CN116162153A - Monoclonal antibody of hepatitis B virus surface antigen and application thereof - Google Patents
Monoclonal antibody of hepatitis B virus surface antigen and application thereof Download PDFInfo
- Publication number
- CN116162153A CN116162153A CN202211063471.8A CN202211063471A CN116162153A CN 116162153 A CN116162153 A CN 116162153A CN 202211063471 A CN202211063471 A CN 202211063471A CN 116162153 A CN116162153 A CN 116162153A
- Authority
- CN
- China
- Prior art keywords
- seq
- amino acid
- hepatitis
- monoclonal antibody
- 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
- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 64
- 239000000427 antigen Substances 0.000 title claims abstract description 64
- 102000036639 antigens Human genes 0.000 title claims abstract description 64
- 108091007433 antigens Proteins 0.000 title claims abstract description 64
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 61
- 238000001262 western blot Methods 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000003053 immunization Effects 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 63
- 238000001514 detection method Methods 0.000 claims description 56
- 108090000623 proteins and genes Proteins 0.000 claims description 48
- 102000004169 proteins and genes Human genes 0.000 claims description 42
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 34
- 125000003729 nucleotide group Chemical group 0.000 claims description 31
- 239000002773 nucleotide Substances 0.000 claims description 30
- 238000006467 substitution reaction Methods 0.000 claims description 25
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 150000007523 nucleic acids Chemical class 0.000 claims description 18
- 239000013598 vector Substances 0.000 claims description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 16
- 241000699670 Mus sp. Species 0.000 claims description 15
- 230000035772 mutation Effects 0.000 claims description 13
- 239000012620 biological material Substances 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 9
- 238000012217 deletion Methods 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 8
- 238000003780 insertion Methods 0.000 claims description 8
- 230000037431 insertion Effects 0.000 claims description 8
- 238000002649 immunization Methods 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 238000004113 cell culture Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 210000004989 spleen cell Anatomy 0.000 claims description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 4
- 238000003113 dilution method Methods 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 230000000670 limiting effect Effects 0.000 claims description 4
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 4
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 230000000890 antigenic effect Effects 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 208000002672 hepatitis B Diseases 0.000 abstract description 13
- 210000003527 eukaryotic cell Anatomy 0.000 abstract description 7
- 210000004408 hybridoma Anatomy 0.000 abstract description 7
- 239000000523 sample Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 239000012528 membrane Substances 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000010166 immunofluorescence Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 108010032595 Antibody Binding Sites Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 239000012474 protein marker Substances 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 208000037581 Persistent Infection Diseases 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 101710142246 External core antigen Proteins 0.000 description 1
- 241000285366 HBV genotype D Species 0.000 description 1
- -1 HBsAg particles Chemical class 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 description 1
- 229940124736 hepatitis-B vaccine Drugs 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—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
- C12N2535/00—Supports or coatings for cell culture characterised by topography
-
- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention discloses a monoclonal antibody of hepatitis B virus surface antigen and application thereof, wherein a hybridoma cell strain expressing the hepatitis B virus surface antigen is obtained by immunizing a mouse with the antigen and screening. The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the monoclonal antibody are respectively shown in SEQ ID NO. 1-3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the monoclonal antibody are respectively shown in SEQ ID NO.4, KVS and SEQ ID NO. 5; the monoclonal antibody can be used for detecting eukaryotic cell-derived HBsAg, different genotypes of HBsAg and HBsAg mutant by Western blot, is suitable for detecting various types of samples, and opens up a new method for detecting hepatitis B virus and treating hepatitis B diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a monoclonal antibody of hepatitis B virus surface antigen and application thereof.
Background
Monoclonal antibodies are highly homogeneous antibodies raised against only one specific epitope by a single B cell clone. In view of the above characteristics of monoclonal antibodies, they have been rapidly used in a variety of fields of medicine, such as diagnostic reagents for laboratory test medicine, techniques for enzyme-linked immunosorbent assays, immunohistochemistry, flow cytometry, etc., protein purification or targeted therapy of tumors and radioimmunoimaging techniques. However, monoclonal antibodies have limitations such as when detecting viral proteins by immunoblotting (Western Blot), antibodies recognizing viral antigens often recognize conformational epitopes on the antigen, and thus monoclonal antibodies are difficult to apply to Western Blot detection.
Acute and chronic infections caused by hepatitis b virus (hepatitis B virus, HBV) are global public health problems that seriously jeopardize human health. Along with the inoculation of hepatitis B vaccine, the spread of virus is greatly controlled. But China is still the most countries of the world with hepatitis B patients. The current clinical drugs for treating chronic hepatitis B, including interferon and nucleoside/nucleotide analogues, although effective in inhibiting HBV replication, cannot completely clear the virus and may develop drug resistance.
The hepatitis B surface antigen-HBsAg not only can be used as the envelope protein of HBV to participate in the journey of complete viral particles, but also can form non-infectious subviral particles with host lipid, namely HBsAg particles, and has three forms of large, medium and small. Wherein small HBsAg is encoded by 226 amino acids and is the major component responsible for the production of protective antibodies by the body, and wherein there is an immunodominant region called the "a" epitope, located at amino acids 124-147. There are 4 transmembrane regions on HBsAg, and the "a" epitope located between transmembrane region 2 and transmembrane region 3 is exposed on the surface of the viral particle and is the major choice of the immunoreactive fragment of current HBsAg-associated antibodies. Because of the natural choice, survival time and spatial pressure of the healthy individuals during HBV chronic infection, a series of mutants will appear, and the "a" epitope is the region with higher mutation frequency of HBsAg. Thus, whether or not the HBsAg antibody against this region can bind to the mutated HBsAg and whether or not all types of mutation can be covered is problematic.
In the current scientific research and clinical detection of HBV, the detection of HBsAg is an important index for measuring HBV replication. At present, the commercialized HBsAg ELISA kit can meet the requirements of clinically detecting HBsAg in serum and other body fluids and detecting the HBsAg level in cell supernatant in scientific research. ELISA detection antibodies involving HBsAg recognize more than a spatial conformational epitope of HBsAg and rarely recognize a linear epitope of HBsAg. There is currently a lack of effective, simple and accurate HBsAg antibodies for Western blot detection of HBsAg protein levels on the market.
Disclosure of Invention
The invention provides a monoclonal antibody of hepatitis B virus surface antigen and application thereof, which are used for solving the problems in the prior art.
The scheme of the invention is as follows:
a monoclonal antibody of hepatitis B virus surface antigen, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises a heavy chain complementarity determining region HCDR1 composed of amino acid sequences shown as SEQ ID NO.1, a heavy chain complementarity determining region HCDR2 composed of amino acid sequences shown as SEQ ID NO.2 and a heavy chain complementarity determining region HCDR3 composed of amino acid sequences shown as SEQ ID NO. 3;
the light chain variable region comprises a light chain complementarity determining region LCDR1 composed of the amino acid sequences shown in SEQ ID NO.4, a light chain complementarity determining region LCDR2 composed of the amino acid sequences KVS and a light chain complementarity determining region LCDR3 composed of the amino acid sequences shown in SEQ ID NO. 5.
As a preferable technical scheme, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.6 or the amino acid sequence with the similarity of the sequence shown as SEQ ID NO.6 being more than or equal to 80%, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.7 or the amino acid sequence with the similarity of the sequence shown as SEQ ID NO.7 being more than or equal to 80%.
As a preferred embodiment, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, which are one of any isotype and subclass.
The monoclonal antibodies described above consist of heavy chain sequences and corresponding light chain sequences, the variable regions of both the light and heavy chains determining the binding recognition and specificity for an antigen, the specificity of an antibody being determined by the structural complementarity of the antibody binding site and the epitope, the antibody binding site consisting of residues derived primarily from the highly variable region or Complementarity Determining Regions (CDRs), and the CDRs being joined by insertion of Framework Regions (FR) which can be readily determined by one skilled in the art after knowing the amino acid sequences of the CDRs. Thus, the function and application of the monoclonal antibody S1705 of the present invention can be basically achieved as long as the CDR sequences of the found heavy chain sequence and light chain sequence are unchanged. Thus, the specific sequences of the monoclonal antibodies of the invention are not limited to the above specific heavy and light chain sequence variable regions, which are only monoclonal antibody sequences specifically employed in one implementation of the invention.
As a preferable technical scheme, the protein composed of the amino acid sequence with the similarity of more than or equal to 80% with the sequence shown as SEQ ID NO.6 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 6; the protein composed of the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.7 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 7; in the case of said substitution, the protein consisting of an amino acid sequence which is 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% identical to the reference sequence SEQ ID NO.6, SEQ ID NO.7 corresponds to a homologous sequence derived from a species different from the reference sequence; such substitutions include conservative substitutions, which are substitutions in which one amino acid is substituted by another amino acid having similar structure and/or chemical properties, and non-conservative substitutions. The sequences of the heavy or light chain variable regions differ from the reference sequences only by conservative amino acid substitutions.
The invention also discloses a method for preparing the monoclonal antibody of the hepatitis B virus surface antigen, which comprises the following steps:
the HBsAg protein expressed by CHO is used for three times of immunization on the balb/c mice, 100 mu g of protein is mixed with an equal volume of Freund adjuvant each time, and the abdominal cavities of the mice are injected in multiple points; two weeks after the third immunization, taking spleen cells of the mice to fuse with myeloma cell line SP2/0, culturing in a 96-well cell culture plate by a limiting dilution method, taking culture supernatant after two weeks, performing Western Blot detection, and finally screening to obtain antibody S1705 obtained by a cell clone, wherein HBsAg protein is detected.
As a preferred technical scheme, the monoclonal antibody can also be prepared by adopting a conventional gene recombination method, and can be a humanized antibody; or immunizing the mice again with the hybridoma cell strain obtained by screening to obtain the hepatitis B surface antibody monoclonal antibody.
The invention also discloses a biological material related to the monoclonal antibody of the hepatitis B virus surface antigen, which is characterized in that the biological material is selected from one of the following A) to D):
a) A nucleic acid molecule encoding the monoclonal antibody;
b) An expression cassette, a recombinant vector, a recombinant cell line comprising the nucleic acid molecule as described in A);
c) An antigenic peptide recognized by the monoclonal antibody;
d) A derivative of the monoclonal antibody;
the nucleotide sequence for encoding the HCDR1 is shown as SEQ ID NO.8, the nucleotide sequence for encoding the HCDR2 is shown as SEQ ID NO.9, the nucleotide sequence for encoding the HCDR3 is shown as SEQ ID NO.10, the nucleotide sequence for encoding the LCDR1 is shown as SEQ ID NO.11, the nucleotide sequence for encoding the LCDR2 is shown as SEQ ID NO.16, and the nucleotide sequence for encoding the LCDR2 is shown as SEQ ID NO.16: aaagtttcc, the nucleotide sequence encoding said LCDR3 is shown in SEQ ID NO. 12.
The nucleotide sequence for encoding the heavy chain variable region is shown as SEQ ID NO.13, and the nucleotide sequence for encoding the light chain variable region is shown as SEQ ID NO. 14;
the above specific nucleic acid sequences are only nucleic acid sequences specifically employed in one implementation of the present invention, and, depending on the degeneracy of the codons, several nucleic acid sequences encoding the same heavy chain variable region sequence or light chain variable region sequence (e.g., conservative nucleotide sequence variants derived from degenerated and silent variants of the genetic code, nucleotide substitutions, deletions and additions are also included) may be encoded, with the exception of the nucleic acid sequences defined above, within the scope of the present invention.
As a preferable technical scheme, the monoclonal antibody recognizes a linear epitope of the hepatitis B virus surface antigen, and the recognized epitope is between amino acids 25-50 of the hepatitis B virus surface antigen.
The antigen peptide is positioned on the inner side of a lipid membrane of the hepatitis B virus surface antigen and positioned between 17 th amino acid and 66 th amino acid of a transmembrane region of the hepatitis B virus surface antigen, and the amino acid sequence of the antigen peptide is shown as SEQ ID NO. 15.
An expression cassette containing a nucleic acid molecule encoding an antibody refers to a DNA capable of expressing the antibody in a host cell, which DNA may include not only a promoter that initiates transcription of the antibody gene, but also a terminator that terminates transcription of the antibody gene.
As a preferred technical scheme, the vector is one of a plasmid, a cosmid, a phage and a viral vector; a vector is a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed, and the vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried by the vector in the host cell.
Host cells refer to cells into which vectors are introduced, and include prokaryotic cells, fungal cells, insect cells, animal cells, etc., such as E.coli, yeast cells, S2 drosophila cells, BHK cells, CHO cells, HEK293 cells. The expression cassette, recombinant vector and recombinant cell line described above can be prepared by methods conventional in the art.
As a preferred embodiment, the derivatives of the monoclonal antibodies include one of antibody Fab fragments, single chain antibodies or fusion antibodies thereof, and the derivatives can be prepared by conventional methods in the art.
The amino acid sequences and the coding nucleotide sequences of the variable regions of the heavy chain and the light chain of the monoclonal antibody S1705 are shown in the following table:
TABLE 1 sequence information Table
The invention also discloses application of the monoclonal antibody related biological material of the hepatitis B virus surface antigen, which is characterized by comprising one or more of the following applications:
the application in preparing products for detecting and diagnosing hepatitis B virus;
the application of the composition in preparing medicines for treating and preventing diseases caused by hepatitis B virus.
As a preferable technical scheme, the product for detecting and diagnosing the hepatitis B virus is used for detecting hepatitis B virus surface antigens or mutants thereof with different genotypes.
As a preferred technical scheme, the hepatitis b virus comprises A, B, C, D genotype hepatitis b virus; and/or the mutation site of the mutant is G145R.
As a preferred embodiment, the above-mentioned product may be a diagnostic reagent (diagnostic kit), a detection reagent (detection kit), or the like, which may further include other reaction reagents.
As a preferable technical scheme, the method for detecting the hepatitis B virus surface antigen or the mutant thereof comprises flow detection, tissue immunofluorescence detection, western blot detection and ELISA detection.
As a preferred technical scheme, the flow detection is used for detecting the surface antigen of the hepatitis B virus or the mutant thereof presented on the surface of the cell, the tissue immunofluorescence detection is used for detecting the surface antigen of the hepatitis B virus or the mutant thereof in a primary sample, the Western blot detection or ELISA detection comprises the use of the monoclonal antibody for detecting the surface antigen of the hepatitis B virus or the mutant thereof which is derived from eukaryotic cells and the surface antigen of the hepatitis B virus of different genotypes or the mutant thereof, and the ELISA detection also comprises the use of the antigen peptide for detecting the monoclonal antibody. It is understood that the above detection method may also be any suitable detection method in the art.
In the above detection methods, the sample to be detected encompasses a variety of sample types obtained from a subject and can be used in diagnosis or detection, including but not limited to liquid samples of blood and other biological origin, solid tissue samples, clinical samples, cells in culture medium, cell supernatants, cell lysates, serum, plasma, biological fluids, tissue samples, and the like.
The invention also discloses a product for detecting and diagnosing hepatitis B virus.
As a preferred embodiment, the monoclonal antibody may be used as a coating antibody or a secondary enzyme-labeled antibody (detection antibody).
The invention discloses a monoclonal antibody of hepatitis B virus surface antigen, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain complementarity determining region HCDR1 composed of amino acid sequences shown as SEQ ID NO.1, a heavy chain complementarity determining region HCDR2 composed of amino acid sequences shown as SEQ ID NO.2 and a heavy chain complementarity determining region HCDR3 composed of amino acid sequences shown as SEQ ID NO. 3; the light chain variable region comprises a light chain complementarity determining region LCDR1 composed of the amino acid sequences shown in SEQ ID NO.4, a light chain complementarity determining region LCDR2 composed of the amino acid sequences KVS and a light chain complementarity determining region LCDR3 composed of the amino acid sequences shown in SEQ ID NO. 5.
The invention has the advantages that:
based on ELISA detection antibodies and neutralizing antibodies which are not lack against HBsAg in the market, in order to fill up the gap of antibodies for detecting HBV surface antigens by Western blot in the market, the invention screens out a monoclonal antibody which can meet the requirements of scientific research and clinical detection and has high immune response, recognizes linear epitopes of the HBV surface antigens, can be used for detecting Western blot molecules of different serotypes of HBsAg of different genotypes, and is used for other treatment methods of hepatitis B diseases.
Drawings
FIG. 1 is a graph showing experimental results of epitope identification in example 2 of the present invention;
FIG. 2 is a graph showing the result of detecting HBsAg antigen by Western blot using the antibody S1705 of example 3 of the present invention;
FIG. 3 is a graph showing the result of Western blot detection antibody S1705 in example 4 of the present invention;
FIG. 4 is a graph showing the result of Western blot detection of HBsAg mutant in example 5 of the present invention;
FIG. 5 is a graph showing the results of flow assay of HBsAg on the surface of HepAD38, a positive cell line for hepatitis B, in example 6 of the present invention;
FIG. 6 is a graph showing the results of flow assay of HBV replicating mouse primary hepatocyte surface HBsAg in example 6 of the invention;
FIG. 7 is a graph showing the result of immunofluorescence detection of HBsAg in primary samples of patients with hepatitis B in example 7 of the present invention.
Detailed Description
In order to overcome the defects, the invention provides a monoclonal antibody of hepatitis B virus surface antigen and application thereof, which are used for solving the problems in the background technology.
A monoclonal antibody of hepatitis B virus surface antigen, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises a heavy chain complementarity determining region HCDR1 composed of amino acid sequences shown as SEQ ID NO.1, a heavy chain complementarity determining region HCDR2 composed of amino acid sequences shown as SEQ ID NO.2 and a heavy chain complementarity determining region HCDR3 composed of amino acid sequences shown as SEQ ID NO. 3;
the light chain variable region comprises a light chain complementarity determining region LCDR1 composed of the amino acid sequences shown in SEQ ID NO.4, a light chain complementarity determining region LCDR2 composed of the amino acid sequences KVS and a light chain complementarity determining region LCDR3 composed of the amino acid sequences shown in SEQ ID NO. 5.
The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.6 or the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.7 or the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO. 7.
The monoclonal antibodies also include heavy and light chain constant regions, which are one of any isotype and subclass.
The monoclonal antibodies described above consist of heavy chain sequences and corresponding light chain sequences, the variable regions of both the light and heavy chains determining the binding recognition and specificity for an antigen, the specificity of an antibody being determined by the structural complementarity of the antibody binding site and the epitope, the antibody binding site consisting of residues derived primarily from the highly variable region or Complementarity Determining Regions (CDRs), and the CDRs being joined by insertion of Framework Regions (FR) which can be readily determined by one skilled in the art after knowing the amino acid sequences of the CDRs. Thus, the function and application of the monoclonal antibody S1705 of the present invention can be basically achieved as long as the CDR sequences of the found heavy chain sequence and light chain sequence are unchanged. Thus, the specific sequences of the monoclonal antibodies of the invention are not limited to the above specific heavy and light chain sequence variable regions, which are only monoclonal antibody sequences specifically employed in one implementation of the invention.
As a preferable technical scheme, the protein composed of the amino acid sequence with the similarity of more than or equal to 80% with the sequence shown as SEQ ID NO.6 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 6; the protein composed of the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.7 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 7; in the case of said substitution, the protein consisting of an amino acid sequence which is 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% identical to the reference sequence SEQ ID NO.6, SEQ ID NO.7 corresponds to a homologous sequence derived from a species different from the reference sequence; such substitutions include conservative substitutions, which are substitutions in which one amino acid is substituted by another amino acid having similar structure and/or chemical properties, and non-conservative substitutions. The sequences of the heavy or light chain variable regions differ from the reference sequences only by conservative amino acid substitutions.
The invention also discloses a method for preparing the monoclonal antibody of the hepatitis B virus surface antigen, which comprises the following steps:
the HBsAg protein expressed by CHO is used for three times of immunization on the balb/c mice, 100 mu g of protein is mixed with an equal volume of Freund adjuvant each time, and the abdominal cavities of the mice are injected in multiple points; two weeks after the third immunization, taking spleen cells of the mice to fuse with myeloma cell line SP2/0, culturing in a 96-well cell culture plate by a limiting dilution method, taking culture supernatant after two weeks, performing Western Blot detection, and finally screening to obtain antibody S1705 obtained by a cell clone, wherein HBsAg protein is detected.
As a preferred technical scheme, the monoclonal antibody can also be prepared by adopting a conventional gene recombination method, and can be a humanized antibody; or immunizing the mice again with the hybridoma cell strain obtained by screening to obtain the hepatitis B surface antibody monoclonal antibody.
The invention also discloses a biological material related to the monoclonal antibody of the hepatitis B virus surface antigen, which is characterized in that the biological material is selected from one of the following A) to D):
a) A nucleic acid molecule encoding the monoclonal antibody;
b) An expression cassette, a recombinant vector, a recombinant cell line comprising the nucleic acid molecule as described in A);
c) An antigenic peptide recognized by the monoclonal antibody;
d) A derivative of the monoclonal antibody;
the nucleotide sequence for encoding the HCDR1 is shown as SEQ ID NO.8, the nucleotide sequence for encoding the HCDR2 is shown as SEQ ID NO.9, the nucleotide sequence for encoding the HCDR3 is shown as SEQ ID NO.10, the nucleotide sequence for encoding the LCDR1 is shown as SEQ ID NO.11, the nucleotide sequence for encoding the LCDR2 is shown as SEQ ID NO.16, and the nucleotide sequence for encoding the LCDR2 is shown as SEQ ID NO.16: aaagtttcc, the nucleotide sequence encoding said LCDR3 is shown in SEQ ID NO. 12.
The nucleotide sequence for encoding the heavy chain variable region is shown as SEQ ID NO.13, and the nucleotide sequence for encoding the light chain variable region is shown as SEQ ID NO. 14;
the above specific nucleic acid sequences are only nucleic acid sequences specifically employed in one implementation of the present invention, and, depending on the degeneracy of the codons, several nucleic acid sequences encoding the same heavy chain variable region sequence or light chain variable region sequence (e.g., conservative nucleotide sequence variants derived from degenerated and silent variants of the genetic code, nucleotide substitutions, deletions and additions are also included) may be encoded, with the exception of the nucleic acid sequences defined above, within the scope of the present invention.
The monoclonal antibody recognizes a linear epitope of the hepatitis B virus surface antigen, and the recognition epitope is between 25 th amino acid and 50 th amino acid of the hepatitis B virus surface antigen.
The antigen peptide is positioned on the inner side of a lipid membrane of the hepatitis B virus surface antigen and positioned between 17 th amino acid and 66 th amino acid of a transmembrane region of the hepatitis B virus surface antigen, and the amino acid sequence of the antigen peptide is shown as SEQ ID NO. 15.
An expression cassette containing a nucleic acid molecule encoding an antibody refers to a DNA capable of expressing the antibody in a host cell, which DNA may include not only a promoter that initiates transcription of the antibody gene, but also a terminator that terminates transcription of the antibody gene.
The vector is one of plasmid, cosmid, phage and virus vector; a vector is a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed, and the vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried by the vector in the host cell.
Host cells refer to cells into which vectors are introduced, and include prokaryotic cells, fungal cells, insect cells, animal cells, etc., such as E.coli, yeast cells, S2 drosophila cells, BHK cells, CHO cells, HEK293 cells. The expression cassette, recombinant vector and recombinant cell line described above can be prepared by methods conventional in the art.
The derivatives of the monoclonal antibodies comprise one of antibody Fab fragments, single chain antibodies or fusion antibodies thereof, and can be prepared by adopting a conventional method in the field.
The invention also discloses application of the monoclonal antibody related biological material of the hepatitis B virus surface antigen, which is characterized by comprising one or more of the following applications:
the application in preparing products for detecting and diagnosing hepatitis B virus;
the application of the composition in preparing medicines for treating and preventing diseases caused by hepatitis B virus.
The product for detecting and diagnosing the hepatitis B virus is used for detecting hepatitis B virus surface antigens or mutants thereof with different genotypes.
The hepatitis B virus comprises A, B, C, D genotype hepatitis B virus; and/or the mutation site of the mutant is G145R.
The above-mentioned products may be diagnostic reagents (diagnostic kits), detection reagents (detection kits), etc., which may also include other reaction reagents.
The method for detecting the hepatitis B virus surface antigen or the mutant thereof comprises flow detection, tissue immunofluorescence detection, western blot detection and ELISA detection.
The flow detection is used for detecting the surface antigen of the hepatitis B virus or the mutant thereof presented on the surface of the cell, the tissue immunofluorescence detection is used for detecting the surface antigen of the hepatitis B virus or the mutant thereof in a primary sample, the Western blot detection or ELISA detection comprises the use of the monoclonal antibody for detecting the surface antigen of the hepatitis B virus or the mutant thereof which is derived from eukaryotic cells and the surface antigen of the hepatitis B virus or the mutant thereof with different genotypes, and the ELISA detection also comprises the use of the antigen peptide for detecting the monoclonal antibody. It is understood that the above detection method may also be any suitable detection method in the art.
In the above detection methods, the sample to be detected encompasses a variety of sample types obtained from a subject and can be used in diagnosis or detection, including but not limited to liquid samples of blood and other biological origin, solid tissue samples, clinical samples, cells in culture medium, cell supernatants, cell lysates, serum, plasma, biological fluids, tissue samples, and the like.
The invention also discloses a product for detecting and diagnosing hepatitis B virus.
The monoclonal antibody can be used as a coating antibody or an enzyme-labeled secondary antibody (detection antibody).
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.
EXAMPLE 1 preparation of monoclonal antibody S1705
In the embodiment, the HBsAg protein expressed by CHO is adopted to immunize a balb/c mouse for three times, a mouse hybridoma cell strain is obtained through cell fusion, and then the antibody is purified, and a monoclonal antibody S1705 which can be used for detecting hepatitis B surface protein by Western blot is obtained from tens of antibodies, and the specific method comprises the following steps:
the balb/c mice were immunized three times with CHO-expressed HBsAg protein, 100ug protein each time mixed with an equal volume of Freund's adjuvant, and the mice were injected intraperitoneally. Two weeks after the third immunization, mice were sacrificed, the spleens of the mice were isolated, and spleen cells were obtained after dissociation. Myeloma cells SP2/0 and spleen cells are mixed according to the ratio of 1:10, and are uniformly mixed for 120 seconds by adding PEG4000 preheated at 42 ℃ to carry out cell fusion, then the mixture is cultured in a 96-well cell culture plate by a limiting dilution method, and culture supernatant is taken after two weeks and is subjected to Western Blot detection. A hybridoma cell line under the condition of protein denaturation can be identified, and the hybridoma cell line is named S1705. Then, the S1705 cell line was expanded to 5T 75 cell culture flasks, after 5-7 days of culture, the supernatant was collected to about 50ml, collected in a 50ml centrifuge tube, and centrifuged at 8000rpm for 15 minutes. The supernatant was collected in a fresh centrifuge tube and passed through a 0.22 μm filter. To 50ml of supernatant, 200. Mu.l of Protein A/G beads were added and the mixture was subjected to a spin mixer overnight at 4 ℃. The next day, the centrifuge tube was removed, centrifuged at 1500rpm/min and at 4℃for 2min. 40ml of the supernatant was discarded, protein A/G beads in the well-mixed centrifuge tube were blown off and transferred to an affinity column. The chromatographic column is washed by cold PBS for 10 times, the last time the lower port is plugged by a plug, the cold PBS is soaked for a while and then is discharged, and then the lower port is plugged. The affinity purification column was mounted in a 15ml centrifuge tube (400. Mu.l of 1M Tris9.0 was added to the tube) and eluted by adding 3.6ml Elution Buffer. The eluate was transferred to a 30KD desalting filtration column, centrifuged at 4000rpm/min for 15min. 4ml of pre-chilled PBS was added to wash the filter tube once, and the tube was added to a desalting filter column at 4000rpm/min and centrifuged for 15min. The above steps are repeated once. Antibody S1705 was transferred to EP tube and stored at 4 degrees.
The sequence of the antibody S1705 was identified: the amino acid sequences of heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 6; the amino acid sequences of heavy chain complementarity determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region are shown in SEQ ID NO.4, KVS and SEQ ID NO.5, respectively, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7. The antibody S1705 further comprises a heavy chain constant region and a light chain constant region, and the nucleotide sequences encoding the respective constant regions are as follows:
example 2 epitope identification of monoclonal antibody S1705
This example demonstrates the identification of the epitope of monoclonal antibody S1705 prepared in example 1 by the synthesis of a peptide library, as follows:
the binding epitope of the S1705 antibody was identified by synthesis of a pool of HBsAg peptides of different segments but overlapping amino acids. The library of synthesized HBsAg peptides was dissolved in DMSO solution, and then 96-well plates were coated at 100 μl per well at a concentration of 10 μg/ml overnight. PBST plates were washed 3 times followed by 2% bsa blocking for 2 hours at room temperature. PBST wash plate 3 times, antibody S1705 at 1: diluted in a proportion of 1000, added to the well plate at a volume of 50. Mu.l/well and incubated for 1 hour at room temperature. PBST wash plates 3 times, secondary anti IgG-HRP at 1:5000 dilution, 50 μl/well volume added to the well plate, and incubated for 1 hr at room temperature. PBST plates were washed 5 times, and plates were read by adding color developing solution.
The experimental results of the epitope identification are shown in FIG. 1, and the recognition epitope of the antibody S1705 is confirmed to be between 25 th amino acid and 50 th amino acid of HBsAg; further researches show that the antigen peptide aimed by the antibody S1705 is positioned on the inner side of a lipid membrane with extremely low incidence rate of the escape mutation of the HBsAg and positioned between transmembrane regions of the small HBsAg (17 th amino acid-66 th amino acid, the amino acid sequence is shown as SEQ ID NO. 15), and the recognition epitopes are quite conserved on the HBsAg with ten HBV different genotypes.
In the protocol, a library of HBsAg peptide fragments with overlapping amino acid sequences was synthesized for identifying the epitope of monoclonal antibody S1705. The antibody binding epitope is detected through the HBsAg peptide segment, and the recognition epitope of S1705 is confirmed to be between 25 th amino acid and 50 th amino acid of the HBsAg; and found that the antigen peptide against which the S1705 antibody is directed is located inside the lipid membrane where the incidence of the escape mutation of HBsAg is extremely low, between the transmembrane regions of small HBsAg (amino acid 17-amino acid 66, recognition site 17-66 determined from the results of FIG. 1). Further, the recognition epitope is found to be very conserved on HBsAg of ten HBV different genotypes, and Western blot also proves that the antibody can detect HBsAg of 4 different genotypes.
EXAMPLE 3 Western blot detection of eukaryotic cell-derived HBsAg
In this example, the monoclonal antibody S1705 prepared in example 1 was used to detect eukaryotic cell-derived HBsAg by Western blot, and the specific experimental procedure is as follows:
the HBsAg sample used for detection was s protein expressed by CHO cells (ProSpec, cat.HBS-875), 2ug of protein was taken, an appropriate amount of protein binding buffer was added, half of the sample was taken for protein denaturation (HBs denaturation) in a water bath, and half was not denatured (HBs was not denatured). Huh7 cells were harvested, and a control sample (Huh 7 lysate) was prepared by adding the protein lysate and the protein binding buffer, and bathing in water for 10 minutes.
Western blot detection: after the gel is prepared, an electrophoresis tank is arranged, the inner cavity is filled with 1 XPAGE electrophoresis buffer solution, and a comb is carefully pulled out; the outer well was filled with 1 XPAGE running buffer to 1/2. And adding 8-15 mu l of protein samples into the loading holes in sequence, and supplementing by using a protein loading buffer when the protein marker is insufficient in volume. And (3) carrying out electrophoresis at constant pressure, concentrating the gel part by 90V, and increasing the voltage to 110-120V after the sample enters the separation gel. The electrophoresis time is determined according to the molecular weight of the protein to be detected, and the protein marker is generally required to be electrophoresed, and bromophenol blue is electrophoresed to the bottom of the separation gel. After electrophoresis is completed, the glass plate is taken down and pried off, and the gel is cut according to the size of the target protein; nitrocellulose membranes were cut according to gel size, and both the gel and membrane were immersed in a transfer buffer. Starting from one side of the positive electrode, placing a layer of foam cushion, a piece of thick filter paper, a layer of nitrocellulose membrane, gel, a piece of thick filter paper and a layer of foam cushion in sequence, taking note that no bubbles exist among the gel, the membrane and the filter paper, fixing a clamping plate, putting the clamping plate into a membrane transferring instrument, covering a cap, and taking note of the electrode direction. And (3) switching on a power supply, and carrying out constant current 300mA and film transfer for 1 hour and 15 minutes. After completion of the transfer, the membrane was blocked in PBST (blocking solution) containing 5% nonfat dry milk for 1h. Antibody S1705 as primary antibody, at 1:500 was diluted with a blocking solution, dropped onto a Parafilm membrane in a wet box, and nitrocellulose membrane was back-off onto the drop, and incubated overnight at 4℃in the wet box. The PBST was then washed 3 times for 10 minutes each. Use blocking liquid to 1:2000, the goat anti-mouse secondary antibody is diluted in proportion, and the secondary antibody is incubated in a reverse buckling method and is placed at room temperature for 1-2 hours. The PBST was washed 3 times for 10 minutes each. The luminous liquid is prepared at present. Sticking a preservative film in a film clamp, placing the film on the preservative film, dripping the luminescent liquid on the film in a darkroom, incubating for about 1 minute, sucking redundant luminescent liquid by using water absorption paper, covering the preservative film on the film, placing a film on the preservative film, closing the film clamp, and determining the tabletting time according to whether the protein is easy to detect. And (3) putting the film into a developing solution for developing, and transferring the film into a fixing solution when a target strip appears. And (5) after the luminescence is finished, air-drying the film, marking the protein marker position, and scanning pictures on an adesaine imaging scanner.
The result of Western blot detection of HBsAg antigen by antibody S1705 obtained from the hybridoma is shown in FIG. 2, which shows that antibody S1705 can detect the band of HBsAg, indicating that antibody S1705 can detect eukaryotic cell-derived HBsAg.
Example 4 Western blot detection of HBsAg for HBV of different genotypes
In this example, the expression of HBsAg of HBV of different genotypes was detected by Western blot using the monoclonal antibody S1705 prepared in example 1, which was transfected with HBV plasmids of different genotypes (genotype A/B/C/D) by Huh7 cells, after 48 hours, RIPA lysate was used to lyse the cells, the supernatant was centrifuged, and 2 Xloading buffer was added, and the sample was loaded after denaturation. The specific experimental procedure is as follows:
preparation of HBsAg protein samples: huh7 cells (10% foetal calf serum, 100U/ml penicillin, DMEM medium with 0.1mg/ml streptomycin) were seeded one day in 24 well plates in advance and transfected when cells had attached to the wall and cell density to 80%. Each well of transfected pUC19-HBV1.3 plasmid had a mass of 0.5. Mu.g. The HBV plasmids of the 4 genotypes transferred are pUC19-HBV-A2, pUC19-HBV-B5, pUC19-HBV-C2 and pUC19-HBV-D3 respectively, and the negative control is pUC19 empty plasmid and commercial HBsAg is used as a positive control. 1/20 of the medium volume Opi-Mem (25. Mu.l if the total medium volume is 500. Mu.l) was placed in a 1.5ml centrifuge tube and plasmid was added (P3000 reagent 0.5. Mu.l was added at this stage when lipofectamine 3000 was used for transfection); 1/20 of the culture medium volume Opi-Mem was placed in a 1.5ml centrifuge tube, and 0.5. Mu.l of lipofectamine 3000 transfection reagent or 2. Mu.l of PEI transfection reagent was added thereto and left for about 5 minutes. The plasmid-mixed Opi-Mem was added to the Opi-Mem mixed with transfection reagent and the vortex machine was gently shaken and left at room temperature for 10-15 minutes after flash off. Cells were harvested 48-72 hours after transfection, 50-80. Mu.l RIPA buffer was added, lysed on ice for 5-15 minutes, the samples were transferred to a 1.5ml centrifuge tube, 1/5 volume of 6 Xprotein loading buffer was added, mixed well, boiled in water bath for 10 minutes, and immediately placed on ice. Small amounts of supernatant were collected to detect HBsAg and HBeAg levels (kit is ambu) and the success of transfection was verified.
The Western blot protocol was performed as described in example 3, and the experimental results are shown in FIG. 3, which shows that antibody S1705 can detect bands of each HBsAg, indicating that antibody S1705 can detect HBsAg of different genotypes.
EXAMPLE 5 Western blot detection of HBsAg mutant
In this example, the monoclonal antibody S1705 prepared in example 1 was used for Western blot detection of HBsAg mutant. Huh7 cells transfected with a cell sample of the mutation site G145R plasmid (pUC 19-HBV 1.3-G145R), cells were lysed by RIPA lysate 48 hours after transfection, the supernatant was centrifuged, added with 2 Xloading buffer, denatured and loaded, and the HBsAg mutant protein sample was prepared by the method described in example 4.
The Western blot protocol was carried out using the protocol described in example 3, and the experimental results are shown in FIG. 4, which shows that antibody S1705 can detect the HBsAg band of the G145R site mutation, indicating that antibody S1705 can be used to detect the HBsAg mutant.
Example 6-flow detection of HBsAg presented on cell surface
In this example, the monoclonal antibody S1705 prepared in example 1 was used for flow assay of HBsAg presented on the cell surface, and the specific experimental procedure is as follows:
cell samples derived from cell lines were derived from HepAD38 cells, HBV stably replicating cell lines (HBV genotype D). HepAD38 was digested from the petri dish, washed with PBS buffer, centrifuged for 5min, the supernatant was discarded, the FACS solution was added to resuspend the cells, divided into experimental and control groups, and transferred to a 1.5ml centrifuge tube. Primary cell lines were derived from mouse liver cells successfully modeled by high pressure tail vein, the liver cells were ground, filtered, resuspended with FACS fluid, and the supernatant was discarded after centrifugation, and divided into two tubes of experimental and control groups. 1, the method comprises the following steps: 500, antibody S1705 was diluted and added to the experimental group samples and incubated at room temperature for 2 hours. An appropriate amount of FACS liquid was added, centrifuged at 10000rpm for 5 minutes, the supernatant was discarded, and the above steps were repeated 3 times. An appropriate amount of FACS fluid was added to 1:200, adding into the experimental group and the control group, and incubating for 1 hour at room temperature in a dark place. An appropriate amount of FACS liquid was added, centrifuged at 10000rpm for 5 minutes, the supernatant was discarded, and the above steps were repeated 3 times. Cells were resuspended and subjected to flow assays (Attune NxT, thermo Fisher).
The results of the experiments on HepAD38 cells and mouse liver cells are shown in fig. 5 and 6, respectively, and show that the HBsAg level of HepAD38 cells and HBsAg level of HBV replicating mouse liver cells can be detected by using the antibody S1705 flow.
EXAMPLE 7 tissue immunofluorescence detection of HBsAg in primary samples
In this example, the monoclonal antibody S1705 prepared in example 1 was used to detect HBsAg in the primary sample by tissue immunofluorescence, and tissue sections of hepatitis B, liver cancer and paracancer were purchased from Miss Biotech Inc., the specific experimental procedure is as follows:
the sections were placed on slides and fixed using immunostaining fixative. The immune blocking solution was added and blocked overnight in a 4 degree refrigerator. Antibody S1705 at 1:500, drop-wise onto glass slides and incubate at room temperature for 2 hours. The wash was washed 5 minutes and 3 times in total. 1, the method comprises the following steps: 2000, the fluorescent secondary antibody is diluted in proportion and dripped on a glass slide, and incubated for 1 hour at room temperature in a dark place. The wash was washed 5 minutes and 3 times in total. The encapsulation was performed with an encapsulation tablet, and a laser scanning confocal microscope (Leica, SP 8).
The results of the above experiment are shown in FIG. 7, which shows that antibody S1705 can be used to detect HBsAg levels in different tissue samples.
From the above examples, it is known that the monoclonal antibody S1705 obtained by screening of the present invention can be used for detecting linear epitopes of hepatitis B surface protein, the antigen peptide aimed at is located inside the lipid membrane of HBsAg and between the transmembrane regions of small HBsAg; the antibody S1705 can be used for Western blot detection of eukaryotic cell-derived HBsAg, HBsAg with different genotypes and HBsAg mutant; the antibody S1705 can be used for detecting the HBsAg presented on the cell surface in a flow mode, can be used for detecting the HBsAg in a primary sample by tissue immunofluorescence, is suitable for detecting various types of samples, and opens up a new method for detecting hepatitis B virus and treating hepatitis B diseases.
The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A monoclonal antibody to hepatitis b virus surface antigen, characterized in that: monoclonal antibodies include heavy chain variable regions and light chain variable regions;
the heavy chain variable region comprises a heavy chain complementarity determining region HCDR1 composed of amino acid sequences shown as SEQ ID NO.1, a heavy chain complementarity determining region HCDR2 composed of amino acid sequences shown as SEQ ID NO.2 and a heavy chain complementarity determining region HCDR3 composed of amino acid sequences shown as SEQ ID NO. 3;
the light chain variable region comprises a light chain complementarity determining region LCDR1 composed of the amino acid sequences shown in SEQ ID NO.4, a light chain complementarity determining region LCDR2 composed of the amino acid sequences KVS and a light chain complementarity determining region LCDR3 composed of the amino acid sequences shown in SEQ ID NO. 5.
2. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a hepatitis b virus surface antigen: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.6 or the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.7 or the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO. 7.
3. A monoclonal antibody to hepatitis b virus surface antigen according to claim 1, wherein: the monoclonal antibodies also include heavy chain constant regions and light chain constant regions.
4. A monoclonal antibody to hepatitis b virus surface antigen according to claim 2, wherein: the protein composed of the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.6 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 6; the protein composed of the amino acid sequence with the similarity of more than or equal to 80 percent with the sequence shown as SEQ ID NO.7 contains mutation such as deletion, insertion or substitution compared with the reference sequence shown as SEQ ID NO. 7; in the case of said substitution, the protein consisting of an amino acid sequence which is 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% identical to the reference sequence SEQ ID NO.6, SEQ ID NO.7 corresponds to a homologous sequence derived from a species different from the reference sequence; such substitutions include conservative substitutions, which are substitutions in which one amino acid is substituted by another amino acid having similar structure and/or chemical properties, and non-conservative substitutions.
5. A method for preparing a monoclonal antibody to hepatitis b virus surface antigen as claimed in claim 1, comprising the steps of:
the HBsAg protein expressed by CHO is used for three times of immunization on the balb/c mice, 100 mu g of protein is mixed with an equal volume of Freund adjuvant each time, and the abdominal cavities of the mice are injected in multiple points; two weeks after the third immunization, taking spleen cells of the mice to fuse with myeloma cell line SP2/0, culturing in a 96-well cell culture plate by a limiting dilution method, taking culture supernatant after two weeks, performing Western Blot detection, and finally screening to obtain antibody S1705 obtained by a cell clone, wherein HBsAg protein is detected.
6. A biological material related to monoclonal antibodies to hepatitis b virus surface antigens according to claim 1, wherein said biological material is selected from one of the following a) to D):
a) A nucleic acid molecule encoding the monoclonal antibody;
b) An expression cassette, a recombinant vector, a recombinant cell line comprising the nucleic acid molecule as described in A);
c) An antigenic peptide recognized by the monoclonal antibody;
d) A derivative of the monoclonal antibody;
the nucleotide sequence for encoding the HCDR1 is shown as SEQ ID NO.8, the nucleotide sequence for encoding the HCDR2 is shown as SEQ ID NO.9, the nucleotide sequence for encoding the HCDR3 is shown as SEQ ID NO.10, the nucleotide sequence for encoding the LCDR1 is shown as SEQ ID NO.11, the nucleotide sequence for encoding the LCDR2 is shown as SEQ ID NO.16, and the nucleotide sequence for encoding the LCDR3 is shown as SEQ ID NO. 12.
The nucleotide sequence for encoding the heavy chain variable region is shown as SEQ ID NO.13, and the nucleotide sequence for encoding the light chain variable region is shown as SEQ ID NO. 14; the amino acid sequence of the antigen peptide is shown as SEQ ID NO. 15.
7. The monoclonal antibody-related biomaterial of hepatitis b virus surface antigen of claim 6, wherein: the vector is one of plasmid, cosmid, phage and virus vector.
8. The monoclonal antibody-related biomaterial of hepatitis b virus surface antigen of claim 6, wherein: the derivatives of the monoclonal antibodies comprise one of antibody Fab fragments, single chain antibodies or fusion antibodies thereof, and can be prepared by adopting a conventional method in the field.
9. Use of a biological material related to monoclonal antibodies to hepatitis b virus surface antigen according to claim 6, comprising one or more of the following applications:
the application in preparing products for detecting and diagnosing hepatitis B virus;
the application of the composition in preparing medicines for treating and preventing diseases caused by hepatitis B virus.
10. A product for detecting and diagnosing hepatitis b virus according to claim 1 or 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211063471.8A CN116162153A (en) | 2022-09-01 | 2022-09-01 | Monoclonal antibody of hepatitis B virus surface antigen and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211063471.8A CN116162153A (en) | 2022-09-01 | 2022-09-01 | Monoclonal antibody of hepatitis B virus surface antigen and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116162153A true CN116162153A (en) | 2023-05-26 |
Family
ID=86418823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211063471.8A Pending CN116162153A (en) | 2022-09-01 | 2022-09-01 | Monoclonal antibody of hepatitis B virus surface antigen and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116162153A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117024578A (en) * | 2023-07-19 | 2023-11-10 | 重庆明道捷测生物科技有限公司 | IgG antibody for capturing HBV core particles and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007014267A (en) * | 2005-07-07 | 2007-01-25 | Toyama Univ | MONOCLONAL ANTIBODY AGAINST HBs ANTIGEN OF HEPATITIS B VIRUS, GENE AND PEPTIDE RELATING THERETO, METHOD FOR ASSAYING HEPATITIS B VIRUS, METHOD FOR DIAGNOSIS OF HEPATITIS B AND METHOD FOR TREATMENT OF HEPATITIS B |
| CN101037476A (en) * | 2006-03-15 | 2007-09-19 | 复旦大学 | Fusion protein for preparing hepatitis B vaccine and its carrier |
| CN101928344A (en) * | 2002-10-17 | 2010-12-29 | 根马布股份公司 | The human monoclonal antibodies of anti-CD20 |
| WO2013185558A1 (en) * | 2012-06-11 | 2013-12-19 | 厦门大学 | Polypeptides and antibodies for treating hbv infection and related diseases |
| CN106008708A (en) * | 2015-03-25 | 2016-10-12 | 复旦大学 | Monoclonal antibody of human hepatitis B virus X protein and use thereof |
| CN108018267A (en) * | 2017-12-27 | 2018-05-11 | 复旦大学 | One kind targeting apparent gene regulation and control anti-hepatitis B virus molecule HBx1C and its application |
| CN108137675A (en) * | 2015-10-07 | 2018-06-08 | 胡默波斯生物医学公司 | Strongly neutralize the antibody and its purposes of hepatitis type B virus |
| CN112079932A (en) * | 2020-09-22 | 2020-12-15 | 浙江康佰裕生物科技有限公司 | Chimeric antigen receptor for treating liver cancer and application thereof |
| CN113138276A (en) * | 2020-01-19 | 2021-07-20 | 厦门万泰凯瑞生物技术有限公司 | Method for detecting HBcAg and antibody |
| CN114540312A (en) * | 2022-03-16 | 2022-05-27 | 河南省农业科学院 | Preparation method and application of monoclonal antibody of Marek's disease virus (MrV) pp38 in serum type 1 |
-
2022
- 2022-09-01 CN CN202211063471.8A patent/CN116162153A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928344A (en) * | 2002-10-17 | 2010-12-29 | 根马布股份公司 | The human monoclonal antibodies of anti-CD20 |
| JP2007014267A (en) * | 2005-07-07 | 2007-01-25 | Toyama Univ | MONOCLONAL ANTIBODY AGAINST HBs ANTIGEN OF HEPATITIS B VIRUS, GENE AND PEPTIDE RELATING THERETO, METHOD FOR ASSAYING HEPATITIS B VIRUS, METHOD FOR DIAGNOSIS OF HEPATITIS B AND METHOD FOR TREATMENT OF HEPATITIS B |
| CN101037476A (en) * | 2006-03-15 | 2007-09-19 | 复旦大学 | Fusion protein for preparing hepatitis B vaccine and its carrier |
| WO2013185558A1 (en) * | 2012-06-11 | 2013-12-19 | 厦门大学 | Polypeptides and antibodies for treating hbv infection and related diseases |
| CN106008708A (en) * | 2015-03-25 | 2016-10-12 | 复旦大学 | Monoclonal antibody of human hepatitis B virus X protein and use thereof |
| CN108137675A (en) * | 2015-10-07 | 2018-06-08 | 胡默波斯生物医学公司 | Strongly neutralize the antibody and its purposes of hepatitis type B virus |
| CN108018267A (en) * | 2017-12-27 | 2018-05-11 | 复旦大学 | One kind targeting apparent gene regulation and control anti-hepatitis B virus molecule HBx1C and its application |
| CN113138276A (en) * | 2020-01-19 | 2021-07-20 | 厦门万泰凯瑞生物技术有限公司 | Method for detecting HBcAg and antibody |
| CN112079932A (en) * | 2020-09-22 | 2020-12-15 | 浙江康佰裕生物科技有限公司 | Chimeric antigen receptor for treating liver cancer and application thereof |
| CN114540312A (en) * | 2022-03-16 | 2022-05-27 | 河南省农业科学院 | Preparation method and application of monoclonal antibody of Marek's disease virus (MrV) pp38 in serum type 1 |
Non-Patent Citations (1)
| Title |
|---|
| 张小艳;龚劲松;张波;刘静华;黄永国;张春燕;李方和;: "乙肝病毒表面抗原G145R变异单克隆抗体的制备及其初步鉴定", 细胞与分子免疫学杂志, no. 08, 18 August 2008 (2008-08-18) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117024578A (en) * | 2023-07-19 | 2023-11-10 | 重庆明道捷测生物科技有限公司 | IgG antibody for capturing HBV core particles and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6127043B2 (en) | AGR2 blocking antibody and use thereof | |
| CN107022030B (en) | Monoclonal antibody for detecting alpha-fetoprotein, kit and application | |
| WO2016173558A1 (en) | Preparation and use of anti-norovirus gii.4 type murine monoclonal antibody | |
| CA2879185A1 (en) | Method for detecting cancer | |
| WO2016173559A1 (en) | Preparation and use of murine monoclonal antibody against gi.1 norovirus | |
| CN110627901B (en) | Monoclonal antibody of influenza virus matrix protein M1 and application thereof | |
| CN116162153A (en) | Monoclonal antibody of hepatitis B virus surface antigen and application thereof | |
| CN108794630A (en) | Mouse-anti-human T IM3 protein monoclonal antibodies prepare and its immunohistochemistry purposes | |
| WO2019045086A1 (en) | USE FOR SPECIFIC ANTIBODY FOR ACTIVE OR LATENT TGF-β1 | |
| CN113227148A (en) | anti-GPC 3 antibody, antigen-binding fragment thereof, and medicinal use thereof | |
| EP4198054A1 (en) | Anti-hmmw antibody, composition containing same, nucleic acid molecule encoding same, and use thereof | |
| WO2017107131A1 (en) | Tp recombinant antigen, and preparation method and application thereof | |
| CN111018989A (en) | anti-PD-L1 monoclonal antibody and application thereof in preparation of anti-cancer drugs | |
| CN109593131B (en) | Monoclonal antibody for resisting 14-3-3 eta protein and application thereof | |
| CN106674350B (en) | Preparation and application of liver cancer marker single-chain antibody | |
| AU2005290997B2 (en) | Novel cancer associated antibodies and antigens and their use in cancer diagnosis | |
| CN111778257A (en) | Genetically modified CD96 protein, monoclonal antibody thereof and application | |
| CN106589128B (en) | Preparation and application of liver cancer marker monoclonal antibody | |
| CN109897109B (en) | Monoclonal antibody of antitumor marker protein and application thereof | |
| EP4170021A1 (en) | Mouse anti-mcr-1 protein hybridoma cell strain, monoclonal antibody, and application | |
| KR102241520B1 (en) | An antibody having immune reactivity to Foot-and-Mouth Disease Virus type O and composition for detecting FMDV type O comprising the same | |
| CN112724253B (en) | Antibody of anti-human vault protein and application thereof | |
| CN114853889B (en) | Monoclonal antibodies against human GPR48 and uses thereof | |
| CN113087802B (en) | Kit for detecting cancer by combining nucleic acid detection with antibody | |
| CN114213542B (en) | CPS-I antibodies and uses 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 |