CN117050174B - Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same - Google Patents
Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same Download PDFInfo
- Publication number
- CN117050174B CN117050174B CN202311316201.8A CN202311316201A CN117050174B CN 117050174 B CN117050174 B CN 117050174B CN 202311316201 A CN202311316201 A CN 202311316201A CN 117050174 B CN117050174 B CN 117050174B
- Authority
- CN
- China
- Prior art keywords
- antibody
- seq
- heavy chain
- light chain
- kit
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 101710159910 Movement protein Proteins 0.000 title claims abstract description 36
- 238000002965 ELISA Methods 0.000 claims abstract description 46
- 101000581815 Homo sapiens Regenerating islet-derived protein 3-alpha Proteins 0.000 claims abstract description 40
- 102100027336 Regenerating islet-derived protein 3-alpha Human genes 0.000 claims abstract description 30
- 201000010099 disease Diseases 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 239000012472 biological sample Substances 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 11
- 238000003118 sandwich ELISA Methods 0.000 claims description 11
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims 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 claims description 8
- 241001529936 Murinae Species 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 8
- 229910052751 metal Inorganic materials 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 8
- 238000003127 radioimmunoassay Methods 0.000 claims description 8
- 206010017758 gastric cancer Diseases 0.000 claims description 7
- 210000002381 plasma Anatomy 0.000 claims description 7
- 206010033645 Pancreatitis Diseases 0.000 claims description 6
- 208000008469 Peptic Ulcer Diseases 0.000 claims description 6
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 6
- 201000002313 intestinal cancer Diseases 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 208000011906 peptic ulcer disease Diseases 0.000 claims description 6
- 238000003745 diagnosis Methods 0.000 claims description 5
- 208000010643 digestive system disease Diseases 0.000 claims description 5
- 201000011549 stomach cancer Diseases 0.000 claims description 5
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 4
- 229960005156 digoxin Drugs 0.000 claims description 4
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 4
- 238000003018 immunoassay Methods 0.000 claims description 4
- 238000001114 immunoprecipitation Methods 0.000 claims description 4
- 238000002493 microarray Methods 0.000 claims description 4
- 238000001262 western blot Methods 0.000 claims description 4
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000011230 binding agent Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 150000004696 coordination complex Chemical class 0.000 claims 2
- 208000018522 Gastrointestinal disease Diseases 0.000 claims 1
- 208000018685 gastrointestinal system disease Diseases 0.000 claims 1
- 102000049256 human REG3A Human genes 0.000 abstract description 11
- 102100027361 Lithostathine-1-alpha Human genes 0.000 description 23
- 239000000243 solution Substances 0.000 description 16
- 238000000034 method Methods 0.000 description 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 3
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 210000004989 spleen cell Anatomy 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003722 extracellular fluid Anatomy 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 102000002086 C-type lectin-like Human genes 0.000 description 1
- 108050009406 C-type lectin-like Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091058544 REG family proteins Proteins 0.000 description 1
- 101710175687 Regenerating islet-derived protein 3-alpha Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- VXIVSQZSERGHQP-UHFFFAOYSA-N chloroacetamide Chemical compound NC(=O)CCl VXIVSQZSERGHQP-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000010200 validation analysis Methods 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
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/062—Gastritis or peptic ulcer disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/067—Pancreatitis or colitis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides an antibody combination that can be used for detecting regenerated islet-derived protein 3A (REG 3A) based on ELISA. Each antibody in the antibody combination provided by the invention can specifically bind to REG3A with high affinity, and can form a double-antibody sandwich mode, so that qualitative and quantitative detection of human REG3A can be performed. Correspondingly, the invention also provides an ELISA detection kit containing the antibody combination.
Description
Technical Field
The present invention relates to the field of biological detection, and in particular, to an antibody combination and a kit comprising the same, which can be used for detecting REG3A in a biological sample.
Background
Regenerated islet-derived protein 3A (Regenerating islet-derived protein III-alpha, REG 3A) belongs to the REG protein family and is a secreted calcium-dependent lectin protein. REG3A has a molecular weight of 19kDa and a total length of 175 amino acids, including signal peptide (1-26 aa), propeptide (27-37 aa), C-type lectin-like domain (47-173 aa).
REG3A is known to have various roles in anti-inflammatory, antibacterial, cell proliferation, apoptosis, tissue repair, etc. REG3A expression is seen in various tissues of human body, including pancreas, small intestine, liver, skin, and gastrointestinal tract, etc. However, the levels of this protein are very low in healthy tissues (including pancreas and skin), while they are highly expressed in tissue lesions. Recent evidence is increasing that there are varying degrees of REG3A expression in patients with diabetes, inflammatory Bowel Disease (IBD), and cancer. Also for this reason, a great deal of research has been conducted on the correlation between REG3A and different disease types. Studies have shown that mRNA/protein expression of REG3A is significantly elevated in inflammation or cancer of stomach, intestine, pancreas as well as in IBD inflammatory bowel disease, and thus REG3A has important clinical detection significance.
The double-antibody sandwich ELISA has the advantages of simple operation, high efficiency, reliability and suitability for rapid screening of a large number of samples, and particularly, the double-sandwich ELISA based on the monoclonal antibody is more stable and has stronger specificity than the double-sandwich ELISA based on the polyclonal antibody. Accordingly, attempts may be made to develop antibodies or antibody combinations capable of specifically binding to REG3A and allowing effective detection against REG3A in biological samples (such as serum), thereby preparing detection or diagnostic reagents that can qualitatively or quantitatively detect the presence or concentration of REG3A in biological samples.
Disclosure of Invention
In order to solve the above technical problems, it is an object of the present invention to provide a novel reagent and method capable of detecting the presence or concentration of REG3A in serum with high sensitivity and specificity.
Specifically, it is an object of the present invention to provide an antibody combination targeting different epitopes of regenerated islet-derived protein 3A (REG 3A), each of which is required to bind to human REG3A with high affinity and specificity, and can form a double antibody sandwich pattern, thereby enabling qualitative and quantitative detection of REG3A.
Based on the above antibody combinations, it is another object of the present invention to provide ELISA kits comprising the antibody combinations, such as double antibody sandwich ELISA kits, for qualitative and quantitative detection of REG3A in a sample to be tested.
The technical scheme of the invention is as follows.
In one aspect, the invention provides an antibody combination comprising:
(i) An antibody 1, the antibody 1 comprising a Heavy Chain (HC) and a Light Chain (LC), wherein the heavy chain comprises a heavy chain CDR1 (H-CDR 1) shown in SEQ ID No. 5, a heavy chain CDR2 (H-CDR 2) shown in SEQ ID No. 6, and a heavy chain CDR3 (H-CDR 3) shown in SEQ ID No. 7, and the light chain comprises a light chain CDR1 (L-CDR 1) shown in SEQ ID No. 11, a light chain CDR2 (L-CDR 2) shown in SEQ ID No. 12, and a light chain CDR3 (L-CDR 3) shown in SEQ ID No. 13; and
(ii) An antibody 2, the antibody 2 comprising a Heavy Chain (HC) and a Light Chain (LC), wherein the heavy chain comprises a heavy chain CDR1 (H-CDR 1) shown in SEQ ID No. 17, a heavy chain CDR2 (H-CDR 2) shown in SEQ ID No. 18, and a heavy chain CDR3 (H-CDR 3) shown in SEQ ID No. 19, and the light chain comprises a light chain CDR1 (L-CDR 1) shown in SEQ ID No. 23, a light chain CDR2 (L-CDR 2) shown in SEQ ID No. 24, and a light chain CDR3 (L-CDR 3) shown in SEQ ID No. 25.
In the context of the present invention, the antibodies 1 and 2 are binding agents capable of specifically binding to regenerated islet-derived protein 3A (REG 3A).
Preferably, the heavy chain of antibody 1 comprises a heavy chain variable region (VH), the light chain comprises a light chain variable region (VL), and the heavy chain variable region of antibody 1 comprises the amino acid sequence shown in SEQ ID No. 8, the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 14; and/or the heavy chain of the antibody 2 comprises a heavy chain variable region (VH), the light chain comprises a light chain variable region (VL), and the heavy chain variable region of the antibody 2 comprises the amino acid sequence shown in SEQ ID No. 20, the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 26.
Preferably, the antibodies 1 and 2 are selected from the group consisting of murine antibodies, chimeric antibodies and humanized antibodies. Preferably, the antibodies 1 and 2 are selected from the group consisting of monoclonal antibodies, fab, fv and ScFv formats.
Preferably, in the antibody combination provided by the invention, the antibody 1 and/or antibody 2 is a monoclonal antibody consisting of two heavy chains and two light chains, preferably a murine IgG1/Kappa isotype or a murine IgG2a/Kappa isotype. In this regard, the structure of the antibody is shown in FIG. 1.
According to a specific embodiment of the invention, the heavy chain of said antibody 1 comprises the amino acid sequence shown in SEQ ID NO. 1 and the light chain comprises the amino acid sequence shown in SEQ ID NO. 2; and/or the heavy chain of the antibody 2 comprises an amino acid sequence shown as SEQ ID NO. 3, and the light chain comprises an amino acid sequence shown as SEQ ID NO. 4.
According to a specific embodiment of the invention, the antibody 1 and/or antibody 2 is provided with a detectable label. Preferably, antibody 2 is provided with a detectable label. Such as enzyme labels (e.g., horseradish peroxidase), radiolabels, luminescent labels, chromogenic labels, haptens (e.g., digoxin, biotin), metal complexes, and metals (e.g., colloidal gold).
In another aspect, the present invention provides the use of the antibody combination in the preparation of a reagent for detecting regenerated islet-derived protein 3A (REG 3A). Alternatively, the present invention provides the use of the antibody combination in the preparation of a reagent for aiding in the diagnosis of a regenerated islet-derived protein 3A (REG 3A) -related disease or a reagent for predicting the risk of developing a regenerated islet-derived protein 3A (REG 3A) -related disease. Preferably, in the context of the present invention, the regenerated islet-derived protein 3A (REG 3A) is human regenerated islet-derived protein 3A (REG 3A) (Genbank Acc. No.: NM-002580).
In the context of the present invention, the REG 3A-related diseases are characterized by elevated REG3A expression. Preferably, the REG 3A-related disease is a digestive system disease. Preferably, the disease is a digestive tract disease, such as stage 1, stage 2, stage 3 or stage 4 bowel cancer; stomach cancer; pancreatitis; peptic ulcer; inflammatory bowel disease.
In the context of the present invention, the reagent may be a reagent capable of detecting the presence or concentration of REG3A in a biological sample from a subject. In the context of the present invention, "concentration" refers to the detectable amount of the protein in a biological sample, and "concentration" is used interchangeably herein with "level" or "amount". According to a specific embodiment of the present invention, the presence or concentration of REG3A is detected in whole blood, serum or plasma.
The above detection results may be used to aid in diagnosing whether the subject has a regenerated islet-derived protein 3A (REG 3A) related disease or to predict whether the subject is at risk for developing the related disease. In the context of the present invention, the subject is a mammal, preferably a primate, more preferably a human; the biological sample is one or more selected from whole blood, plasma, serum, blood cells, ascites, lymph, saliva, sputum, sweat, urine, mucus, interstitial fluid, tissue biopsies and cells from a subject, preferably whole blood, serum or plasma.
Preferably, the reagent is useful in a detection method selected from the group consisting of: chemiluminescent immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), western blotting, antibody microarray, immunoprecipitation, radioimmunoassay (RIA), and the like. Preferably, the reagent is a detection reagent for an ELISA, for example a detection reagent for a double antibody sandwich ELISA, wherein the ELISA is used to detect regenerated islet-derived protein 3A in whole blood, serum or plasma. Preferably, in the ELISA, the antibody 1 is used as a capture antibody (coated antibody) and the antibody 2 is used as a detection antibody.
In another aspect, the invention also provides a kit comprising the antibody combination of the invention.
The kit is used to detect the presence or concentration of REG3A in a biological sample from a subject, and thus can be used to aid in diagnosing whether the subject has, or is at risk for developing, a regenerated islet-derived protein 3A (REG 3A) related disease. Preferably, the regenerated islet-derived protein 3A is human regenerated islet-derived protein 3A. The subject is a mammal, preferably a primate, more preferably a human; the biological sample is one or more selected from whole blood, plasma, serum, blood cells, ascites, lymph, saliva, sputum, sweat, urine, mucus, interstitial fluid, tissue biopsies and cells from a subject, preferably whole blood, serum or plasma. The REG 3A-related diseases are characterized by elevated REG13A expression. Preferably, the REG 3A-related disease is a digestive system disease. Preferably, the disease is a digestive tract disease, such as stage 1, stage 2, stage 3 or stage 4 bowel cancer; stomach cancer; pancreatitis; peptic ulcer; inflammatory bowel disease.
The kit can be used in a detection method selected from the group consisting of: chemiluminescent immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), western blotting, antibody microarray, immunoprecipitation, radioimmunoassay (RIA), and the like. Preferably, the kit is an ELISA detection kit, e.g. a double antibody sandwich ELISA detection kit, comprising the antibody combination according to the invention. Preferably, the ELISA detection kit comprises the antibody 1 as a capture antibody and the antibody 2 as a detection antibody. Preferably, antibody 2 is provided with a detectable label. Such as enzyme labels (e.g., horseradish peroxidase), radiolabels, luminescent labels, chromogenic labels, haptens (e.g., digoxin, biotin), metal complexes, and metals (e.g., colloidal gold).
More preferably, the detection kit further comprises other reagents required for detecting the presence or concentration of REG3A in a biological sample by ELISA. For example, the detection kit further comprises one or more, even all, of the following: a calibrator and/or quality control for regenerated islet-derived protein 3A; an antibody diluent; a washing liquid; a sealing liquid; a sample diluent to be tested; an ELISA plate; developing solution; and (5) stopping liquid.
In yet another aspect, the present invention provides a method for detecting regenerated islet-derived protein 3A (REG 3A) in a biological sample from a subject, the method comprising: the presence or concentration of regenerated islet-derived protein 3A in a biological sample from a subject is detected using an antibody combination or kit of the invention.
Alternatively, the present invention provides a method for aiding in the diagnosis of a regenerated islet-derived protein 3A (REG 3A) -related disease or for predicting the risk of developing a regenerated islet-derived protein 3A (REG 3A) -related disease, the method comprising: the concentration of regenerated islet-derived protein 3A in a biological sample from a subject is detected using the antibody combination or kit of the invention. And, the method further comprises comparing the detected concentration of regenerated islet-derived protein 3A to a reference level, a higher concentration of REG3A in the biological sample being indicative of the subject suffering from or at risk of suffering from a regenerated islet-derived protein 3A (REG 3A) -related disease.
In this aspect, islet-derived protein 3A (REG 3A), REG 3A-related diseases, subjects, biological samples, detection methods, and the like are regenerated as defined above.
In the above-described methods provided by the present invention, the reference level refers to a value determined by comprehensive consideration of ROC analysis and clinical requirements of healthy and disease populations in terms of the concentration of regenerated islet-derived protein 3A (REG 3A) in biological samples.
According to a specific embodiment of the invention, the method is performed using an ELISA detection kit provided by the invention, such as a double antibody sandwich ELISA detection kit. According to a specific embodiment of the present invention, the method may comprise the steps of:
(1) Coating a capture antibody (antibody 1) on an ELISA plate, and sealing;
(2) Diluting a sample to be tested;
(3) Respectively adding a sample to be detected into different plate holes of the ELISA plate, optionally adding a calibrator and/or a quality control material, adding a labeled detection antibody (antibody 2), and incubating;
(4) Washing the plate;
(5) Detecting, e.g., developing, the label on the detection antibody;
(6) Terminating the reaction;
(7) Reading results, such as OD values;
(8) And (3) taking the concentration of the calibrator as an abscissa, taking the detection result of the calibrator, such as an OD value, as an ordinate, manufacturing a standard curve, and then calculating the concentration of the regenerated islet-derived protein 1 alpha in the sample based on the detection result of the sample to be detected.
Specifically, the present invention provides an antibody combination against human regenerated islet-derived protein 3A (REG 3A), each antibody in the antibody combination being capable of binding to REG3A with high affinity and specificity and binding to different epitopes of the protein, thus forming a diabody sandwich pattern, and thus being combinable for qualitative and quantitative detection of REG3A. Correspondingly, the invention also provides an ELISA detection method adopting the antibody combination and a corresponding detection kit.
Experiments prove that the pairing of the antibody 1 (capture antibody) and the antibody 2 (detection antibody) provided by the invention can accurately, specifically and qualitatively and quantitatively detect the human REG3A in serum. The inventors of the present invention have verified using a large number of serum samples including those from stage 1 to stage 4 intestinal cancer, gastric cancer, pancreatitis, peptic ulcer, inflammatory bowel disease patients. The results show that the detection sensitivity of the double-antibody sandwich ELISA on the serum sample of the patient can reach more than 70% by adopting the antibody combination provided by the invention, and the detection result is very accurate, so that the antibody combination and the ELISA (for example, double-antibody sandwich ELISA) detection method and the kit based on the antibody combination have good clinical application value.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a schematic diagram of the structure of an antibody in the antibody combination provided by the invention when the antibody is a monoclonal antibody.
FIGS. 2A to 2E are ROC curves plotted according to REG3A detection results in different serum samples, wherein:
fig. 2A: apparent healthy vs. intestinal cancer ROC curve;
fig. 2B: apparent healthy vs gastric carcinoma ROC curve;
fig. 2C: apparent healthy vs pancreatitis ROC curve;
fig. 2D: apparent healthy vs peptic ulcer ROC curve;
fig. 2E: apparent healthy vs inflammatory bowel disease ROC curve.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.
The human REG3A recombinant protein used in the examples was prepared by recombination according to (Genbank Acc. No.: NM-002580, 27-173 aa).
Example 1Acquisition of monoclonal antibody to regenerated islet-derived protein 3A (REG 3A)
Immunization: the human REG3A recombinant protein is used for immunizing mice for 3-4 times by adopting a subcutaneous multipoint injection mode. And taking a small amount of mouse blood samples for potency determination after immunization, and selecting the spleens of mice with high potency for next fusion.
Cell fusion: grinding spleen cells of a mouse, sieving with a cell sieve, centrifuging, cleaning with a culture solution, and preparing spleen lymphocyte suspension; the myeloma cells and the spleen cells are mixed according to a certain proportion, and a fusion agent is added to fuse the myeloma cells and the spleen cells.
Screening: the hybridoma cells are selected by culturing in HAT selection medium, and the culture supernatant is assayed by ELISA to select hybridoma cells producing antibodies specifically recognizing human REG3A. Cloning the positive hybridoma cells obtained by screening by a limiting dilution method to obtain stable monoclonal hybridoma cells.
Antibody production: and (3) performing amplification culture on the selected hybridoma cells, taking culture supernatant, and purifying by adopting an affinity chromatography mode to obtain the monoclonal antibody.
5 monoclonal antibodies were obtained and tested as murine IgG1/Kappa isotype or murine IgG2a/Kappa isotype, with the antibody information shown in Table 1 and the structure shown in FIG. 1.
Examples2Determination of the titers of monoclonal antibodies
The human REG3A recombinant protein was formulated into a coating solution of 1. Mu.g/mL with 50mmol/L of a carbonic acid buffer of pH9.6, added to the ELISA plate at 100. Mu.L/well, and coated overnight (16-20 h) at 2-8 ℃.
Blocking solution (10 mmol/L phosphate buffer pH 7.4+1% bovine serum albumin+0.05Tween 20) was added to the ELISA plate at 250. Mu.L/well, incubated at 37℃for 2h, and the remaining solution in the ELISA plate was discarded after completion.
The 5 monoclonal antibodies obtained in example 1 were diluted to 1000ng/mL with a diluent (10 mmol/L phosphate buffer pH 7.4+0.5% bovine serum albumin+0.05% Tween 20+0.1% Proclin 300), and then diluted again in a double ratio, and the diluted solution was added to the above ELISA plate at 100. Mu.L/well, incubated at 37℃for 1 hour, and after the completion, the plate was washed 3 times.
To the above ELISA plate was added a dilution of HRP-labeled goat anti-mouse antibody (available from Thermo Scientific, cat# 31430) at 100. Mu.L/well, incubated at 37℃for 1h, and the plate was washed 3 times after the completion.
Adding TMB substrate solution into the ELISA plate at 100 mu L/hole, incubating for 15min at 37 ℃, and then adding sulfuric acid at 0.2mol/L at 50 mu L/hole to terminate the reaction; the sample is read by an enzyme-labeled instrument at a main wavelength of 450nm and a secondary wavelength of 620 nm.
The experimental results are shown in Table 2, which indicate that 5 antibodies can effectively recognize the human REG3A recombinant protein, and the titer is 512000 or > 512000.
Example 3Determination of monoclonal antibody titers after HRP labeling
The 5 monoclonal antibodies obtained in example 1 were individually HRP-labeled by sodium periodate method.
The human REG3A recombinant protein was formulated into a coating solution of 1. Mu.g/mL with 50mmol/L of a carbonic acid buffer of pH9.6, added to the ELISA plate at 100. Mu.L/well, and coated overnight (16-20 h) at 2-8 ℃.
Blocking solution (10 mmol/L phosphate buffer pH 7.4+1% bovine serum albumin+0.05Tween 20) was added to the ELISA plate at 250. Mu.L/well, incubated at 37℃for 2h, and the remaining solution in the ELISA plate was discarded after completion.
5 strains of HRP-labeled monoclonal antibody were diluted 1000-fold with a diluent (10 mmol/L phosphate buffer pH 7.4+0.5% bovine serum albumin+0.05% Tween 20+0.1% Proclin 300), and then diluted again, the diluent was added to the above ELISA plate at 100. Mu.L/well, incubated at 37℃for 1 hour, and after the completion, the plate was washed 3 times.
Adding TMB substrate solution into the ELISA plate at 100 mu L/hole, incubating for 15min at 37 ℃, and then adding sulfuric acid at 0.2mol/L at 50 mu L/hole to terminate the reaction; and then reading at the main wavelength of 450nm and the auxiliary wavelength of 620nm by using an enzyme-labeled instrument.
The experimental results are shown in table 3, which shows that the affinity of the monoclonal antibodies 10G9H12 and 13C1C4 is poor after HRP labeling, and the monoclonal antibodies cannot be used; the titers were highest after the 11C2A10 labeling with HRP, and the titers of 26A12C11 and 6C12F8 were slightly lower, but could be used.
From this, it was determined that monoclonal antibodies 11C2a10, 26a12C11 and 6C12F8 were HRP-labeled and were paired with other antibodies as detection antibodies.
Example 4Screening and validation of capture and detection antibodies
The 5 monoclonal antibodies obtained in example 1 were used as capture antibodies, and were paired with HRP-labeled monoclonal antibodies, respectively, to determine the detection effect on the antigen human REG3A recombinant protein.
The 5 antibodies were prepared into 1. Mu.g/mL coating solutions with 50mmol/L of a carbonic acid buffer solution at pH9.6, and then added into the ELISA plates at 100. Mu.L/well, respectively, and coated overnight (16-20 h) at 2-8deg.C, and after the completion, the plates were washed 2 times and then dried by pipetting.
Blocking solution (10 mmol/L phosphate buffer pH 7.4+1% bovine serum albumin+0.05Tween 20) was added to the ELISA plate at 250. Mu.L/well, incubated at 37℃for 2h, and the remaining solution in the ELISA plate was discarded after completion.
Human REG3A recombinant protein was diluted to a concentration of 500ng/mL in antigen diluent (10 mmol/L pH7.4 phosphate buffer+0.5% bovine serum albumin+0.1% 2-chloroacetamide+0.1% 4-acetaminophen+0.1% triton X100+0.1% Proclin 300), then added to the above ELISA plate at 100. Mu.L/well, incubated at 37℃for 1h, and after the completion the plate was washed 3 times.
Detection antibodies 11C2A10-HRP, 26A12C11-HRP and 6C12F8-HRP were added to the ELISA plate at 100. Mu.L/well, incubated for 1h at 37℃and the plate was washed 3 times after the completion.
Adding TMB substrate solution into the ELISA plate at 100 mu L/hole, incubating for 15min at 37 ℃, and then adding sulfuric acid at 0.2mol/L at 50 mu L/hole to terminate the reaction; the sample is read by an enzyme-labeled instrument at a main wavelength of 450nm and a secondary wavelength of 620 nm.
The experimental results are shown in Table 4, and demonstrate that "the capture antibody 13C1C4+ detection antibody 11C2A10-HRP" is the optimal antibody pair.
Monoclonal antibodies 13C1C4 and 11C2a10 were sequenced to obtain antibody sequences.
(1) 13C1C4
(1-1) heavy chain (SEQ ID NO. 1 (Signal peptide removed)):
MAVLGLLFCLVTFPSCVLSQVQLRQSGPGLVQPSQSLSITCTVSGFSLS RYGVH WVRQSPGKGLEWLG V IWSGGSTDYNAVFIS RLSITKDNSKSQVFFKMNSLQVNDTAIYYCAR GGNWYFDV WGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
(1-2) light chain (SEQ ID NO. 2 (Signal peptide removed)):
MRVLAELLGLLLFCFLGVRCDIQMNQSPSSLSASLGDTITITC HASQNINVWLS WYQQKPGNIPKLLIY KASRLHT GVPSRFSGSGSGTGFTLTISSLQPEDIATYYC QQGQSYPLT FGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
(2) 11C2A10
(2-1) heavy chain (SEQ ID NO. 3 (Signal peptide removed)):
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFT RFLMH WVKQKPGQGLEWIG Y INPYNDGTNYNEKFKG KATLTSDKSSSAAYMELSSLTSEDSAVYYCAR DWYFDY WGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
(2-2) light chain (SEQ ID NO. 4 (Signal peptide removed)):
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISC RTSESVDSYGNSFMH WYQQKPGQPPK LLIY RASNLES GIPARFSGSGSRTDFTLTINPVEADDVATYYC QQSNEEPYM FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
example 5Detection of REG3A in different samples with Capture antibody 13C1C4+ detection antibody 11C2A10-HRP
Serum samples were obtained from the affiliated tumor hospital at the university of chinese and the zhujiang hospital at the university of south medical science from the following groups of people: 715 apparent healthy subjects, 2034 intestinal cancer patients (of which period 1 is 366, period 2 is 714, period 3 is 585, and period 4 is 369), 67 gastric cancer patients, 33 pancreatitis patients, 109 peptic ulcer patients, and 79 inflammatory bowel disease patients.
ELISA kit is prepared by adopting the combination of the capture antibody 13C1C4+ detection antibody 11C2A10-HRP, and the serum sample is detected. And (3) taking apparent healthy people as a control group, comparing the apparent healthy people with data of each disease group respectively, and performing ROC analysis. The results showed that the serum REG3A content of the disease group was significantly different from that of the apparent healthy group (P value < 0.0001). And calculating a Johnson index according to the ROC analysis result to obtain a recommended positive reference value, and corresponding diagnosis sensitivity and diagnosis specificity. The results are shown in Table 5, FIGS. 2A to 2E.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.
Claims (38)
1. An antibody combination, comprising:
(i) An antibody 1, the antibody 1 comprising a Heavy Chain (HC) and a Light Chain (LC), wherein the heavy chain comprises a heavy chain CDR1 (H-CDR 1), a heavy chain CDR2 (H-CDR 2), and a heavy chain CDR3 (H-CDR 3), the heavy chain CDR1 is as shown in SEQ ID No. 5, the heavy chain CDR2 is as shown in SEQ ID No. 6, and the heavy chain CDR3 is as shown in SEQ ID No. 7; the light chain comprises a light chain CDR1 (L-CDR 1), a light chain CDR2 (L-CDR 2) and a light chain CDR3 (L-CDR 3), the light chain CDR1 is shown as SEQ ID NO. 11, the light chain CDR2 is shown as SEQ ID NO. 12 and the light chain CDR3 is shown as SEQ ID NO. 13; and
(ii) An antibody 2, the antibody 2 comprising a Heavy Chain (HC) and a Light Chain (LC), wherein the heavy chain comprises a heavy chain CDR1 (H-CDR 1), a heavy chain CDR2 (H-CDR 2), and a heavy chain CDR3 (H-CDR 3), the heavy chain CDR1 is as shown in SEQ ID No. 17, the heavy chain CDR2 is as shown in SEQ ID No. 18, and the heavy chain CDR3 is as shown in SEQ ID No. 19; the light chain comprises a light chain CDR1 (L-CDR 1), a light chain CDR2 (L-CDR 2) and a light chain CDR3 (L-CDR 3), the light chain CDR1 is shown as SEQ ID NO. 23, the light chain CDR2 is shown as SEQ ID NO. 24 and the light chain CDR3 is shown as SEQ ID NO. 25.
2. The antibody combination of claim 1, wherein antibody 1 and antibody 2 are binding agents capable of specifically binding to regenerated islet-derived protein 3A (REG 3A).
3. The antibody combination of claim 1, wherein the heavy chain of antibody 1 comprises a heavy chain variable region (VH), the light chain comprises a light chain variable region (VL), and the heavy chain variable region of antibody 1 comprises the amino acid sequence shown in SEQ ID No. 8, the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 14; and, a step of, in the first embodiment,
the heavy chain of antibody 2 comprises a heavy chain variable region (VH), the light chain comprises a light chain variable region (VL), and the heavy chain variable region of antibody 2 comprises the amino acid sequence shown in SEQ ID No. 20, the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 26.
4. The antibody combination according to claim 3, wherein the antibodies 1 and 2 are selected from the group consisting of murine antibodies, chimeric antibodies and humanized antibodies.
5. The antibody combination according to claim 3, wherein the antibodies 1 and 2 are selected from the group consisting of monoclonal antibodies, fab, fv and ScFv antibody formats.
6. The antibody combination of claim 5, wherein antibody 1 and antibody 2 are both monoclonal antibodies consisting of two identical heavy chains and two identical light chains.
7. The antibody combination of claim 6, wherein both antibody 1 and antibody 2 are murine IgG1/Kappa isotype or murine IgG2a/Kappa isotype.
8. The antibody combination according to claim 6, wherein the heavy chain of antibody 1 comprises the amino acid sequence shown in SEQ ID No. 1 and the light chain comprises the amino acid sequence shown in SEQ ID No. 2; and, the heavy chain of the antibody 2 comprises an amino acid sequence shown in SEQ ID NO. 3, and the light chain comprises an amino acid sequence shown in SEQ ID NO. 4.
9. The antibody combination according to any one of claims 1 to 8, wherein the antibody 1 or antibody 2 is provided with a detectable label.
10. The antibody combination of claim 9, wherein the antibody 2 is detectably labeled.
11. The antibody combination of claim 10, wherein the detectable label is an enzyme label, a radiolabel, a luminescent label, a chromogenic label, a hapten, a metal complex, or a metal.
12. The antibody combination of claim 11, wherein the enzyme label is horseradish peroxidase.
13. The antibody combination of claim 11, wherein the hapten is digoxin or biotin.
14. The antibody combination of claim 11, wherein the metal is colloidal gold.
15. Use of the antibody combination of any one of claims 1 to 14 in the preparation of a reagent for detecting regenerated islet-derived protein 3A (REG 3A).
16. Use of the antibody combination of any one of claims 1 to 14 in the manufacture of a reagent for aiding in the diagnosis of a regenerated islet-derived protein 3A (REG 3A) -related disease or a reagent for predicting the risk of developing a regenerated islet-derived protein 3A (REG 3A) -related disease.
17. The use according to claim 15 or 16, wherein the regenerated islet-derived protein 3A is human regenerated islet-derived protein 3A.
18. The use according to claim 16, wherein the REG 3A-related disease is characterized by elevated REG3A expression.
19. The use according to claim 18, wherein the REG3A related disease is a digestive system disease.
20. The use according to claim 19, wherein the digestive system disease is a digestive tract disease.
21. The use according to claim 20, wherein the gastrointestinal disorder is stage 1, stage 2, stage 3 or stage 4 bowel cancer; stomach cancer; pancreatitis; peptic ulcer; inflammatory bowel disease.
22. Use according to claim 15 or 16, characterized in that the reagent is used in a detection method selected from the group consisting of: chemiluminescent immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), western blotting, antibody microarray, immunoprecipitation and Radioimmunoassay (RIA).
23. The use according to claim 22, wherein the reagent is a detection reagent for ELISA.
24. The use according to claim 23, wherein the detection reagent is a detection reagent for a double antibody sandwich ELISA.
25. The use according to claim 23 or 24, wherein the ELISA is for detection of regenerated islet-derived protein 3A in whole blood, serum or plasma.
26. The use according to claim 25, wherein in the ELISA the antibody 1 is used as a capture antibody and the antibody 2 is used as a detection antibody.
27. A kit comprising the antibody combination of any one of claims 1 to 14.
28. The kit of claim 27, wherein the kit is a kit for use in a detection method selected from the group consisting of: chemiluminescent immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), western blotting, antibody microarray, immunoprecipitation and Radioimmunoassay (RIA).
29. The kit of claim 28, wherein the kit is an ELISA detection kit.
30. The kit of claim 29, wherein the detection kit is a double antibody sandwich ELISA detection kit.
31. The kit of claim 30, wherein the kit comprises the antibody 1 as a capture antibody and the antibody 2 as a detection antibody.
32. The kit of claim 31, wherein the antibody 2 is detectably labeled.
33. The kit of claim 32, wherein the detectable label is an enzyme label, a radiolabel, a luminescent label, a chromogenic label, a hapten, a metal complex, or a metal.
34. The kit of claim 33, wherein the enzyme label is horseradish peroxidase.
35. The kit of claim 33, wherein the hapten is digoxin or biotin.
36. The kit of claim 33, wherein the metal is colloidal gold.
37. The kit of any one of claims 27 to 36, further comprising other reagents required for detecting the presence or concentration of REG3A in a biological sample using ELISA.
38. The kit of claim 37, wherein the additional reagents are one or more of the following:
regenerated islet-derived protein 3A calibrator and/or quality control; an antibody diluent; a washing liquid; a sealing liquid; a sample diluent to be tested; an ELISA plate; developing solution; and (5) stopping liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311316201.8A CN117050174B (en) | 2023-10-12 | 2023-10-12 | Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311316201.8A CN117050174B (en) | 2023-10-12 | 2023-10-12 | Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117050174A CN117050174A (en) | 2023-11-14 |
| CN117050174B true CN117050174B (en) | 2023-12-19 |
Family
ID=88655851
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311316201.8A Active CN117050174B (en) | 2023-10-12 | 2023-10-12 | Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117050174B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365384A (en) * | 2017-07-26 | 2017-11-21 | 中国药科大学 | A kind of targeting Reg3A single-chain antibody |
| CN113248609A (en) * | 2021-07-14 | 2021-08-13 | 深圳市盛波尔生命科学技术有限责任公司 | Antibody combination for regenerated islet-derived protein 1alpha and detection kit comprising same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200240990A1 (en) * | 2019-01-24 | 2020-07-30 | University Of Kentucky Research Foundation | Reg3a and reg family member biomarkers and methods for diagnosis and treatment of cancer |
-
2023
- 2023-10-12 CN CN202311316201.8A patent/CN117050174B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365384A (en) * | 2017-07-26 | 2017-11-21 | 中国药科大学 | A kind of targeting Reg3A single-chain antibody |
| CN113248609A (en) * | 2021-07-14 | 2021-08-13 | 深圳市盛波尔生命科学技术有限责任公司 | Antibody combination for regenerated islet-derived protein 1alpha and detection kit comprising same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117050174A (en) | 2023-11-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113248609B (en) | Antibody combination for regenerated islet-derived protein 1alpha and detection kit comprising same | |
| AU2011204652B2 (en) | Progastrin and liver pathologies | |
| US10126312B2 (en) | Diagnostic method for urinary tract infection | |
| EP3349011B1 (en) | Liver cancer test method | |
| CN111487419B (en) | Application of KL-6 in children neocoronary pneumonia | |
| WO2005043165A2 (en) | Specific method for cancer detection | |
| WO2011133770A2 (en) | Salivary protein markers for detection of breast cancer | |
| SG192593A1 (en) | Methods and kits for predicting the risk of respiratory failure, renal failure or thrombopenia in a septic patient by measuring endocan levels in blood | |
| US20170146541A1 (en) | Detection of high-risk intraductal papillary mucinous neoplasm and pancreatic adenocarcinoma | |
| JP2018533724A (en) | Use of laminin 2 to diagnose hepatocellular carcinoma and pancreatic cancer | |
| EP2906951A1 (en) | Diagnostic method for colorectal cancer | |
| RU2671578C2 (en) | Method for predicting risk of getting cancer or diagnosing cancer in female subject | |
| WO2020158858A1 (en) | Method for immunologically analyzing free aim in biological specimen, and method for detecting nash in subject | |
| CN117050174B (en) | Antibody combination against regenerated islet-derived protein 3A and detection kit comprising same | |
| CN108139404B (en) | Antibody specifically recognizing and binding to REIC/Dkk-3 protein of active structure, and monitoring of cancer therapy using the anti-REIC/Dkk-3 antibody | |
| AU2015215008B2 (en) | Composition and method for detecting malignant neoplastic disease | |
| KR102018209B1 (en) | Diagnosis composition of gastric cancer and diagnosis method of gastric cancer using the same | |
| US20210140966A1 (en) | Immunoassay for detecting tumor pyruvate kinase m2 | |
| CN115819548B (en) | Marker and method for detecting inflammation-related diseases | |
| US20190285633A1 (en) | Point of care assays | |
| CN115280146A (en) | Method for assaying fragment containing human type IV collagen 7S domain and kit for use in the assay method | |
| OA19228A (en) | Point of care assays | |
| WO2009090880A1 (en) | Method for diagnosis of diabetes | |
| WO2015031614A1 (en) | Soluble cmet assay |
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 |