JP6505749B2 - 肺疾患および他の疾患用の生化学マーカー - Google Patents
肺疾患および他の疾患用の生化学マーカー Download PDFInfo
- Publication number
- JP6505749B2 JP6505749B2 JP2016563251A JP2016563251A JP6505749B2 JP 6505749 B2 JP6505749 B2 JP 6505749B2 JP 2016563251 A JP2016563251 A JP 2016563251A JP 2016563251 A JP2016563251 A JP 2016563251A JP 6505749 B2 JP6505749 B2 JP 6505749B2
- Authority
- JP
- Japan
- Prior art keywords
- binding partner
- peptide
- elastin
- immunological binding
- sample
- 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title description 5
- 201000010099 disease Diseases 0.000 title description 4
- 210000004072 lung Anatomy 0.000 title description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 97
- 108010014258 Elastin Proteins 0.000 claims description 79
- 102000016942 Elastin Human genes 0.000 claims description 78
- 229920002549 elastin Polymers 0.000 claims description 78
- 230000027455 binding Effects 0.000 claims description 53
- 239000012634 fragment Substances 0.000 claims description 52
- 230000001900 immune effect Effects 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 17
- 102000035195 Peptidases Human genes 0.000 claims description 16
- 108091005804 Peptidases Proteins 0.000 claims description 16
- 238000003776 cleavage reaction Methods 0.000 claims description 16
- 230000007017 scission Effects 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 16
- 239000000872 buffer Substances 0.000 claims description 15
- 102000007079 Peptide Fragments Human genes 0.000 claims description 14
- 108010033276 Peptide Fragments Proteins 0.000 claims description 14
- 238000003556 assay Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 230000009870 specific binding Effects 0.000 claims description 11
- 238000003018 immunoassay Methods 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000002860 competitive effect Effects 0.000 claims description 3
- 238000002967 competitive immunoassay Methods 0.000 claims description 3
- 230000001575 pathological effect Effects 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 238000004166 bioassay Methods 0.000 claims description 2
- 235000019833 protease Nutrition 0.000 claims description 2
- 210000003296 saliva Anatomy 0.000 claims description 2
- 210000002700 urine Anatomy 0.000 claims description 2
- 230000036039 immunity Effects 0.000 claims 1
- 239000006226 wash reagent Substances 0.000 claims 1
- 230000009257 reactivity Effects 0.000 description 25
- 101000851058 Homo sapiens Neutrophil elastase Proteins 0.000 description 21
- 102000052502 human ELANE Human genes 0.000 description 21
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 19
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 15
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 206010041823 squamous cell carcinoma Diseases 0.000 description 15
- 239000004365 Protease Substances 0.000 description 14
- 101000990912 Homo sapiens Matrilysin Proteins 0.000 description 12
- 102100030417 Matrilysin Human genes 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 235000019419 proteases Nutrition 0.000 description 9
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 6
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 4
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000005192 partition Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 3
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 2
- 101000577881 Homo sapiens Macrophage metalloelastase Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 208000004852 Lung Injury Diseases 0.000 description 2
- 206010069363 Traumatic lung injury Diseases 0.000 description 2
- VEVRNHHLCPGNDU-MUGJNUQGSA-O desmosine Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(CC[C@H](N)C(O)=O)=C(CCC[C@H](N)C(O)=O)C(CC[C@H](N)C(O)=O)=C1 VEVRNHHLCPGNDU-MUGJNUQGSA-O 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 231100000515 lung injury Toxicity 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102100033167 Elastin Human genes 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 101000851054 Homo sapiens Elastin Proteins 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 102100027998 Macrophage metalloelastase Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001094 effect on targets Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 102000054289 human ELN Human genes 0.000 description 1
- 102000052074 human MMP7 Human genes 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- RGXCTRIQQODGIZ-UHFFFAOYSA-O isodesmosine Chemical group OC(=O)C(N)CCCC[N+]1=CC(CCC(N)C(O)=O)=CC(CCC(N)C(O)=O)=C1CCCC(N)C(O)=O RGXCTRIQQODGIZ-UHFFFAOYSA-O 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- 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
- G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- 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
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
- G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
- G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
- G01N2333/96411—Serine endopeptidases (3.4.21)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
- G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
- G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
- G01N2333/96419—Metalloendopeptidases (3.4.24)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
- G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7052—Fibrosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
免疫化用に以下のペプチドを選択した。
標準的な手順に従って、C末端エピトープの場合はN末端で、N末端エピトープの場合はC末端でKLHに結合する言及した免疫原ペプチドを用いて、200μLの乳化抗原(免疫化1回あたり50μg)で腹部中に皮下接種して6頭の4〜6週齢のBalb/Cマウスを免疫化した。安定した力価レベルが得られるまで、免疫化を続けた。
抗体は、免疫化に用いた非結合形態の相同ペプチド(選択ペプチド)を用いて、ペプチド特異性に基づいて選択することによって、さらにエラスチン配列の1アミノ酸だけ切断部位を越えて拡張したペプチドの伸長バージョンを除外することによって、ネオエピトープ特異性を有する抗体のみを確実に更なる分析のために選択した。
96ウェルストレプトアビジンプレートを、コーターバッファ中に溶解したスクリーニングペプチドでコーティングし、20℃にて30分間インキュベートした。インキュベーション後、プレートを洗浄バッファ(20mMトリス、50mM NaCl、pH7.2)中で5回洗浄した。20μLのペプチド(選択、除外、またはナンセンス)またはヒトサンプルを適切なウェルに2連で加えた後に、100μLのPOD結合モノクローナル抗体を加えてから、プレートをシェーカー上で1時間インキュベートした。洗浄後、100μLテトラメチルベンジジン(TMB)(Kem−En−Tec)を加え、プレートを暗所で20℃にて15分間インキュベートした。
図1は、NB590抗体に対する、遊離および伸長ペプチドの反応性を示す。吸光度の低下が、遊離ペプチド濃度の上昇と共に見られる。これは、ウェル内でスクリーニングペプチドの代わりに遊離ペプチドとの反応が増大したことによる。これは、伸長ペプチドには見られなかった。というのも、安定したB/B0レベルにより、NB590−01抗体と伸長ペプチドとの間で反応が非常に低かった、または反応がなかったことが示されたためである。
図4は、NB592抗体に対する、遊離および伸長ペプチドの反応性を示す。吸光度の低下が、遊離ペプチド濃度の上昇と共に見られる。これは、ウェル内でスクリーニングペプチドの代わりに遊離ペプチドとの反応が増大したことによる。これは、伸長ペプチドには見られなかった。というのも、安定したB/B0レベルにより、伸長ペプチドとNB592抗体との間で反応が非常に低かった、または反応がなかったことが示されたためである。
図7は、NB593抗体に対する、遊離および伸長ペプチドの反応性を示す。吸光度の低下が、遊離ペプチド濃度の上昇と共に見られる。これは、ウェル内でスクリーニングペプチドの代わりに遊離ペプチドとの反応が増大したことによる。これは、伸長ペプチドには見られなかった。というのも、安定したB/B0レベルにより、NB593抗体間で反応が非常に低かった、または反応がなかったことが示されたためである。
図10は、NB595抗体に対する、遊離および伸長ペプチドの反応性を示す。吸光度の低下が、遊離ペプチド濃度の上昇と共に見られる。これは、ウェル内でスクリーニングペプチドの代わりに遊離ペプチドとの反応が増大したことによる。これは、伸長ペプチドには見られなかった。というのも、安定したB/B0レベルにより、NB595抗体との反応が非常に低かった、または反応がなかったことが示されたためである。
図13は、NB599抗体に対する、遊離した伸長ペプチドの反応性を示す。吸光度の低下が、遊離ペプチド濃度の上昇と共に見られる。これは、ウェル内でスクリーニングペプチドの代わりに遊離ペプチドとの反応が増大したことによる。吸光度の低下は、伸長ペプチドについて、有意により低かった。というのも、B/B0レベルにより、ペプチドとNB599抗体との反応が弱いことが示されたためである。
Claims (11)
- インビボでエラスチンをプロテイナーゼにより切断部位で切断することによって前記部位に形成されたネオエピトープを含むペプチドフラグメントの定量化のためのバイオアッセイ方法であって、前記ペプチドフラグメントを含むサンプルを、前記ネオエピトープに対して特異的な結合親和性を有する免疫学的結合パートナーと接触させるステップと、前記サンプル中のペプチドフラグメントへの、前記免疫学的結合パートナーの結合のレベルを判定するステップとを含み、前記免疫学的結合パートナーが、末端配列
前記免疫学的結合パートナーが、配列………FGPGVVと反応するならば、………FGPGVVG(配列番号5)と特異的に反応せず、………VPGLGVと反応するならば、………VPGLGVG(配列番号6)と特異的に反応せず、IKAPKL……と反応するならば、PIKAPKL……(配列番号7)と特異的に反応しない、方法。 - 前記免疫学的結合パートナーが、特異的な結合親和性を有するモノクローナル抗体またはモノクローナル抗体のフラグメントである、請求項1に記載の方法。
- 前記免疫学的結合パートナーおよび競合剤が前記サンプルの存在下でインキュベートされて、前記競合剤が前記サンプル中の前記ペプチドフラグメントと競合して前記免疫学的結合パートナーに結合する競合免疫アッセイとして行われる、請求項1または2に記載の方法。
- 前記競合剤が、前記ネオエピトープを明らかにするような、合成ペプチドであるか、またはエラスチンの切断によって形成される精製された天然ペプチドである、請求項3に記載の方法。
- 前記免疫学的結合パートナーと、前記免疫学的結合パートナーが結合するペプチドフラグメント中に含有されるペプチド配列に対して特異的な結合親和性を有する更なる免疫学的結合パートナーとを前記サンプルの存在下でインキュベートして、その両方を前記サンプル中の前記ペプチドフラグメントに一緒に結合させるサンドイッチ免疫アッセイとして行われる、請求項1から4のいずれか1項に記載の方法。
- 前記サンプルが、尿、血清、血液、血漿、または唾液のサンプルである、請求項1から5のいずれか1項に記載の方法。
- 前記サンプルが、患者由来のサンプルであり、前記ペプチドフラグメントの前記結合の前記判定レベルを、(a)比較可能な健常な個体および/または(b)病的状態に特有の値と比較することをさらに含む、請求項1から6のいずれか1項に記載の方法。
- エラスチンから誘導された末端配列
配列………FGPGVVと反応するならば、………FGPGVVG(配列番号5)と特異的に反応せず、………VPGLGVと反応するならば、………VPGLGVG(配列番号6)と特異的に反応せず、IKAPKL……と反応するならば、PIKAPKL……(配列番号7)と特異的に反応しない、免疫学的結合パートナー。 - モノクローナル抗体またはその結合フラグメントである、請求項8に記載の免疫学的結合パートナー。
- 請求項9に記載のモノクローナル抗体を産生する細胞株。
- 請求項8または9のいずれか1項に記載の免疫学的結合パートナーと、
前記免疫学的結合パートナーと結合する競合剤と
を含む免疫アッセイキットであって、場合によっては、洗浄試薬、バッファ、反応停止試薬、酵素標識、酵素標識基質、較正標準、抗マウス抗体、および前記キットを用いてアッセイを行うための説明書の1つまたは複数を含む、キット。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1400472.5 | 2014-01-13 | ||
GB201400472A GB201400472D0 (en) | 2014-01-13 | 2014-01-13 | Biochemical Markers for pulmonary and other diseases |
PCT/EP2015/050269 WO2015104342A1 (en) | 2014-01-13 | 2015-01-08 | Biochemical markers for pulmonary and other diseases |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2017504040A JP2017504040A (ja) | 2017-02-02 |
JP6505749B2 true JP6505749B2 (ja) | 2019-04-24 |
Family
ID=50191196
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2016563251A Active JP6505749B2 (ja) | 2014-01-13 | 2015-01-08 | 肺疾患および他の疾患用の生化学マーカー |
Country Status (8)
Country | Link |
---|---|
US (2) | US10001490B2 (ja) |
EP (1) | EP3094979B1 (ja) |
JP (1) | JP6505749B2 (ja) |
KR (1) | KR102307378B1 (ja) |
DK (1) | DK3094979T3 (ja) |
ES (1) | ES2821884T3 (ja) |
GB (1) | GB201400472D0 (ja) |
WO (1) | WO2015104342A1 (ja) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3139639A1 (en) | 2015-09-04 | 2017-03-08 | Music Group IP Ltd. | Method for determining a connection order of nodes on a powered audio bus |
GB201802070D0 (en) * | 2018-02-08 | 2018-03-28 | Nordic Bioscience As | Elastin assay |
EA202192033A1 (ru) * | 2019-01-22 | 2021-10-21 | Клемсон Юниверсити Рисерч Фаундейшн | Антитела к эластину и способы их применения |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4976734A (en) | 1985-10-31 | 1990-12-11 | Uab Research Foundation | Stimulation of chemotaxis by chemotactic peptides |
US8101717B2 (en) * | 2006-11-13 | 2012-01-24 | The University Of Sydney | Use of tropoelastin for repair or restoration of tissue |
EP2538221A3 (en) | 2007-11-05 | 2013-04-24 | Nordic Bioscience A/S | Biochemical markers for CVD risk assessment |
ES2635494T3 (es) * | 2009-03-30 | 2017-10-04 | Nordic Bioscience A/S | Ensayo de biomarcador de fibrosis |
WO2011094343A1 (en) * | 2010-01-26 | 2011-08-04 | The Trustees Of Columbia University In The City Of New York | Methods of validating candidate compounds for use in treating copd and other diseases |
-
2014
- 2014-01-13 GB GB201400472A patent/GB201400472D0/en not_active Ceased
-
2015
- 2015-01-08 KR KR1020167019716A patent/KR102307378B1/ko active IP Right Grant
- 2015-01-08 JP JP2016563251A patent/JP6505749B2/ja active Active
- 2015-01-08 EP EP15701300.4A patent/EP3094979B1/en active Active
- 2015-01-08 DK DK15701300.4T patent/DK3094979T3/da active
- 2015-01-08 WO PCT/EP2015/050269 patent/WO2015104342A1/en active Application Filing
- 2015-01-08 ES ES15701300T patent/ES2821884T3/es active Active
- 2015-01-08 US US15/111,062 patent/US10001490B2/en active Active
-
2018
- 2018-05-31 US US15/994,786 patent/US20180267060A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
DK3094979T3 (da) | 2020-10-12 |
US10001490B2 (en) | 2018-06-19 |
EP3094979A1 (en) | 2016-11-23 |
WO2015104342A1 (en) | 2015-07-16 |
US20180267060A1 (en) | 2018-09-20 |
ES2821884T3 (es) | 2021-04-28 |
US20160334416A1 (en) | 2016-11-17 |
KR102307378B1 (ko) | 2021-10-01 |
JP2017504040A (ja) | 2017-02-02 |
KR20160108358A (ko) | 2016-09-19 |
GB201400472D0 (en) | 2014-02-26 |
EP3094979B1 (en) | 2020-08-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6095702B2 (ja) | 線維症バイオマーカアッセイ | |
EP4150337A1 (en) | Calprotectin assay | |
WO2008054724A9 (en) | Monoclonal antibodies against osteopontin | |
JP6505749B2 (ja) | 肺疾患および他の疾患用の生化学マーカー | |
JP6175058B2 (ja) | チチンタンパク質のネオエピトープアッセイによる心血管イベントのインビトロ評価 | |
ES2900363T3 (es) | Ensayo de colágeno tipo VII alfa 1 | |
CN108026522B (zh) | 特异性纯化的抗普莱晒谱星抗体 | |
JP2000515854A (ja) | 脳タンパク質s―100の存在の測定方法 | |
JP2014520825A5 (ja) | ||
KR102131860B1 (ko) | 아르기닌이 메틸화된 ggt1에 특이적으로 결합하는 대장암 진단용 바이오마커 조성물 | |
US20240125802A1 (en) | Immunoassay for Detecting Eosinophilic Esophagitis | |
JP7425735B2 (ja) | エラスチンアッセイ | |
US20240085428A1 (en) | Assay for Detecting Collagen XI Biomarkers | |
CN114746755A (zh) | 测量蛋白酶介导的iv型胶原蛋白降解的新表位特异性测定 | |
WO2019121925A1 (en) | Tumstatin assay | |
JP2024525130A (ja) | Xx型コラーゲンのアッセイ | |
WO2023148165A1 (en) | Method for diagnosing collagen degradatation associated disease | |
KR20230145319A (ko) | 콜라겐 xviii 바이오마커 검출용 검정 | |
CN113710699A (zh) | Xxiii型胶原蛋白测定 | |
JP2007127472A (ja) | グリシル化ガストリン(glycine−extendedgastrin)の分析方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20170921 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20180516 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180706 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181009 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190226 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190327 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6505749 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |