CN116718762A - Polypeptide sample stabilizer - Google Patents
Polypeptide sample stabilizer Download PDFInfo
- Publication number
- CN116718762A CN116718762A CN202310680237.8A CN202310680237A CN116718762A CN 116718762 A CN116718762 A CN 116718762A CN 202310680237 A CN202310680237 A CN 202310680237A CN 116718762 A CN116718762 A CN 116718762A
- Authority
- CN
- China
- Prior art keywords
- stabilizer
- percent
- polypeptide
- blood collection
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 65
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 62
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 60
- 239000003381 stabilizer Substances 0.000 title claims abstract description 33
- 210000004369 blood Anatomy 0.000 claims abstract description 35
- 239000008280 blood Substances 0.000 claims abstract description 35
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 9
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims abstract description 9
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 claims abstract description 6
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 claims abstract description 6
- 108010052968 leupeptin Proteins 0.000 claims abstract description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 5
- FRTNIYVUDIHXPG-UHFFFAOYSA-N acetic acid;ethane-1,2-diamine Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.NCCN FRTNIYVUDIHXPG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 239000003755 preservative agent Substances 0.000 claims abstract description 5
- 230000002335 preservative effect Effects 0.000 claims abstract description 5
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 claims abstract description 3
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- HLWRUJAIJJEZDL-UHFFFAOYSA-M sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate Chemical group [Na+].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC([O-])=O HLWRUJAIJJEZDL-UHFFFAOYSA-M 0.000 claims description 2
- JZBRFIUYUGTUGG-UHFFFAOYSA-J tetrapotassium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].[K+].[K+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O JZBRFIUYUGTUGG-UHFFFAOYSA-J 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 14
- 238000000338 in vitro Methods 0.000 abstract description 10
- 210000002966 serum Anatomy 0.000 abstract description 5
- 230000006641 stabilisation Effects 0.000 abstract description 3
- 238000011105 stabilization Methods 0.000 abstract description 3
- 102400000921 Gastrin Human genes 0.000 description 9
- 108010066264 gastrin 17 Proteins 0.000 description 8
- GKDWRERMBNGKCZ-RNXBIMIWSA-N gastrin-17 Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 GKDWRERMBNGKCZ-RNXBIMIWSA-N 0.000 description 8
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 description 6
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 description 6
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 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 6
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 description 6
- 108010075254 C-Peptide Proteins 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 description 3
- 108010052343 Gastrins Proteins 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000000087 stabilizing effect Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102000005593 Endopeptidases Human genes 0.000 description 2
- 108010059378 Endopeptidases Proteins 0.000 description 2
- 108020001621 Natriuretic Peptide Proteins 0.000 description 2
- 102000004571 Natriuretic peptide Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000692 natriuretic peptide Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- BWVGWYGBCIARRP-UHFFFAOYSA-N 2-(hydroxymethyl)-2-(propylamino)propane-1,3-diol Chemical compound CCCNC(CO)(CO)CO BWVGWYGBCIARRP-UHFFFAOYSA-N 0.000 description 1
- SETPGGFWNLJETP-UHFFFAOYSA-N 3-amino-2-(hydroxymethyl)pentane-1,3,5-triol Chemical compound OCCC(O)(N)C(CO)CO SETPGGFWNLJETP-UHFFFAOYSA-N 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 210000000712 G cell Anatomy 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 239000003629 gastrointestinal hormone Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000004206 stomach function Effects 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a polypeptide sample stabilizer, which comprises the following components in percentage by mass: ethylenediamine tetraacetic acid or ethylenediamine tetraacetic acid salt: 1 to 3 percent; n-ethylmaleimide: 1 to 3 percent; cyclodextrin: 1 to 2 percent; leupeptin: 1 to 3 percent; 0.02 to 0.1 percent of preservative; the balance of acid buffer solution with pH of 2.0-4.0. The polypeptide sample stabilizer breaks through the limitation of the traditional stabilizer on the types, can be applied to the stabilization of various polypeptide samples, improves the stability of polypeptides in-vitro blood, and ensures that the polypeptides are not easy to decompose in vitro (serum/plasma/whole blood), thereby improving the accuracy of the detection result of the polypeptides.
Description
Technical Field
The invention belongs to the technical field of medical in-vitro diagnosis, and particularly relates to a polypeptide sample stabilizer.
Background
Peptides (peptides) are compounds formed by peptide bonds of alpha-amino acids, which are also proteolytic intermediates. The peptide has important biological functions, is an important physiological regulator for human body, can comprehensively regulate the physiological functions of the human body, and can strengthen and exert the physiological activity of the human body.
In the metabolic pathway of the human body, polypeptides are key intermediates thereof, and their content in body fluids can reflect many human body functional states. In the field of in vitro diagnosis, many pathological markers for disease detection are polypeptide antigens, and a certain disease can be directly reflected according to the increase or decrease of the polypeptide antigens in body fluid.
Wherein, gastrin-17 which reflects the stomach function state consists of 17 amino acids, and gastrointestinal hormone secreted by G cells of the antrum and the duodenum plays an important role in regulating the digestive tract function and maintaining the structural integrity, and the content of gastrin reflects an important index of the gastric mucosa injury condition. Natriuretic Peptide (BNP), which is responsive to cardiovascular function, can be used as an auxiliary diagnostic indicator for congestive heart failure and mild cardiac dysfunction.
The C peptide is a single-chain polypeptide consisting of 31 amino acids (AA 33-63), and is an accompanying product of cleavage of proinsulin into insulin in the course of insulin biosynthesis, and evidence shows that the C peptide can prevent the progress of long-term complications of type I diabetes as a substitute for insulin.
The polypeptide pathological markers are also a lot, and with the continuous development of diagnostic reagents, the human serum detection using the polypeptide pathological markers as core indexes belongs to a noninvasive, painless, safe and economic detection method. The current methods for polypeptide detection mainly comprise an enzyme-linked immunosorbent assay, a fluorescence chromatography method, a magnetic particle chemiluminescence method and the like.
The mechanism of the change of the polypeptides in vitro (serum/plasma/whole blood) is not exactly concluded, but studies have shown that the change of the polypeptides in vitro may be caused by multiple reasons, and the more extensive expression includes the following:
first kind: endopeptidases and exopeptidases in the sample can further degrade polypeptide antigens.
Second kind: in vitro aggregation of polypeptides also results in structural changes in the polypeptides, which can be undetectable by detection reagents.
Third kind: complement proteins in the sample can also degrade polypeptides.
For whatever reason, the polypeptides in the sample can be changed rapidly after blood collection, and the storage time is only tens of minutes in theory at 2-8 ℃, and the degradation speed is faster along with the temperature rise, which means that the detection must be completed in a very short time from blood collection, but many hospitals cannot detect in time in a short time in practical work. This also results in the clinical situation that the detection results of polypeptides are inaccurate and the patients cannot be accurately reflected.
Many studies have been made by those skilled in the art on the case where polypeptides are unstable in isolated blood.
The Chinese patent publication No. CN115702637A discloses a protective agent for a blood sample of gastrin 17, and proposes that 2-di (2-hydroxyethyl) amino-2-hydroxymethyl-1, 3-propanediol and/or-1, 3-bis [ (trimethylol) methylamino ] propane are used as a stabilizer, so that degradation of polypeptides can be effectively avoided, the preservation time of the blood sample of polypeptides is greatly prolonged, and the accuracy of detection results is ensured. The mechanism by which this patent works to inhibit the degradation of gastrin 17 with only one buffer is not clear.
Chinese patent publication No. CN1950398A discloses "stabilization of peptide", which shows that the use of DMSO can extend the storage time of gastrin in serum or plasma samples from 3h to 72h, but DMSO as an organic solvent can cause red blood cells to be broken, resulting in errors in the detection results.
The existing stabilizing agents are only aimed at stabilizing gastrin polypeptides in blood samples, the pertinence is single, and the stabilizing effect is not ideal.
Disclosure of Invention
The invention aims to provide a stabilizer for polypeptide samples, which breaks through the limitation of the traditional stabilizer on types, can be applied to the stabilization of various polypeptide samples, improves the stability of the polypeptides in isolated blood, and ensures that the polypeptides are not easy to decompose in vitro (serum/plasma/whole blood), thereby improving the accuracy of the detection result of the polypeptides.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a polypeptide sample stabilizer is characterized by comprising the following components in percentage by mass: ethylenediamine tetraacetic acid or ethylenediamine tetraacetic acid salt: 1 to 3 percent; n-ethylmaleimide: 1 to 3 percent; cyclodextrin: 1 to 2 percent; leupeptin: 1 to 3 percent; 0.02 to 0.1 percent of preservative; the balance of acid buffer solution with pH of 2.0-4.0.
Preferably, the ethylenediamine tetraacetic acid salt is ethylenediamine tetraacetic acid sodium salt or ethylenediamine tetraacetic acid potassium salt.
Preferably, the acidic buffer is one or more of glycine-hydrochloric acid, citric acid-sodium citrate and acetic acid-sodium acetate buffer.
Preferably, the leupeptin is one or more of hydrochloride and sulfate.
Preferably, the preservative is one or more of sodium azide, P300 and Bro.
Through researches, the in vitro degradation of polypeptide molecules is determined by multiple factors, and the main influencing factors are related to the structure of the polypeptide, the type of sample, the storage condition and the like.
The acidic buffer solution can enable the polypeptide with higher isoelectric point to have positive charges, thereby reducing electrostatic adsorption among polypeptides and ensuring the dispersibility of the polypeptides. The cyclodextrin molecule has a slightly conical hollow cylindrical three-dimensional ring structure, the outer edge of the cyclodextrin molecule is hydrophilic, the inner cavity of the cyclodextrin molecule is hydrophobic, a stable microenvironment can be provided, and polypeptide aggregation caused by the combination of hydrophobic bonds can be effectively avoided for polypeptide with stronger hydrophobicity. The acid system is matched with cyclodextrin, so that the polypeptide deactivation caused by physical and chemical actions (electrostatic adsorption, hydrophobic combination and the like) can be effectively solved.
EDTA, as a metal chelator, is capable of inhibiting a variety of metalloenzyme activities. The single enzyme inhibitor has a good stabilizing effect on a certain polypeptide, EDTA, NEMI and leupeptin are added in a compound way to fundamentally improve the stability of the polypeptide, the activity of bioactive substances such as endopeptidase, complement and the like in blood can be effectively inhibited by utilizing the combined effect of cyclodextrin and an acidic buffer solution, the stability of different kinds of polypeptides can be improved from multiple angles by the synergic effect of various inhibitors, one of the stabilizing agents can be used as an independent stabilizing agent, and the stabilizing agent is added into a blood collection tube filled with blood after blood collection and is uniformly mixed.
Secondly, in the production process of the blood collection tube, the stabilizer is added into the blood collection tube to be used as a component part of the blood collection tube, and the blood collection tube is directly used for collecting blood when blood needs to be collected.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, by adding various stabilizers and matching the acid system with cyclodextrin, the inactivation of the polypeptide caused by physical and chemical actions (electrostatic adsorption, hydrophobic combination and the like) can be effectively solved, and the stability of the polypeptide in an in-vitro sample is fundamentally improved.
The obtained stabilizer has the advantages of strong universality, strong realizability and low cost, can detect the stability of polypeptide pathological markers (such as gastrin-17, natriuretic peptide and the like) in blood samples, is simple and convenient to operate, and can adapt to detection of different methodologies (POCT, enzyme immunity and magnetic particles).
Detailed Description
The invention is further illustrated below with reference to examples.
Specific components of the stabilizers of examples and comparative examples of the present invention are shown in Table 1;
the stabilizers and the stabilizers of the present invention were respectively added to blood samples containing polypeptides at different concentrations, and then the samples were placed in the same environment at room temperature (18-25 ℃) and were measured for the content of gastrin-17 (G-17), brain Natriuretic Peptide (BNP) and C-peptide (BNP) in the blood samples by using a gastrin-17 (G-17) chemiluminescent detection kit, brain Natriuretic Peptide (BNP) and C-peptide (C-P) chemiluminescent detection kit, which were developed from the peak at room temperature (18-25 ℃) for different periods of time, respectively, and the results are shown in table 2, respectively.
As can be seen from the data in Table 2, the stability of the polypeptide in the isolated blood sample can be improved well by adding the stabilizer of the invention.
Comparative example 1 was stored for 4 hours at room temperature, the activity of polypeptides was reduced by more than 40%, whereas the test group added with the stabilizer of the present invention, the sample was left for 7 days, and the content of polypeptides in the blood sample was not significantly reduced.
As can be seen from the data of comparative examples 2/3/4/5, the addition of a single component can improve the stability of polypeptides to some extent, and the effects of the components are different for different types of polypeptides. Compared with the test group, the compound synergistic effect of the components of the test group can obviously improve the stability of the polypeptide and can improve the universality of the stabilizer.
The foregoing description is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims, and all changes in construction or process equivalents that may be employed in the practice of the invention, or in other related art, are intended to be encompassed by the invention as claimed herein
/>
/>
Claims (7)
1. The polypeptide sample stabilizer is characterized by comprising the following components in percentage by mass: ethylenediamine tetraacetic acid or ethylenediamine tetraacetic acid salt: 1 to 3 percent; n-ethylmaleimide: 1 to 3 percent; cyclodextrin: 1 to 2 percent; leupeptin: 1 to 3 percent; 0.02 to 0.1 percent of preservative; the balance of acid buffer solution with pH of 2.0-4.0.
2. The polypeptide sample stabilizer according to claim 1, wherein the ethylenediamine tetraacetic acid salt is ethylenediamine tetraacetic acid sodium salt or ethylenediamine tetraacetic acid potassium salt.
3. The polypeptide sample stabilizer according to claim 1, wherein the acidic buffer is one or more of glycine-hydrochloric acid, citric acid-sodium citrate, acetic acid-sodium acetate buffer.
4. The peptide sample stabilizer according to claim 1, wherein the leupeptin is one or more of hydrochloride and sulfate.
5. The polypeptide sample stabilizer according to claim 1, wherein the preservative is one or more of sodium azide, P300, bro.
6. A method of using the stabilizer for polypeptide samples according to any one of claims 1 to 5, wherein the stabilizer is obtained by uniformly mixing the components for later use, and the stabilizer is added into a blood collection tube filled with blood after blood collection, and uniformly mixed.
7. A method of using the stabilizer for a polypeptide sample as set forth in any one of claims 1 to 5, wherein the stabilizer is added to a blood collection tube during the production of the blood collection tube, and the blood collection tube is directly used for collecting blood when blood collection is required as a constituent of the blood collection tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310680237.8A CN116718762A (en) | 2023-06-09 | 2023-06-09 | Polypeptide sample stabilizer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310680237.8A CN116718762A (en) | 2023-06-09 | 2023-06-09 | Polypeptide sample stabilizer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116718762A true CN116718762A (en) | 2023-09-08 |
Family
ID=87867323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310680237.8A Pending CN116718762A (en) | 2023-06-09 | 2023-06-09 | Polypeptide sample stabilizer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116718762A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2096336A1 (en) * | 1991-09-30 | 1993-03-31 | Andreas Bergmann | Method of stabilizing endogenic, physiologically active peptides |
| US20040067889A1 (en) * | 2002-06-19 | 2004-04-08 | Alexander Belenky | Stabilization of brain natriuretic peptide (BNP) in blood samples, methods and compositions related thereto |
| CN1739789A (en) * | 2005-05-11 | 2006-03-01 | 北京双鹭药业股份有限公司 | Somatostation a aqua prepn and its prepn process and application |
| CN101022822A (en) * | 2004-08-24 | 2007-08-22 | 第一阿斯比奥制药株式会社 | Liquid preparation of physiologically active peptide |
| CN108279307A (en) * | 2018-01-30 | 2018-07-13 | 郑州安图生物工程股份有限公司 | Protection and the heparin tube for stablizing gastrin-releasing peptide precursor in serum or blood plasma |
| CN108333360A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Gastrin-releasing peptide precursor dilution and its application and kit |
| CN110221006A (en) * | 2019-07-25 | 2019-09-10 | 深圳健科医学检验实验室 | It is a kind of for the pre-treating method of sulfydryl polypeptide biological sample and to the detection method of the biological sample |
| CN110596367A (en) * | 2019-09-06 | 2019-12-20 | 广州科方生物技术股份有限公司 | Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof |
| CN112165951A (en) * | 2018-04-16 | 2021-01-01 | 默克专利股份有限公司 | Protein formulation additives for improved thermal stability |
| CN113727700A (en) * | 2019-04-26 | 2021-11-30 | 伊莱利利公司 | Method for preparing stable peptide preparation |
| CN115702637A (en) * | 2021-08-13 | 2023-02-17 | 郑州安图生物工程股份有限公司 | Protective agent for gastrin 17 blood sample |
-
2023
- 2023-06-09 CN CN202310680237.8A patent/CN116718762A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2096336A1 (en) * | 1991-09-30 | 1993-03-31 | Andreas Bergmann | Method of stabilizing endogenic, physiologically active peptides |
| WO1993007489A1 (en) * | 1991-09-30 | 1993-04-15 | Henning Berlin Gmbh Chemie- Und Pharmawerk | Method of stabilizing endogenic, physiologically active peptides |
| US20040067889A1 (en) * | 2002-06-19 | 2004-04-08 | Alexander Belenky | Stabilization of brain natriuretic peptide (BNP) in blood samples, methods and compositions related thereto |
| CN101022822A (en) * | 2004-08-24 | 2007-08-22 | 第一阿斯比奥制药株式会社 | Liquid preparation of physiologically active peptide |
| CN1739789A (en) * | 2005-05-11 | 2006-03-01 | 北京双鹭药业股份有限公司 | Somatostation a aqua prepn and its prepn process and application |
| CN108333360A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Gastrin-releasing peptide precursor dilution and its application and kit |
| CN108279307A (en) * | 2018-01-30 | 2018-07-13 | 郑州安图生物工程股份有限公司 | Protection and the heparin tube for stablizing gastrin-releasing peptide precursor in serum or blood plasma |
| CN112165951A (en) * | 2018-04-16 | 2021-01-01 | 默克专利股份有限公司 | Protein formulation additives for improved thermal stability |
| CN113727700A (en) * | 2019-04-26 | 2021-11-30 | 伊莱利利公司 | Method for preparing stable peptide preparation |
| CN110221006A (en) * | 2019-07-25 | 2019-09-10 | 深圳健科医学检验实验室 | It is a kind of for the pre-treating method of sulfydryl polypeptide biological sample and to the detection method of the biological sample |
| CN110596367A (en) * | 2019-09-06 | 2019-12-20 | 广州科方生物技术股份有限公司 | Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof |
| CN115702637A (en) * | 2021-08-13 | 2023-02-17 | 郑州安图生物工程股份有限公司 | Protective agent for gastrin 17 blood sample |
Non-Patent Citations (4)
| Title |
|---|
| MATILAINEN, LAURA: "The effect of cyclodextrins on chemical and physical stability of glucagon and characterization of glucagon/γ-CD inclusion complexes", J PHARM SCI, vol. 97, no. 7, 31 July 2008 (2008-07-31) * |
| 元英进: "《现代制药工艺学 上》", 31 July 2004, 化学工业出版社, pages: 310 - 313 * |
| 李范珠: "《药剂学 新世纪》", vol. 2, 31 July 2020, 中国中医药出版社, pages: 318 - 320 * |
| 梁运霞等: "β-环糊精在药剂学上的作用", 黑龙江畜牧兽医, no. 8, 31 December 2007 (2007-12-31), pages 112 - 113 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Müller et al. | Hyperglucagonemia in diabetic ketoacidosis: its prevalence and significance | |
| Walfish | Evaluation of three thyroid-function screening tests for detecting neonatal hypothyroidism | |
| Burch et al. | Improved method for measurement of delta-aminolevulinic acid dehydratase activity of human erythrocytes | |
| Mellinkoff et al. | Relationship between serum amino acid concentration and fluctuations in appetite | |
| Hale et al. | Metabolic effects of bicarbonate in the treatment of diabetic ketoacidosis. | |
| Du Vigneaud et al. | CARBOHYDRATE UTILIZATION. I. RATE OF DISAPPEARANCE OF ɗ-GLUCOSE FROM THE BLOOD. | |
| JUNGAS et al. | Studies on the metabolism of adipose tissue: the stimulation of oxygen consumption by TSH preparations in relation to growth hormone and other pituitary fractions | |
| Vallance-Owen et al. | Studies on insulin antagonism in plasma | |
| Tyson et al. | Endocrine-metabolic response to acute starvation in human gestation | |
| CN110596367A (en) | Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof | |
| Gjesdal et al. | Effects of fasting on plasma and platelet-free fatty acids and platelet function in healthy males | |
| JP3273154B2 (en) | Methods for stabilizing endogenous, physiologically active peptides | |
| CN116718762A (en) | Polypeptide sample stabilizer | |
| JP5090348B2 (en) | Monitoring of cardiac natriuretic peptide during diagnosis, treatment and treatment of heart disease | |
| Eggleston et al. | Optical rotation of the molybdate complex of dextro-isocitric acid | |
| JP4183197B2 (en) | Method for measuring glycated albumin | |
| BAYER | Effect of heparin on serum free thyroxine linked to post‐heparin lipolytic activity | |
| Ahmed et al. | Biochemical study on diabetic nephropathy patients | |
| VANNUCCHI et al. | Assessment of zinc nutritional status of pellagra patients | |
| JP4442340B2 (en) | Antibody, adsorbent containing the same, and immunoassay reagent | |
| Persijn | A rapid enzymatic assay for methotrexate in serum | |
| CN117030685A (en) | Cortisol calibration quality control product preservation solution and cortisol chemiluminescence detection kit | |
| Sonal et al. | A STUDY OF OXIDATIVE STRESS AND STATUS OF SERUM ZINC & COPPER IN SUBJECTS WITH HYPOTHYROIDISM | |
| Pal et al. | Status of lipid peroxidation and role of serum zinc and copper in subjects with hypothyroidism | |
| Dekker et al. | Indomethacin stimulates glucose production in adults with uncomplicated falciparum malaria |
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 |