CN115505575A - Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application - Google Patents
Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application Download PDFInfo
- Publication number
- CN115505575A CN115505575A CN202211463230.2A CN202211463230A CN115505575A CN 115505575 A CN115505575 A CN 115505575A CN 202211463230 A CN202211463230 A CN 202211463230A CN 115505575 A CN115505575 A CN 115505575A
- Authority
- CN
- China
- Prior art keywords
- antibody
- canine
- hybridoma cell
- monoclonal antibody
- tibgao
- 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.)
- Granted
Links
- 102100021596 Interleukin-31 Human genes 0.000 title claims abstract description 91
- 101710181613 Interleukin-31 Proteins 0.000 title claims abstract description 90
- 241000282465 Canis Species 0.000 title claims abstract description 76
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 34
- 230000003248 secreting effect Effects 0.000 title description 3
- 238000001514 detection method Methods 0.000 claims abstract description 31
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 22
- 238000003118 sandwich ELISA Methods 0.000 claims abstract description 22
- 229960002685 biotin Drugs 0.000 claims abstract description 11
- 235000020958 biotin Nutrition 0.000 claims abstract description 11
- 239000011616 biotin Substances 0.000 claims abstract description 11
- 206010012438 Dermatitis atopic Diseases 0.000 claims abstract description 10
- 201000008937 atopic dermatitis Diseases 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 238000002965 ELISA Methods 0.000 claims description 16
- 208000003251 Pruritus Diseases 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 206010020751 Hypersensitivity Diseases 0.000 claims description 4
- 230000007815 allergy Effects 0.000 claims description 4
- 206010015150 Erythema Diseases 0.000 claims description 3
- 208000026935 allergic disease Diseases 0.000 claims description 3
- 231100000321 erythema Toxicity 0.000 claims description 3
- 208000024891 symptom Diseases 0.000 claims description 3
- 230000003902 lesion Effects 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 claims 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 210000004962 mammalian cell Anatomy 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 230000027455 binding Effects 0.000 abstract description 21
- 238000000034 method Methods 0.000 abstract description 17
- 206010003445 Ascites Diseases 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000010494 dissociation reaction Methods 0.000 description 8
- 230000005593 dissociations Effects 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 239000000427 antigen Substances 0.000 description 7
- 230000003053 immunization Effects 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 201000004624 Dermatitis Diseases 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 241000282472 Canis lupus familiaris Species 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000007803 itching Effects 0.000 description 4
- 230000001823 pruritic effect Effects 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 102000015617 Janus Kinases Human genes 0.000 description 3
- 108010024121 Janus Kinases Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000002808 molecular sieve Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 3
- 201000004384 Alopecia Diseases 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 101000586302 Homo sapiens Oncostatin-M-specific receptor subunit beta Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100021594 Interleukin-31 receptor subunit alpha Human genes 0.000 description 2
- 101710131691 Interleukin-31 receptor subunit alpha Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102100030098 Oncostatin-M-specific receptor subunit beta Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003979 eosinophil Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 102000000021 Chemokine CCL1 Human genes 0.000 description 1
- 108010055288 Chemokine CCL1 Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 101001043821 Homo sapiens Interleukin-31 Proteins 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 208000006877 Insect Bites and Stings Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 208000028004 allergic respiratory disease Diseases 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 208000037896 autoimmune cutaneous disease Diseases 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 208000024963 hair loss Diseases 0.000 description 1
- 230000003676 hair loss Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 230000011268 leukocyte chemotaxis Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 210000002856 peripheral neuron Anatomy 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 201000004335 respiratory allergy Diseases 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 231100000430 skin reaction Toxicity 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000003594 spinal ganglia Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- C07K2317/90—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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/91—Cell lines ; Processes using cell lines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
- G01N2800/202—Dermatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
技术领域technical field
本发明属于兽医、医药生物领域,具体涉及两株分泌犬IL-31单克隆抗体的杂交瘤细胞株及其应用。The invention belongs to the fields of veterinary medicine and medical biology, and in particular relates to two hybridoma cell lines secreting canine IL-31 monoclonal antibody and application thereof.
背景技术Background technique
特应性皮炎(Atopic dermatitis, AD)是一种引起皮肤过敏性瘙痒的炎症。在犬中,AD能够引起严重的瘙痒,并伴随着继发性脱发和红斑。全球约有450万只狗患有这种慢性终身疾病。而发病率也在逐年增加。Atopic dermatitis (AD) is an inflammation that causes allergic itching of the skin. In dogs, AD can cause severe pruritus with secondary alopecia and erythema. About 4.5 million dogs worldwide suffer from this chronic, lifelong condition. And the incidence rate is also increasing year by year.
白细胞介素-31 (IL-31)是2004年被克隆出来的细胞因子。它主要是由被激活T辅助细胞(Th2)产生。作为一种神经调节因子,在皮肤炎症和瘙痒中扮演重要角色。IL-31结合由IL-31受体α(IL-31RA)和肿瘤抑制素M受体β(OSMRβ)组成的异源二聚体,IL-31的信号能够激活三种重要的信号通路:Janus激酶(JAK)/转录激活因子(STAT),磷酸肌醇3-激酶(P13K)/AKT和丝裂原激酶(ERK)/MAPK,导致细胞发生生物学变化。这些变化包括免疫细胞趋化性、促炎细胞因子和趋化因子分泌,皮肤瘙痒反应,及部分由于细胞增殖、分化而导致的皮肤屏障破坏。IL-31的表达在AD患者中明显升高,能够诱导趋化因子CCL1,CCL17和CCL22。这些趋化因子招募更多的T细胞到受感染的皮肤,从而分泌更多的IL-31。研究者们已经发现IL-31RA/OSMRβ共受体在巨噬细胞、肥大细胞、嗜酸性粒细胞、嗜碱性粒细胞、角质细胞和外周神经元的背根神经节中都有表达[Gibbs BF, Patsinakidis N, Raap U. Roleof the pruritic cytokine IL-31in autoimmune skin diseases. Front Immunol.2019;10:1383.]。Interleukin-31 (IL-31) is a cytokine that was cloned in 2004. It is mainly produced by activated T helper cells (Th2). As a neuromodulator, it plays an important role in skin inflammation and pruritus. IL-31 binds to a heterodimer composed of IL-31 receptor α (IL-31RA) and tumor inhibin M receptor β (OSMRβ), and IL-31 signaling can activate three important signaling pathways: Janus Kinase (JAK)/activator of transcription (STAT), phosphoinositide 3-kinase (P13K)/AKT and mitogen kinase (ERK)/MAPK, lead to biological changes in cells. These changes include immune cell chemotaxis, secretion of pro-inflammatory cytokines and chemokines, pruritic skin responses, and skin barrier disruption due in part to cell proliferation and differentiation. The expression of IL-31 is significantly elevated in AD patients and can induce the chemokines CCL1, CCL17 and CCL22. These chemokines recruit more T cells to infected skin, which secrete more IL-31. Investigators have found that the IL-31RA/OSMRβ co-receptor is expressed in macrophages, mast cells, eosinophils, basophils, keratinocytes, and the dorsal root ganglia of peripheral neurons [Gibbs BF , Patsinakidis N, Raap U. Role of the pruritic cytokine IL-31 in autoimmune skin diseases. Front Immunol. 2019;10:1383.].
IL-31参与了皮炎、瘙痒性皮损、过敏和呼吸道过敏。IL-31在瘙痒性过敏皮肤部位产量增高,可以诱发各种物种,如老鼠、猴子和狗的抓挠行为。有研究表明,当IL-31通过皮内,皮下或静脉在犬中给药时能在几分钟到几小时内诱发强烈的瘙痒行为。过表达IL-31的转基因小鼠会出现皮肤炎症、瘙痒、严重皮炎和脱发。皮下注射IL-31会引起炎症细胞、中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞浸润,导致表皮增厚和真皮层增生。进一步研究表明IL-31与人的特应性皮炎引起的皮肤炎症和瘙痒相关[Lai T, Wu D, Li W, et al.Interleukin-31expression and relationto disease severity in human asthma. SciRep. 2016;6(1):22835.; West NR, Hegazy AN, Owens BMJ, et al. Oncostatin Mdrivesintestinal inflammation and predicts response to tumor necrosisfactor-neutralizingtherapy in patients with inflammatory boweldisease. Nat Med.2017;23(5):579–589.]。IL-31 is involved in dermatitis, pruritic lesions, allergies and respiratory allergies. Increased production of IL-31 at sites of pruritic allergic skin induces scratching behavior in various species such as mice, monkeys and dogs. Studies have shown that when IL-31 is administered intradermally, subcutaneously or intravenously in dogs, it can induce intense itching behavior within minutes to hours. Transgenic mice overexpressing IL-31 developed skin inflammation, pruritus, severe dermatitis, and hair loss. Subcutaneous injection of IL-31 induces infiltration of inflammatory cells, neutrophils, eosinophils, lymphocytes, and macrophages, resulting in thickening of the epidermis and hyperplasia of the dermis. Further studies have shown that IL-31 is related to skin inflammation and itching caused by human atopic dermatitis [Lai T, Wu D, Li W, et al. Interleukin-31 expression and relation to disease severity in human asthma. SciRep. 2016; 6( 1):22835.; West NR, Hegazy AN, Owens BMJ, et al. Oncostatin Mdrivesintestinal inflammation and predicts response to tumor necrosisfactor-neutralizingtherapy in patients with inflammatory bowel disease. Nat Med.2017;23(5):579–589.] .
目前,已有公司研制出针对犬IL-31引起的信号传导的治疗药物,如硕腾公司推出的JAK小分子抑制剂爱波克和犬IL-31单克隆抗体药物Cytopoint。然而这些针对IL-31信号传导的药物价格较为昂贵,需做到精准治疗,方可避免盲目使用,减少宠物持有者的支出费用。此外,有研究发现,将体外重组的IL-31作为疫苗,可少量多次的接种到动物体内,以此来预防特应性皮炎的发生[Olomski F, Fettelschoss V, Jonsdottir S, et al.Interleukin 31 in insect bite hypersensitivity-Alleviating clinical symptomsby active vaccination against itch. Allergy. 2020 Apr;75(4):862-871.]。IL-31引起的犬特应性皮炎的对症精准治疗,以及重组犬IL-31蛋白在表达系统中的产量确定等均需要配套相应的IL-31检测试剂。At present, some companies have developed therapeutic drugs targeting signal transduction caused by canine IL-31, such as the JAK small molecule inhibitor Aipoc and the canine IL-31 monoclonal antibody drug Cytopoint launched by Zoetis. However, these drugs targeting IL-31 signal transduction are relatively expensive, and precise treatment is required to avoid blind use and reduce the expenses of pet owners. In addition, studies have found that recombinant IL-31 in vitro can be used as a vaccine to prevent atopic dermatitis in small amounts and multiple times [Olomski F, Fettelschoss V, Jonsdottir S, et al.Interleukin 31 in insect bite hypersensitivity-Alleviating clinical symptoms by active vaccination against itch. Allergy. 2020 Apr;75(4):862-871.]. The symptomatic and precise treatment of canine atopic dermatitis caused by IL-31, as well as the determination of the production of recombinant canine IL-31 protein in the expression system, all require corresponding IL-31 detection reagents.
发明内容Contents of the invention
本发明第一个目的是提供了两株分泌犬IL-31的单克隆抗体的杂交瘤细胞株,分别为杂交瘤细胞株TIBGAO-2D102和杂交瘤细胞株TIBGAO-1C6。已于2022年5月26日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址为中国北京市朝阳区北辰西路1号院3号),其中杂交瘤细胞株TIBGAO-2D102保藏编号为CGMCC No.45175,杂交瘤细胞株TIBGAO-1C6保藏编号为CGMCC No.45174。The first object of the present invention is to provide two hybridoma cell lines secreting canine IL-31 monoclonal antibody, namely hybridoma cell line TIBGAO-2D102 and hybridoma cell line TIBGAO-1C6. On May 26, 2022, it was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China), and the hybridoma cell line TIBGAO-2D102 was preserved under the number CGMCC No.45175, and the deposit number of the hybridoma cell line TIBGAO-1C6 is CGMCC No.45174.
本发明第二个目的是提供了两株结合犬IL-31的单克隆抗体,2D102和1C6,这两株抗体分别由上述两株杂交瘤细胞株分泌产生。ELISA结果表明这两株单抗均可特异性结合犬IL-31蛋白。The second object of the present invention is to provide two monoclonal antibodies binding to canine IL-31, 2D102 and 1C6, which are secreted and produced by the above two hybridoma cell lines respectively. ELISA results showed that the two monoclonal antibodies could specifically bind to canine IL-31 protein.
本发明提供所述的单克隆抗体在犬IL-31引起的相关疾病检测或诊断试剂中的应用。The present invention provides the application of the monoclonal antibody in detection or diagnosis reagents for related diseases caused by canine IL-31.
具体地,所述犬IL-31相关疾病是有瘙痒、皮损、红斑、过敏为症状的特应性皮炎。优选地,所述的检测方法是双抗夹心ELISA检测方法。Specifically, the canine IL-31-related disease is atopic dermatitis with symptoms of itching, skin lesions, erythema, and allergies. Preferably, the detection method is a double-antibody sandwich ELISA detection method.
本发明还提供所述的单克隆抗体在制备检测犬IL-31蛋白制剂中的应用。The invention also provides the application of the monoclonal antibody in the preparation and detection of canine IL-31 protein preparation.
本发明的第五个目的是提供一种双抗夹心ELISA检测试剂盒,该试剂盒用于双抗夹心ELISA检测方法,其包含由杂交瘤细胞株TIBGAO-2D102分泌的结合犬IL-31的单克隆抗体2D102,以及生物素标记的由杂交瘤细胞株TIBGAO-1C6分泌的结合犬IL-31的单克隆抗体1C6。The fifth object of the present invention is to provide a double-antibody sandwich ELISA detection kit, which is used for the double-antibody sandwich ELISA detection method, which comprises a single antibody binding to canine IL-31 secreted by the hybridoma cell line TIBGAO-2D102 Cloned antibody 2D102, and biotin-labeled canine IL-31-binding monoclonal antibody 1C6 secreted by the hybridoma cell line TIBGAO-1C6.
具体地,是以杂交瘤细胞株TIBGAO-2D102分泌的单克隆抗体2D102为捕获抗体,以生物素标记、由杂交瘤细胞株TIBGAO-1C6分泌的单克隆抗体1C6为检测抗体。Specifically, the monoclonal antibody 2D102 secreted by the hybridoma cell line TIBGAO-2D102 was used as the capture antibody, and the biotin-labeled monoclonal antibody 1C6 secreted by the hybridoma cell line TIBGAO-1C6 was used as the detection antibody.
优选地,上述双抗夹心ELISA检测方法中,所述的捕获抗体是包被于酶标板上,所述捕获抗体的最佳包被量为2μg/孔,所述检测抗体的工作浓度为2μg/ml。Preferably, in the above double-antibody sandwich ELISA detection method, the capture antibody is coated on a microtiter plate, the optimal coating amount of the capture antibody is 2 μg/well, and the working concentration of the detection antibody is 2 μg /ml.
与现有技术相比,本发明具有如下有益效果:Compared with the prior art, the present invention has the following beneficial effects:
1、单克隆抗体2D102和1C6可与犬IL-31结合,相比于已经上市的结合犬IL-31的犬源化抗体Cytopoint具有更高的亲和力和更慢的解离速率,可应用于犬IL-31或与IL-31相关的疾病的诊断,以及犬IL-31蛋白在不同表达系统中产量的检测,以及犬IL-31的实验室检测。1. Monoclonal antibodies 2D102 and 1C6 can bind to canine IL-31, and have higher affinity and slower dissociation rate than Cytopoint, a caninized antibody that binds to canine IL-31 that has been marketed, and can be applied to canine Diagnosis of IL-31 or IL-31-related diseases, detection of canine IL-31 protein production in different expression systems, and laboratory detection of canine IL-31.
2、本发明的两株杂交瘤细胞株可以稳定分泌单克隆抗体,所分泌的两株抗体的配对效果较好,适用于双抗体夹心ELISA检测方法。2. The two hybridoma cell lines of the present invention can stably secrete monoclonal antibodies, and the pairing effect of the two secreted antibodies is better, which is suitable for the detection method of double-antibody sandwich ELISA.
3、本发明的两株杂交瘤细胞株所分泌的两株抗体应用双抗夹心ELISA不受犬血清的干扰,应用犬IL-31相关疾病的检测具有较高的灵敏度。3. The two antibodies secreted by the two hybridoma cell lines of the present invention are not interfered by canine serum by double-antibody sandwich ELISA, and have higher sensitivity in the detection of canine IL-31-related diseases.
4、本发明涉及到的小鼠杂交瘤细胞株及其分泌的单克隆抗体在犬IL-31检测试剂的研制,以及与犬IL31相关的疾病诊断试剂研制方面具有重要的应用前景。4. The mouse hybridoma cell line and the monoclonal antibody secreted by the present invention have important application prospects in the development of canine IL-31 detection reagents and the development of canine IL31-related disease diagnostic reagents.
5、本发明中的双抗夹心ELISA,具有操作简单,结果清晰等优点。5. The double-antibody sandwich ELISA of the present invention has the advantages of simple operation and clear results.
附图说明Description of drawings
图1 是本发明通过两株杂交瘤细胞(TIBGAO-2D102,TIBGAO-1C6)经腹水制备的抗体2D102(A)和1C6(B)分别经SuperdexTM200 10/30 GL分子筛层析纯化结果;Figure 1 shows the purification results of antibodies 2D102 (A) and 1C6 (B) prepared by two strains of hybridoma cells (TIBGAO-2D102, TIBGAO-1C6) through ascites, respectively, and purified by Superdex TM 200 10/30 GL molecular sieve chromatography;
图2 是本发明制备的两种抗体及犬源化抗体Cytopoint分别与重组犬和猫IL-31的ELISA结合图。其中,A为2D102、1C6、Cytopoint及无关对照抗体与重组犬IL-31蛋白的结合,B为2D102、1C6与无关对照抗体与重组猫IL-31蛋白的结合;Figure 2 is the ELISA binding diagrams of the two antibodies prepared by the present invention and the caninized antibody Cytopoint to recombinant canine and cat IL-31 respectively. Among them, A is the binding of 2D102, 1C6, Cytopoint and irrelevant control antibody to recombinant canine IL-31 protein, B is the binding of 2D102, 1C6 and irrelevant control antibody to recombinant cat IL-31 protein;
图3为本发明中两株杂交瘤细胞经小鼠腹水纯化得到的两种抗体2D102和1C6及商业化Cytopoint抗体经表面等离子共振的方法测定的与重组犬IL-31的亲和力。其中,A为三个抗体与重组犬IL-31的结合常数(ka);B为三个抗体与重组犬IL-31的解离常数(kd);C为三个抗体与重组犬IL-31的亲和力常数(KD)。所有结果都是三次试验数据的统计值。其中*代表p<0.05,**代表p<0.01,***代表p<0.001。Figure 3 shows the affinities of two antibodies 2D102 and 1C6 obtained from two strains of hybridoma cells purified from mouse ascites and commercialized Cytopoint antibodies to recombinant canine IL-31 determined by surface plasmon resonance. Among them, A is the association constant (ka) of the three antibodies and recombinant canine IL-31; B is the dissociation constant (kd) of the three antibodies and recombinant canine IL-31; C is the three antibodies and recombinant canine IL-31 affinity constant (KD). All results are statistical values of data from three experiments. Where * represents p<0.05, ** represents p<0.01, *** represents p<0.001.
图4是本发明中两株杂交瘤细胞经小鼠腹水纯化得到的两种抗体夹心ELISA结合图。其中,A显示2D102为固定抗体,生物素标记的1C6为检测抗体;B显示1C6为固定抗体,生物素标记的2D102为检测抗体;Fig. 4 is a sandwich ELISA combination diagram of two antibody hybridoma cells purified from mouse ascites in the present invention. Among them, A shows that 2D102 is an immobilized antibody, and biotin-labeled 1C6 is a detection antibody; B shows that 1C6 is an immobilized antibody, and biotin-labeled 2D102 is a detection antibody;
图5为本发明应用的双抗夹心ELISA方法检测重组犬IL-31的浓度。其中,A是用本发明中的双抗夹心ELISA方法检测用PBS稀释获得的不同浓度的重组犬IL-31后所作的标准曲线;B为本发明中的双抗夹心ELISA方法检测犬血清(已用商业化试剂盒检测重组犬IL-31为阴性)稀释的不同浓度的重组犬IL-31所作的标准曲线。Figure 5 shows the concentration of recombinant canine IL-31 detected by the double-antibody sandwich ELISA method used in the present invention. Wherein, A is the standard curve made after using the double-antibody sandwich ELISA method in the present invention to detect different concentrations of recombinant canine IL-31 obtained by dilution with PBS; B is the double-antibody sandwich ELISA method in the present invention to detect canine serum (already The standard curve made by different concentrations of recombinant canine IL-31 diluted with commercial kits (recombinant canine IL-31 was negative).
生物材料保藏信息:Biological material deposit information:
本发明的小鼠杂交瘤细胞株TIBGAO-1C6已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏单位简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101。保藏日期为2022年5月26日,保藏号为CGMCC No.45174;该菌株的分类命名:小鼠杂交瘤细胞。The mouse hybridoma cell line TIBGAO-1C6 of the present invention has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee. The preservation unit is referred to as CGMCC. Institute, zip code 100101. The preservation date is May 26, 2022, and the preservation number is CGMCC No.45174; the taxonomic designation of the strain: mouse hybridoma cell.
本发明的小鼠杂交瘤细胞株TIBGAO-2D102已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏单位简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101。保藏日期为2022年5月26日,保藏号为CGMCC No.45175;该菌株的分类命名:小鼠杂交瘤细胞。The mouse hybridoma cell line TIBGAO-2D102 of the present invention has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee. The preservation unit is referred to as CGMCC. Institute, zip code 100101. The preservation date is May 26, 2022, and the preservation number is CGMCC No.45175; the taxonomic designation of the strain: mouse hybridoma cell.
具体实施方式detailed description
为更进一步阐述本发明所采取的技术手段及其效果,以下结合附图并通过具体实施方式来进一步说明本发明的技术方案,实施例中所采用的所有试剂均为市场上可以购得的,或者可以按照文本或已知的方法制备。In order to further illustrate the technical means and effects thereof adopted by the present invention, the technical scheme of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods. All reagents adopted in the examples are available on the market. Alternatively it can be prepared according to the text or known methods.
实施例1 鼠单克隆抗体的制备及纯化Example 1 Preparation and purification of mouse monoclonal antibody
将纯化的重组犬IL-31蛋白作为抗原,分五次,皮下多点免疫6-8周的雌性BALB/c小鼠。免疫剂量为30μg/次,前四次免疫间隔时间为14天。首次免疫时,使用弗氏完全佐剂;第2-4次免疫时,使用弗氏不完全佐剂。在第3次免疫后第7天,取尾静脉血检测血清抗体效价,选取效价最高小鼠进行融合。在融合前3天进行冲刺免疫,即第五次免疫,此次是将100μg免疫原液注入小鼠腹腔。取该小鼠的脾细胞与骨髓瘤细胞SP2/0按10:1比进行融合,通过HAT筛选培养基选择培养,用有限稀释法获得杂交瘤细胞株。将重组犬IL-31利用pH9.6的碳酸盐缓冲液中包被在酶标板中,25 ng/孔,利用ELISA的方法筛选与抗原结合能力较高的单克隆抗体细胞株共15株,将这15株抗体进行竞争ELISA配对,得到具有竞争结合犬IL-31的两株单克隆抗体,即2D102和1C6。Purified recombinant canine IL-31 protein was used as antigen, and subcutaneously immunized female BALB/c mice of 6-8 weeks in five times. The immunization dose was 30 μg/time, and the interval between the first four immunizations was 14 days. For the first immunization, Freund's complete adjuvant was used; for the second to fourth immunization, Freund's incomplete adjuvant was used. On the seventh day after the third immunization, the tail vein blood was taken to detect the serum antibody titer, and the mouse with the highest titer was selected for fusion. Sprint immunization was performed 3 days before fusion, that is, the fifth immunization, and this
利用退役BALB/c小鼠进行单克隆抗体的制备。首先,腹腔注射腹水专用佐剂,0.4ml/只。在接种佐剂后的10-14天,腹腔注射杂交瘤细胞,接种剂量是30万细胞/只。在注射细胞后的10-15天,收集小鼠的腹水,通过Protein A进行IgG的富集纯化,而后通过SuperdexTM200 10/300 GL分子筛进一步纯化,获得形式均一的2D102和1C6的IgG蛋白,分子筛显示IgG在16-17 ml出峰(图1中A,B)。SDS-PAGE显示,这两株抗体在还原条件(loadingbuffer中加入二硫苏糖醇,+DTT)的情况下约为50 kDa大小的的重链和25 kDa大小的轻链(图1中 A,B)。在非还原条件(loading buffer中加入不加DTT,-DTT)下,重链和轻链由二硫键连接形成一个四肽链分子异源二聚体,即IgG,分子量在150 kDa(图1中A,B)。Production of monoclonal antibodies was performed using decommissioned BALB/c mice. First, intraperitoneal injection of special adjuvant for ascites, 0.4ml/cause. 10-14 days after the inoculation of the adjuvant, the hybridoma cells were injected intraperitoneally, and the inoculation dose was 300,000 cells/monkey. 10-15 days after the injection of the cells, the ascites of the mice was collected, and IgG was enriched and purified by Protein A, and then further purified by Superdex TM 200 10/300 GL molecular sieves to obtain IgG proteins of uniform 2D102 and 1C6, Molecular sieves showed that IgG peaked at 16-17 ml (A, B in Figure 1). SDS-PAGE showed that the heavy chain of the two antibodies was about 50 kDa in size and the light chain of 25 kDa in size (A in Figure 1, B). Under non-reducing conditions (without adding DTT, -DTT in the loading buffer), the heavy chain and the light chain are connected by disulfide bonds to form a heterodimer of tetrapeptide chain molecules, namely IgG, with a molecular weight of 150 kDa (Figure 1 in A, B).
实施例2 单克隆抗体与重组犬IL-31和重组猫IL-31的结合活性测定Example 2 Determination of the binding activity of monoclonal antibodies to recombinant canine IL-31 and recombinant cat IL-31
利用间接ELISA的方法将纯化的重组犬IL-31和重组猫IL-31-Fc融合蛋白分别利用pH9.6的碳酸盐包被液固定在96孔ELISA板上,200 ng/孔,4℃过夜。接着将酶标板放置于室温,摇床30 min,使其恢复到室温,弃去酶标板中的液体,用PBS洗三次,每次5 min。而后,加入封闭液(PBS+2% BSA),摇床孵育1 h。用PBST(PBS +0.05% Tween-20)将96孔酶标板洗三次,每次5 min。随后,加入不同稀释浓度的单克隆抗体,100 μl/孔,室温,摇床振荡1 h。用PBST将酶标板洗三次,每次5 min。继而加入1:5000稀释的HRP标记的羊抗鼠IgG或者兔抗狗IgG,室温,摇床振荡1 h。用PBST将酶标板洗三次后加入TMB,显色5 min左右,而后加入2M的H2SO4终止反应。用酶标仪在450 nm波长下读数。结果显示,2D102与重组犬IL-31可特异性的结合,EC50为0.01 μg/ml,1C6与重组犬IL-31蛋白可特异性结合,EC50为0.02 μg/ml,Cytopoint与重组犬IL-31蛋白EC50为0.14 μg/ml(图2中A)。而2D102和1C6均与重组猫IL-31-Fc融合蛋白无结合,(图2中B)。结果表明这两株杂交瘤细胞株分泌的抗体能特异性的结合重组犬IL-31蛋白,结合力高于商业化抗体Cytopoint。Purified recombinant canine IL-31 and recombinant feline IL-31-Fc fusion protein were immobilized on 96-well ELISA plates with pH 9.6 carbonate coating solution by indirect ELISA method, 200 ng/well, 4°C overnight. Then place the microplate at room temperature and shake it for 30 minutes to return to room temperature. Discard the liquid in the microplate and wash it three times with PBS for 5 minutes each time. Then, add blocking solution (PBS+2% BSA), and incubate on a shaking table for 1 h. Wash the 96-well ELISA plate three times with PBST (PBS +0.05% Tween-20), 5 min each time. Subsequently, monoclonal antibodies of different dilution concentrations were added, 100 μl/well, at room temperature, shaken for 1 h. The plate was washed three times with PBST, 5 min each time. Then add 1:5000 dilution of HRP-labeled goat anti-mouse IgG or rabbit anti-dog IgG, shake at room temperature for 1 h. Wash the ELISA plate three times with PBST, add TMB, develop color for about 5 min, and then add 2M H 2 SO 4 to terminate the reaction. Read with a microplate reader at a wavelength of 450 nm. The results showed that 2D102 could specifically bind to recombinant canine IL-31 with an EC 50 of 0.01 μg/ml, 1C6 could specifically bind to recombinant canine IL-31 protein with an EC 50 of 0.02 μg/ml, and Cytopoint could specifically bind to recombinant canine IL-31 The EC 50 of -31 protein was 0.14 μg/ml (A in Figure 2). However, both 2D102 and 1C6 had no binding to the recombinant cat IL-31-Fc fusion protein (B in Figure 2). The results showed that the antibodies secreted by the two hybridoma cell lines could specifically bind to the recombinant canine IL-31 protein, and the binding force was higher than that of the commercial antibody Cytopoint.
实施例3单克隆抗体与重组犬IL-31的结合动力学测定Example 3 The binding kinetics determination of monoclonal antibody and recombinant canine IL-31
采用表面等离子共振的方法测定抗体与抗原结合的动力学参数。首先,在CM5芯片表面通过氨基偶联的方式固定SA,然后将生物素标记的重组犬IL-31通过与SA结合固定在芯片上。而后,将固定的重组犬IL-31蛋白与作为流动相的、不同浓度的2D102、1C6和Cytopoint抗体结合。结果如图3和表1所示,2D102、1C6、Cytopoint的结合常数(ka)接近,分别为1.27±(0.02)×105 M-1s-1、1.64±(0.41)×105M-1s-1和1.78±(0.89)×105M-1s-1。2D102和1C6与重组犬IL-31结合解离的动力学中,解离常数(kd)分别为8.58±(6.18)×10-7s-1和2.93±(1.42)×10-6s-1。Cytopoint抗体的解离常数为1.22±(0.45)×10-4s-1。商业化抗体Cytopoint与抗原结合后再解离的速率与2D102、1C6相比,有显著意义差异(p<0.05),说明Cytopoint与抗原结合后的解离速度要比本发明中的两个抗体快。2D102、1C6和Cytopoint与重组犬IL-31的亲和力常数(KD)分别为6.81±(4.96)×10-12 M、2.80±(1.55)×10-11M和8.28±(3.01)×10-11 M。2D102与重组犬IL-31的亲和力KD值与商业化抗体Cytopoint相比,有显著意义差异(p<0.05),说明该抗体与抗原结合的亲和力高于Cytopoint。The kinetic parameters of antibody-antigen binding were determined by surface plasmon resonance. First, SA was immobilized on the surface of the CM5 chip by amino coupling, and then biotin-labeled recombinant canine IL-31 was immobilized on the chip by combining with SA. Then, the immobilized recombinant canine IL-31 protein was combined with different concentrations of 2D102, 1C6 and Cytopoint antibodies as the mobile phase. The results are shown in Figure 3 and Table 1. The binding constants (ka) of 2D102, 1C6, and Cytopoint are close, being 1.27±(0.02)×10 5 M -1 s -1 , 1.64±(0.41)×10 5 M -1 , respectively . 1 s −1 and 1.78±(0.89)×10 5 M −1 s −1 . The dissociation constants (kd) of 2D102 and 1C6 binding to recombinant canine IL-31 were 8.58±(6.18)×10 -7 s -1 and 2.93±(1.42)×10 -6 s -1 respectively . The dissociation constant of Cytopoint antibody is 1.22±(0.45)×10 -4 s -1 . Compared with 2D102 and 1C6, the dissociation rate of the commercial antibody Cytopoint after binding to the antigen is significantly different (p<0.05), indicating that the dissociation rate of Cytopoint after binding to the antigen is faster than the two antibodies in the present invention . The affinity constants (KD) of 2D102, 1C6 and Cytopoint to recombinant canine IL-31 are 6.81±(4.96)×10 -12 M, 2.80±(1.55)×10 -11 M and 8.28±(3.01)×10 -11 , respectively M. Compared with the commercial antibody Cytopoint, the KD value of the affinity between 2D102 and recombinant canine IL-31 is significantly different (p<0.05), indicating that the antibody has a higher affinity for antigen binding than Cytopoint.
表1 三次试验ka、kd和KD数据统计后的具体数值Table 1 The specific values of ka, kd and KD after three experiments
该结果表明2D102和1C6与重组犬IL-31是快结合、慢解离的模式,与商业化抗体Cytopoint相比,在解离速率方面具有较高的优势。并且2D102相比于Cytopoint具有更高的亲和力。The results indicate that 2D102 and 1C6 bind to recombinant canine IL-31 in a fast binding and slow dissociation mode, which has a higher dissociation rate compared with the commercial antibody Cytopoint. And 2D102 has a higher affinity than Cytopoint.
实施例4双抗夹心ELISA方法的建立The establishment of
在96孔的酶标板上经pH9.6的碳酸盐包被液固定一株单克隆抗体,经验证,捕获抗体最佳固定量为2 μg/孔,4℃过夜。接着将酶标板放置于室温,摇床30 min,使其恢复到室温,弃去ELISA板中的液体,用PBS洗三次,每次5 min。而后,加入封闭液(PBS+2% BSA),摇床孵育1 h。用PBST将96孔板洗三次,每次5 min。随后,以2 μg/ml的重组犬IL-31作为检测抗原加入到酶标板中,100 μl/孔,室温,摇床振荡1 h。用PBST将ELISA板洗三次,每次5 min。继而加入不同浓度梯度的生物素标记的另一株单克隆抗体,100μl/孔,室温,摇床振荡1 h。用PBST将酶标板洗三次,每次5 min。然后加入1:10000稀释的HRP标记的链霉亲和素(streptavidin),室温,摇床振荡1 h。用PBST将酶标板洗三次后每孔50 μl加入TMB,显色5min左右,而后每孔50 μl加入2 M的H2SO4终止反应。用酶标仪在450 nm波长下读数。结果显示,当2D102为捕获抗体,生物素标记的1C6为检测抗体时,随着1C6浓度的增加,OD450 的值增加(图4中A)。同样,以1C6为捕获抗体,生物素标记的2D102为检测抗体时,随着2D102的浓度增加,OD450的值在增加(图4中B)。这些结果表明2D102和1C6这两株单克隆抗体与犬IL-31的结合表位不一样,这两个抗体之间不存在竞争结合犬IL-31的关系。根据双抗夹心ELISA的检测结果,也确定了这两个配对抗体的最适工作浓度,即捕获抗体的最佳包被量为2 μg/孔,生物素标记的检测抗体的最适工作浓度为2 μg/ml。A monoclonal antibody was immobilized on a 96-well ELISA plate with pH 9.6 carbonate coating solution. It was verified that the optimal amount of capture antibody immobilized was 2 μg/well, overnight at 4°C. Then place the ELISA plate at room temperature and shake it for 30 min to return to room temperature, discard the liquid in the ELISA plate, and wash with PBS three times for 5 min each time. Then, add blocking solution (PBS+2% BSA), and incubate on a shaking table for 1 h. The 96-well plate was washed three times with PBST, 5 min each time. Subsequently, 2 μg/ml recombinant canine IL-31 was added to the microtiter plate as the detection antigen, 100 μl/well, at room temperature, shaken for 1 h. Wash the ELISA plate three times with PBST, 5 min each time. Then add another strain of monoclonal antibody labeled with biotin in different concentration gradients, 100 μl/well, shake at room temperature for 1 h on a shaker. The plate was washed three times with PBST, 5 min each time. Then add 1:10000 diluted HRP-labeled streptavidin (streptavidin), shake at room temperature for 1 h. After washing the ELISA plate three times with PBST, 50 μl of TMB was added to each well, and the color was developed for about 5 minutes, and then 50 μl of 2 M H 2 SO 4 was added to each well to terminate the reaction. Read with a microplate reader at a wavelength of 450 nm. The results showed that when 2D102 was used as the capture antibody and biotin-labeled 1C6 was used as the detection antibody, the OD450 value increased as the concentration of 1C6 increased (A in Figure 4). Similarly, when 1C6 was used as the capture antibody and biotinylated 2D102 was used as the detection antibody, the OD450 value increased as the concentration of 2D102 increased (B in Figure 4). These results indicated that the two monoclonal antibodies 2D102 and 1C6 had different binding epitopes to canine IL-31, and there was no competition between the two antibodies for binding to canine IL-31. According to the detection results of the double-antibody sandwich ELISA, the optimal working concentration of the two paired antibodies was also determined, that is, the optimal coating amount of the capture antibody was 2 μg/well, and the optimal working concentration of the biotin-labeled detection antibody was 2 μg/ml.
实施例5双抗夹心ELISA检出线的确定Determination of the detection line of the double-antibody sandwich ELISA of embodiment 5
在该实施例中,利用实施例3中确定的双抗夹心ELISA方法来测定该方法检测犬IL-31的最低检出值。首先,用包被液固定捕获抗体2D102,2 μg/孔,4℃过夜。而后,用封闭液封闭1 h。接着,加入用PBS稀释的不同浓度的重组犬IL-31蛋白,分别是25.6 ng/ml,12.8ng/ml,6.4 ng/ml,3.2 ng/ml,1.6 ng/ml, 0.8 ng/ml,0.4 ng/ml,0.2 ng/ml。100μl/孔,室温,摇床振荡1 h。用PBST将ELISA板洗三次,每次5 min。继而加入2 μg/ml生物素标记的1C6检测抗体,100μl/孔,室温,摇床振荡1 h。用PBST将酶标板洗三次,每次5 min。然后加入1:10000稀释的HRP标记的streptavidin,室温,摇床振荡1 h。用PBST将酶标板洗三次后每孔加入100 μl TMB,显色5 min左右,而后每孔加入100 μl H2SO4(2 M)终止反应。用酶标仪在450 nm波长下读数。结果显示,犬IL-31蛋白在0.2~25.6 ng/ml的浓度区间时,该发明建立的双抗夹心ELISA方法的检出结果同犬IL-31蛋白的浓度呈线性关系,以此绘制标准曲线,标准曲线方程为y=-0.0037x2+0.1891x+0.0731,R2=0.9973。最低检出的犬IL-31的浓度为400 pg/ml(OD450值为0.13)。In this example, the double-antibody sandwich ELISA method determined in Example 3 was used to determine the minimum detection value of the method for detecting canine IL-31. First, capture antibody 2D102 was immobilized with coating solution, 2 μg/well, overnight at 4°C. Then, block with blocking solution for 1 h. Next, different concentrations of recombinant canine IL-31 protein diluted with PBS were added, which were 25.6 ng/ml, 12.8 ng/ml, 6.4 ng/ml, 3.2 ng/ml, 1.6 ng/ml, 0.8 ng/ml, 0.4 ng/ml, 0.2 ng/ml. 100 μl/well, shake for 1 h at room temperature. Wash the ELISA plate three times with PBST, 5 min each time. Then add 2 μg/ml biotin-labeled 1C6 detection antibody, 100 μl/well, shake at room temperature for 1 h on a shaker. The plate was washed three times with PBST, 5 min each time. Then add 1:10000 diluted HRP-labeled streptavidin, shake at room temperature for 1 h. After washing the ELISA plate three times with PBST, add 100 μl TMB to each well, develop color for about 5 minutes, and then add 100 μl H 2 SO 4 (2 M) to each well to terminate the reaction. Read with a microplate reader at a wavelength of 450 nm. The results show that when the concentration of canine IL-31 protein is in the range of 0.2-25.6 ng/ml, the detection result of the double-antibody sandwich ELISA method established by the invention has a linear relationship with the concentration of canine IL-31 protein, and the standard curve is drawn accordingly , the standard curve equation is y=-0.0037x 2 +0.1891x+0.0731, R 2 =0.9973. The lowest detected concentration of canine IL-31 was 400 pg/ml (OD450 value 0.13).
为确定该发明建立的双抗夹心ELISA方法在测定临床犬血清学样本时有无血清本身造成的背景或干扰,该发明中使用犬的血清(通过商业化试剂盒检测为重组犬IL-31阴性),而非PBS缓冲液,作为稀释液,加入了终浓度分别为25 ng/ ml,12.5 ng/ml,6.25 ng/ml,3.125 ng/ml,1.56 ng/ml,0.78 ng/ml,0.39 ng/ml的重组犬IL-31蛋白。而后,采用该发明建立的双抗夹心ELISA方法测定了这些样品的响应值。结果如图5中B所示,该发明建立的双抗夹心ELISA方法的检出结果同用犬血清稀释的重组犬IL-31蛋白的浓度仍呈线性关系,所作标准曲线方程为y=-0.0044x2+0.2063x+0.1097,R2=0.9918。最低检出的重组犬IL-31的浓度为390.6 pg/ml (OD450值为0.1)。该结果表明,使用犬血清作为稀释液得到的结果(图5中B)与PBS作为稀释液的结果(图5中A)一致,证明该发明建立的双抗夹心ELISA方法不受犬血清的干扰,具有较好灵敏度。In order to determine whether the double-antibody sandwich ELISA method established by this invention has background or interference caused by the serum itself in the determination of clinical canine serological samples, the canine serum (detected as recombinant canine IL-31 negative by commercial kits) is used in this invention ), instead of PBS buffer, as a diluent, the final concentrations were 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.125 ng/ml, 1.56 ng/ml, 0.78 ng/ml, 0.39 ng /ml of recombinant canine IL-31 protein. Then, the response values of these samples were determined by using the double-antibody sandwich ELISA method established by the invention. As a result, as shown in Figure 5, B, the detection result of the double-antibody sandwich ELISA method established by the invention is still in a linear relationship with the concentration of the recombinant canine IL-31 protein diluted with canine serum, and the standard curve equation is y=-0.0044 x 2 +0.2063x+0.1097, R 2 =0.9918. The lowest detected concentration of recombinant canine IL-31 was 390.6 pg/ml (OD450 value 0.1). The results show that the results obtained using canine serum as the diluent (B in Figure 5) are consistent with the results obtained with PBS as the diluent (A in Figure 5), proving that the double-antibody sandwich ELISA method established by this invention is not interfered by canine serum , with better sensitivity.
Claims (14)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211463230.2A CN115505575B (en) | 2022-11-16 | 2022-11-16 | Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211463230.2A CN115505575B (en) | 2022-11-16 | 2022-11-16 | Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115505575A true CN115505575A (en) | 2022-12-23 |
CN115505575B CN115505575B (en) | 2023-03-24 |
Family
ID=84514554
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211463230.2A Active CN115505575B (en) | 2022-11-16 | 2022-11-16 | Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115505575B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101522713A (en) * | 2006-09-01 | 2009-09-02 | 津莫吉尼蒂克斯公司 | Variable region sequences of IL-31 monoclonal antibodies and methods of use |
CN103890009A (en) * | 2011-07-21 | 2014-06-25 | 佐蒂斯有限责任公司 | Interleukin-31 monoclonal antibody |
CN110563844A (en) * | 2019-09-04 | 2019-12-13 | 华中农业大学 | Polyclonal antibody against canine interleukin 31 receptor and application thereof |
CN114829396A (en) * | 2019-12-20 | 2022-07-29 | 英特维特国际股份有限公司 | Bispecific caninized antibodies and bispecific binding partners for the treatment of atopic dermatitis |
-
2022
- 2022-11-16 CN CN202211463230.2A patent/CN115505575B/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101522713A (en) * | 2006-09-01 | 2009-09-02 | 津莫吉尼蒂克斯公司 | Variable region sequences of IL-31 monoclonal antibodies and methods of use |
CN103044550A (en) * | 2006-09-01 | 2013-04-17 | 津莫吉尼蒂克斯公司 | IL-31 monoclonal antibodies and methods of use |
CN103890009A (en) * | 2011-07-21 | 2014-06-25 | 佐蒂斯有限责任公司 | Interleukin-31 monoclonal antibody |
CN110563844A (en) * | 2019-09-04 | 2019-12-13 | 华中农业大学 | Polyclonal antibody against canine interleukin 31 receptor and application thereof |
CN114829396A (en) * | 2019-12-20 | 2022-07-29 | 英特维特国际股份有限公司 | Bispecific caninized antibodies and bispecific binding partners for the treatment of atopic dermatitis |
CN114829397A (en) * | 2019-12-20 | 2022-07-29 | 英特维特国际股份有限公司 | Bispecific caninized antibodies for the treatment of atopic dermatitis |
Non-Patent Citations (3)
Title |
---|
SOHEI OYAM: "Cynomolgus monkey model of interleukin-31-induced scratching depicts blockade of human interleukin-31 receptor A by a humanized monoclonal antibody", 《 EXPERIMENTAL DERMATOLOGY》 * |
YUXIN ZHENG: "Development and characterization of a novel mouse anti-canine oncostatin M receptor beta monoclonal antibody", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
向娟: "IL-31及其受体在特应性皮炎发病中的作用", 《国际皮肤性病学杂质》 * |
Also Published As
Publication number | Publication date |
---|---|
CN115505575B (en) | 2023-03-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2016203590B2 (en) | Interleukin-31 monoclonal antibody | |
US7087396B2 (en) | Monoclonal antibody and method and kit for immunoassay of soluble human ST2 | |
CN110616192B (en) | Monoclonal antibody of anti-human neurofilament light chain (NEFL) and application thereof | |
KR20140047628A (en) | Assays for detecting neutralizing autoantibodies to biologic therapy with tnf alpha | |
JPS6357599A (en) | Canine heart filaria vaccine and diagnostic test | |
KR20150088890A (en) | Assays for detecting neutralizing autoantibodies to biologic therapy | |
CN112142841A (en) | A novel anti-human IL-17A monoclonal antibody, a kit comprising the same and a detection method thereof | |
CN111004324A (en) | Calprotectin monoclonal antibody and application thereof | |
CN101062949B (en) | Recombinant anti human IgE monoclonal antibody and preparation method and usage thereof | |
CN115505575B (en) | Two hybridoma cell lines secreting canine IL-31 monoclonal antibody and their application | |
CN118388645B (en) | Anti-human interleukin-4 antibody and its application | |
CN115925963B (en) | Bispecific antibodies that bind HBP | |
CN116813765B (en) | Specific antibody for Rab8 protein and preparation method and application thereof | |
CA2924405C (en) | Biomarkers for tuberculosis | |
CN118362735A (en) | A dust mite specific IgE antibody detection kit | |
CN117720657A (en) | Anti-human interleukin 12P70 antibody and application thereof | |
CN117986360B (en) | Specific antibody of IL18 protein and preparation method and application thereof | |
CN117327186B (en) | Bispecific antibodies that bind MMP3 proteins and uses thereof | |
CN116063482B (en) | C-reactive protein (CRP) urine detection kit | |
CN116179497A (en) | Monoclonal antibody against human IL-17A protein and application thereof | |
Lee et al. | Detection of Anti‐Cytokine Autoantibodies and Clinical Applications | |
CN120157758A (en) | Monoclonal antibody against Porphyromonas gingivalis FimA and its application | |
CN116926016A (en) | Hybridoma cell strain secreting canine OSMR beta monoclonal antibody and application thereof | |
CN119331094A (en) | Anti-CD25 monoclonal antibody, preparation method and application thereof | |
CN119874914A (en) | Antibody A3 for resisting programmed death receptor 1 and application 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 |