vectors containing the heavy chain constant region (FIG. 2) and the light chain constant region (FIG. 3) of the rabbit monoclonal antibodies were routinely linearized with NheI and XbaI restriction enzymes, respectively.
1.6.3, purifying the PCR product amplified in the step 1.4, and respectively constructing a heavy chain variable region gene and a light chain variable region gene into corresponding mammal expression vectors by adopting a homologous recombination mode; after sequencing verification, the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies are transfected into 293F cells together; and (3) carrying out transfection for 72-96 hours to obtain the rabbit monoclonal antibody which contains recombinant recognition mouse interleukin IL-6 in the culture supernatant.
Purifying recombinant rabbit monoclonal antibody recognizing Mouse IL-6 from transfected culture medium supernatant by using protein A affinity gel resin, verifying antibody purity by using 12% SDS-PAGE gel electrophoresis, subpackaging after verification, and preserving at-20deg.C for later use.
1.7 monoclonal antibody screening and identification
After a plurality of MouseIL-6 rabbit monoclonal antibodies are obtained, the rabbit monoclonal antibodies are firstly subjected to preliminary identification and screening, including identification of antibody affinity and identification of antigen recognition epitopes, and the specific method is as follows:
1.7.1 screening of monoclonal antibodies:
the affinity of the obtained rabbit monoclonal antibody was preliminarily determined using a gate biomolecular interaction analyzer from Probe Life. Wherein the material used is recombinant Mouse IL-6 protein, the concentration used is 150nM and 75nM, and the concentration of the rabbit monoclonal antibody is 2 mug/mL; by comparing the affinities of the respective antibodies, an affinity constant of 1X 10 or less is selected from the above -9 Is a human antibody.
1.7.2, identification of antigen recognition epitopes:
the obtained rabbit monoclonal antibody was subjected to a pairing reaction using a gate biomolecular interaction analyzer from Probe Life company to test its recognized epitope determinant.
Wherein the material used is recombinant Mouse IL-6 protein, the concentration is 5 mug/mL, the concentration of the first rabbit monoclonal antibody is 2 mug/mL, and the concentration of the second rabbit monoclonal antibody is 5 mug/mL.
Two antibodies recognizing different epitope determinants were selected from the two antibodies by analyzing the pairing data between the two antibodies, designated rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G9, respectively. Affinity constants of the 12E1 monoclonal antibody and the 2G9 monoclonal antibody are as follows:
affinity constants of 12E1 mab and 2G9 mab (Kassay)
K off (1/s) K on (1/Ms) K D (M)
12E1 1.85×10 -6 5.63×10 6 3.29×10 -13
2G9 2.72×10 -5 4.59×10 6 5.93×10 -12
The affinity of the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 was measured, and the measurement results are shown in the table, FIG. 4 and FIG. 5, and the results show that: dissociation coefficient, binding coefficient and binding constant of rabbit monoclonal antibody 12E1 and recombinant Mouse IL-6 were 1.85X10, respectively -6 、5.63×10 6 、3.29×10 -13 The method comprises the steps of carrying out a first treatment on the surface of the Dissociation coefficient, binding coefficient and binding constant of rabbit monoclonal antibody 2G9 and recombinant Mouse IL-6 were 2.72X10 respectively -5 、4.59×10 6 、5.93×10 -12 The rabbit monoclonal antibodies 12E1 and 2G9 are shown to have high affinity with recombinant Mouse IL-6, respectively.
And compared with the affinity constant of the human IL-6A B rabbit monoclonal antibody disclosed in CN112175080B (see table below), the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 have more affinity with the patent literature, and especially the rabbit monoclonal antibody 12E1 has about 10-1000 times higher affinity than the corresponding A and B.
CN112175080B rabbit monoclonal antibody A and B affinity constant (Kassay)
K off (1/s) K on (1/Ms) K D (M)
A 3.28×10 -4 4.19×10 5 7.80×10 -10
B 1.34×10 -5 3.57×10 6 3.75×10 -12
The results of the epitope determination of rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G9 are shown in FIG. 6. As can be seen from fig. 6, the rabbit monoclonal antibodies 12E1 and 2G9 recognize different epitopes of Mouse IL-6, respectively, and thus both can be used for ELISA pairing.
Nucleotide sequencing was performed on the selected rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G9, respectively, and the sequencing work was completed by Jin Kairui Biotechnology Co.
Wherein, the nucleotide sequence of the full length of the light chain of the rabbit monoclonal antibody 12E 1:
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTCCTGCTCTGGCTGCCCGGAGCCAGGTGCGATATAGTCCTCACGCAAACTCCGGCGTCTGTCAGCGCAGCCGTCGGTGGCACTGTTACGATAAAATGTCAGGCCTCAGAAGACATCTATTCAAATCTCGCATGGTATCAACAAAAACCCGGACAGCCTCCGAAGCTGCTCATTTACGGGGCCTCAACAATGGCTTCAGGCGTCAGCTCAAGATTCAAAGGTAGTGGGTCAGGCACGGAATTCAACCTGATTATCAGTGACTTGGAGTGCGCTGACGCCACGACGTATTACTGTCAGGGGACAATCTATTTTTCCGGGGAGAATACCTTTGGCGGGGGTACAAAGGTTGTTGTTGAGGGAGACCCTGTGGCCCCTACGGTGCTCATATTTCCCCCGGCGGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG(SEQ ID NO:5)。
nucleotide sequence of the light chain variable region of rabbit monoclonal antibody 12E 1:
GGGCTCCTCCTGCTCTGGCTGCCCGGAGCCAGGTGCGATATAGTCCTCACGCAAACTCCGGCGTCTGTCAGCGCAGCCGTCGGTGGCACTGTTACGATAAAATGTCAGGCCTCAGAAGACATCTATTCAAATCTCGCATGGTATCAACAAAAACCCGGACAGCCTCCGAAGCTGCTCATTTACGGGGCCTCAACAATGGCTTCAGGCGTCAGCTCAAGATTCAAAGGTAGTGGGTCAGGCACGGAATTCAACCTGATTATCAGTGACTTGGAGTGCGCTGACGCCACGACGTATTACTGTCAGGGGACAATCTATTTTTCCGGGGAGAATACCTTTGGCGGGGGTACAAAGGTTGTTGTTGAGGGAGACCCTGTGGCCCCTACGGTGCTCATATTTCCCCCGGCG(SEQ ID NO:6)。
nucleotide sequence of the full length heavy chain of rabbit monoclonal antibody 2G 9:
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTTGCCGTGCTGAAGGGAGTGCAGTGCCAAAGTCTGGAAGAATCAGGCGGGCGCTTGGTCACGCCGGGCACTCCCCTGACCCTGACTTGCACAGTGTCCGGTTTCTCTCTCTCTCGCTACTGGATGTCTTGGGTTCGCCAGGCACCTGGTAAAGGACTGGAATGGATAGGCTATATATACGGAGGTAGTGGTACAACTTGGTTTGCGAGCTGGGCGAAGGGAAGATTTACTATCTCAAAGACCTCTACGACTGTCGATTTGAAAATCACCAGCCCGACTACGGAGGACACCGCAACATACTTTTGCGCACGCGACTATGTCGATTATGATTCTGATATTGGATTTTACAACCTGTGGGGACCGGGAACTTTGGTGACTGTCAGCAGCGGACAACCGAAAGCACCCAGTGTTTTTCCTTTGGCTCCCTGTTGCGGAGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAA(SEQ ID NO:7)。
nucleotide sequence of the heavy chain variable region of rabbit monoclonal antibody 2G 9:
GTTGCCGTGCTGAAGGGAGTGCAGTGCCAAAGTCTGGAAGAATCAGGCGGGCGCTTGGTCACGCCGGGCACTCCCCTGACCCTGACTTGCACAGTGTCCGGTTTCTCTCTCTCTCGCTACTGGATGTCTTGGGTTCGCCAGGCACCTGGTAAAGGACTGGAATGGATAGGCTATATATACGGAGGTAGTGGTACAACTTGGTTTGCGAGCTGGGCGAAGGGAAGATTTACTATCTCAAAGACCTCTACGACTGTCGATTTGAAAATCACCAGCCCGACTACGGAGGACACCGCAACATACTTTTGCGCACGCGACTATGTCGATTATGATTCTGATATTGGATTTTACAACCTGTGGGGACCGGGAACTTTGGTGACTGTCAGCAGCGGACAACCGAAAGCACCCAGTGTTTTTCCTTTGGCTCCCTGTTGCGGAGACACA(SEQ IDNO:8)。
wherein, the amino acid sequences of the rabbit monoclonal antibodies are respectively:
full length sequence of rabbit monoclonal antibody 12E1 light chain:
MDTRAPTQLLGLLLLWLPGARCDIVLTQTPASVSAAVGGTVTIKCQASEDIYSNLAWYQQKPGQPPKLLIYGASTMASGVSSRFKGSGSGTEFNLIISDLECADATTYYCQGTIYFSGENTFGGGTKVVVEGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:9)。
full length sequence of rabbit monoclonal antibody 12E1 heavy chain:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEWIGYIDTESGGTYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGGDSPFYPYTAWGNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:10)。
rabbit monoclonal antibody 12E1 light chain variable region sequence:
MDTRAPTQLLGLLLLWLPGARCDIVLTQTPASVSAAVGGTVTIKCQASEDIYSNLAWYQQKPGQPPKLLIYGASTMASGVSSRFKGSGSGTEFNLIISDLECADATTYYCQGTIYFSGENTFGGGTKVVVE(SEQ ID NO:11)。
rabbit monoclonal antibody 12E1 heavy chain variable region sequence:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEWIGYIDTESGGTYYATWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGGDSPFYPYTAWGNLWGPGTLVTVSS(SEQ ID NO:12)。
amino acid sequence of complementarity determining region VL-12E1-CDR1 of the light chain variable region of rabbit monoclonal antibody 12E 1: EDIYSNLAW (SEQ ID NO: 13).
Amino acid sequence of complementarity determining region VL-12E1-CDR2 of the light chain variable region of rabbit monoclonal antibody 12E 1: LIYGASTMASGV (SEQ ID NO: 14).
Amino acid sequence of complementarity determining region VL-12E1-CDR3 of the light chain variable region of rabbit monoclonal antibody 12E 1: QGTIYFSGENTF (SEQ ID NO: 15).
Amino acid sequence of complementarity determining region VL-12E1-CDR1 of heavy chain variable region of rabbit monoclonal antibody 12E 1: FSLSSYAMI (SEQ ID NO: 16).
Amino acid sequence of complementarity determining region VL-12E1-CDR2 of heavy chain variable region of rabbit monoclonal antibody 12E 1: WIGYIDTESGGTYYATWAK (SEQ ID NO: 17).
Amino acid sequence of complementarity determining region VL-12E1-CDR3 of heavy chain variable region of rabbit monoclonal antibody 12E 1: YFCARGGDSPFYPYTAWGNL (SEQ ID NO: 18).
Full length sequence of rabbit monoclonal antibody 2G9 light chain:
MDTRAPTQLLGLLLLWLPGARCAYDMTQTPDSVEVAVGGTVTINCQASQSINNQLSWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQTGSSGNVDNPFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:19)。
full length sequence of rabbit monoclonal antibody 2G9 heavy chain:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSRYWMSWVRQAPGKGLEWIGYIYGGSGTTWFASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDYVDYDSDIGFYNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:20)。
rabbit monoclonal antibody 2G9 light chain variable region sequence:
MDTRAPTQLLGLLLLWLPGARCAYDMTQTPDSVEVAVGGTVTINCQASQSINNQLSWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQTGSSGNVDNPFGGGTEVVVK(SEQ ID NO:21)。
rabbit monoclonal antibody 2G9 heavy chain variable region sequence:
METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSRYWMSWVRQAPGKGLEWIGYIYGGSGTTWFASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDYVDYDSDIGFYNLWGPGTLVTVSS(SEQ ID NO:22)。
amino acid sequence of complementarity determining region VL-2G9-CDR1 of light chain variable region of rabbit monoclonal antibody 2G 9: QSINNQLSW (SEQ ID NO: 23).
Amino acid sequence of complementarity determining region VL-2G9-CDR2 of the light chain variable region of rabbit monoclonal antibody 2G 9: LIYKASTLASGV (SEQ ID NO: 24).
Amino acid sequence of complementarity determining region VL-2G9-CDR3 of the light chain variable region of rabbit monoclonal antibody 2G 9: QQTGSSGNVDNPF (SEQ ID NO: 25).
Amino acid sequence of complementarity determining region VL-2G9-CDR1 of heavy chain variable region of rabbit monoclonal antibody 2G 9: FSLSRYWMS (SEQ ID NO: 26).
Amino acid sequence of complementarity determining region VL-2G9-CDR2 of heavy chain variable region of rabbit monoclonal antibody 2G 9: WIGYIYGGSGTTWFASWAK (SEQ ID NO: 27).
Amino acid sequence of complementarity determining region VL-2G9-CDR3 of heavy chain variable region of rabbit monoclonal antibody 2G 9: YFCARDYVDYDSDIGFYNL (SEQ ID NO: 28).
The sequencing alignment results are shown in fig. 7 and 8: the light chain variable region VL and heavy chain variable region VH sequence identity of rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G9 were 75% and 82%, respectively.
Example 2
The embodiment establishes a double antibody sandwich method ELISA detection method aiming at rabbit monoclonal antibodies 12E1 and 2G9 of Mouse IL-6 protein, and comprises the following steps:
2.1 coating: rabbit monoclonal antibody 12E1 (prepared in example 1) was diluted to 2. Mu.g/mL with 1 XPBS, vortexed, added to 96-well microwell plates at 100. Mu.L/well, covered with cover plate membrane, and incubated in a refrigerator at 4℃for 16-20h.
2.2 washing the plates: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.3 closing: e013 blocking solution (90 mL of the prepared PBS reagent was added to a beaker, 5g of Skim mill, 1g of BSA and 0.05mL of Tween-20 were added, respectively, and the mixture was thoroughly mixed to supplement ddH 2 O to 100mL of total volume, storing at room temperature or 4 ℃ for use, and preparing at present) adding 200 mu L/well into a plate hole, covering a cover plate film, sealing for 2h at 37 ℃, discarding sealing liquid after sealing, drying an ELISA plate after beating, drying for 0.5-2h in a baking oven at 37 ℃, and taking out for standby.
2.4 protein addition: the standard sample (recombinant Mouse IL-6 protein, yinqiao, 50136-MNAE_LC13DE 1604) and the sample to be tested are diluted with a diluent (PBS buffer with pH of 7.2) at the following concentration: 100. 50,25,12.5,6.25, 1.56,0pg/mL, then added sequentially to the ELISA plate at 100. Mu.L/well, covered with cover plate membrane and incubated for 2h at 37 ℃.
2.5 washing the plates: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.6 preparation of rabbit monoclonal antibody 2G 9-biotin:
2.6.1 anti-Mouse IL-6 rabbit monoclonal antibody 2G9 was formulated as a 1mg/mL solution and NHS-LC-biotin (N-succinimidyl 6-biotin aminocaproic acid, available from Thermo Inc.) was formulated as a 60mg/mL solution using DMSO (dimethyl sulfoxide).
2.6.2, 200. Mu.L of 1mg/mL of anti-Mouse IL-6 rabbit monoclonal antibody 2G9 solution is taken, and 10. Mu.L of 60mg/mL of NHS-LC-biotin solution is added; after mixing, the mixture was left at room temperature for 30min, and then 50. Mu.L of 500mM Tris-HCl solution of pH=9.0 was added to terminate the reaction.
2.6.3 finally, 4mL of 1 XPBS buffer with pH=7.4 was added and centrifuged with a 30kDa exclusion limit to remove excess biotin molecules and allow for equilibration of the buffer system.
Adding a detection antibody: after the rabbit monoclonal antibody 2G9-biotin was diluted to 0.1. Mu.g/mL, the diluted solution was added to an ELISA plate in sequence at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 1 hour.
2.7 washing the plates: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.8 adding SA-HRP: 100 XSA-HRP concentrate was 100 XSA diluted, and then added to an ELISA plate in order of 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.
2.9 washing the plates: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.10 adding TMB color development liquid: TMB color development solution is added into an ELISA plate in sequence at 100 mu L/well, a cover plate film is covered, and incubation is carried out for 15min at 37 ℃.
2.11 after incubation, the microplate was removed, 50. Mu.L of stop solution was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
The standard curve drawn according to the detection result is shown in fig. 9. The results show that the antibody pair specifically recognizes Mouse IL-6 and that the sensitivity can be as low as 1.56pg/mL.
Example 3
The performance of the rabbit monoclonal antibodies 12E1 and 2G9 against murine interleukin IL-6 was tested in this example. The specific method comprises the following steps:
3.1, specific detection of rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G 9:
the specificity of the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 against the mouse interleukin-6 protein was detected with 3 interleukin proteins similar to the mouse interleukin-6 protein, respectively, using the double antibody sandwich ELISA method of example 2, the concentration of each standard protein was 100pg/mL. The 3 kinds of interleukin proteins are Mouse interleukin IL-1 (Mouse IL-1), human interleukin IL-6 (Human IL-6) and Mouse interleukin IL-11 (Mouse IL-11), respectively.
The detection results are shown in fig. 10, and as can be seen from fig. 10, when the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 prepared by the invention are used for carrying out double-antibody sandwich ELISA detection on different interleukin proteins, the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 are only combined with the mouse interleukin IL-6 and do not generate any cross reaction with other interleukins; the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 prepared by the invention have high specificity to the mouse interleukin IL-6.
3.2, test the thermal stability of rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G 9:
3.2.1. coating: the rabbit monoclonal antibody 12E1 is diluted to 1 mug/ml by 1 XPBS, and after being mixed evenly by a vortex meter, 100 mug/well is added into a 96-well micro-well plate, a cover plate film is covered, and the mixture is placed in a refrigerator at 4 ℃ for incubation for 16-20h.
3.2.2. Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 300. Mu.l was added, and after standing for 40s, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
3.2.3. Closing: e013 blocking solution (90 mL of the formulated PBS reagent was added to a beaker, 5g of Skim mill, 1g of BSA and 0, respectively05mL Tween-20 was mixed well to supplement dH 2 O to 100mL of total volume) is added into the plate hole at 200 μl/well, a cover plate film is covered, the sealing is carried out for 2h at 37 ℃, sealing liquid is discarded after the sealing is finished, and the ELISA plate is dried after being patted and is placed into a baking oven at 37 ℃ for 0.5-2h.
3.2.4. Thermal destruction: dividing the coated ELISA plate, lyophilized protein, and 100×concentrated detection antibody into three parts, respectively placing at-20deg.C, 4deg.C, and 37deg.C, sealing, preserving for 7 days, and taking out for testing.
3.2.5. Protein adding: the Mouse IL-6 protein stored under different conditions is dissolved by a diluent, and then diluted, wherein the diluted concentration is as follows: 100,50,25,12.5,6.25,3.12,1.56,0pg/mL, then 100. Mu.l/well were added sequentially to the ELISA plates stored at the corresponding temperature, covered with cover plate membrane, and incubated at 37℃for 2h.
3.2.6. Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.l was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
3.2.7. Adding a detection antibody: after 2G9-biotin stored under different conditions is subjected to 100 Xdilution, the diluted 2G9-biotin is sequentially added into an ELISA plate stored at a corresponding temperature by 100 mu l/well, covered with a cover plate film and incubated for 1h at 37 ℃.
8. Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.l was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
9. Adding SA-HRP: 100 XSA-HRP concentrate was 100 XSA diluted, and then added to an ELISA plate in order of 100. Mu.l/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.
10. Washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.l was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
11. Adding TMB color development liquid: TMB color development solution is added into an ELISA plate in sequence at 100 mu l/well, a cover plate film is covered, and incubation is carried out for 15min at 37 ℃.
12. After the incubation was completed, the microplate was removed, 50. Mu.l of stop solution was added to each well, and immediately reading was performed with an microplate reader at 450 nm.
The statistical results are shown in FIG. 11, and the standard curve equation established by using four parameters of the Logistic curve for the results of measuring the IL-6 concentration of the mouse interleukin after the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 are treated at-20 ℃ is as follows:
Y 2 ＝0.100+4.98X 2 1.205 /(X 2 1.205 +297.82)(R 2 ＝0.9966)，
Y 2 is a correction value for absorbance at-20 ℃ (y2=od 450nm -OD 630nm ),X 2 Is the concentration of Mouse IL-6.
The standard curve equation established after 4 ℃ treatment is
Y3＝0.097+9.69X 2 1.068 /(X21.068+389.62)(R 2 ＝0.9993)，
Y3 is a correction value for the absorbance value at 4 ℃ (y3=od 450nm -OD 630nm ),X 2 Is the concentration of Mouse IL-6;
the standard curve equation established after 37 ℃ treatment is
Y4＝0.092+75.65X 2 0.960 /(X 2 0.960 +2591.89)(R 2 ＝0.9984)，
Y4 is a correction value for the absorbance value at 37 ℃ (y4=od 450nm -OD 630nm )；X 2 Is the concentration of Mouse IL-6.
Through data calculation, the coefficient of variation of the concentration of the Mouse IL-6 protein detected by the rabbit monoclonal antibodies 12E1 and 2G9 which are respectively subjected to the treatment of-20 ℃ and 4 ℃ and 37 ℃ is less than 10% (see figure 11), which shows that the heat stability of the anti-Mouse IL-6 rabbit monoclonal antibodies 12E1 and 2G9 prepared by the invention is higher.
In addition, it is worth noting that the team of inventors also compared the rabbit monoclonal antibodies 12E1 and 2G9 prepared according to the present invention with the partial anti-interleukin-6 antibodies disclosed in the prior art, found that:
patent document CN112175080B discloses an anti-human interleukin-6 high affinity rabbit monoclonal antibody and application, which uses the amino acid sequences of rabbit monoclonal antibodies a and B and develops an enzyme-linked immunosorbent assay method, but the binding epitopes of the rabbit monoclonal antibodies a and B and Mouse IL-6 are single.
Patent document CN112279913B discloses an anti-human IL-6 monoclonal antibody and application thereof, wherein the monoclonal antibody A, B is K D (M) was 7.8X10 respectively -10 3.75X10 -12 . The invention adopts the rabbit monoclonal antibodies 12E1 and 2G9 to test K in the K assay D (M), 12E1 was 3.29×10 -13 2G9 is 5.93×10 -12 The specificity is higher.
Meanwhile, the mouse IL-6 recombinant protein is adopted as an immunogen to develop antibodies, and the antibodies are different from the human IL-6 and canine IL-6 recombinant proteins in antigen sequences in the patent documents, so that the specificity of the finally optimized and screened mouse interleukin IL-6 rabbit monoclonal antibodies 12E1 and 2G9 is more excellent.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. 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 (10)

1. A Mouse IL-6 rabbit monoclonal antibody pair, wherein the Mouse IL-6 rabbit monoclonal antibody pair consists of rabbit monoclonal antibody 12E1 and rabbit monoclonal antibody 2G 9;
the sequence of the complementarity determining region of the rabbit monoclonal antibody 12E1 is shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 respectively;
the sequence of the complementarity determining region of the rabbit monoclonal antibody 2G9 is shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28 respectively.
2. The Mouse IL-6 rabbit monoclonal antibody pair of claim 1, wherein the sequence of the light chain variable region of rabbit monoclonal antibody 12E1 is shown in SEQ ID No. 11; and/or
The sequence of the heavy chain variable region of the rabbit monoclonal antibody 12E1 is shown as SEQ ID NO. 12.
3. The Mouse IL-6 rabbit monoclonal antibody pair of claim 2, wherein the full length sequence of the light chain of rabbit monoclonal antibody 12E1 is shown in SEQ ID No. 9; and/or the full-length sequence of the heavy chain of the rabbit monoclonal antibody 12E1 is shown as SEQ ID NO. 10.
4. A Mouse IL-6 rabbit monoclonal antibody pair according to any one of claims 1 to 3, wherein the sequence of the light chain variable region of rabbit monoclonal antibody 2G9 is as shown in SEQ ID No. 21; and/or the sequence of the heavy chain variable region of the rabbit monoclonal antibody 2G9 is shown as SEQ ID NO. 22.
5. The Mouse IL-6 rabbit monoclonal antibody pair of claim 4, wherein the full length sequence of the light chain of rabbit monoclonal antibody 2G9 is shown in SEQ ID No. 19; and/or the full-length sequence of the heavy chain of the rabbit monoclonal antibody 2G9 is shown as SEQ ID NO. 20.
6. Use of a Mouse IL-6 rabbit monoclonal antibody pair as defined in any one of claims 1 to 5 in the preparation of a reagent or kit for detecting Mouse IL-6.
7. The use according to claim 6, wherein the reagent or kit is for enzyme-linked immunosorbent assay of Mouse IL-6, wherein the rabbit monoclonal antibody 12E1 is used as a capture antibody and the rabbit monoclonal antibody 2G9 is used as a marker antibody.
8. A reagent or kit for detecting Mouse IL-6, comprising the rabbit monoclonal antibody 12E1 and the rabbit monoclonal antibody 2G9 of any one of claims 1 to 5.
9. A reagent or kit for detecting Mouse IL-6 as claimed in claim 8, wherein said Mouse IL-6 is selected from at least one of recombinant Mouse IL-6, cell secreted Mouse IL-6.
10. A method of preparing a Mouse IL-6 rabbit monoclonal antibody pair of any one of claims 1 to 5, comprising the steps of:
loading the heavy chain gene and the light chain gene of the Mouse IL-6 rabbit monoclonal antibody pair of any one of claims 1 to 5 onto expression vectors, respectively, and transfecting 293F cells;
culturing to obtain a supernatant containing the Mouse IL-6 rabbit monoclonal antibody pair;
purifying to obtain the final product.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101754772A (en) * 2007-05-21 2010-06-23 奥尔德生物制药公司 At antibody of IL-6 and uses thereof
CN111867627A (en) * 2018-01-04 2020-10-30 维塔里斯股份有限公司 Use of an anti-IL-6 antibody, such as clarizazumab (Clazakizumab), for desensitizing a solid organ transplant recipient and/or for preventing, stabilizing, or alleviating antibody-mediated rejection (ABMR)
CN112175080A (en) * 2020-10-22 2021-01-05 优睿赛思(武汉)生物科技有限公司 Human interleukin-6 resistant high affinity rabbit monoclonal antibody and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101754772A (en) * 2007-05-21 2010-06-23 奥尔德生物制药公司 At antibody of IL-6 and uses thereof
CN111867627A (en) * 2018-01-04 2020-10-30 维塔里斯股份有限公司 Use of an anti-IL-6 antibody, such as clarizazumab (Clazakizumab), for desensitizing a solid organ transplant recipient and/or for preventing, stabilizing, or alleviating antibody-mediated rejection (ABMR)
CN112175080A (en) * 2020-10-22 2021-01-05 优睿赛思(武汉)生物科技有限公司 Human interleukin-6 resistant high affinity rabbit monoclonal antibody and application

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