CN103290097B - Gamma-glutamoyl transferase detection reagent - Google Patents
Gamma-glutamoyl transferase detection reagent Download PDFInfo
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- CN103290097B CN103290097B CN201310200473.1A CN201310200473A CN103290097B CN 103290097 B CN103290097 B CN 103290097B CN 201310200473 A CN201310200473 A CN 201310200473A CN 103290097 B CN103290097 B CN 103290097B
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- damping fluid
- detection reagent
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- glutamyl transferase
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 51
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 102000004357 Transferases Human genes 0.000 title claims abstract description 15
- 108090000992 Transferases Proteins 0.000 title claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 239000003085 diluting agent Substances 0.000 claims abstract description 18
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 6
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 6
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 6
- 239000000872 buffer Substances 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- 239000012530 fluid Substances 0.000 claims description 38
- 238000013016 damping Methods 0.000 claims description 34
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 6
- SUMDYPCJJOFFON-UHFFFAOYSA-N isethionic acid Chemical compound OCCS(O)(=O)=O SUMDYPCJJOFFON-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 6
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 claims description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims description 5
- 239000011668 ascorbic acid Substances 0.000 claims description 5
- 229960005070 ascorbic acid Drugs 0.000 claims description 5
- 229930182470 glycoside Natural products 0.000 claims description 5
- 150000002338 glycosides Chemical class 0.000 claims description 5
- 230000002452 interceptive effect Effects 0.000 claims description 5
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical class COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 claims description 5
- 229920000136 polysorbate Polymers 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 claims description 4
- 239000011814 protection agent Substances 0.000 claims description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 4
- 229960005486 vaccine Drugs 0.000 claims 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 claims description 3
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 3
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims 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 claims 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 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 229960003280 cupric chloride Drugs 0.000 claims description 3
- YAGKRVSRTSUGEY-UHFFFAOYSA-N ferricyanide Chemical compound [Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-N 0.000 claims description 3
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 claims description 3
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 claims description 3
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 3
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 239000004302 potassium sorbate Substances 0.000 claims description 3
- 235000010241 potassium sorbate Nutrition 0.000 claims description 3
- 229940069338 potassium sorbate Drugs 0.000 claims description 3
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 3
- 239000004299 sodium benzoate Substances 0.000 claims description 3
- 235000010234 sodium benzoate Nutrition 0.000 claims description 3
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 3
- HALGLMAADGHVSV-UHFFFAOYSA-N 1-aminopropane-2-sulfonic acid Chemical compound NCC(C)S(O)(=O)=O HALGLMAADGHVSV-UHFFFAOYSA-N 0.000 claims 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 claims description 2
- UZRZNAWBVOGJAG-UHFFFAOYSA-N 2-hydroxy-1-(methylamino)propane-1-sulfonic acid Chemical compound CNC(C(C)O)S(O)(=O)=O UZRZNAWBVOGJAG-UHFFFAOYSA-N 0.000 claims description 2
- ISILBGFTARENPI-UHFFFAOYSA-N 3-amino-1,4-dihydroxypentane-3-sulfonic acid Chemical compound CC(O)C(N)(S(O)(=O)=O)CCO ISILBGFTARENPI-UHFFFAOYSA-N 0.000 claims description 2
- NHQAUSOKYWFWHO-UHFFFAOYSA-N 4-aminobutane-2-sulfonic acid Chemical compound OS(=O)(=O)C(C)CCN NHQAUSOKYWFWHO-UHFFFAOYSA-N 0.000 claims description 2
- QFOHBWFCKVYLES-UHFFFAOYSA-N Butylparaben Chemical compound CCCCOC(=O)C1=CC=C(O)C=C1 QFOHBWFCKVYLES-UHFFFAOYSA-N 0.000 claims description 2
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 claims description 2
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- XPJVKCRENWUEJH-UHFFFAOYSA-N Isobutylparaben Chemical compound CC(C)COC(=O)C1=CC=C(O)C=C1 XPJVKCRENWUEJH-UHFFFAOYSA-N 0.000 claims description 2
- CMHMMKSPYOOVGI-UHFFFAOYSA-N Isopropylparaben Chemical compound CC(C)OC(=O)C1=CC=C(O)C=C1 CMHMMKSPYOOVGI-UHFFFAOYSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims 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 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- QDPMLKBAQOZXEF-UHFFFAOYSA-N ethanesulfonic acid;sodium Chemical compound [Na].CCS(O)(=O)=O QDPMLKBAQOZXEF-UHFFFAOYSA-N 0.000 claims description 2
- 239000004403 ethyl p-hydroxybenzoate Substances 0.000 claims description 2
- 235000010228 ethyl p-hydroxybenzoate Nutrition 0.000 claims description 2
- 229940043351 ethyl-p-hydroxybenzoate Drugs 0.000 claims description 2
- NUVBSKCKDOMJSU-UHFFFAOYSA-N ethylparaben Chemical compound CCOC(=O)C1=CC=C(O)C=C1 NUVBSKCKDOMJSU-UHFFFAOYSA-N 0.000 claims description 2
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 claims description 2
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 claims description 2
- 229960003415 propylparaben Drugs 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 235000010288 sodium nitrite Nutrition 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000004108 freeze drying Methods 0.000 abstract description 2
- 235000019154 vitamin C Nutrition 0.000 abstract description 2
- 239000011718 vitamin C Substances 0.000 abstract description 2
- 239000003755 preservative agent Substances 0.000 abstract 2
- 230000002335 preservative effect Effects 0.000 abstract 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 abstract 1
- 229930003268 Vitamin C Natural products 0.000 abstract 1
- 239000000654 additive Substances 0.000 abstract 1
- 230000000996 additive effect Effects 0.000 abstract 1
- 230000001681 protective effect Effects 0.000 abstract 1
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- 239000004094 surface-active agent Substances 0.000 abstract 1
- 238000012123 point-of-care testing Methods 0.000 description 20
- 238000005516 engineering process Methods 0.000 description 13
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- 125000002642 gamma-glutamyl group Chemical group 0.000 description 7
- 238000000034 method Methods 0.000 description 6
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- 238000012360 testing method Methods 0.000 description 5
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- 239000008280 blood Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 4
- FGMRHNYMZYMARX-UHFFFAOYSA-N 3-amino-2-nitrobenzoic acid Chemical class NC1=CC=CC(C(O)=O)=C1[N+]([O-])=O FGMRHNYMZYMARX-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
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- IEQAICDLOKRSRL-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-dodecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO IEQAICDLOKRSRL-UHFFFAOYSA-N 0.000 description 1
- AHFFWTMLFDZSOF-QMMMGPOBSA-N 53602-84-9 Chemical compound OC(=O)[C@@H](N)CCC(=O)NC1=CC=C([N+]([O-])=O)C(C(O)=O)=C1 AHFFWTMLFDZSOF-QMMMGPOBSA-N 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a gamma-glutamoyl transferase detection reagent which comprises a diluent and a reaction reagent, wherein the diluent is a buffer liquid, a surfactant, a preservative, a debilirubin and a vitamin C oxidase, and the reaction reagent is a buffer liquid, a bi-glycoside peptide, L-gamma-glutamoyl-3-hydroxyl-4-nitroaniline, a primer protector, a preservative and a freeze-drying protective additive. The detection reagent disclosed by the invention has good sensitivity, accuracy, precision and linearity, and can completely satisfy the clinical examination requirement.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of gamma-glutamyl based transferase detection reagent for POCT analyser.
Background technology
Hepatopathy is the great disease of a kind of common hazardness, its timely Treatment and diagnosis is played an important role for the prevention of disease and diagnosis and treatment, gamma-glutamyl based transferase (γ-glutamyltransferase, γ-GGT) be distributed widely in tissue, in kidney at most, secondly be pancreas and liver, in normal human serum, γ-GGT is mainly from liver, it is in acute hepatitis, chronic active hepatitis and liver cirrhosis only slightly raise when losing compensatory, but when obstructive jaundice, because of acatharsia, adverse current enters blood, during primary hepatocarcinoma, synthesize hyperfunction in liver, all can significantly raise, even reach normal more than 10 times, alcoholic γ-GGT also obviously raises, contribute to diagnosing alcoholic liver disease.Current γ-GGT generally adopts various large automatic Biochemical Analyzer to detect, but because large automatic Biochemical Analyzer equipment price is high, and complicated operation, operator need have relevant expertise and accept corresponding training, use complementary conditions to require high, need be equipped with voltage stabilized source, water purification machine etc., and floor space is large, maintenance cost is high, needs professional's time-based maintenance, and therefore basic medical unit or household person all do not have condition to buy and use.In addition, large hospital patient is many, detects loaded down with trivial details, the long flow path of formality, waiting time long, and this also brings the huge time to patient and bears, and the more important thing is that patient is affected adversely treatment because not diagnosing in time, even can lose one's life.
Real-time test (point-of-caretesting, POCT) is an emerging technical field, and principal feature is rapid results, easy and simple to handle, easily uses and miniaturization.Along with diagnosis and the progress of ancillary technique, and people are to the requirement of the understanding of disease and treatment level raising, and POCT receives publicity gradually.In fact POCT becomes the important research project of external diagnosis reagent and instrument field gradually at European & American Market, existing many products put goods on the market, but this kind of POCT instrument has a major defect at present, be exactly that analyser and reagent consumptive material are expensive especially, for China, there is huge primary care system like this, and using amount of reagent country greatly, instrument and the matched reagent consumptive material of American-European exploitation at present obviously all face the challenge.
Microfluidic chip technology is one of of paramount importance cutting edge technology in the 21 century world, it is integrated into basic operation units such as sample preparation involved in the fields such as biological and chemical, reaction, separation, detection and cell cultures, sorting, cracking on the chip of a piece tens square centimeters (even less), network is formed by microchannel, running through whole system with controlled fluid, is a kind of technology of the various functions replacing standard biologic or chemical laboratory.Microfluidic chip technology is incorporated into POCT equipment, start the new situation of POCT development, can make laboratory was complicated in the past whole blood quantitatively, the step such as blood cell serum is separated, serum-dilution and Simultaneous Determination, complete in the on-line automatic equalization of chip, reach multiple mark synchronous detection object.POCT analyser in conjunction with microfluidic chip technology has split hair caccuracy concurrently, lowly needs blood volume, simple to operate, detection reagent consumption is few, low cost and other advantages, the POCT instrument of Sheng Yi company limited as international in Bao Sheng, the Piccolo of Abaxis company, the Afinion etc. of Axis-Shield company, this type of POCT analyser all can realize a small amount of sample can analyze, easy and simple to handle, without crossed contamination, can automated job, be highly suitable for China and there is huge primary care system like this and using amount of reagent is national greatly.
Along with Eleventh Five-Year Plan plan purchasing and reinforcement for three grades and second-grade hospital infrastructure device, current middle rank possesses good software and hardware facilities to go to the hospital, but particularly its full-automatic biochemical testing instruments facility of the unit such as commune hospital, clinic is generally not enough for basic medical unit, also do not have the inspection professional of enough numbers, therefore foundation operation POCT analytical system that is simple and easy, cheap, real-time report has important meaning to the effect of the vast basic hospital of performance in disease prevention and diagnosis and treatment.And provide a kind of determination of amylase reagent that can be used for the POCT analyser of above-mentioned introducing microfluidic chip technology to become problem demanding prompt solution.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, a kind of gamma-glutamyl based transferase detection reagent that can be used for the POCT analyser introducing microfluidic chip technology is provided.
The gamma-glutamyl based transferase detection reagent that the present invention can be used for above-mentioned POCT analyser (introducing microfluidic chip technology) realizes by following technical scheme.
Can be used for a gamma-glutamyl based transferase detection reagent for POCT analyser, comprise diluent and reaction reagent, wherein diluent consists of the following composition:
Damping fluid (pH6.5-7.5) 0.01-1.0mol/L,
Tensio-active agent 0.1-10.0%(mass percent),
Sanitas 0.1-10.0%(mass percent),
Remove bilirubin agent interfering 1-10mol/L or 1-100KU/L,
Ascorbic acid oxidase 1-100KU/L;
Wherein said reaction reagent consists of the following composition:
Damping fluid (pH7.0-8.0) 0.1-1.0mol/L,
Two glycosides peptide 0.1-1mol/L,
L-γ-glutamyl-3-hydroxyl-4-N-methyl-p-nitroaniline 10-100mmol/L,
Substrate Protection agent 1-100mmol/L,
Sanitas 0.1-1g/L,
Lyophilized vaccine 10-30g/L.
Wherein said damping fluid can be the amino damping fluid of trishydroxymethyl, glycine-NaOH buffer, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer, N-tri-(methylol) methylamino--2-hydroxy-propanesulfonic acid damping fluid, N-tri-(methylol) methyl-2-amino ethanesulfonic acid buffer, piperazine-N, two (2-hydroxyethanesulfonic acid) damping fluid of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonate buffer, two (2-hydroxyethyl) amino-2-hydroxy-propanesulfonic acid damping fluid of 3-, 3-(ring is amine)-2-hydroxyl-1-propanesulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid damping fluid, 3-(ring is amine)-1-propanesulfonic acid damping fluid, 3-morpholine propanesulfonic acid damping fluid, one or more of N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid.
Described tensio-active agent is nonionogenic tenside, as being selected from TWEEN series (as tween (TWEEN) 20, polysorbate40, polysorbate60, tween 80), SPAN series is (as class of department (SPAN) 20, class 40 of department, class 60 of department, class 80 of department), TRITON series is (as TritonX-100, TritonX-114, TritonX-405) in concrete material one or more (namely can be TWEEN series, SPAN series or TRITON serial in the concrete material of one, or more than one concrete material in often kind of series, or the concrete material of one in two kinds or more series).
The described bilirubin agent interfering that goes is one or more in yellow prussiate of potash, high-potassium ferricyanide, bilirubin oxidase.
Described sanitas is selected from the concrete material of one in potassium sorbate, Sodium Benzoate, Sodium Nitrite, proclin series sanitas (as Proclin300) or the concrete material of one in nipagin esters (as methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester).
Described Substrate Protection agent be selected from dimethyl sulfoxide (DMSO), ethylene glycol, glycerine, cupric chloride, copper sulfate, boric acid one or several.
Described lyophilized vaccine be selected from trehalose, sucrose, bovine serum albumin, tween 80, triton x-100, fatty alcohol-polyoxyethylene ether (Brij-35) one or several.
It is liquid state that described γ-GGT measures diluent in reagent, and reaction reagent is dry powder.
The preparation method that described γ-GGT measures the diluent of reagent is as follows: mix after described agent formulations is added distilled water according to formula rate and stir evenly.
The preparation method that described γ-GGT measures the reaction reagent of reagent is as follows: mix after described agent formulations is added distilled water according to formula rate and stir evenly, 0.5-10 μ l reaction reagent is joined reaction detection groove, volatilizes 24-72h through freeze-drying (industry routine techniques) or 2-8 DEG C.
The test condition that described γ-GGT measures reagent is as follows: temperature: 37 DEG C; Detect predominant wavelength 405nm, commplementary wave length 750nm.
Microfluidic chip technology of the present invention is integrated into by basic operation unit on the chip of a piece tens square centimeters (even less), forms network by microchannel, runs through a kind of technology of whole system with controlled fluid.It is two-layer up and down that its feature is that chip is generally divided into, there is the through hole for application of sample on upper strata, the difform fluid channel etc. that lower floor comprises sample cell, dilution liquid bath, sample amounts groove, diluent quantitative slot, reservoir, multiple reaction detection groove, one group of self-inspection groove for system compensation, one group of overflow groove, many groups being preinstalled with reaction reagent control fluid flowing.Its detection method generally comprises following steps: sample solution and diluent are injected in described sample cell and dilution liquid bath through respective through hole by (1); (2) chip described in starter motor rotation; (3) sample solution realizes solid-liquid separation with quantitative under centrifugal action, and diluent enters diluent quantitative slot simultaneously; (4) quantitative sample and diluent flow into tempering tank and mix; (5) mixed liquid enters reaction detection groove and reaction reagent reacts; (6) in reaction detection groove, in situ detection is carried out by the test set supporting with chip.
The measuring method that described γ-GGT measures reagent is as follows: 5-20 μ l sample is joined sample cell, 20-100 μ l diluent is joined dilution liquid bath, starter motor, records absorbance A 1, record absorbance A 2 after continuing reaction 5-9min after 37 DEG C of reaction 1min.
The reaction principle that described γ-GGT measures reagent is: sample first carries out mixing hatching with diluent; to remove the interfering substances such as bilirubin, vitamins C and blood fat in sample; after mixed solution enters reaction detection groove; with the L-gamma-glutamyl-3-carboxyl-p-Nitroaniline in reaction reagent for substrate; glycylglycine is the acceptor of gamma-glutamyl; under the catalysis of γ-GGT in the sample, glutamyl is transferred on glycylglycine molecule, generates amino-2 nitrobenzoic acids of yellow 5-simultaneously.And amino-2 nitrobenzoic acids of the 5-generated have maximum absorption band at 405nm wavelength place, and the activity of γ-GGT is proportional in the speed that generates of amino-2 nitrobenzoic acids of 5-and serum, therefore by under mensuration 405nm wavelength, the speed indicator that absorbancy increases calculates the vigor of γ-GGT.
Advantage of the present invention and beneficial effect: reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet Clinical Laboratory requirement completely.Reagent of the present invention can be used for the POCT analyser introducing microfluidic chip technology, thus realization operation is simple and easy, cheap, the foundation of the POCT analytical system of real-time report.
Accompanying drawing explanation
The analyte that Fig. 1 is diluted to different concns with the high density serum specimen come from hospital's collection detects, the linear result figure of γ-GGT.
The GGT end value that Fig. 2 and automatic clinical chemistry analyzer measure compares result figure, and wherein X-axis represents determination data in automatic clinical chemistry analyzer Hitachi 7180, and Y-axis represents determination data on POCT analyser.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.
Following experimental technique if no special instructions, is ordinary method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
Embodiment 1
Diluent:
Amino damping fluid (pH7.0) 0.1mol/L of trishydroxymethyl
TWEEN?80?5%
Proclin300?0.1%
Bilirubin oxidase 1KU/L
Ascorbic acid oxidase 1KU/L
Reaction reagent:
Amino damping fluid (pH7.5) 0.15mol/L of trishydroxymethyl
Two glycosides peptide 0.1mol/L
L-γ-glutamyl-3-hydroxyl-4-N-methyl-p-nitroaniline 10mmol/L
Cupric chloride 10mmol/L
Potassium sorbate 0.1g/L
Bovine serum albumin 5g/L
Embodiment 2
Diluent:
Amino damping fluid (pH7.5) 0.5mol/L of trishydroxymethyl
TRITON?X-100?5%
Methyl p-hydroxybenzoate 1%
Yellow prussiate of potash 1mol/L
Ascorbic acid oxidase 2KU/L
Reaction reagent:
3-morpholine propanesulfonic acid damping fluid (pH7.0) 0.2mol/L
Two glycosides peptide 0.2mol/L
L-γ-glutamyl-3-hydroxyl-4-N-methyl-p-nitroaniline 20mmol/L
Boric acid 50 mmol/L
Proclin300?1g/L
Trehalose 10g/L
Embodiment 3
Diluent:
3-morpholine propanesulfonic acid damping fluid (pH6.5) 1.0mol/L
SPAN?20?10%
Sodium Benzoate 1%
High-potassium ferricyanide 10mol/L
Ascorbic acid oxidase 10KU/L
Reaction reagent:
Two (2-hydroxyethanesulfonic acid) damping fluid (pH8.0) 1mol/L of piperazine-N, N-
Two glycosides peptide 1mol/L
L-γ-glutamyl-3-hydroxyl-4-N-methyl-p-nitroaniline 100mmol/L
DMSO?100?mmol/L
Proclin300?1g/L
Fatty alcohol-polyoxyethylene ether 10g/L
Be described below in conjunction with the performance of form to the embodiment of the present invention 1 gained reagent.
1, precision
Table 1, precision assessment result
2, linear
The analyte being diluted to different concns with the high density serum specimen come from hospital's collection detects, and γ-GGT linearly the results are shown in Fig. 1..
3, methodology Comparability test
Compare with the GGT end value that automatic clinical chemistry analyzer measures, result is as Fig. 2, wherein X-axis represents determination data in automatic clinical chemistry analyzer Hitachi 7180, POCT analyser (the Taiwan Bao Sheng world of microfluidic chip technology is introduced in Y-axis representative, Amishield, TMO-100) upper determination data.
4, detection sensitivity
The appraisal procedure of the detection sensitivity standard deviation of 10-20 dummy signal strength, and the standard deviation of 3-15 least significant non-zero sample signal strength, calculate gained with statistical software EP Evaluator release6.Experimental result shows reagent of the present invention good sensitivity, can meet the improvement of U.S. clinical laboratory completely and amend legislation.
Table 2 reagent detection sensitivity
From above-mentioned detected result, reagent of the present invention has good sensitivity, accuracy, precision and linear, can meet Clinical Laboratory requirement completely.
Claims (8)
1. a γ
-glutamyl transferase detection reagent, is characterized in that:
This reagent comprises diluent and reaction reagent, and wherein diluent consists of the following composition:
PH of buffer 6.5-7.5 0.01-1.0 mol/L,
Tensio-active agent 0.1-10.0% mass percent,
Sanitas 0.1-10.0% mass percent,
Remove bilirubin agent interfering 1-10 mol/L or 1-100 KU/L,
Ascorbic acid oxidase 1-100 KU/L;
Wherein said reaction reagent consists of the following composition:
PH of buffer 7.0-8.0 0.1-1.0 mol/L,
Two glycosides peptide 0.1-1 mol/L,
L-γ-glutamyl-3-hydroxyl-4-N-methyl-p-nitroaniline 10-100 mmol/L,
Substrate Protection agent 1-100 mmol/L,
Sanitas 0.1-1 g/L,
Lyophilized vaccine 10-30 g/L;
Described tensio-active agent be selected from TWEEN series, SPAN series, TRITON serial in one or more of concrete material.
2. γ according to claim 1
-glutamyl transferase detection reagent, is characterized in that: described damping fluid is the amino damping fluid of trishydroxymethyl, glycine-NaOH buffer, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid buffer, N-tri-(methylol) methylamino--2-hydroxy-propanesulfonic acid damping fluid, N-tri-(methylol) methyl-2-amino ethanesulfonic acid buffer, two (2-hydroxyethanesulfonic acid) damping fluid of piperazine-N, N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morpholine) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonate buffer, two (2-hydroxyethyl) amino-2-hydroxy-propanesulfonic acid damping fluid of 3-, 3-(ring is amine)-2-hydroxyl-1-propanesulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid damping fluid, 3-(ring is amine)-1-propanesulfonic acid damping fluid, 3-morpholine propanesulfonic acid damping fluid, one or more of N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid.
3. γ according to claim 1
-glutamyl transferase detection reagent, is characterized in that: the described bilirubin agent interfering that goes is one or more in yellow prussiate of potash, high-potassium ferricyanide, bilirubin oxidase.
4. γ according to claim 1
-glutamyl transferase detection reagent, is characterized in that: described sanitas is the one in a kind of or parabens in potassium sorbate, Sodium Benzoate, Sodium Nitrite, proclin series sanitas.
5. γ according to claim 4
-glutamyl transferase detection reagent, is characterized in that: described proclin series sanitas is Proclin300.
6. γ according to claim 4
-glutamyl transferase detection reagent, is characterized in that: described nipagin esters is the one in methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propylparaben, butyl p-hydroxybenzoate, p-Hydroxybenzoic acid isopropyl ester, p-Hydroxybenzoic acid isobutyl ester.
7. γ according to claim 1
-glutamyl transferase detection reagent, is characterized in that: described Substrate Protection agent is one or several in dimethyl sulfoxide (DMSO), ethylene glycol, glycerine, cupric chloride, copper sulfate, boric acid.
8. γ according to claim 1
-glutamyl transferase detection reagent, is characterized in that: described lyophilized vaccine is one or several in trehalose, sucrose, bovine serum albumin, tween 80, triton x-100, fatty alcohol-polyoxyethylene ether.
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Non-Patent Citations (2)
| Title |
|---|
| 1118例健康人尿GGT参考值调查;张宪等;《中国医科大学学报》;20040630;第33卷(第3期);269-270 * |
| 常规法测定不同GGT催化活性的不确定度评定;沈蕾等;《临床检验杂志》;20111231;第29卷(第9期);650-652 * |
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