CN118255885B - Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof - Google Patents
Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof Download PDFInfo
- Publication number
- CN118255885B CN118255885B CN202410497847.9A CN202410497847A CN118255885B CN 118255885 B CN118255885 B CN 118255885B CN 202410497847 A CN202410497847 A CN 202410497847A CN 118255885 B CN118255885 B CN 118255885B
- Authority
- CN
- China
- Prior art keywords
- antibody
- seq
- amino acid
- variable region
- human leptin
- 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.)
- Active
Links
- 101001063991 Homo sapiens Leptin Proteins 0.000 title claims abstract description 64
- 102000049953 human LEP Human genes 0.000 title claims abstract description 64
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 47
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 27
- 150000007523 nucleic acids Chemical class 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 20
- 108020004707 nucleic acids Proteins 0.000 claims description 17
- 102000039446 nucleic acids Human genes 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 15
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 9
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 5
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 5
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 4
- 239000003547 immunosorbent Substances 0.000 claims description 2
- 102000016267 Leptin Human genes 0.000 abstract description 44
- 108010092277 Leptin Proteins 0.000 abstract description 44
- 230000008901 benefit Effects 0.000 abstract description 11
- 238000003118 sandwich ELISA Methods 0.000 abstract description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 52
- 229940039781 leptin Drugs 0.000 description 38
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 37
- 210000004027 cell Anatomy 0.000 description 26
- 238000000034 method Methods 0.000 description 22
- 102000036639 antigens Human genes 0.000 description 20
- 108091007433 antigens Proteins 0.000 description 20
- 230000027455 binding Effects 0.000 description 19
- 239000000427 antigen Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 17
- 238000002965 ELISA Methods 0.000 description 16
- 239000007788 liquid Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 12
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 230000035945 sensitivity Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 6
- 239000011616 biotin Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 201000008482 osteoarthritis Diseases 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- UVGHPGOONBRLCX-NJSLBKSFSA-N (2,5-dioxopyrrolidin-1-yl) 6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O UVGHPGOONBRLCX-NJSLBKSFSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 206010033307 Overweight Diseases 0.000 description 2
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 2
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000012151 immunohistochemical method Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 102000005861 leptin receptors Human genes 0.000 description 2
- 108010019813 leptin receptors Proteins 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000000225 synapse Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 241000270730 Alligator mississippiensis Species 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 101000959794 Homo sapiens Interferon alpha-2 Proteins 0.000 description 1
- 101000960969 Homo sapiens Interleukin-5 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108090001041 N-Methyl-D-Aspartate Receptors Proteins 0.000 description 1
- 102000004868 N-Methyl-D-Aspartate Receptors Human genes 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000003486 adipose tissue brown Anatomy 0.000 description 1
- 210000000593 adipose tissue white Anatomy 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- -1 anti-obesity factor Chemical compound 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000008355 cartilage degradation Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000003937 effect on alzheimer disease Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 230000019637 foraging behavior Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 102000055228 human IL5 Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 208000018937 joint inflammation Diseases 0.000 description 1
- 210000001985 kidney epithelial cell Anatomy 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000011242 neutrophil chemotaxis Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000006286 nutrient intake Nutrition 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 102000013498 tau Proteins Human genes 0.000 description 1
- 108010026424 tau Proteins Proteins 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a monoclonal antibody, an antibody pair and a detection kit aiming at human leptin and application thereof. The monoclonal antibody is a first antibody or a second antibody, the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are respectively shown in SEQ ID NO.5-7, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown in SEQ ID NO. 8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15-17, respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NO.18-20, respectively. The antibody provided by the invention has the advantages of strong specificity, high affinity and the like, and can be used for developing a double-antibody sandwich ELISA detection system and stably, rapidly and efficiently detecting the leptin protein at an extremely trace level in a sample.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a monoclonal antibody, an antibody pair and a detection kit aiming at human leptin and application thereof.
Background
Leptin (Leptin, LEP), also known as Leptin, anti-obesity factor, leptin, is a hormone-like protein, produced mainly by adipocytes of white adipose tissue, and also brown adipose tissue, placenta (syncytial germ layer), ovary, skeletal muscle, stomach (lower region of gastric basal gland), mammary epithelial cells, bone marrow, gastric master cells, and P/D1 cells, combine to Leptin. The human Leptin (Leptin, LEP) gene is located on chromosome seven of human, the coded product contains 167 amino acids, the 21 amino acids at the amino terminal are signal peptides, 117-167aa predicts a pair of disulfide bonds, and the predicted protein structure is mainly alpha helix.
Studies have found that in many physiological and pathological situations, human leptin levels change. Leptin acts primarily on the Leptin receptor (LEPR) of the central nervous system and activates this receptor to transmit signals downstream, thereby regulating the behavior and metabolism of the organism. When the body fat of an individual is reduced or is in a low-energy state, the content of leptin in serum can be obviously reduced, so that the foraging behavior is stimulated, and the self energy consumption is reduced; conversely, when body fat of an organism increases, leptin content in serum increases, thereby inhibiting feeding and accelerating metabolism. Leptin has a major function of antagonizing insulin action in peripheral tissues, including inhibiting its secretion, promoting the use of fat by somatic cells as a metabolic raw material, and the like. Leptin is very sensitive to nutrient status in vivo, and after exercise and during hunger, leptin content is very low, which can cause hunger sensation, sex hormone drop, thyroxine and growth hormone drop, etc., so as to reduce nutrient consumption in vivo. Leptin has a degree of influence on innate immunity, including promoting neutrophil chemotaxis, activating macrophage phagocytosis, promoting inflammation, and inhibiting regulatory T cells, promoting helper T cell innate immunity, etc. Leptin-deficient individuals are more susceptible to infection, while those with excess leptin are more susceptible to autoimmunity. Leptin also increases cortical bone content, reduces cancellous bone content, and increases overall bone density. Degenerative arthritis and obesity are closely related, and obese individuals are more prone to develop degenerative arthritis, not only due to overweight mechanical loading, but also due to excessive manifestations of soluble factors, leptin and pro-inflammatory cytokines are responsible for joint inflammation and cartilage destruction. There is growing evidence that leptin acts as a cartilage degradation factor in the pathogenesis of degenerative arthritis and as a potential biomarker of the progression of the disease, suggesting that leptin and the mechanisms of regulation and signaling may be a novel and promising target for the treatment of degenerative arthritis. In studies in mice, leptin was found to regulate excitatory synaptic activation via stimulation of the NMDA receptor, leptin activates the PI3K/mTOR pathway of neurons, and leptin long subtype receptor LepRb activation stimulates mTOR activation and subsequent synaptogenesis, allowing old synapses to mature and new synapses to occur simultaneously. Furthermore, it has been shown that leptin reduces the content of β -amyloid and Tau protein and thus may have a soothing effect on alzheimer's disease and may affect its pathophysiology.
In view of the important regulatory functions of leptin, detection of individual leptin levels has a very important clinical guidance value for the occurrence, development, diagnosis and prognosis of related diseases, and the use of leptin-like drugs also requires the cooperation of related pharmacokinetic monitoring. Investigation shows that the average concentration of leptin in normal non-obese Chinese men is (2.15+/-1.46) mug/L, and in female women is (7.86+/-3.60) mug/L; average leptin concentration in overweight or obese men was (4.87.+ -. 3.47) μg/L and women was (16.59.+ -. 6.92) μg/L. Leptin levels are associated with metabolism, work and rest of the individual and other levels of leptin. Currently, leptin detection methods rely primarily on the ability of leptin antibodies to specifically bind to leptin in the test sample. Therefore, the development of a stable anti-interference human leptin protein detection antibody has very important significance. However, the anti-leptin antibodies with better specificity and higher sensitivity in the market at present are fewer, and cannot meet the requirement of high-sensitivity detection.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a monoclonal antibody, an antibody pair and a detection kit aiming at human leptin and application thereof, and the antibody provided by the invention has the advantages of strong specificity, high affinity and the like, and can be used for developing a double-antibody sandwich ELISA detection system, so that trace leptin protein in a sample can be stably, rapidly and efficiently detected, and the antibody has the advantages of high selectivity, specificity, wide sensitivity and linear range, good accuracy and the like.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
In a first aspect the present invention provides a monoclonal antibody directed against human leptin, either as a first antibody or as a second antibody, wherein: the amino acid sequences of the complementarity determining regions CDR1-3 on the light chain variable region of the first antibody are shown as SEQ ID NO.5-7 respectively, and the amino acid sequences of the complementarity determining regions CDR1-3 on the heavy chain variable region are shown as SEQ ID NO.8-10 respectively; the amino acid sequences of the CDRs 1-3 of the second antibody light chain variable region are shown as SEQ ID NO.15-17, respectively, and the amino acid sequences of the CDRs 1-3 of the heavy chain variable region are shown as SEQ ID NO.18-20, respectively.
Further, the amino acid sequence of the first antibody light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4; the amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14.
Further, the first antibody and the second antibody further comprise a light chain variable region and a heavy chain variable region, the light chain constant regions of the first antibody and the second antibody are both kappa chains, and the heavy chain constant regions are both of the IgG type.
Further, the amino acid sequence of the first antibody light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2; the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12.
Further, the first antibody or the second antibody is a full-length antibody or an antibody fragment; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
In a second aspect, the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody as described above, a recombinant vector or a host cell comprising said nucleic acid molecule.
In a third aspect the invention provides a monoclonal antibody pair directed against human leptin consisting of a first antibody and a second antibody as described above.
In a fourth aspect, the invention provides the use of a monoclonal antibody or antibody pair directed against human leptin as described above in the preparation of a kit for detecting human leptin.
In a fifth aspect the invention provides a kit for detecting human leptin, the kit comprising a monoclonal antibody directed against human leptin as described above or a monoclonal antibody pair directed against human leptin as described above.
Further, the kit is a double-antibody sandwich ELISA kit, and the kit comprises a first antibody and a second antibody, wherein the first antibody is used as a capture antibody, and the second antibody with a detectable label is modified to be used as a detection antibody.
The invention has the advantages and positive effects that:
1. The first antibody and the second antibody with the CDR sequences can specifically identify and bind human leptin, have the advantages of strong specificity, high affinity and the like, the affinity constant of the binding of the first antibody and the recombinant expressed human leptin protein is 1.54 multiplied by 10 -8 (M), the affinity constant of the binding of the second antibody and the recombinant expressed human leptin protein is 1.92 multiplied by 10 -9 (M), and the invention is suitable for the requirements of immunodiagnosis reagents on antibody raw materials with high sensitivity and high specificity.
2. The invention uses the first antibody as the capture antibody and the second antibody as the detection antibody to develop a double-antibody sandwich ELISA detection system, can stably, rapidly and efficiently detect the trace leptin protein in the sample, has the detection limit as low as 12.389pg/mL, has the advantages of high selectivity, specificity, wide sensitivity and linearity range, good accuracy and the like, and has important significance in clinical diagnosis and scientific research application.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of a vector used for constructing a rabbit monoclonal antibody expression vector according to example 1 of the present invention, from left to right, pRB322 vector map carrying light chain constant region and heavy chain constant region, respectively;
FIG. 2 is a graph showing the affinity of rabbit monoclonal antibody A for binding to human leptin according to example 2 of this invention;
FIG. 3 is a graph showing the affinity of rabbit monoclonal antibody B for binding to human leptin according to example 2 of this invention;
FIG. 4 is a standard curve of the ELISA method based on rabbit monoclonal antibodies A and B in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added.
The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" is considered to include the following: (i) A, (ii) B, and (iii) A and B.
The terms "first," "second," and the like, are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order, it being understood that such uses may be interchanged where appropriate.
The terms "rabbit monoclonal antibody", "antibody" and "mab" and the like have the same meaning and are used interchangeably, as not specifically indicated, to refer to antibodies that specifically bind human Leptin (LEP). The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity.
A typical antibody molecule (full length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs and FR can be identified according to Kabat definition, chothia definition, a combination of both Kabat definition and Chothia definition, abM definition, contact definition, IMGT unique number definition and/or conformational definition, or any CDR determination method known in the art. As used herein, is defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.
The terms "monoclonal antibody" or "mab" and the like are used interchangeably and refer to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. "monoclonal antibodies" are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as limiting the source or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a monoclonal antibody aiming at human leptin, wherein the rabbit monoclonal antibody is a first antibody or a second antibody, the rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs) respectively named as CDR1, CDR2 and CDR3; wherein:
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.
The invention uses commercialized human leptin protein (from ABclonal, with the product number of RP 00028) as immunogen to immunize rabbits, and prepares the first antibody and the second antibody with the CDR sequences, the obtained antibody can specifically identify and bind the recombinant expressed human LEP or the natural human LEP protein secreted in cells and serum, the specificity of the antibody is strong, the affinity is high, the affinity constant of the binding of the first antibody and the recombinant expressed human leptin protein is 1.54 multiplied by 10 -8 (M), the affinity constant of the binding of the second antibody and the recombinant expressed human leptin protein is 1.92 multiplied by 10 -9 (M), and the invention is suitable for the requirements of immunodiagnosis reagents on high-sensitivity and high-specificity antibody raw materials. The invention also discloses a double-antibody sandwich enzyme-linked immunosorbent assay (double-antibody sandwich ELISA) system which is developed by utilizing the two antibodies, wherein the first antibody is used as a capture antibody and the second antibody is used as a detection antibody, and the recombinant leptin protein with various concentrations is quantitatively detected, so that the very trace leptin protein in serum and cell culture medium samples can be stably, rapidly and efficiently detected, the detection limit is as low as 12.389pg/mL, and the double-antibody sandwich ELISA system has the advantages of high selectivity, specificity, wide sensitivity and linearity range, good accuracy and the like, and has important significance in clinical diagnosis and scientific research application.
Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the first antibody is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region (VH) of the first antibody is shown as SEQ ID NO. 4. The amino acid sequence of the light chain variable region (VL) of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 14.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region and a heavy chain constant region, CL and VL comprising the light chain and CH and VH comprising the heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA C REIGN was searched for CL via IMGT online database (www.imgt.org).
Specifically, the amino acid sequence of the light chain (L chain) of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 2. The amino acid sequence of the light chain (L chain) of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain (H chain) is shown as SEQ ID NO. 12. The first antibody and the second antibody have light chain constant regions of kappa chains and heavy chain constant regions of IgG type.
It should be noted that the rabbit monoclonal antibody of the present invention may be a full-length antibody (having a typical Y-type molecular structure) or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full-length form of the rabbit monoclonal antibody, specifically, an antibody fragment includes CDR regions as described above, more preferably has variable regions as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, particularly to the same epitope, as the full-length antibody.
Such antibody fragments include, but are not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) 2 fragment consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) The sc (Fv) 2 fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via a linker or the like. These antibody fragments can be obtained by conventional techniques in the art.
Yet another embodiment of the invention provides a nucleic acid molecule encoding a first antibody and/or a second antibody as described above, a recombinant vector or a host cell comprising said nucleic acid molecule.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody. The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.
The starting vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and thus, in the context of the present invention, vectors are used interchangeably with plasmids. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is well known in the art and will not be described in detail herein.
Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the rabbit monoclonal antibodies of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.
Transfection or transformation of the recombinant vector into a host cell according to the present invention is carried out using conventional techniques. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after the exponential growth phase and treated with CaCl 2 or MgCl 2. Microinjection, electroporation, or liposome encapsulation may also be used if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomycete, salmonella typhimurium, yeast, drosophila S2 or Sf9 cells, CHO, COS7, 293 series cells, etc. After obtaining host cells transfected or transformed with the vectors described above, the cells are cultured under appropriate conditions to express the monoclonal antibodies, which are then isolated to obtain purified antibodies.
In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322 and the host cell is a human kidney epithelial cell (293F cell).
In another embodiment of the invention there is provided a monoclonal antibody pair directed against human leptin consisting of a first antibody and a second antibody as described above.
The advantages of the monoclonal antibodies against human leptin over the prior art are the same as those described above and are not described in detail herein.
In a further embodiment the invention provides the use of a monoclonal antibody or antibody pair directed against human leptin as described above in the preparation of a kit for detecting human leptin.
The advantages of the monoclonal antibody or antibody directed against human leptin in the preparation of a kit for detecting human leptin are the same as those of the monoclonal antibody directed against human leptin described above with respect to the prior art, and will not be described in detail herein.
Based on the same inventive concept as described above, the embodiments of the present invention also provide a kit for detecting human leptin, the kit comprising the first antibody and/or the second antibody as described above.
The first antibody and the second antibody may be used alone, or may be used together, or may be used in combination. In the detection, for example, when the first antibody and/or the second antibody are used separately or in combination, the first antibody and/or the second antibody are used as the primary antibody or the capture antibody, and the sample to be detected is contacted with the first antibody and/or the second antibody, followed by detection of the antibody. In some embodiments, the first antibody and/or the second antibody may be conjugated to a detectable label, and qualitative or quantitative detection of LEP may be achieved by analyzing the change in the identifiable signal produced by the detectable label. In other embodiments, the primary and/or secondary antibodies (as primary or capture antibodies) to the human LEP are not labeled, and the detectable label is conjugated to a secondary antibody (as a detection antibody) or other molecule that binds to the primary antibody, e.g., if the anti-human LEP antibody is a rabbit IgG antibody, the secondary antibody may be an anti-rabbit IgG antibody, thereby producing a change in the recognizable signal by the conjugation of the detectably labeled secondary antibody. When the antibodies are paired, one of the first antibody and the second antibody is used as a primary antibody or a capture antibody, and the other is used as a secondary antibody or a detection antibody.
Preferably, the kit comprises a first antibody as a capture antibody (or primary antibody) and a second antibody as a detection antibody (or secondary antibody), and the second antibody is modified with a detectable label. The kit has good detection sensitivity and specificity due to the inclusion of the antibody pair.
Such detectable labels for producing identifiable signal changes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.
In a preferred embodiment, the detectable label is biotin.
The detection methods described above employ conventional immunological methods including, but not limited to: enzyme Immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISPOT), immunohistochemical method (IHC), immunofluorescence method (IF), immunoblotting method (WB), flow Cytometry (FC), etc., and thus the kit may be an enzyme immunoassay kit, an enzyme-linked immunosorbent kit, an immunohistochemical kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.
The immunodetection sample comprises recombinantly expressed human LEP, or LEP secreted by the cell, or LEP in human serum.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
EXAMPLE 1 preparation of Rabbit monoclonal antibodies A and B
The immunogens and antigen proteins used in this example were Human Leptin (LEP) expressed by e.coli (from ABclonal, cat No. RP 00028) and high affinity anti-Human LEP rabbit monoclonal antibodies a and B were successfully developed based on single B lymphocyte screening and culture monoclonal antibody development techniques.
The antibodies A and B were sequenced and the sequencing was performed by Jin Kairui Biotechnology Inc., the Amino Acid (AA) sequences of which are shown in tables 1-2, respectively. For convenience of description, light chain Complementarity Determining Regions (CDRs) 1-3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, and heavy chain Complementarity Determining Regions (CDRs) 1-3 are denoted by HCDR1, HCDR2 and HCDR3, respectively.
TABLE 1 sequence information for Rabbit monoclonal antibody A of this example
TABLE 2 sequence information of rabbit monoclonal antibody B of this example
The preparation method of the rabbit monoclonal antibodies A and B specifically comprises the following steps:
1. Immunization of animals
3 New Zealand white rabbits were immunized with Human LEP protein (from ABclonal, cat. No. RP 00028) as an immunogen, 300 μg of each white rabbit was immunized, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant to prepare an emulsifier before the first immunization, and injected subcutaneously in the abdomen and back of the rabbits at multiple points. 150 μg of immunogen is mixed with an equal amount of incomplete Freund's adjuvant every 3 weeks after the primary immunization to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting. Serum samples of rabbits were collected after three immunizations, titers against Human LEP were determined by ELISA after dilution, rabbits with high serum titers were boosted by subcutaneous multipoint injection with 300 μg immunogen once, animals were sacrificed three days later and spleens were taken.
2. Separation of B lymphocytes from spleen and B lymphocyte sorting
The method comprises the steps of separating B lymphocytes in spleen by adopting a conventional method, then separating to obtain antigen-specific B lymphocytes, and carrying out related methods, namely, a method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication No. CN110016462A, publication No. 2019-07-16) and an in-vitro culture system and application of B lymphocytes (publication No. CN111518765A, publication No. 2020-08-11).
3. Cloning of genes encoding Rabbit monoclonal antibodies
Positive clones were identified by antigen-coated ELISA of the cultured B lymphocyte supernatant. Cells of positive clones were collected and lysed, and RNA was extracted according to the Quick-RNA TM MicroPrep kit instructions (purchased from ZYMO, cat. No. R1051) and reverse transcribed into cDNA. The cDNA is used as a template, a natural paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) are amplified from the cDNA of the corresponding positive clone by adopting a PCR method, and a plurality of clones are selected for sequencing, and the sequencing work is completed by Jin Kairui biotechnology company. Primer pair sequences (5 '-3') for amplifying VL and VH genes are shown below, and F and R represent forward and reverse primers, respectively:
VL-F: TGAATTCGAGCTCGGTACCCATGGACACGAGGGCCCCCAC (see SEQ ID NO. 21);
VL-R: CACACACACGATGGTGACTGTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 22);
VH-F: TGAATTCGAGCTCGGTACCCATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 23);
VH-R: GTAGCCTTTGACCAGGCAGCCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 24).
The PCR reaction system is as follows: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2X Gloria HiFi (from ABclonal self-produced under the trade designation RK 20717) and 6.5. Mu. L H 2 O; the PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.
4. Production and purification of Rabbit monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies recognizing recombinant Human LEP proteins, the heavy chain gene and the light chain gene of the rabbit monoclonal antibodies are respectively loaded on a mammal expression vector pBR322, the used vector carries a rabbit monoclonal antibody light chain constant region (CL) and a heavy chain constant region (CH) gene, the expression patterns are shown in figure 1, pRB322 origin and f1 origin are replication promoters in escherichia coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a promoter in eukaryote, SV40 PAterminator is a tailing signal, LIGHT CHAIN constant is a nucleic acid sequence of the light chain constant region (left graph), HEAVY CHAIN constant is a nucleic acid sequence of the heavy chain constant region (right graph). CL, CH genes CL is obtained by searching IMGT online database (www.imgt.org), searching rabbit source IGG GAMMA C REIGN for CH, searching rabbit source IGG KAPPA C REIGN. The variable region genes containing the signal peptide obtained by amplification in the steps are respectively constructed to the upstream of CL and CH genes corresponding to the expression vector pBR322, so that the light chain gene and heavy chain gene expression vectors are obtained, and the successful construction of the expression vectors is verified by sequencing.
The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as a rabbit monoclonal antibody against Human interferon alpha 2 and its use (publication No. CN116063487A, publication No. 2023-05-05) and a light chain variable region upstream of a signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" and a heavy chain variable region upstream of a signal peptide "METGLRWLLLVAVLKGVQC" of a high affinity Human IL-5 rabbit monoclonal antibody and its use (publication No. CN115819578A, publication No. 2023-03-21).
And (3) transfecting the expression vector which is verified to be correct by sequencing and contains the light chain gene and the heavy chain gene into 293F cells, and culturing for 72-96 hours after transfection to obtain the recombinant rabbit monoclonal antibody which contains the recognition of the Human LEP in the culture supernatant. Recombinant rabbit monoclonal antibodies recognizing the Human LEP protein were purified from the culture supernatant using protein a affinity gel resin (purchased from world and cat No. SA 023100). The purity of the antibody is verified to be more than 95% by using 12% SDS-PAGE gel electrophoresis, and the purified antibody is split-packed and then stored at a low temperature of-20 ℃ for standby.
Example 2 affinity test of Rabbit monoclonal antibodies A and B
The affinity of the obtained rabbit monoclonal antibody was measured using a Gator biomolecular interaction analyzer from Probe Life. The antigen is recombinant Human LEP protein, the concentration is 769.23nM and 333.333nM, and the concentration of the rabbit monoclonal antibody is 2 mug/mL; by comparing the affinities of the antibodies, the antibodies with better affinities are selected, namely the antibodies A and B of the invention. The affinity curves of antibodies a and B are shown in fig. 2-3, respectively, wherein the ordinate represents the change in thickness of the conjugate after binding of the probe to the antibody and protein, the abscissa represents the binding time, the dark gray curve is a real-time binding numerical curve, and the light gray curve is a fitted average curve. The affinity constants calculated by curve fitting are shown in table 3.
TABLE 3 determination of affinity-related parameters for Rabbit monoclonal antibodies A and B
| Rabbit monoclonal antibodies | Koff(1/s) | Kon(1/Ms) | KD(M) |
| A | 1.93×10-3 | 1.25×105 | 1.54×10-8 |
| B | 9.02×10-5 | 4.70×104 | 1.92×10-9 |
The dissociation coefficient K off in table 3 is a constant for characterizing the dissociation rate of an antibody from an antigen, the binding coefficient K on is a constant for characterizing the binding rate of an antibody to its target, and the affinity constant K D is a ratio of K off/Kon, representing the equilibrium dissociation constant between an antibody and its antigen. As can be seen from Table 3, the affinity constant K D for the rabbit monoclonal antibody A and the Human LEP protein was 1.54X10 -8 (M), and the affinity constant K D for the rabbit monoclonal antibody B and the Human LEP protein was 1.92X10 -9 (M), indicating that the rabbit monoclonal antibody A, B has high affinity with recombinant Human LEP, respectively.
Example 3 method for establishing double antibody sandwich ELISA adsorption based on rabbit monoclonal antibodies A and B
The embodiment establishes a double-antibody sandwich method ELISA detection method aiming at rabbit monoclonal antibodies A and B of the Human LEP protein, and comprises the following steps:
1) Coating the capture antibody A: diluting the antibody A into 2 mug/mL by using 1 XPBS, uniformly mixing by using a vortex instrument, adding into a 96-well micro-pore plate by using 100 mug/well, covering a cover plate film, and placing in a refrigerator at 4 ℃ for incubation for 16-20h;
2) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed once by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper;
3) Closing: adding E013 blocking solution (containing 2% BSA, 5% sucrose, 0.05% Tween and 0.1%proclin300,pH 7.2% in 1 XPBS) into the plate holes at a ratio of 200 μl/hole, covering with cover plate film, blocking at 37deg.C for 2h, discarding blocking solution, drying the ELISA plate, oven drying at 37deg.C for 0.5-2h, and taking out;
4) Adding antigen protein: diluting the recombinant Human LEP protein with a diluent to the concentration of: 2000. 1000, 500, 250, 125, 62.5, 31.25, 0pg/mL, then sequentially adding into the ELISA plate at 100 mu L/hole, covering a cover plate film, and incubating at 37 ℃ for 2h;
5) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper;
6) Adding detection antibody B: diluting the biotin-labeled rabbit monoclonal antibody B (B-biotin) to 0.1 mu g/mL, sequentially adding 100 mu L/hole into an ELISA plate, covering a cover plate film, and incubating at 37 ℃ for 1h;
The B-biotin processing method comprises the following steps: preparing an antibody B into a solution with the concentration of 1mg/mL, and preparing NHS-LC-biotin into a solution with the concentration of 60mg/mL by using DMSO; 200 mu L of 1mg/mL of antibody B solution is taken, and 10 mu L of 60mg/mL of NHS-LC-biotin solution is added; after mixing, the mixture was left at room temperature for 30min, and then 50. Mu.g of 500mM Tris solution (pH 9.0) was added to stop the reaction; finally adding a large amount of 1 XPBS buffer solution with pH of 7.4, centrifuging by using a centrifugal column with the exclusion limit of 30KD, and removing redundant biotin molecules and balancing a buffer solution system;
7) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper;
8) Adding SA-HRP: 100 xSA-HRP (horseradish peroxidase labeled streptavidin, available from Wuhan Sanying biotechnology Co., ltd., product No. SA 00001-0) concentrate is diluted 100 times, and added into an ELISA plate sequentially at 100 μl/hole, covered with a cover plate film, and incubated at 37deg.C for 0.5 hr;
9) Washing the plate: after the incubation is completed, the liquid in the holes is discarded, the plate is washed three times by 1 XPBST, 300 mu L of sample is added, the liquid in the holes is discarded after standing for 40s, and the liquid in the holes is dried on the flat paper;
10 Adding TMB color development liquid: adding 3,3', 5' -tetramethyl benzidine (TMB) color development liquid into an ELISA plate at a concentration of 100 mu L/hole, covering a cover plate film, and incubating at 37 ℃ for 15min;
11 Reading: after the incubation was completed, the microplate was removed, 50. Mu.L of stop solution (1 mol/L hydrochloric acid) was added to each well, and immediately reading was performed with an microplate reader.
Sensitivity of the double antibody sandwich method ELISA method: the corrected value Y1 of absorbance (y1=od 450nm-OD630nm) is plotted on the ordinate with the raman LEP protein concentration on the abscissa, see fig. 4. The lowest Human LEP concentration with the average absorbance value greater than the average absorbance value of the triplicate blank control was the sensitivity of the dual antibody sandwich enzyme-linked immunosorbent assay (see table 4). Experimental results show that the detection sensitivity of the established double-antibody sandwich method ELISA method based on the anti-Human LEP protein rabbit monoclonal antibodies A and B reaches 12.389pg/mL, and the high-sensitivity and high-reliability detection of trace Human LEP proteins in a sample to be detected can be satisfied.
TABLE 4 sensitivity test data for Rabbit monoclonal antibodies A and B
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A monoclonal antibody directed against human leptin, wherein the monoclonal antibody is a first antibody or a second antibody, wherein:
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively;
The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.
2. The monoclonal antibody directed against human leptin according to claim 1, wherein the amino acid sequence of the first antibody light chain variable region is shown in SEQ ID No.3 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4;
The amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14.
3. The monoclonal antibody directed against human leptin according to claim 2, wherein said first antibody or said second antibody further comprises a light chain constant region and a heavy chain constant region, said light chain constant region being a kappa chain and said heavy chain constant region being of the IgG type.
4. A monoclonal antibody directed against human leptin according to claim 3, characterized in that the amino acid sequence of the first antibody light chain is shown in SEQ ID No.1 and the amino acid sequence of the heavy chain is shown in SEQ ID No. 2;
the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12.
5. The monoclonal antibody directed against human leptin according to claim 1, wherein the first antibody or the second antibody is a full-length antibody or an antibody fragment;
The antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
6. A nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody or the second antibody of any one of claims 1-5.
7. A monoclonal antibody pair directed against human leptin, consisting of a first antibody as set forth in any one of claims 1-5 and a second antibody.
8. Use of a monoclonal antibody directed against human leptin according to any one of claims 1 to 5 or a monoclonal antibody directed against human leptin according to claim 7 for the preparation of a kit for detecting human leptin.
9. A kit for detecting human leptin, comprising a monoclonal antibody directed against human leptin according to any one of claims 1-5 or a monoclonal antibody pair directed against human leptin according to claim 7.
10. The kit for detecting human leptin according to claim 9, wherein the kit is a double-antibody sandwich enzyme-linked immunosorbent kit, the kit comprising a first antibody and a second antibody, and the first antibody is a capture antibody, and the second antibody to which a detectable label is coupled is a detection antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410497847.9A CN118255885B (en) | 2024-04-24 | 2024-04-24 | Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410497847.9A CN118255885B (en) | 2024-04-24 | 2024-04-24 | Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118255885A CN118255885A (en) | 2024-06-28 |
| CN118255885B true CN118255885B (en) | 2024-10-08 |
Family
ID=91612937
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410497847.9A Active CN118255885B (en) | 2024-04-24 | 2024-04-24 | Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118255885B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110183530A (en) * | 2019-06-21 | 2019-08-30 | 深圳市亚辉龙生物科技股份有限公司 | Leptin immunogene, hybridoma, monoclonal antibody, polyclonal antibody and application |
| CN111225925A (en) * | 2017-10-03 | 2020-06-02 | 中外制药株式会社 | anti-HLA-DQ 2.5 antibodies |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3313887A2 (en) * | 2015-06-26 | 2018-05-02 | MAB Discovery GmbH | Monoclonal anti-il-1racp antibodies |
-
2024
- 2024-04-24 CN CN202410497847.9A patent/CN118255885B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111225925A (en) * | 2017-10-03 | 2020-06-02 | 中外制药株式会社 | anti-HLA-DQ 2.5 antibodies |
| CN110183530A (en) * | 2019-06-21 | 2019-08-30 | 深圳市亚辉龙生物科技股份有限公司 | Leptin immunogene, hybridoma, monoclonal antibody, polyclonal antibody and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118255885A (en) | 2024-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112166121B (en) | Monoclonal antibody of nerve growth factor, and coding gene and application thereof | |
| CN109096395A (en) | Blocking-up type CD47 nano antibody and application thereof | |
| WO2021017071A1 (en) | Anti-human st2 antibody and use thereof | |
| CN117362431B (en) | Anti-mouse interleukin 10 rabbit monoclonal antibody and application thereof | |
| US20140243265A1 (en) | Antibodies to modified human igf-1/e peptides | |
| CN116396384B (en) | Rabbit monoclonal antibody aiming at mouse adiponectin, application of rabbit monoclonal antibody and double-antibody sandwich method ELISA (enzyme-linked immunosorbent assay) kit | |
| CN118255885B (en) | Monoclonal antibody, antibody pair and detection kit for human leptin and application thereof | |
| JP2020510841A (en) | Human erythroferon antibodies and uses thereof | |
| WO2021213245A1 (en) | Antibody or antigen-binding fragment, preparation method and pharmaceutical uses therefor | |
| CN118206657A (en) | Antibody of galectin-3 and application thereof | |
| CN114874310B (en) | IgG epitope peptide of whey allergen beta-lactoglobulin | |
| CN115286715B (en) | anti-CD3 nano antibody or antigen binding portion thereof and preparation method thereof | |
| CN113912716B (en) | Antibodies against alpha-synuclein antigens and uses thereof | |
| CN118440198B (en) | Anti-human CD69 protein rabbit monoclonal antibody and application thereof | |
| CN110317271B (en) | Heavy/light chain variable region of PINP recombinant antibody, coding gene and recombinant antibody | |
| CN118344476B (en) | Human C reactive protein monoclonal antibody, antibody pair, and detection reagent or kit and application thereof | |
| CN113150158A (en) | Antibody pair for detecting RANKL content in serum and application thereof | |
| CN117487018B (en) | Rabbit monoclonal antibody for resisting human epididymis secretion protein 4 and application thereof | |
| CN118440198A (en) | Anti-human CD69 protein rabbit monoclonal antibody and application thereof | |
| CN118085075B (en) | Anti-human alpha fetoprotein rabbit monoclonal antibody and application thereof | |
| CN118561998A (en) | Anti-sheep interleukin 6 antibody, antibody pair and application thereof | |
| CN118344476A (en) | Human C reactive protein monoclonal antibody, antibody pair, and detection reagent or kit and application thereof | |
| CN118255883A (en) | Monoclonal antibody, antibody pair and kit aiming at human opsin-like protein 1 and application thereof | |
| CN114891085B (en) | IgE epitope peptide of whey allergen beta-lactoglobulin | |
| US20100028917A1 (en) | A neuroglobin enzyme-linked immunosorbent assay kit and the use of it |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |