CN116284416A - Monoclonal antibody for resisting endogenous PINK1 protein and application thereof - Google Patents
Monoclonal antibody for resisting endogenous PINK1 protein and application thereof Download PDFInfo
- Publication number
- CN116284416A CN116284416A CN202310026939.4A CN202310026939A CN116284416A CN 116284416 A CN116284416 A CN 116284416A CN 202310026939 A CN202310026939 A CN 202310026939A CN 116284416 A CN116284416 A CN 116284416A
- Authority
- CN
- China
- Prior art keywords
- chain variable
- monoclonal antibody
- pink1
- variable region
- amino acid
- 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
- 101000605835 Homo sapiens Serine/threonine-protein kinase PINK1, mitochondrial Proteins 0.000 title claims abstract 18
- 102100038376 Serine/threonine-protein kinase PINK1, mitochondrial Human genes 0.000 title claims abstract 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 52
- 210000004027 cell Anatomy 0.000 claims description 43
- 230000004927 fusion Effects 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 230000014509 gene expression Effects 0.000 claims description 8
- 210000004989 spleen cell Anatomy 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 5
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 101150072531 10 gene Proteins 0.000 claims description 3
- 241001529936 Murinae Species 0.000 claims description 2
- 210000004102 animal cell Anatomy 0.000 claims description 2
- 239000012620 biological material Substances 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 8
- 229920001184 polypeptide Polymers 0.000 abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 8
- 238000012216 screening Methods 0.000 abstract description 5
- 210000004408 hybridoma Anatomy 0.000 abstract description 4
- 230000009465 prokaryotic expression Effects 0.000 abstract description 4
- 101100190541 Caenorhabditis elegans pink-1 gene Proteins 0.000 abstract description 3
- 241000282567 Macaca fascicularis Species 0.000 abstract description 3
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 241000588724 Escherichia coli Species 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 description 33
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000001262 western blot Methods 0.000 description 9
- 238000011532 immunohistochemical staining Methods 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 6
- 102000045222 parkin Human genes 0.000 description 6
- 101000619542 Homo sapiens E3 ubiquitin-protein ligase parkin Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 238000007664 blowing Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000003125 immunofluorescent labeling Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000282693 Cercopithecidae Species 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 101000981464 Homo sapiens Presenilins-associated rhomboid-like protein, mitochondrial Proteins 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 102100024056 Presenilins-associated rhomboid-like protein, mitochondrial Human genes 0.000 description 3
- 102000044159 Ubiquitin Human genes 0.000 description 3
- 108090000848 Ubiquitin Proteins 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 210000003470 mitochondria Anatomy 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 210000001700 mitochondrial membrane Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 108010042046 Mitochondrial processing peptidase Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 108010081575 PTEN-induced putative kinase Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 2
- 238000011091 antibody purification Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000025608 mitochondrion localization Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102100030013 Endoribonuclease Human genes 0.000 description 1
- 101710199605 Endoribonuclease Proteins 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 description 1
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 description 1
- 101150066884 Pink1 gene Proteins 0.000 description 1
- 102000015499 Presenilins Human genes 0.000 description 1
- 108010050254 Presenilins Proteins 0.000 description 1
- 101710113029 Serine/threonine-protein kinase Proteins 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000004642 autophagic pathway Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007711 cytoplasmic localization Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 208000018620 early-onset Parkinson disease Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 210000004884 grey matter Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108700007244 parkin Proteins 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a monoclonal antibody of an anti-endogenous PINK1 protein and application thereof, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 1-3 respectively; the amino acid sequences of the light chain variable regions are respectively shown as SEQ ID No. 4-5 and LVS. The monoclonal antibody can be used for identifying and detecting the PINK1 protein and is used for non-clinical diagnosis; can also be used for preparing reagents and kits for identifying and detecting the PINK1 protein. According to the invention, a PINK1 gene sequence fragment is constructed artificially, introduced into escherichia coli for prokaryotic expression, a PINK1 polypeptide fragment is collected and purified, a mouse is immunized by the PINK1 polypeptide fragment, a monoclonal antibody is prepared, and finally, a hybridoma cell strain 2E7B6 capable of specifically binding with a PINK1 protein is obtained through screening. The antibody can be specifically combined with PINK1 proteins of human and cynomolgus monkeys, and has good experimental performance.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody for resisting endogenous PINK1 protein and application thereof.
Background
PINK1 (PTEN-reduced kinase 1) is a serine/threonine protein kinase whose protein structure comprises an N-terminal mitochondrial localization sequence (mitochondrial targeting Sequence, MTS) linked to a transmembrane sequence (Transmembrane segment, TMS) and a serine/threonine kinase functional domain. It has been found that mutations in PINK1 at certain sites are associated with early onset parkinson's disease.
The metabolic conditions of PINK1 in vivo include two different modes, physiological and stress. Normally, full-length PINK1 is directed to the mitochondria by a mitochondrial localization sequence (MTS) and anchored to the inner mitochondrial membrane, mitochondrial processing peptidases (mitochondrial processing peptidase, MPP) in the mitochondrial matrix cleave off the localization sequence, and then presenilin-related diamond proteins (presenilin associated rhomboid like protein, PARL) cleave the transmembrane region at amino acid position 104, freeing the PINK1 kinase active region from the mitochondrial surface, free from the cytoplasm, and then degraded by the N-terminal regulated pathway.
When mitochondria are damaged or stimulated by the outside world, the mitochondrial membrane depolarizes, so that the PINK1 is positioned in disorder, only the outer membrane is positioned, the inner membrane cannot be accessed, and the MPP and the PARL cannot be excised at corresponding sites, so that the PINK1 cannot be dissociated and degraded. At this time, the abnormally located full-length PINK1 protein recruits E3Ubiquitin ligase (E3 Ubiquitin ligase) Parkin protein to the outer mitochondrial membrane, phosphorylates at the Parkin S65 site to activate Parkin, and at the same time, can activate Parkin through phosphorylating the Ubiquitin (Ubiquitin) molecule S65 site and then through Ubiquitin S65. Activated Parkin acts as an amplifier, triggering downstream PINK1/Parkin dependent autophagy pathways, clearing damaged mitochondria.
Knowledge of the structure and function of Pink1 is continually refreshed, but there are still many problems that need to be further confirmed. Because of the lack of specific and sensitive PINK1 antibodies, a great deal of research on the PINK1 antibodies is remained in vitro for over-expression or expressed in a stress state through chemical drug stimulation, and the research on the structure and the function of endogenous PINK1 proteins is not deep.
At present, commercial PINK1 antibodies are mostly polyclonal antibodies after knockout verification, and compared with monoclonal antibodies, the polyclonal antibodies have some limitations in use, for example, the differences among the batches of the polyclonal antibodies are larger; the non-specific impurity bands are more in the immunoblotting experiment, the background is deeper in the immunostaining experiment, and the like.
Disclosure of Invention
The invention aims to prepare the monoclonal antibody which has stronger specificity, higher recognition efficiency and simpler and more convenient acquisition method and can recognize endogenous human and monkey PINK1 proteins, and the monoclonal antibody is used for PINK1 protein detection for non-clinical diagnosis purposes.
The aim of the invention is achieved by the following technical scheme:
the heavy chain variable regions VHCDR1, VHCDR2 and VHCDR3 of the anti-endogenous PINK1 protein monoclonal antibody are respectively shown in SEQ ID No. 1-3; the amino acid sequences of the light chain variable region VLCDR1 and VLCDR3 are respectively shown in SEQ ID No. 4-5, and the amino acid sequence of the light chain variable region VLCDR2 is LVS;
the gene sequences encoding the heavy chain variable region and the light chain variable region amino acid sequences of the anti-endogenous PINK1 protein monoclonal antibody are also within the protection scope of the invention;
further, the gene sequences ntCDR1, ntCDR2, ntCDR3 of SEQ ID No. 6-8 are used to encode the amino acid sequences of the heavy chain variable regions VHCDR1, VHCDR2, VHCDR3, respectively; SEQ ID Nos. 9 to 10 gene sequences ntCDR1 and ntCDR3 are used for encoding the amino acid sequences of the light chain variable regions VLCDR1 and VLCDR3 respectively, and the gene sequence ctggtgtgtct (ntCDR 2) is used for encoding the amino acid sequence of the light chain variable region VLCDR 2;
the anti-endogenous PINK1 protein monoclonal antibody is a murine monoclonal antibody.
The anti-endogenous PINK1 protein monoclonal antibody is secreted by a monoclonal cell strain 2E7B 6;
the monoclonal cell strain 2E7B6 is obtained by fusing immune mouse spleen cells and mouse myeloma cells.
The anti-endogenous PINK1 protein monoclonal antibody can also be any one of the following antibodies:
(a) A single chain antibody obtained by linking any heavy chain variable region amino acid and any light chain variable region amino acid;
(b) A fusion antibody comprising the single chain antibody of (a);
(c) An intact antibody comprising any heavy chain variable region amino acid and any light chain variable region amino acid;
(d) Monoclonal antibodies produced by monoclonal cell line 2E7B6.
Biological materials related to the monoclonal antibodies of the invention are also within the scope of the invention, which are any of the following:
(1) An expression cassette comprising a gene sequence encoding the heavy chain variable region and the light chain variable region amino acid sequences of the anti-endogenous PINK1 protein monoclonal antibody;
(2) A recombinant vector containing gene sequences encoding the heavy chain variable region and the light chain variable region amino acid sequences of the anti-endogenous PINK1 protein monoclonal antibody;
(3) A recombinant vector comprising the expression cassette of (1);
(4) A transgenic animal cell line comprising a gene sequence encoding the heavy chain variable region and the light chain variable region amino acid sequences of the anti-endogenous PINK1 protein monoclonal antibody;
(5) A microorganism comprising a gene sequence encoding the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-endogenous PINK1 protein monoclonal antibody.
The anti-endogenous PINK1 protein monoclonal antibody can be used for identifying and detecting PINK1 proteins and is used for non-clinical diagnosis; preferably for the identification and detection of human and non-human primate PINK1 proteins;
the anti-endogenous PINK1 protein monoclonal antibody can be used for Western immunoblotting detection (WB), immunohistochemical staining (IHC) and immunofluorescence chemical staining (IF) of PINK1 proteins;
the non-human primate includes cynomolgus monkey, macaque, etc.
The anti-endogenous PINK1 protein monoclonal antibody can be used for preparing a reagent and a kit for identifying and detecting the PINK1 protein.
Compared with the prior art, the invention has the following advantages and effects:
according to the invention, a PINK1 gene sequence fragment is constructed artificially, introduced into escherichia coli for prokaryotic expression, a PINK1 polypeptide fragment is collected and purified, a mouse is immunized by the PINK1 polypeptide fragment, a monoclonal antibody is prepared, and finally, a hybridoma cell strain 2E7B6 capable of specifically binding with a PINK1 protein is obtained through screening. The antibody can be specifically combined with PINK1 proteins of human and cynomolgus monkeys, and has better experimental performance in experiments such as western immunoblotting (WB), immunohistochemical staining (IHC), immunofluorescence chemical staining (IF) and the like.
Drawings
FIG. 1 is a graph showing the results of in vitro expression of the PINK1 protein polypeptide (175 aa-250 aa); wherein lane M 1 Is a marker; lane 1 is a human PINK1 protein polypeptide (175 aa-250 aa); lane 2 is BSA.
FIG. 2 is a culture diagram of hybridoma monoclonal cell selection 2E7B6.
FIG. 3 is a SDS-PAGE of purified antibodies; wherein lane M is marker; lane 1 is antibody secreted by cell line 2E7B6.
FIG. 4 is a graph showing the Western Blotting (WB) results of the 2E7B6 antibody.
FIG. 5 is a graph of the results of immunohistochemical staining (IHC) of the 2E7B6 antibody.
FIG. 6 is a graph showing the results of immunofluorescent staining (IF) with the 2E7B6 antibody.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 prokaryotic expression vector construction
1.1 in vitro expression and purification of human PINK1 protein polypeptide (175 aa-250 aa). Total RNA was extracted from human somatic cells (skin fibroblasts), reverse transcribed into cDNA, and the gene sequence corresponding to amino acids 175 to 250 was sequenced by specific primers, primers were designed to amplify PINK1 (175 aa-250 aa), primers were as follows: upstream primer atgcctacattgccccagaac, downstream primer agccaaggccactcggctcgc.
1.2 upstream primer added BamHI cleavage site and downstream primer added HindIII cleavage site, PCR amplification was performed and gel recovery was performed. The gel recovered product and pET30a vector were digested simultaneously with BamHI and HindIII and ligated, and transferred into Ecoli DH5a competence for expression vector expansion. Specifically, ecoli DH5a competent cells were taken out from a-80℃refrigerator, thawed on ice, added with 2ul of recombinant plasmid to 100ul of competent cells, incubated on ice for 30 minutes, then incubated on ice for 45 seconds in a 42℃water bath, then incubated on ice for 2 minutes, then added with 1ml of antibiotic-free LB medium, shaken on a 37℃shaker for 1 hour, finally plated on a kanamycin-containing plate, and incubated overnight in a 37℃incubator.
1.3 colony sequencing, selecting the receptor bacteria successfully introduced with the recombinant plasmid, performing amplification culture and extracting the plasmid.
EXAMPLE 2 PINK1 protein polypeptide induced expression, purification and characterization
2.1 transferring the amplified plasmid of interest into BL21 prokaryotic expression vector system. For specific procedures, refer to example 1, step 1.2.
2.2 picking up monoclonal colonies, shaking (4 ml) for 4 hours at 37 ℃ with a shaking table of 250rpm, transferring to a large shaking table (500 ml), adding 1mM inducer IPTG (isopropyl thiogalactoside) when the OD value reaches 0.6-0.8, and culturing overnight.
2.3 collecting bacterial liquid, cracking and extracting protein, purifying and enriching the protein through nickel column affinity, and performing SDS-PAGE identification, wherein the PINK1 protein is correctly expressed as shown in figure 1.
EXAMPLE 3 monoclonal antibody preparation
3.1 immunization of animals.
5 BALB/c mice were selected, and PINK1 protein was mixed with Freund's adjuvant and immunized by subcutaneous injection at 50 ug/day, and the immunization was boosted once for 2-3 weeks. Blood collection detection, namely determining the titer of the antiserum against the PINK1 by an indirect ELISA method.
3.2 cell electrofusion
3.2.1 preparation of myeloma cells: one week prior to fusion, SP2/0 cells were grown expanded in DMEM medium containing 10% FBS. By the time of fusion, the cells grew to approximately 6T 25 cell flasks, and SP2/0 cells were collected on the day of fusion into 50mL centrifuge tubes, 1500rpm, and centrifuged for 5min. The supernatant was discarded, then 20mL of DMEM basal medium was added, the cells were blown off and counted.
3.2.2 preparation of mouse spleen cells: mice with serum ELISA titers greater than 8000 post-four-immunization were selected (i.e., ELISA remained positive after 8000-fold serum dilution). Mice to be fused were euthanized on the day of fusion by cervical dislocation. Soaking in 75% alcohol for 5min. Spleens were aseptically removed and placed in petri dishes with 10mL DMEM basal medium. The screen was placed in another plate, the spleen was transferred to the screen and the spleen was ground with the syringe core. DMEM was added to the screen and the screen was rinsed to allow more spleen cells to collect in the dish. The cells were transferred to a 50mL centrifuge tube, and the spleen cells were washed once with serum-free DMEM and centrifuged at 1500rpm for 5min. Spleen cell counts were collected.
3.2.3 cell fusion: myeloma cells and spleen cells are mixed, preferably such that the ratio of myeloma cells to spleen cells is 1:2. The cells were placed in 50ml centrifuge tubes, diluted with DMEM basal medium, and centrifuged at 1500rpm for 5min. The supernatant was discarded. Shaking the centrifuge tube to homogenize the cellsThe cells were washed once with the electrofusion solution and then centrifuged at 1500rpm for 5min. The supernatant was discarded. Dilution of cells to 2X 10 with electrofusion 7 2ml of cell suspension is added into the fusion pool for electrofusion on-machine experiment, and the fusion pool is kept stand for 10min after electric shock. HAT DMEM medium was then added. The fused cells were plated into 96-well plates at 100ul per well. The cell culture plate is then placed in CO 2 Culturing in an incubator. The hybridoma cell cloning efficiency is over 50 percent, a small amount of cell fragments are contained, and the cell growth state is good after 6 days of fusion. Screening assays were started 10 days after fusion.
3.2.4 fusion cell screening: on the day before detection, 1ug/ml antigen was coated with PBS on ELISA plates overnight. The ELISA detection is carried out by sucking 100ul of cell supernatant per well in the next day, and positive wells are judged according to ELISA results (positive wells are judged when the OD value of a sample well/the OD value of a negative well is more than or equal to 2.1). And (3) picking the positive holes detected by the whole plate by using a single-channel pipette, and performing a second confirmation detection to further confirm the positive holes. The positive well cells after the determination were subcloned.
3.2.5 cell subcloning: blowing cells in the positive holes, counting, adding N/4mL of DMEM culture medium (N is a cell counting result) into a centrifuge tube, taking 100ul of cell suspension into the centrifuge tube, keeping 1mL after blowing evenly, adding DMEM to 4mL, blowing evenly, and keeping 100ul (about 2 drops) at the bottom of the tube. Adding DMEM to 5ml into a centrifuge tube, uniformly mixing, dropwise adding into the first three rows of a 96-well plate, keeping about 1.8-2ml of DMEM to 5ml of DMEM at the bottom of each drip tube, uniformly blowing, dropwise adding into D, E, F three rows of the 96-well plate, keeping about 1.5-1.8ml of DMEM to about 2.8-3ml of DMEM at the bottom of each drip tube, uniformly blowing, dropwise adding into G, H rows of the 96-well plate, dropwise adding into each drip, observing under a microscope after 7-10 days, detecting a hole with clone growth, marking a monoclonal hole, picking positive monoclonal cells as far as possible for subcloning again, and picking out the monoclonal holes for enlarging culture strains after 100% of positivity is detected.
3.2.6 subclone screening: the specific steps are the same as 3.2.4.
3.2.7 antibody expression: the monoclonal cell line 2E7B6 (the culture diagram is shown in FIG. 2) is selected for preparing the ascites antibody of the mice. The 2E7B6 monoclonal cell strain was inoculated into the abdominal cavity of a mouse, and ascites was collected after 7 to 10 days for antibody purification. Optionally: in this step, the 2E7B6 monoclonal cell line may be cultured in vitro, and the culture supernatant may be collected.
3.2.8 antibody purification: loading Protein A+G agarose gel medium into an affinity purification chromatographic column, slowly loading 2E7B6 cell supernatant, eluting with alkaline eluting buffer solution after antibody combination to obtain the required purified antibody, immediately dialyzing in PBS at 4deg.C overnight, and measuring purity and concentration every other day (as shown in figure 3, 0.52 mg/mL).
3.2.9 antibody identification: the 2E7B6 monoclonal cell strain is sent to Nanjde Tex bioengineering Co.Ltd, the light and heavy chain variable regions and the non-variable regions are respectively subjected to gene sequencing, and amino acid backtracking is carried out through a universal codon table to determine the amino acid sequences of the light and heavy chain variable regions and the non-variable regions.
The amino acid sequences of the heavy chain variable regions VHCDR1, VHCDR2 and VHCDR3 of the monoclonal antibody obtained by the 2E7B6 monoclonal cell strain are respectively shown in SEQ ID No. 1-3; the amino acid sequences of the VLCDR1 and VLCDR3 of the obtained monoclonal antibody light chain variable region are respectively shown in SEQ ID No. 4-5, and the amino acid sequence of the VLCDR2 of the light chain variable region is LVS;
the gene sequences ntCDR1, ntCDR2 and ntCDR3 of SEQ ID No. 6-8 are respectively used for encoding amino acid sequences of heavy chain variable regions VHCDR1, VHCDR2 and VHCDR 3; SEQ ID Nos. 9 to 10 gene sequences ntCDR1, ntCDR3 are used for encoding the amino acid sequences of the light chain variable regions VLCDR1, VLCDR3, respectively, and the gene sequence ctggtgtgtct (ntCDR 2) is used for encoding the amino acid sequence of the light chain variable region VLCDR 2.
EXAMPLE 4 Western blot application of monoclonal antibodies
4.1 taking normal control monkey and PINK1 knock-down monkey models of cerebral cortex tissue (see references Yang W, liu Y, tu Z, et al CRISPR/Cas9-mediated PINK1 deletion leads to neurodegeneration in rhesus monkeys [ J ]. Cell research,2019,29 (4): 334-336. Animal model previously constructed in the subject group), SDS-PAGE was performed after total protein extraction under conditions of 120V for 90 minutes.
4.2 transfer proteins to nitrocellulose membrane by electrotransfer after electrophoresis was completed at 100v for 60 min.
4.3 after the film transfer, taking out the nitrocellulose film, and sealing with 5% skim milk powder sealing liquid for 1 hour.
4.4 monoclonal antibodies were added and incubated overnight at 4℃on a shaker.
4.5 wash three times with TBST solution for 10 minutes each.
4.6 adding horseradish peroxidase (HRP) labeled rabbit anti-mouse secondary antibody, shaking table at room temperature for 1 hr.
4.7 wash three times with TBST solution for 10 minutes each.
4.8, evenly incubating the developing solution and developing by an automatic developing instrument.
The experimental result is shown in fig. 4, the comparison effect of the antibody is obviously improved compared with that of similar polyclonal antibodies of the N company, and the antibody has more specific recognition effect and higher recognition efficiency on the target strip under the same antibody concentration.
EXAMPLE 5 immunohistochemical use of monoclonal antibodies
5.1 incubation of monkey brain cortex tissue brain sections with 3% hydrogen peroxide for 10min, followed by PBS infiltration and washing off residual hydrogen peroxide
5.2 adding enough citric acid antigen retrieval liquid into the staining jar to fully infiltrate the brain slice, and placing the staining jar into a boiling water bath at 100 ℃ to carry out antigen retrieval.
5.3 after natural cooling, rinsing with PBS for 3 times, adding donkey serum blocking solution (containing 0.3% Triton-X100) for blocking and membrane permeation for 1 hr.
5.4 PINK1 antibody was added and incubated overnight at 4 ℃.
5.5 rinsing 3 times with PBS, dripping rabbit anti-mouse HRP secondary antibody, and incubating for 1 hour at room temperature.
5.6 rinsing with PBS for 3 times, dripping DAB color development liquid, and stopping color development when the brown yellow changes and the color depth is proper.
5.7 sequentially placing the slices into 80% ethanol, 95% ethanol, 100 % ethanol 1, 100% ethanol 2, xylene 1 and xylene 2, and dehydrating and drying.
5.8 neutral resin seals and observations under microscope.
The experimental results are shown in fig. 5, wherein the dark brown color is colored cells, and it can be seen that the neural cells can be well identified in the gray matter area, and the glial cells can be well identified in the white matter area.
EXAMPLE 6 immunofluorescence application of monoclonal antibodies
6.1 refer to example 5, steps 5.2-5.4;
6.2 rinsing 3 times with PBS, dripping rabbit anti-mouse fluorescent secondary antibody, and incubating for 1 hour at room temperature.
6.3 rinsing 3 times with PBS, and nuclear counterstaining with DAPI was performed.
6.4 washing off DAPI, anti-quench caplets
6.5 microscopic observations.
The experimental results are shown in FIG. 6, which shows that cells can be well stained and that PINK1 exhibits cytoplasmic localization.
In general, the invention obtains the monoclonal strain 2E7B6 by immunizing mice with specific PINK1 antigen fragments and carrying out monoclonal screening, and the monoclonal strain has better experimental performance in experiments such as western immunoblotting (WB), immunohistochemical staining (IHC), immunofluorescence chemical staining (IF) and the like, thereby laying a good foundation for the subsequent study of protein functions and pathogenic mechanisms of PINK1 gene related diseases.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An anti-endogenous PINK1 protein monoclonal antibody, which is characterized in that: the amino acid sequences of the heavy chain variable regions VHCDR1, VHCDR2 and VHCDR3 are respectively shown in SEQ ID No. 1-3; the amino acid sequences of the light chain variable region VLCDR1 and VLCDR3 are respectively shown in SEQ ID No. 4-5, and the amino acid sequence of the light chain variable region VLCDR2 is LVS.
2. A gene sequence encoding the heavy chain variable region and light chain variable region amino acid sequences of the anti-endogenous PINK1 protein monoclonal antibody of claim 1.
3. The gene sequence according to claim 2, characterized in that: the gene sequences ntCDR1, ntCDR2 and ntCDR3 of SEQ ID No. 6-8 are respectively used for encoding amino acid sequences of heavy chain variable regions VHCDR1, VHCDR2 and VHCDR 3; SEQ ID Nos. 9 to 10 gene sequences ntCDR1, ntCDR3 are used for encoding the amino acid sequences of the light chain variable regions VLCDR1, VLCDR3, respectively, and the gene sequence ctggtgtgtct (ntCDR 2) is used for encoding the amino acid sequence of the light chain variable region VLCDR 2.
4. The anti-endogenous PINK1 protein monoclonal antibody according to claim 1, characterized in that it is a murine monoclonal antibody.
5. The anti-endogenous PINK1 protein monoclonal antibody according to claim 1, which is secreted by monoclonal cell line 2E7B6.
6. The anti-endogenous PINK1 protein monoclonal antibody according to claim 5, characterized in that: the monoclonal cell strain 2E7B6 is obtained by fusing immune mouse spleen cells and mouse myeloma cells.
7. The anti-endogenous PINK1 protein monoclonal antibody according to claim 1, characterized in that it is also any one of the following antibodies:
(a) A single chain antibody obtained by ligating any one of the heavy chain variable region amino acids of claim 1 with any one of the light chain variable region amino acids;
(b) A fusion antibody comprising the single chain antibody of (a);
(c) An intact antibody comprising any of the heavy chain variable region amino acids and any of the light chain variable region amino acids of claim 1;
(d) Monoclonal antibodies produced by monoclonal cell line 2E7B6.
8. A biological material associated with the monoclonal antibody according to any one of claims 1-7, characterized by any one of the following:
(1) An expression cassette comprising the gene sequence of claim 2 or 3;
(2) A recombinant vector comprising the gene sequence of claim 2 or 3;
(3) A recombinant vector comprising the expression cassette of (1);
(4) A transgenic animal cell line comprising the gene sequence of claim 2 or 3;
(5) A microorganism comprising the gene sequence of claim 2 or 3.
9. Use of an anti-endogenous PINK1 protein monoclonal antibody according to any of claims 1-8 for the identification and detection of PINK1 proteins for non-clinical diagnostic purposes.
10. Use of the anti-endogenous PINK1 protein monoclonal antibody according to any one of claims 1-8 in the preparation of reagents and kits for identifying and detecting PINK1 protein.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310026939.4A CN116284416A (en) | 2023-01-09 | 2023-01-09 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
| CN202311700819.4A CN117777297A (en) | 2023-01-09 | 2023-12-11 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310026939.4A CN116284416A (en) | 2023-01-09 | 2023-01-09 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116284416A true CN116284416A (en) | 2023-06-23 |
Family
ID=86791335
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310026939.4A Pending CN116284416A (en) | 2023-01-09 | 2023-01-09 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
| CN202311700819.4A Pending CN117777297A (en) | 2023-01-09 | 2023-12-11 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311700819.4A Pending CN117777297A (en) | 2023-01-09 | 2023-12-11 | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN116284416A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776154A (en) * | 2012-08-22 | 2012-11-14 | 天津三箭生物技术有限公司 | Mouse anti-human beta-Tubulin monoclonal antibody and hybridoma cell strain for secreting same |
| CN105001327A (en) * | 2015-02-12 | 2015-10-28 | 福州迈新生物技术开发有限公司 | Anti-p16 monoclonal antibody and preparation method and application thereof |
| CN113072642A (en) * | 2021-04-25 | 2021-07-06 | 福州迈新生物技术开发有限公司 | Monoclonal antibody of anti-OCT 3/4 protein and cell strain, preparation method and application thereof |
-
2023
- 2023-01-09 CN CN202310026939.4A patent/CN116284416A/en active Pending
- 2023-12-11 CN CN202311700819.4A patent/CN117777297A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776154A (en) * | 2012-08-22 | 2012-11-14 | 天津三箭生物技术有限公司 | Mouse anti-human beta-Tubulin monoclonal antibody and hybridoma cell strain for secreting same |
| CN105001327A (en) * | 2015-02-12 | 2015-10-28 | 福州迈新生物技术开发有限公司 | Anti-p16 monoclonal antibody and preparation method and application thereof |
| CN113072642A (en) * | 2021-04-25 | 2021-07-06 | 福州迈新生物技术开发有限公司 | Monoclonal antibody of anti-OCT 3/4 protein and cell strain, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117777297A (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112661842B (en) | anti-Ki-67 specific monoclonal antibody and application thereof | |
| CN113150137B (en) | Preparation method and application of NDM-1 monoclonal antibody | |
| EP3543258B1 (en) | Tem-1 diagnostic antibodies | |
| CN104497142B (en) | The monoclonal antibody of CP4 EPSPS albumen | |
| CN102232087A (en) | Antibodies to modified human IGF-1/E peptides | |
| CN113150138B (en) | KPC-2 monoclonal antibody, and preparation method and application thereof | |
| CN111996173B (en) | Hybridoma cell strain, preparation method and application thereof, monoclonal antibody and application thereof | |
| US20200166512A1 (en) | Composition and methods for detecting cancer | |
| CN116462758B (en) | Anti-human PTPRZ1 monoclonal antibody and application thereof in cell flow | |
| CN109535255B (en) | Anti-human CD26 antibody and application thereof in detection kit | |
| CN116284416A (en) | Monoclonal antibody for resisting endogenous PINK1 protein and application thereof | |
| CN116143909B (en) | anti-HIV-1P 24 antibody and preparation method and application thereof | |
| KR20140065370A (en) | Antibody for colorectal cancer marker | |
| CN115785275B (en) | Anti-plasmodium antibody and application thereof | |
| LIU et al. | Prokaryotic expression, ascitic polyclonal antibody preparation and identification of cashmere goat Izumo1 | |
| CN113150139A (en) | PBP2a monoclonal antibody and preparation method and application thereof | |
| KR20070109347A (en) | Hybridomas producing monoclonal antibodies against human ctrp1 and the monoclonal antibodies produced by the hybridomas | |
| CN114395040B (en) | Regenerated protein REG1A monoclonal antibody and application thereof | |
| CN114990074B (en) | Hybridoma cell strain, anti-human fumarate hydratase monoclonal antibody and application thereof | |
| CN113687067B (en) | Detection kit for AChR (AchR-alpha-amino acid) single subunit antibody and application thereof | |
| KR100493932B1 (en) | Monoclonal antibody recognizing resistin, production method and use thereof | |
| CN117659187B (en) | Anti-human NUT protein rabbit monoclonal antibody and application thereof | |
| EP1130030A1 (en) | Human erythroid differentiation related factor | |
| CN109709337B (en) | Immunohistochemical detection kit for human CD26 and clinical application thereof | |
| CN107132358B (en) | Bovine-derived creatine kinase isoenzyme double-antibody sandwich ELISA rapid detection kit |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20230623 |