CN117106091A - Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit - Google Patents
Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit Download PDFInfo
- Publication number
- CN117106091A CN117106091A CN202310893824.5A CN202310893824A CN117106091A CN 117106091 A CN117106091 A CN 117106091A CN 202310893824 A CN202310893824 A CN 202310893824A CN 117106091 A CN117106091 A CN 117106091A
- Authority
- CN
- China
- Prior art keywords
- antibody
- seq
- monoclonal antibody
- amino acid
- acid sequence
- 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.)
- Pending
Links
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 37
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 37
- 239000013598 vector Substances 0.000 claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 11
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 33
- 238000004113 cell culture Methods 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 4
- 239000013599 cloning vector Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000000427 antigen Substances 0.000 description 41
- 108091007433 antigens Proteins 0.000 description 40
- 102000036639 antigens Human genes 0.000 description 40
- 239000003153 chemical reaction reagent Substances 0.000 description 31
- 238000001514 detection method Methods 0.000 description 28
- 230000027455 binding Effects 0.000 description 26
- 238000009739 binding Methods 0.000 description 26
- 239000000243 solution Substances 0.000 description 24
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 21
- 239000012634 fragment Substances 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 13
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 13
- 229920001184 polypeptide Polymers 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 12
- 241001529936 Murinae Species 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 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 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 229940027941 immunoglobulin g Drugs 0.000 description 10
- 239000004816 latex Substances 0.000 description 10
- 229920000126 latex Polymers 0.000 description 10
- 238000006467 substitution reaction Methods 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 9
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 239000004471 Glycine Substances 0.000 description 6
- 206010070834 Sensitisation Diseases 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 230000008313 sensitization Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229910001868 water Inorganic materials 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 108091006027 G proteins Proteins 0.000 description 3
- 102000030782 GTP binding Human genes 0.000 description 3
- 108091000058 GTP-Binding Proteins 0.000 description 3
- 241000581650 Ivesia Species 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000012089 stop solution Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 208000017667 Chronic Disease Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000012409 standard PCR amplification Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101000763311 Mus musculus Thrombomodulin Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000006334 disulfide bridging Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- -1 probody Proteins 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Reproductive Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides an anti-human immunoglobulin G4 monoclonal antibody, a preparation method, an isolated nucleic acid molecule, a vector and a kit, wherein the monoclonal antibody is an antibody 5B11; or a mixture of antibody 5B11 and antibody 33B 3; antibody 5B11 comprises VH CDR1, VH CDR2 and VH CDR3 with amino acid sequences shown as SEQ ID NO.2-4 and VL CDR1, VL CDR2 and VL CDR3 with amino acid sequences shown as SEQ ID NO. 5-7; antibody 33B3 comprises the VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 shown in SEQ ID NO.10-12 and SEQ ID NO.13-15, respectively. The monoclonal antibody provided by the invention has high affinity and high specificity.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to the field of antibodies and in vitro detection. In particular to an anti-human immunoglobulin G4 monoclonal antibody, a preparation method, an isolated nucleic acid molecule, a vector and a kit.
Background
Immunoglobulin G (IgG) is the highest content immunoglobulin in human serum, and generally accounts for about 75-80% of total immunoglobulin, and is the main antibody component in blood and extracellular fluid, and plays an important role in immunity in human body. IgG is divided into four subtypes, igG1 about 60%, igG2 about 25%, igG3 about 10% and IgG4 about 5%. The distinction between IgG subtypes is reflected in several important biological functions, such as antigen recognition, complement activation, and binding to cell surface receptors.
IgG4 molecules are the most popular topics in the world biology world at present, particularly the research and development of diagnostic reagents and antibodies of various subtypes play a positive role in the treatment of various common diseases and difficulties, and are highly valued in the world, and a large amount of funds are invested for research and development. IgG4 responds to chronic diseases and has anti-inflammatory activity, and its unique biological characteristics make it easy to use for the development of therapeutic antibodies that do not require effector functions. Therapeutic antibodies have now made significant progress in the treatment of cancer, tumors, autoimmune diseases, chronic diseases, infectious diseases, allergic diseases and other diseases.
The problems are: in the domestic research on IgG4 antibodies, the quantity of monoclonal antibodies is small, and the suitability of the monoclonal antibodies for detecting the concentration of IgG4 in human serum is poor.
Disclosure of Invention
The object of the present invention is to provide a monoclonal antibody against human immunoglobulin G4 with high affinity and high specificity, which is a murine antibody with excellent properties, capable of specifically recognizing and binding to immunoglobulin G4 in human serum.
In one aspect, the invention provides an anti-human immunoglobulin G4 monoclonal antibody, which is antibody 5B11; or a mixture of antibody 5B11 and antibody 33B 3;
Antibody 5B11 comprises VH CDR1 shown in SEQ ID No.2, VH CDR2 shown in SEQ ID No.3, VH CDR3 shown in SEQ ID No.4, VL CDR1 shown in SEQ ID No.5, VL CDR2 shown in SEQ ID No.6, and VL CDR3 shown in SEQ ID No. 7;
antibody 33B3 comprises a VH CDR1 shown in SEQ ID NO.10, a VH CDR2 shown in SEQ ID NO.11, a VH CDR3 shown in SEQ ID NO.12, a VL CDR1 shown in SEQ ID NO.13, a VL CDR2 shown in SEQ ID NO.14 and a VL CDR3 shown in SEQ ID NO. 15.
Further, antibody 5B11 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of antibody 5B11 is shown as SEQ ID NO.8, and the amino acid sequence of the light chain variable region of antibody 5B11 is shown as SEQ ID NO. 9.
Further, the antibody 33B3 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of the antibody 33B3 is shown as SEQ ID NO.16, and the amino acid sequence of the light chain variable region of the antibody 33B3 is shown as SEQ ID NO. 17.
In a preferred embodiment, the 3 CDRs contained in the heavy chain variable region and/or the 3 CDRs contained in the light chain variable region are defined by the Chothia numbering system.
Further, the monoclonal antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody; the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab') 2, fv, disulfide-linked Fv, scFv, diabody (diabody) or single domain antibody (sdAb).
Further, the monoclonal antibody further comprises: a heavy chain constant region (CH) of a mammalian immunoglobulin or variant 1 thereof, variant 1 having a substitution, deletion or addition of one or more amino acids compared to the sequence from which it is derived; and, a light chain constant region (CL) of a mammalian immunoglobulin or variant 2 thereof, variant 2 having up to 20 amino acid conservative substitutions compared to the sequence from which it is derived.
In the present invention, a monoclonal antibody may include variant 3, which differs from the antibody from which it is derived only by conservative substitutions of one or more amino acid residues; for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody from which it is derived, and substantially retains the above-described biological function of the antibody from which it is derived.
Further, the heavy chain constant region of the monoclonal antibody is an IgG heavy chain constant region, and the light chain constant region of the monoclonal antibody is a kappa light chain constant region.
Further, the monoclonal antibody is labeled.
The antibody labeling aims to link a label to an antibody, specifically react with an object to be detected to form a multi-component compound, and directly microscopic observation or automatic measurement of test results are carried out by means of a fluorescent microscope, a ray measuring instrument, an enzyme-labeled detector, an electron microscope, a light-emitting immunoassay instrument and other precise instruments. The label in this embodiment is not particularly limited to a certain labeling mode, and specifically, may be an enzyme label, a biotinylation label, a fluorescent label, a colloidal gold label, or the like, and is not limited thereto.
In another aspect, the invention provides an isolated nucleic acid molecule for encoding a monoclonal antibody.
In another aspect, the invention also provides a vector comprising the isolated nucleic acid molecule described above.
Further, the vector is a cloning vector or an expression vector. In a preferred embodiment, the vector of the invention is, for example, a plasmid, a cosmid, a phage, or the like. In a preferred embodiment, the vector is capable of expressing the antibody in vivo in a subject, e.g., a mammal.
In another aspect, the invention also provides a host cell comprising an isolated nucleic acid molecule or vector. Host cells include, but are not limited to: prokaryotic cells such as E.coli cells, eukaryotic cells such as yeast cells, insect cells, plant cells, animal cells such as mammalian cells, and the like. In a preferred embodiment, the host cell of the invention is a mammalian cell, such as CHO, including in particular CHO-K1, CHO-S and CHO DG44.
In another aspect, the present invention also provides a method for preparing an anti-human immunoglobulin G4 monoclonal antibody, comprising: culturing a host cell comprising a vector under conditions that allow expression of the monoclonal antibody; monoclonal antibodies are recovered from the cultured host cell culture.
On the other hand, the invention also provides a kit for detecting the human immunoglobulin G4, which comprises the monoclonal antibody.
In one embodiment, the kit comprises a first mab and a second mab; the first monoclonal antibody and the second monoclonal antibody are matched with a detection sample. Wherein the first mab comprises: VH CDR1 shown in SEQ ID No.2, VH CDR2 shown in SEQ ID No.3, VH CDR3 shown in SEQ ID No. 4; and VL CDR1 shown in SEQ ID NO.5, VL CDR2 shown in SEQ ID NO.6, and VL CDR3 shown in SEQ ID NO. 7. The second mab comprises: VH CDR1 shown in SEQ ID No.10, VH CDR2 shown in SEQ ID No.11, VH CDR3 shown in SEQ ID No. 12; and VL CDR1 shown in SEQ ID NO.13, VL CDR2 shown in SEQ ID NO.14, VL CDR3 shown in SEQ ID NO. 15.
In another embodiment, the kit comprises a first mab or a second mab. The primary and secondary antibodies may also be used alone to detect IgG4.
In another aspect, the invention also provides a method for using the kit, comprising the following steps: diluting a tested sample by using a reagent R1, and adding a reagent R2 for reaction; then using a biochemical analyzer to determine the absorbance at 570 nm; and preparing a standard curve by using the IgG4 standard substance, and calculating the content of IgG4 in the tested sample according to the standard curve and the absorbance value.
Wherein, the reagent R1 is a common buffer solution, which can be 0.05mM Tris buffer solution with pH of 7.4, and the preparation method is as follows: taking 6.057g of Tris; adding 800mL of deionized water, and adding concentrated hydrochloric acid to adjust the pH value to 7.4; deionized water was set to a volume of 1L.
The reagent R2 is prepared by mixing a sensitization source 1 formed by coupling the first monoclonal antibody with latex particles and a sensitization source 2 formed by coupling the second monoclonal antibody with the latex particles.
In another aspect, the invention also provides a method for using the kit, comprising the following steps: diluting a tested sample by using a reagent R1, and adding a reagent R2 for reaction; then using a biochemical analyzer to determine the absorbance at 570 nm; and preparing a standard curve by using the human immunoglobulin G4 standard substance, and calculating the content of the human immunoglobulin G4 in the tested sample according to the standard curve and the absorbance value.
Possible principles of the kit of the invention include: when the corresponding antigen exists in the sample, the sensitization source 1 formed by coupling the first monoclonal antibody and the latex particles and the sensitization source 2 formed by coupling the second monoclonal antibody and the latex particles can be combined with the antigen at the same time, and aggregation reaction occurs. The individual sensitizers are within the wavelength of the incident light and light can pass through. When two sensitizers are concentrated, the transmitted light is reduced, which is proportional to the concentration of the sensitizers and also to the amount of antigen. Qualitative and quantitative analysis of human immunoglobulin G4 was thereby performed.
Wherein, the first monoclonal antibody and the second monoclonal antibody used for constructing the kit of the invention are respectively aimed at different epitopes of human immunoglobulin G4 and have pairing detection effect.
Antibodies of the invention may be derivatized, for example, linked to another molecule. Typically, the derivatives of the antibodies do not affect their binding to immunoglobulin G4. Thus, the antibodies of the invention also include forms of such derivatives. For example, an antibody of the invention may be functionally linked to one or more other molecular groups, such as another antibody, a detection reagent, a pharmaceutical reagent, and/or a protein or polypeptide capable of mediating binding of the antibody or antigen binding fragment to another molecule. Furthermore, the antibodies of the invention may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
Thus, in a preferred embodiment, the antibodies of the invention are conjugated to latex particles. The antibodies of the invention may be conjugated to a detectable label, such as an enzyme, radionuclide, fluorescent dye, luminescent substance, or biotin. The detectable label of the present invention can be any substance that is detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes, radionuclides, fluorescent dyes, luminescent substances, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic beads, and biotin for binding to the above-mentioned label-modified avidin. Patents teaching the use of such markers include, but are not limited to, U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241. The detectable labels described above can be detected by methods known in the art. For example, the radiolabel may be detected using a photographic film or scintillation calculator, and the fluorescent label may be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the coloured label. In certain embodiments, such labels can be suitable for immunological detection, e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, and the like. In certain embodiments, a detectable label as described above may be attached to an antibody of the invention through linkers of different lengths to reduce potential steric hindrance.
Compared with the prior art, the technical effect achieved by adopting the technical scheme is as follows: the monoclonal antibodies provided herein can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention.
Definition of terms
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunology laboratories and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The assignment of amino acids to regions or domains can be carried out by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.
As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The exact boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g. as in the Kabat numbering system, chothia numbering system or IMGT numbering system. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. And, correspondence between different numbering systems is well known to those skilled in the art.
In the present invention, the CDRs contained in the antibodies of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies of the invention are preferably determined by Kabat, chothia or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies of the invention are preferably determined by the Kabat numbering system.
As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG, igA1, igA2, igD, igE or IgM antibodies.
As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies, monodomain antibodies, chimeric antibodies, linear antibodies, nanobodies, probody, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is of IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.
As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain; the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means the fragment obtained after reduction of the disulfide bond joining the two heavy chain fragments of the F (ab') 2 fragment, consisting of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).
As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.
As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.
As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used and may be used for other linkers of the invention. In some cases, disulfide bonds may also exist between VH and VL of scFv.
As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites.
As used herein, the term "single domain antibody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain that retains the ability to specifically bind to the same antigen to which a full length antibody binds. Single domain antibodies are also known as nanobodies.
Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen.
Antigen binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art, and specifically screened in the same manner as for intact antibodies.
In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.
As used herein, the terms "monoclonal antibody," "mAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" merely indicates the character of the antibody as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring preparation of the antibody by any particular method.
The monoclonal antibodies of the invention may be prepared by a variety of techniques, such as hybridoma techniques, recombinant DNA techniques, or phage antibody library techniques.
Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, the specific antigen or epitope thereof may be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography.
As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody and another portion of the light chain or/and heavy chain is derived from another antibody, but which retains binding activity to the antigen of interest in any event. For example, the term "chimeric antibody" may include antibodies (e.g., human murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody and the heavy and light chain variable regions of the antibody are from a second antibody.
As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody and all or part of the non-CDR regions are derived from a human immunoglobulin. Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody may be a mouse, rat, rabbit or non-human primate antibody having the desired properties.
The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chains can be obtained from a murine hybridoma of interest and engineered to contain non-murine immunoglobulin sequences using standard molecular biology techniques.
To prepare chimeric antibodies, the murine immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art. For example, the DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full length light chain gene (as well as Fab light chain gene). The sequences of human light chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is generally preferred.
To prepare humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art. Alternatively, transgenic animals can also be utilized that are capable of producing no endogenous immunoglobulins upon immunization and are capable of producing a fully human antibody repertoire. For example, it has been reported that homozygous deletion of the antibody heavy chain Junction (JH) gene in chimeric and germ-line mutant mice can completely suppress endogenous antibody production, and then transferring an array of human germ-line immunoglobulin genes into germ-line mutant mice will result in the mice producing human antibodies upon encountering antigen stimulation. Non-limiting examples of such transgenic animals include, huMAb mice containing human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and kappa light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and kappa chain loci; or "KM mouse TM" carrying both human heavy chain transgenes and human light chain transchromosomes. Other methods of antibody humanization include phage display techniques.
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with an affinity (KD) of less than about 10-9M, such as less than about 10-9M, 10-10M, 10-11M, or 10-12M or less. Specific binding properties between two molecules can be determined using methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit, then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such an alignment may be achieved by using, for example, a computer program such as the Align program, conveniently by the method of Needleman et al (1970) J.mol.biol.48:443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (jmoibiol. 48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix, and GAP weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.
As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains, acidic side chains, uncharged polar side chains, nonpolar side chains, beta-branched side chains, and aromatic side chains. Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art.
The twenty conventional amino acids referred to herein are written following conventional usage. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
By adopting the technical scheme provided by the invention, the beneficial effects can be achieved: the monoclonal antibody provided by the invention can be specifically combined with IgG4 protein; the double-antibody sandwich immunonephelometry detection method can effectively detect the content of IgG4 protein in human serum, has high sensitivity and wide detection linearity of 0-5.0g/L, and can meet the requirements of clinical detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the linear range measurement of IgG4 provided in the examples of the present invention.
FIG. 2 is a graph showing the correlation between IgG4 detection methods and certain brands of alignment reagents/detection methods according to embodiments of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The main reagents and main instruments of this example are shown in tables 1 and 2, respectively.
Table 1: main reagent
Table 2: main instrument
Antigen:
recombinant IgG4 protein from HEK293 cell expression, and the amino acid sequence is shown in SEQ ID NO. 1.
Experimental animals:
Balb/C mice: balb/C mice, grade SPF (Specific Pathogen Free), were purchased from Shanghai Ling Biotech Inc.
Preparing a buffer solution:
the Phosphate Buffer (PBS) formulation was: naCl,8g; KCl,0.2g; na2HPO4.12H2O, 2.9g; KH2PO4,0.2g; H2O constant volume to 1L
The formula of the 10 XPBST lotion is as follows: 80g of NaCl; KCl,2g; 29g of Na2HPO4.12H2O; KH2PO4,2g; TWEEN-20,5ml; H2O constant volume to 1L
The glycine eluent formula at pH 2.7 is: glycine 1.9g, H2O to 500mL, pH 2.68-2.72.
The glycine eluent formula at pH 1.9 is: glycine 1.9g, H2O to 500mL, pH 1.88-1.92.
Example 1: preparation and screening of IgG4 monoclonal antibodies
The recombinant IgG4 protein (1 mg/ml) was mixed with adjuvants CFA and IFA to prepare immunogens. That is, the recombinant protein is first mixed with CFA in a volume ratio of 1:1 to form immunogen A, and then mixed with IFA in a volume ratio of 1:1 to form immunogen B. Immunogen a is primary and immunogen B is secondary, tertiary and fourth booster. Mice were immunized subcutaneously by 2 mice, tail blood was withdrawn on day 14 after the fourth booster immunization, and tail blood antibody titers were assessed using an indirect ELISA method.
ELISA plates (2. Mu.g/mL) were coated with IgG4 recombinant protein, 100. Mu.L was added to each well and reacted overnight at 4 ℃; plates were washed 3 times with PBST solution and blocked with 5% bsa for 2h at 37 ℃; then, after washing the plate 3 times with PBST solution, mouse tail blood diluted with 5% BSA solution was added for reaction at 37℃for 2 hours; plates were then washed 3 times with PBST solution and goat labeled with HRP at 1:20000 fold dilution was addedAnti-mouse IgG secondary antibody, reacting for 1h at 37 ℃; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added 2 SO 4 ) After mixing, the OD450 value was read on an ELISA reader. Results of the mouse tail blood indirect ELISA evaluation at day 14 post immunization are shown in Table 3.
Table 3: mouse tail blood antibody titer evaluation at day 14 after four immunization with IgG4
| No. 1 mouse | No. 2 mouse | |
| 1/10000 | 1.958 | 1.928 |
| 1/20000 | 1.731 | 1.674 |
| 1/40000 | 1.474 | 1.435 |
| 1/80000 | 1.131 | 1.011 |
| 1/160000 | 0.735 | 0.66 |
| 1/320000 | 0.491 | 0.435 |
| 1/640000 | 0.29 | 0.287 |
| 1/1280000 | 0.187 | 0.188 |
| NC | 0.073 | 0.076 |
Note that: NC is negative control, PBS.
From the results, the antibody titer of the tail blood of two mice recognizing the IgG4 recombinant protein exceeds 1:640000 after four times of immunization, the mice are immunized by impact by using the IgG4 recombinant protein, the spleens of the mice are taken on the 3 rd day after the impact immunization, and the spleen cells and myeloma cells SP2/0-Ag14 are separated for cell fusion. After fusion, more than 3000 hybridoma cell populations were grown in 96-well plates, and the cell culture supernatants in the 96-well plates were evaluated by the indirect ELISA method described above, and monoclonal cell lines capable of secreting monoclonal antibodies that specifically recognized IgG4 protein (excluding cross-reactivity with IgG1/IgG2/IgG3 protein) were screened, and from the screening results, 12 positive clones were selected for the confirmation of screening. The experimental procedure was as follows: the ELISA plate (2. Mu.g/mL) was coated with human IgG4 protein, 100. Mu.L was added to each well and reacted overnight at 4 ℃; plates were washed 3 times with PBST solution and blocked with 5% bsa for 2h at 37 ℃; then, after washing the plate 3 times with PBST solution, adding cell culture supernatant diluted 50 times with PBS solution, and reacting for 2 hours at 37 ℃; the plates were then washed 3 times with PBST solution and goat anti-mouse IgG was added at 1:20000 fold dilution with HRP-labeled Secondary antibody, reacting for 1h at 37 ℃; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H) was added 2 SO 4 ) After mixing, the OD450 values were read on a microplate reader and the results are shown in Table 4.
Table 4: confirmation of Positive clones
| Clone number | 2A4 | 2G6 | 3B2 | 5B11 | 7H8 | 10B4 | 14A7 |
| 1/200 | 1.325 | 1.281 | 1.766 | 1.908 | 1.493 | 1.846 | 2.036 |
| 1/400 | 0.822 | 0.858 | 1.307 | 1.579 | 1.309 | 1.538 | 1.696 |
| 1/800 | 0.544 | 0.553 | 0.901 | 1.284 | 0.913 | 1.21 | 1.301 |
| 1/1600 | 0.319 | 0.327 | 0.533 | 0.859 | 0.576 | 0.88 | 0.912 |
| Clone number | 18D9 | 24C1 | 28G3 | 33B3 | 39C2 | NC | PC |
| 1/200 | 1.958 | 1.928 | 1.848 | 1.87 | 1.66 | 0.076 | 1.863 |
| 1/400 | 1.731 | 1.674 | 1.57 | 1.57 | 1.21 | 0.065 | 1.48 |
| 1/800 | 1.474 | 1.435 | 1.313 | 1.258 | 1.041 | 0.073 | 1.168 |
| 1/1600 | 1.131 | 1.011 | 0.922 | 0.9 | 0.552 | 0.072 | 0.815 |
Note that: NC is negative control, PBS; PC is a positive control, and the serum of the No. 1 mouse is diluted by 1/10000.
Example 2: preparation of purified antibodies
1) Ascites preparation
Ascites was prepared from the 2A4/2G6/3B2/5B11/7H8/10B4/14A7/18D9/24C1/28G3/33B3/39C2 antibody, and approximately 1X 10, respectively 6 The cells were injected into the abdominal cavity of 3 Balb/C mice pre-injected with IFA adjuvant, and after about 10 days, ascites produced by each positive clone was extracted, and then centrifuged at 12000rpm for 15min at 4℃to collect the supernatant for further purification of G protein.
2) Purification of mouse monoclonal antibodies
1mL of the column material coupled with the G protein is added into an empty column, after the column material is washed by PBS solution, 2mL of ascites is diluted by 8mL of PBS and then is put into the column, and then the flowing-through liquid is put into the column again; then, elution was performed with glycine eluent of pH2.7, and one tube (100. Mu.L of a neutralization solution comprising 1M Tris-HCl, 10mM EDTA, 1.5M NaCl, pH8.0-8.38 was previously added) was collected per 1mL of the eluent, and 5 tubes were collected in total; following elution with glycine eluent at pH1.9, one tube (300. Mu.L of neutralization solution added in advance) was collected per 1mL of eluent, and 3 tubes were collected in total; then, the OD280 reading is carried out on each tube of eluent respectively, the eluent with the OD280 more than 0.5 is mixed, the OD280 of the mixed liquid is re-measured after mixing, and the antibody concentration is calculated according to the coefficient of 1.4: antibody concentration = OD280/1.4.
Example 3: screening of purified antibodies
Biotin labeling a part of the murine monoclonal antibody after G protein purification, adding-Bio to the labeled antibody, if the 5B11 antibody after biotin labeling is represented by 5B11-Bio, carrying out antibody pairing screening by using a sandwich ELSIA method, coating an ELISA plate (2 mug/mL) with unlabeled antibody, adding 100 mug of the labeled antibody into each hole, and reacting overnight at 4 ℃; plates were washed 3 times with PBST solution and blocked with 5% bsa for 1.5h at 37 ℃; after washing the plates 3 times with PBST solution, human IgG4 protein (2. Mu.g/mL) was added and incubated at 37℃for 1.5h; washing the plate 3 times by using PBST solution, adding biotin-labeled antibody diluted in a gradient way by 5% BSA solution, and reacting for 1.5 hours at 37 ℃; plates were washed 3 times with PBST solution, and HRP-labeled streptavidin was added at 1:500 dilution and reacted at 37℃for 1h; washing the plate with PBST solution for 5 times, drying, adding 100 mu L TMB developing solution, and reacting at 37 ℃ in a dark place for 15min; then 50. Mu.L of stop solution (2M H2SO 4) was added, and after mixing, the OD450 value was read on a microplate reader. The results of the evaluation of the purified antibodies are shown in Table 5.
Table 5: sandwich ELISA method paired antibody screening partial result
Note that: NC is negative control, PBS.
According to the results of Table 5, antibodies 5B11, 14A7, 33B3 were selected for latex-enhanced immunoturbidimetry paired screening, and the antibodies were coupled to latex particles according to a combination of two pairs 5B11/33B3 and 14A7/33B 3; the coupled latex particles were formulated into detection reagent R2 for subsequent calibrator detection, with the detection results shown in table 6.
The detection method comprises the following steps:
the detecting instrument is as follows: full-automatic biochemical analyzer 7180
The analysis method comprises the following steps: two-point endpoint method
The reaction direction is as follows: ascending reaction
The calibration mode is as follows: spline
Measurement wavelength: 570nm
Measuring temperature: 37 DEG C
Calibration material: reagent R1: reagent r2=2 μl:120 μl
Table 6: preparation of IgG4 detection reagent detection calibrator OD570nm reading by different antibody combination modes
According to the results of Table 6, antibodies 5B11 and 33B3 were selected as starting materials for the IgG 4-content double-antibody sandwich-method turbidimetric immunoassay.
The amino acid sequence of the variable region of the murine monoclonal antibody 5B11 was analyzed by sequencing:
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9.
Further, immunolobin BLAST (IG Blast)
(http:// www.ncbi.nlm.nih.gov/gilast /) on-line analysis, the CDR sequences of the antibody are determined, and the amino acid sequences are respectively: VH CDR1 is shown as SEQ ID NO. 2; VH CDR2 is shown in SEQ ID NO. 3; VH CDR3 is shown in SEQ ID NO. 4; VL CDR1 is shown in SEQ ID NO. 5; VL CDR2 is shown in SEQ ID NO. 6; VL CDR3 is shown in SEQ ID NO. 7.
The amino acid sequence of the variable region of murine monoclonal antibody 33B3 was analyzed by sequencing:
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 17.
Further, the CDR sequences of the antibodies were determined using an online analysis of Immunolobin BLAST (IGBlast), the amino acid sequences of which were: VH CDR1 is shown in SEQ ID NO. 10; VH CDR2 is shown in SEQ ID NO. 11; VH CDR3 is shown in SEQ ID NO. 12; VL CDR1 is shown in SEQ ID NO. 13; VL CDR2 is shown in SEQ ID NO. 14; VL CDR3 is shown in SEQ ID NO. 15.
Example 4: double-antibody sandwich immunonephelometry method for detecting sample IgG4 protein
The method for detecting the content of the immunoglobulin G4 (IgG 4) utilizes immunoglobulin G4 detection reagents R1 and R2 and a calibrator to detect the immunoglobulin G4.
The main components of the detection reagent are as follows:
reagent R1: trimethylolaminomethane buffer
Reagent R2: the sensitization source 1 and 33B3 formed by coupling 5B11 with latex particles and the sensitization source 2 formed by coupling latex particles are mixed and prepared according to a certain proportion.
Calibration material: human immunoglobulin G4.
The detection method comprises the following steps:
the detection instrument used was: full-automatic biochemical analyzer 7180
The analysis method comprises the following steps: two-point endpoint method.
The reaction direction is as follows: lifting reaction;
The calibration mode is as follows: a Spline;
measurement wavelength: 570nm;
measuring temperature: 37 ℃;
sample: reagent R1: reagent r2=2 μl:120 μl;
the method comprises the following steps:
step one, taking 2 mu L of a sample and 120 mu L of a reagent R1, adding the sample and the reagent R1 into a reaction cup, uniformly mixing and incubating for 3min;
step two, adding 120 mu L of reagent R2 into the mixed solution, and uniformly mixing;
step three, respectively reading the first reading point transmission absorbance A1 and the second reading point transmission absorbance A2 at 570nm through a full-automatic biochemical analyzer 7180;
and fifthly, calculating delta A=A2-A1 by software and calculating the concentration of the immunoglobulin G4 in the tested sample according to a calibration curve.
Linear range determination for immunoglobulin G4 reagent: high concentration samples were prepared from IgG4 calibrator, diluted with normal saline at a conventional ratio, each sample was repeatedly assayed 3 times using IgG4 reagents R1, R2 of example 2, the mean was calculated, the regression equation was calculated, and the theoretical value was calculated from the regression equation, and the results were shown in FIG. 1. The result shows that the linear range of the immunoglobulin G4 reagent is 0-5.0G/L, the detection sensitivity is high, the linearity is wide, and the detection requirements of high sensitivity and wide linearity in clinic are met.
The correlation test is carried out on the IgG4 detection method and the reagent detection method for comparing certain known brands in China, 80 serum samples are measured, the correlation analysis is carried out on the measured values, and the result is shown in FIG. 2, wherein the correlation coefficient R of the IgG4 detection method and the reagent detection method for comparing certain known brands in China 2 The = 0.9978 shows that the detection method has good correlation with a detection method of a certain known brand comparison reagent in China.
Example 5:
on the basis of the embodiment 3, the embodiment provides an anti-human immunoglobulin G4 monoclonal antibody, wherein the monoclonal antibody is an antibody 5B11; or a mixture of antibody 5B11 and antibody 33B 3;
antibody 5B11 comprises VH CDR1 shown in SEQ ID No.2, VH CDR2 shown in SEQ ID No.3, VH CDR3 shown in SEQ ID No.4, VL CDR1 shown in SEQ ID No.5, VL CDR2 shown in SEQ ID No.6, and VL CDR3 shown in SEQ ID No. 7;
antibody 33B3 comprises a VH CDR1 shown in SEQ ID NO.10, a VH CDR2 shown in SEQ ID NO.11, a VH CDR3 shown in SEQ ID NO.12, a VL CDR1 shown in SEQ ID NO.13, a VL CDR2 shown in SEQ ID NO.14 and a VL CDR3 shown in SEQ ID NO. 15.
Further, antibody 5B11 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of antibody 5B11 is shown as SEQ ID NO.8, and the amino acid sequence of the light chain variable region of antibody 5B11 is shown as SEQ ID NO. 9.
Further, the antibody 33B3 comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region of the antibody 33B3 is shown as SEQ ID NO.16, and the amino acid sequence of the light chain variable region of the antibody 33B3 is shown as SEQ ID NO. 17.
Further, the heavy chain constant region of the monoclonal antibody is an IgG heavy chain constant region, and the light chain constant region of the monoclonal antibody is a kappa light chain constant region.
Further, the monoclonal antibody is labeled.
Example 6:
this example provides an isolated nucleic acid molecule encoding a monoclonal antibody of any of the examples described above.
Example 7:
this example provides a vector comprising the isolated nucleic acid molecule of the above example.
Further, the vector is a cloning vector or an expression vector.
Example 8:
the present embodiment provides a method for preparing an anti-human immunoglobulin G4 monoclonal antibody according to any one of the above examples, comprising:
culturing a host cell comprising a vector under conditions that allow expression of the monoclonal antibody;
Monoclonal antibodies are recovered from the cultured host cell culture.
Example 9:
the present embodiment provides a kit for detecting human immunoglobulin G4, comprising a monoclonal antibody of any of the above examples.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Claims (10)
1. An anti-human immunoglobulin G4 monoclonal antibody, wherein the monoclonal antibody is
Antibody 5B11; or (b)
A mixture of antibody 5B11 and antibody 33B 3;
the antibody 5B11 comprises a VH CDR1 with an amino acid sequence shown as SEQ ID NO.2, a VH CDR2 with an amino acid sequence shown as SEQ ID NO.3, a VH CDR3 with an amino acid sequence shown as SEQ ID NO.4, a VL CDR1 with an amino acid sequence shown as SEQ ID NO.5, a VL CDR2 with an amino acid sequence shown as SEQ ID NO.6 and a VL CDR3 with an amino acid sequence shown as SEQ ID NO. 7;
the antibody 33B3 comprises a VH CDR1 shown in SEQ ID NO.10, a VH CDR2 shown in SEQ ID NO.11, a VH CDR3 shown in SEQ ID NO.12, a VL CDR1 shown in SEQ ID NO.13, a VL CDR2 shown in SEQ ID NO.14 and a VL CDR3 shown in SEQ ID NO. 15.
2. The monoclonal antibody of claim 1, wherein the antibody 5B11 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region of the antibody 5B11 is shown in SEQ ID No.8, and wherein the amino acid sequence of the light chain variable region of the antibody 5B11 is shown in SEQ ID No. 9.
3. The monoclonal antibody of claim 1, wherein the antibody 33B3 comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region of the antibody 33B3 is shown in SEQ ID No.16, and wherein the amino acid sequence of the light chain variable region of the antibody 33B3 is shown in SEQ ID No. 17.
4. The monoclonal antibody of claim 1, wherein the heavy chain constant region of the monoclonal antibody is an IgG heavy chain constant region and the light chain constant region of the monoclonal antibody is a kappa light chain constant region.
5. The monoclonal antibody of claim 1, wherein the monoclonal antibody is labeled.
6. An isolated nucleic acid molecule encoding the monoclonal antibody of any one of claims 1 to 5.
7. A vector comprising the isolated nucleic acid molecule of claim 6.
8. The vector of claim 7, wherein the vector is a cloning vector or an expression vector.
9. A method of preparing an anti-human immunoglobulin G4 monoclonal antibody according to any one of claims 1-5, comprising:
culturing a host cell comprising the vector of claim 7 under conditions permitting expression of the monoclonal antibody;
recovering the monoclonal antibody from the cultured host cell culture.
10. A kit for detecting human immunoglobulin G4, comprising a monoclonal antibody according to any one of claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310893824.5A CN117106091A (en) | 2023-07-20 | 2023-07-20 | Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310893824.5A CN117106091A (en) | 2023-07-20 | 2023-07-20 | Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117106091A true CN117106091A (en) | 2023-11-24 |
Family
ID=88793741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310893824.5A Pending CN117106091A (en) | 2023-07-20 | 2023-07-20 | Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117106091A (en) |
-
2023
- 2023-07-20 CN CN202310893824.5A patent/CN117106091A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109336973B (en) | Anti-transferrin antibodies and uses thereof | |
| JP7200375B2 (en) | Specific antibody against human anti-Müllerian hormone and use thereof | |
| US20220056112A1 (en) | MONOCLONAL ANTIBODIES AGAINST SARS-CoV-2 NUCLEOCAPSID PROTEIN AND USES THEREOF | |
| CN112409487B (en) | Anti-soluble transferrin receptor antibodies and uses thereof | |
| US20210380719A1 (en) | Rabbit antibodies to human immunoglobulins g | |
| CN114835815B (en) | Pepsinogen I resistant monoclonal antibody, preparation method and application thereof | |
| CN114835814B (en) | Pepsinogen II resistant monoclonal antibody, and preparation method and application thereof | |
| CN117106091A (en) | Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit | |
| CN113234156B (en) | Anti-myoglobin antibody and preparation method and application thereof | |
| CN116813787A (en) | Anti-human immunoglobulin G4 monoclonal antibody, preparation method, carrier and kit | |
| CN117264072B (en) | anti-SN 38 monoclonal antibody and application thereof | |
| WO2019189882A1 (en) | Monoclonal antibody specifically reacting with ncc-st-439 antigen and method for producing same | |
| CN117264071B (en) | Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof | |
| CN117285637B (en) | Anti-idiotype antibody and application thereof | |
| CN116284411B (en) | Antibody against recombinant human coagulation factor VIII-Fc fusion protein and application thereof | |
| CN117304327B (en) | Anti-goat IgG rabbit monoclonal antibody and application thereof | |
| CN117384290B (en) | Human ROBO1 binding molecules and uses thereof | |
| WO2023109785A1 (en) | Antibody and kit for detecting stnfr2 in serum | |
| CN113484526A (en) | Method for detecting biological activity of anti-FcRn antibody or antigen binding fragment thereof | |
| CN115925924A (en) | Antibody for detecting TSP-1 specific peptide fragment and application thereof | |
| CN116813780A (en) | Anti-human CD31 rabbit monoclonal antibody 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 |