CN116718776A - 一种检测ɑ2巨球蛋白的试剂盒 - Google Patents
一种检测ɑ2巨球蛋白的试剂盒 Download PDFInfo
- Publication number
- CN116718776A CN116718776A CN202310603873.0A CN202310603873A CN116718776A CN 116718776 A CN116718776 A CN 116718776A CN 202310603873 A CN202310603873 A CN 202310603873A CN 116718776 A CN116718776 A CN 116718776A
- Authority
- CN
- China
- Prior art keywords
- reagent
- kit
- macroglobulin
- alpha
- concentration
- 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
- 102100033312 Alpha-2-macroglobulin Human genes 0.000 title description 2
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 title description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 74
- 239000003593 chromogenic compound Substances 0.000 claims abstract description 14
- 108090000190 Thrombin Proteins 0.000 claims abstract description 13
- 229960004072 thrombin Drugs 0.000 claims abstract description 13
- 239000004019 antithrombin Substances 0.000 claims abstract description 8
- 239000003085 diluting agent Substances 0.000 claims abstract description 8
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229920000669 heparin Polymers 0.000 claims abstract description 5
- 229960002897 heparin Drugs 0.000 claims abstract description 5
- 239000003755 preservative agent Substances 0.000 claims description 12
- 230000002335 preservative effect Effects 0.000 claims description 12
- 238000002835 absorbance Methods 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000003381 stabilizer Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000011088 calibration curve Methods 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000004094 surface-active agent Substances 0.000 claims description 5
- 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 description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- YDMBNDUHUNWWRP-VJBWXMMDSA-N (2s)-1-[(2r)-2-amino-3-phenylpropanoyl]-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]piperidine-2-carboxamide Chemical compound C([C@@H](N)C(=O)N1[C@@H](CCCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C1=CC=CC=C1 YDMBNDUHUNWWRP-VJBWXMMDSA-N 0.000 claims description 2
- VYLJFJGMPYBPMN-VXKWHMMOSA-N (2s)-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]-1-[2-[(4-methylphenyl)sulfonylamino]acetyl]pyrrolidine-2-carboxamide Chemical compound C1=CC(C)=CC=C1S(=O)(=O)NCC(=O)N1[C@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=2C=CC(=CC=2)[N+]([O-])=O)CCC1 VYLJFJGMPYBPMN-VXKWHMMOSA-N 0.000 claims description 2
- 108010039286 S 2238 Proteins 0.000 claims description 2
- 108010018472 chromozym TH Proteins 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- QQVNCBCBFNWLJX-KCHLEUMXSA-N n-[(2s)-1-[[(2s)-1-[[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]benzamide Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)NC(=O)C=1C=CC=CC=1)C1=CC=CC=C1 QQVNCBCBFNWLJX-KCHLEUMXSA-N 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 31
- 238000000034 method Methods 0.000 abstract description 21
- 238000011002 quantification Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 15
- 239000000872 buffer Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000012496 blank sample Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- -1 polyoxyethylene Polymers 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010091934 Macroglobulins Proteins 0.000 description 2
- 102000018721 Macroglobulins Human genes 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010048998 Acute phase reaction Diseases 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 244000147568 Laurus nobilis Species 0.000 description 1
- 235000017858 Laurus nobilis Nutrition 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 206010033647 Pancreatitis acute Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- 235000005212 Terminalia tomentosa Nutrition 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 201000003229 acute pancreatitis Diseases 0.000 description 1
- 230000004658 acute-phase response Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000007982 barbital buffer Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000000988 hyperfibrinolytic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 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 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229960003766 thrombin (human) Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 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
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及一种检测ɑ2巨球蛋白的试剂盒,该试剂盒包括第一试剂、第二试剂、第三试剂、稀释液和ɑ2巨球蛋白校准品,其中,第一试剂包括凝血酶,第二试剂包括抗凝血酶和肝素,第三试剂包括发色底物,ɑ2巨球蛋白校准品包括已知浓度的ɑ2巨球蛋白。本发明试剂盒的优点在于操作简单,特异性强,定量准确,检测成本低,仪器兼容性强,适合在临床使用。
Description
技术领域
本发明涉及体外诊断试剂技术领域,尤其涉及一种检测ɑ2巨球蛋白的试剂盒。
背景技术
ɑ2巨球蛋白(α2-macroglobuLin,α2MG或AMG)由肝细胞合成,是血浆中分子量最大的蛋白,分子量约为65.2万-80万,正常成人血清中ɑ2巨球蛋白的浓度为:男,1.5-3.5mg/mL,女,1.5-4.7mg/mL。ɑ2巨球蛋白作为一个广谱的蛋白酶抑制剂,它的作用机制是利用空间上的位阻作用将酶分子隔离起来,使其不能和底物发生相互作用,ɑ2巨球蛋白与蛋白酶结合形成复合物后,构象发生改变,可被有关细胞受体识别,迅速的从血循环中被清除,因此它的主要功能是清除组织中内源性或者外源性的蛋白酶以及与其他的一些蛋白酶抑制剂共同调节蛋白水解活性。此外,它也是脑组织免疫炎症反应的一种急性期反应蛋白。ɑ2巨球蛋白还可以通过与免疫炎症因子,细胞因子,生长因子,激素等结合,从而影响这些小分子在体内的平衡,发挥广泛的生理功能。
ɑ2巨球蛋白在机体内发挥重要的作用,因此它的含量变化可以反映机体的生理状态或者病理状态。有文献表明,ɑ2巨球蛋白血浆水平升高在胚胎发育,妊娠和儿童期。不同使其纤维化的患者,其血清中ɑ2巨球蛋白升高程度不同,因此可以通过检测血清中ɑ2巨球蛋白浓度辅助判断肝纤维化分期。ɑ2巨球蛋白的含量对肾病综合征的鉴别诊断极为重要,ɑ2巨球蛋白/白蛋白比率的升高常常指示着肾后性血尿。ɑ2巨球蛋白还可以作为生物标记物用来区分糖尿病患者是否存在并发心肌梗塞。在高血纤维蛋白溶解状态下,白血病和严重肝功能不全在手术后ɑ2巨球蛋白的含量下降,急性胰腺炎患者出现较低的ɑ2巨球蛋白与病情的严重程度有关。在临床中检测ɑ2巨球蛋白含量对疾病的诊断有非常重要的意义。
目前,检测ɑ2巨球蛋白的方法主要为免疫法,如专利CN202111109651.0公开了一种ɑ2巨球蛋白快速检测试剂盒,该试剂盒中包括试剂a(包括磷酸盐缓冲液)、试剂b(包括抗ɑ2巨球蛋白抗体)、标准品溶液和质控溶液,检测时将试剂a与样本混合,孵育,读取吸光值记为I,加入试剂b,混合,孵育,读取吸光值记为II,根据I和II的差值以及标准品溶液中的蛋白浓度和差值计算得到待测样本中ɑ2巨球蛋白的浓度,并进一步通过聚乙二醇和海藻糖协同作用增加线性范围(15-500mg/L),通过加入氯化钾、透明质酸钠提高检测速度(由15min缩减至5min),检测限为15mg/L;专利CN201911231893.X公开了一种高灵敏度人尿液ɑ2-巨球蛋白检测试剂盒,该试剂盒中包括试剂R1(含有缓冲液、盐、促进剂、抗干扰剂和防腐剂,抗干扰剂可为脂肪酸聚氧乙烯酯、聚氧乙烯烷醇酰胺等)、试剂R2(含有缓冲液、抗人ɑ2MG血清、稳定剂和防腐剂)、校准品和质控品,该检测方法中,样本不需人工处理,直接上机测试,适用于全自动生化分析仪,检测限达到2mg/L;专利CN201710117035.7和CN201910679477.X也公开了一种ɑ2巨球蛋白检测试剂盒,也均采用免疫法,通过改进试剂组成以提高检测稳定性和灵敏度。但免疫法受抗体水平的控制,不同的抗体之间存在明显差异,对于检测的准确度较差,低值灵敏度较差。因此,仍需寻找一种操作简单、灵敏度高、准确度高且稳定性强,适宜快速或大规模检测的ɑ2巨球蛋白检测方法。
发明内容
为解决上述技术问题,本发明基于发色底物法构建了一种检测α2巨球蛋白的试剂盒,基于发色底物法解决了目前检测试剂盒普遍存在的检测准确度不高、检测时间长等问题。
本发明的第一个目的是提供一种检测ɑ2巨球蛋白的试剂盒,该试剂盒包括第一试剂、第二试剂、第三试剂、稀释液和ɑ2巨球蛋白校准品:
所述第一试剂包括凝血酶与乙二胺四乙酸(EDTA);
所述第二试剂包括抗凝血酶和肝素;
所述第三试剂包括发色底物;
所述ɑ2巨球蛋白校准品包括已知浓度的ɑ2巨球蛋白。
本发明中,ɑ2巨球蛋白将凝血酶包裹形成一个“笼式结构”,该结构的空间位阻阻碍大分子底物与凝血酶结合,而小分子发色底物则可以自由通过与凝血酶作用产生显色反应,然后通过发生显色反应后溶液的光密度值实现ɑ2巨球蛋白的定性和定量检测。
进一步地,第一试剂中,所述凝血酶选自人凝血酶、牛凝血酶或猪凝血酶,优选为牛凝血酶。
进一步地,第一试剂中还包括缓冲液、离子、稳定剂、表面活性剂和防腐剂中的一种或多种。
进一步地,第二试剂中,所述抗凝血酶可来自牛、猪、人或者重组酶。
进一步地,第二试剂中还包括缓冲液、离子、稳定剂、表面活性剂和防腐剂中的一种或多种。
进一步地,第三试剂中,所述发色底物为可用于测定抗凝血酶活力的发色底物,包括但不限于Bz-Phe-Val-Arg-pNA(S-2160)、H-D-Phe-Pip-Arg-pNA·2HCl(S-2238)、Tos-Gly-Pro-Arg-pNA(Chomozym TH)、H-D-Phe-Pro-Arg-pNA等。
进一步地,第三试剂中还包括赋形剂、缓冲液、稳定剂、表面活性剂和防腐剂中的一种或多种。
进一步地,第三试剂中,所述赋形剂包括但不限于甘露醇、葡萄糖、乳糖、BSA、蔗糖、海藻糖等。
进一步地,所述稀释液包括缓冲液、离子和防腐剂。
进一步地,所述ɑ2巨球蛋白校准品还包括缓冲液、稳定剂、防腐剂和抗氧化剂中的一种或多种,校准品可以制备成液体形式或冻干粉形式。校准品主要用于校准测量系统,评价测量程序或为待测样本赋值。
进一步地,上述各试剂中:
缓冲液可为5-50mM的Tris-HCl缓冲液、2-20mM咪唑缓冲液、10-100mM的HEPES缓冲液、5-25mM的巴比妥缓冲液或其组合,为反应提供适宜的条件;
稳定剂可为0.01~0.5g/L的明胶、2~15g/L的聚乙二醇、0.01~0.5g/L的壳聚糖、10~50g/L的甘氨酸、10~50g/L的木糖醇、20~100g/L的蔗糖、20~80g/L的海藻糖、0.1~5g/L的BSA等;
离子种类可为0.1%~2%的氯化钠、0.1%~0.5%氯化钾、0.05%~0.5%氯化锂、0.05%~0.5%氯化钙等;
表面活性剂可为0.5~15.0g/LTriton、1.0~20.0g/LTween、1.0~15.0g/L月桂醚、2.0~10.0g/L聚氧乙烯烷基苯基醚中的一种或多种,优选吐温-80,浓度为2.0~10.0g/L;
防腐剂可选择任一本领域常用的防腐剂,如叠氮化钠,含量为0.02wt%~0.2wt%时,既可以有效杀菌,又不会产生副作用,可延长试剂盒的有效期;还可以是浓度在0.01%~1.0%的庆大霉素、环丙沙星、ProClin系列防腐剂中的任何一种;
抗氧化剂可为1000U-10000U的SOD、0.1~3g/L的BHA、BHT、PG、茶多酚等;
赋形剂可分为5~30g/L甘露醇,5~30g/L葡聚糖、2~20g/L环糊精、0.1~5g/LBSA等。
优选地,所述第一试剂、第二试剂和第三试剂均为冷冻干燥试剂,即由包含以上所述组分的试剂冷冻干燥制成,检测时,将试剂盒中的冷冻干燥制剂用蒸馏水复溶重建。
进一步地,上述试剂盒中:
所述凝血酶的工作浓度为5-20IU/mL,优选工作浓度为10IU/mL;
所述抗凝血酶的工作浓度为5-15IU/mL,优选的工作浓度为10IU/mL;
所述肝素的工作浓度为0.25-3.0IU/mL,优选的工作浓度为1.5IU/mL;
所述发色底物的工作浓度为0.2-1.7umol/mL,优选工作浓度为0.42umol/mL。在发色底物法检测ɑ2巨球蛋白含量的过程中,作为凝血酶底物,要求具有较高的敏感性和较好的水溶性,才能有效提高检测效率,且具有较宽的线性范围,保证检测结果的准确性。如果底物溶解度过低,试剂接近饱和状态,检测过程中易产生沉淀而导致测定结果的不准确。
本发明中,除特殊说明外,工作浓度指:采用上述试剂盒检测样本ɑ2巨球蛋白的过程中,第一试剂的冷冻干燥剂、第二试剂的冷冻干燥剂和第三试剂的冷冻干燥剂用蒸馏水复溶后,与稀释样本混合,所形成的分析混合物中各组分的浓度被定义为其工作浓度。
进一步地,上述第一试剂、第二试剂和校准品中,缓冲液pH为7.5-8.5,浓度为5-10mmol/L。
进一步地,上述第三试剂中,缓冲液pH为7.5-8.5,浓度为4-6mmol/L。
本发明的第二个目的是提供一种采用上述试剂盒检测ɑ2巨球蛋白的方法,包括以下步骤:
S1、将第一试剂、第二试剂、第三试剂以及ɑ2巨球蛋白校准品进行复溶重建;
S2、用稀释液稀释血浆样本,孵育,与第一试剂混合,孵育,再加入第二试剂,孵育,最后加入第三试剂,孵育,按特定时间间隔测定吸光度并计算吸光度差值;
S3、根据ɑ2巨球蛋白校准品中的蛋白浓度以及吸光度差值制作校准曲线,对待测样本重复上述S2操作,将测定待测样本的吸光度差值代入校准曲线中即可计算出待测样本中ɑ2巨球蛋白的含量。
借由上述方案,本发明至少具有以下优点:
(1)本发明试剂盒测试方法简单准确,组成简单,原料易获得,易于制备。
(2)本发明试剂盒检测ɑ2巨球蛋白含量时,操作简单,适用于多种型号的半自动或全自动凝血分析仪,便于在各级医院,医学生物科研单位等推广使用,从而可促进该检测项目的常规开展。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为本发明试剂盒的校准曲线;
图2为本发明试剂盒线性范围相关性分析。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1检测试剂盒的组成和制备方法
本实施例的ɑ2巨球蛋白检测试剂盒,包含R1试剂、R2试剂、R3试剂,校准品和稀释液,以1mL/瓶分装冻干,具体组成如下:
R1试剂包含:
R2试剂包含:
R3试剂包含:
ɑ2巨球蛋白校准品包含:
ɑ2巨球蛋白稀释液包含:
实施例2检测试剂盒的测定方法
(1)将实施例1所得的ɑ2巨球蛋白R1试剂,R2试剂,R3试剂以及ɑ2巨球蛋白标准品进行复溶重建:每瓶R1试剂用3mL蒸馏水复溶,每瓶R2试剂用3mL蒸馏水复溶,每瓶R3试剂用6mL蒸馏水复溶,每瓶标准品用1mL蒸馏水复溶。
(2)以日本希森美康CA-1500血凝仪操作为例,根据仪器说明,设定分析程序:测定波长为405nm。
取5uL血浆样本,加入稀释液195uL稀释,稀释比例为1:40,在37℃孵育30秒,取50uL稀释后样本,加入25uL R1试剂在37℃孵育90秒,再加入45uL R2试剂37℃孵育90秒,再加入85uL R3试剂在37℃孵育,测定第15秒和第60秒的吸光度差值(ΔOD)。仪器自动将校准品稀释成400%,200%,100%,50%,0%的5个标准点,每管重复测定2次,以吸光度差值(ΔOD)的均值为纵坐标,对应的含量为横坐标,制作“含量-吸光度”校准曲线:Y=0.2057X+0.0248,相关系数r=0.9995,见图1。
取待测样本,同上方法测定样本的ΔOD,代入校准曲线,即可计算出待测样本中ɑ2巨球蛋白的含量。
本试剂盒不仅只适用于日本希森美康CA-1500血凝仪,而且还适用于其他品牌,型号的凝血分析仪,如法国斯塔高STA-R MAX血凝仪,美国贝克曼库尔特ACL TOP血凝仪等,具体参数可以根据仪器不同进行适当调整。
实施例3本发明试剂盒的分析性能评估
(1)线性范围
将接近线性范围上限的高值样本(ɑ2巨球蛋白含量为10mg/ml),用生理盐水分别按2/3,1/3,1/6,1/12的比例稀释,并以生理盐水为空白样本,加上稀释前原样本,共得到6份不同含量的样本,使用实施例1所得的本发明试剂盒,实施例2所述检测方法检测各样本,每个样本重复测定2次,将测定浓度的平均值与理论浓度进行线性回归分析,计算回归方程Y=1.032X-0.0644,相关系数r=0.9991,见图2,表明本发明试剂盒在0-10mg/ml线性范围内相关性较好。
(2)检出限
以5%人血清白蛋白为空白样本,使用实施例1所得本发明试剂盒,实施例2所述的检测方法,重复测定20次,计算空白样本含量均值(X)和标准差(SD),以空白均值加上两倍标准差,计算最低检测限(mg/ml),其结果如表1所示。
表1最低检测极限分析
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种检测ɑ2巨球蛋白的试剂盒,其特征在于,所述试剂盒包括第一试剂、第二试剂、第三试剂、稀释液和ɑ2巨球蛋白校准品:
所述第一试剂包括凝血酶和乙二胺四乙酸;
所述第二试剂包括抗凝血酶和肝素;
所述第三试剂包括发色底物;
所述ɑ2巨球蛋白校准品包括已知浓度的ɑ2巨球蛋白。
2.根据权利要求1所述的试剂盒,其特征在于:所述发色底物为可用于测定抗凝血酶活力的发色底物。
3.根据权利要求2所述的试剂盒,其特征在于:所述发色底物选自Bz-Phe-Val-Arg-pNA、H-D-Phe-Pip-Arg-pNA·2HCl、Tos-Gly-Pro-Arg-pNA、H-D-Phe-Pro-Arg-pNA中的一种或几种。
4.根据权利要求1所述的试剂盒,其特征在于:所述第一试剂、第二试剂或第三试剂中还包括缓冲液、稳定剂、表面活性剂和防腐剂中的一种或多种。
5.根据权利要求1所述的试剂盒,其特征在于:所述第三试剂中还包括赋形剂。
6.根据权利要求1所述的试剂盒,其特征在于:所述试剂盒中,凝血酶的浓度为5-20IU/mL,乙二胺四乙酸浓度为3-15g/L。
7.根据权利要求1所述的试剂盒,其特征在于:所述试剂盒中,抗凝血酶的浓度为5-15IU/mL。
8.根据权利要求1所述的试剂盒,其特征在于:所述试剂盒中,肝素的浓度为0.25-3.0IU/mL。
9.根据权利要求1所述的试剂盒,其特征在于:所述试剂盒中,发色底物的浓度为0.2-1.7umol/mL。
10.根据权利要求1所述的试剂盒,其特征在于,用于检测ɑ2巨球蛋白时包括以下步骤:
S1、将第一试剂、第二试剂、第三试剂以及ɑ2巨球蛋白校准品进行复溶重建;
S2、用稀释液稀释血浆样本,与第一试剂、第二试剂和第三试剂混合孵育,按特定时间间隔测定吸光度并计算吸光度差值;
S3、根据ɑ2巨球蛋白校准品中的蛋白浓度以及吸光度差值制作校准曲线。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310603873.0A CN116718776A (zh) | 2023-05-26 | 2023-05-26 | 一种检测ɑ2巨球蛋白的试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310603873.0A CN116718776A (zh) | 2023-05-26 | 2023-05-26 | 一种检测ɑ2巨球蛋白的试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116718776A true CN116718776A (zh) | 2023-09-08 |
Family
ID=87870691
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310603873.0A Pending CN116718776A (zh) | 2023-05-26 | 2023-05-26 | 一种检测ɑ2巨球蛋白的试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116718776A (zh) |
-
2023
- 2023-05-26 CN CN202310603873.0A patent/CN116718776A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5508202A (en) | Method of determining blood coagulation factor XIII activity and kit of reagents for the determination | |
CN109239061A (zh) | 一种液体型抗凝血酶ⅲ活性测定试剂盒 | |
CN106290907B (zh) | 全血中血清淀粉样蛋白a定量检测试剂以及检测方法 | |
AU730231B2 (en) | Improved thrombin-based assay for antithrombin-III | |
CN106996978A (zh) | α2巨球蛋白检测试剂盒及其制备方法 | |
CN114277089B (zh) | 一种达比加群的检测试剂及试剂盒 | |
CN107153043A (zh) | 一种液体即用型抗凝血酶ⅲ活性测定试剂 | |
WO2008018519A1 (en) | Method for determination of molecular weight of hyaluronic acid | |
CN112285359A (zh) | 一种涎液化糖链抗原测定试剂盒及其检测方法 | |
EP0576038B1 (en) | Method for measuring tissue plasminogen activator, antithrombin III and soluble fibrin | |
CN107782902A (zh) | 一种肌红蛋白单克隆抗体‑酶结合复合物及含有它的检测肌红蛋白含量的试剂盒 | |
CN116718776A (zh) | 一种检测ɑ2巨球蛋白的试剂盒 | |
CN105510572A (zh) | 一种甘氨酰脯氨酸二肽氨基肽酶检测试剂盒 | |
CN114112957B (zh) | 一种脂联素测定试剂盒及其应用 | |
Nilsson et al. | Determination of C1s-C1 inhibitor complexes in plasma by means of an enzyme linked immunosorbent assay. | |
CN114814245A (zh) | 一种基于发色底物法的蛋白c活性测定试剂盒 | |
CN111175515B (zh) | 一种血清淀粉样蛋白a和c反应蛋白的二合一质控物及其制备方法 | |
Jensen et al. | Influence of freeze-drying on the clotting properties of fibrinogen in plasma | |
CN112557665A (zh) | 一种狼疮抗凝物的确认试剂及其制备方法 | |
WO2010101206A1 (ja) | 管理試料の安定化剤、当該安定化剤を含有する管理試料、及び、当該管理試料を含む測定用キット | |
Heidtmann et al. | Assay of complexed alpha 1-antichymotrypsin in plasma | |
CN110133304B (zh) | 组合物、含有该组合物的试剂及其应用 | |
Schellenberg et al. | Carbohydrate-deficient transferrin (CDT) determination by nephelometry using a commercial kit. Analytical and diagnostic aspects | |
US6610291B2 (en) | Ready-to-use ristocetin cofactor test reagent possessing long-term stability | |
CN115219486B (zh) | 一种肝素和低分子肝素抗Xa活性的检测试剂盒及其非疾病诊断检测方法 |
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 |