CN110346316A - It is coupled the method and assay kit of pyruvate oxidation enzymatic determination sialic acid (SIA) - Google Patents
It is coupled the method and assay kit of pyruvate oxidation enzymatic determination sialic acid (SIA) Download PDFInfo
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 33
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 17
- 230000003647 oxidation Effects 0.000 title description 3
- 238000007254 oxidation reaction Methods 0.000 title description 3
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 title description 2
- 238000003149 assay kit Methods 0.000 title description 2
- 230000002255 enzymatic effect Effects 0.000 title description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 8
- 102000005348 Neuraminidase Human genes 0.000 claims abstract 3
- 108010006232 Neuraminidase Proteins 0.000 claims abstract 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 54
- 238000001514 detection method Methods 0.000 claims description 19
- 239000003795 chemical substances by application Substances 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 13
- 210000002966 serum Anatomy 0.000 claims description 8
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 5
- 150000002170 ethers Chemical class 0.000 claims description 5
- 150000001448 anilines Chemical class 0.000 claims description 4
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 claims description 4
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 claims description 4
- 239000011714 flavin adenine dinucleotide Substances 0.000 claims description 4
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 claims description 4
- -1 quinone Amine Chemical class 0.000 claims description 4
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 239000002736 nonionic surfactant Substances 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 150000005846 sugar alcohols Polymers 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 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
- HNXGGWNCFXZSAI-UHFFFAOYSA-N 2-morpholin-2-ylethanesulfonic acid Chemical compound OS(=O)(=O)CCC1CNCCO1 HNXGGWNCFXZSAI-UHFFFAOYSA-N 0.000 claims description 2
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 2
- 238000002835 absorbance Methods 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 239000003755 preservative agent Substances 0.000 claims description 2
- 230000002335 preservative effect Effects 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims 2
- YKYIFUROKBDHCY-ONEGZZNKSA-N (e)-4-ethoxy-1,1,1-trifluorobut-3-en-2-one Chemical group CCO\C=C\C(=O)C(F)(F)F YKYIFUROKBDHCY-ONEGZZNKSA-N 0.000 claims 1
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims 1
- QGZSANLMUAESBT-UHFFFAOYSA-N 3-piperazin-1-ylpropane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN1CCNCC1 QGZSANLMUAESBT-UHFFFAOYSA-N 0.000 claims 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims 1
- 244000309466 calf Species 0.000 claims 1
- 150000001719 carbohydrate derivatives Chemical class 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- AZQWKYJCGOJGHM-UHFFFAOYSA-N para-benzoquinone Natural products O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims 1
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 abstract description 3
- 108010042687 Pyruvate Oxidase Proteins 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract 2
- 238000006555 catalytic reaction Methods 0.000 abstract 2
- 108010084853 neuraminic acid aldolase Proteins 0.000 abstract 2
- 230000008878 coupling Effects 0.000 abstract 1
- 238000010168 coupling process Methods 0.000 abstract 1
- 238000005859 coupling reaction Methods 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 4
- 238000012946 outsourcing Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002075 main ingredient Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- MWFOPMKUGZLPQA-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)propane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CCCN(CC)C1=CC=CC(OC)=C1 MWFOPMKUGZLPQA-UHFFFAOYSA-M 0.000 description 2
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 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 1
- CLHYKAZPWIRRRD-UHFFFAOYSA-N 1-hydroxypropane-1-sulfonic acid Chemical compound CCC(O)S(O)(=O)=O CLHYKAZPWIRRRD-UHFFFAOYSA-N 0.000 description 1
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 1
- ZYVIIEFODRWQGD-UHFFFAOYSA-N 2-piperazin-1-ylethanol;propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O.OCCN1CCNCC1 ZYVIIEFODRWQGD-UHFFFAOYSA-N 0.000 description 1
- VTYZJYSZOUXZEO-UHFFFAOYSA-N 3,4,5-tribromo-2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC(Br)=C(Br)C(Br)=C1O VTYZJYSZOUXZEO-UHFFFAOYSA-N 0.000 description 1
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical group CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-M Aminoacetate Chemical group NCC([O-])=O DHMQDGOQFOQNFH-UHFFFAOYSA-M 0.000 description 1
- 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 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000001621 Mucoproteins Human genes 0.000 description 1
- 108010093825 Mucoproteins Proteins 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 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 1
- 239000002253 acid Substances 0.000 description 1
- 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 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- SVLRFMQGKVFRTB-UHFFFAOYSA-M sodium;3-(3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].COC1=CC(NCC(O)CS([O-])(=O)=O)=CC(OC)=C1 SVLRFMQGKVFRTB-UHFFFAOYSA-M 0.000 description 1
- HDARHUHTZKLJET-UHFFFAOYSA-M sodium;3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 HDARHUHTZKLJET-UHFFFAOYSA-M 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- 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
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention is with neuraminidase and neuraminic acid aldolase, coupling pyruvate oxidase, the method for measuring sialic acid.Principle is: decomposing sialic acid with neuraminidase, generate N-acetyl-neuraminate, N-acetyl-neuraminate generates pyruvic acid under the catalysis of neuraminic acid aldolase, and pyruvic acid generates hydrogen peroxide under the catalysis of pyruvate oxidase, then carries out Trinder reaction solution.The present invention can be made into Application of Sialic Acid Measurement kit simultaneously.
Description
Technical field
The present invention relates to the detection techniques of sialic acid and testing agent for sialic acid box, belong to medicine external diagnosis reagent neck
Domain.
Background technique
Sialic acid (N-acetyl-neuraminate) is the derivative of 9 carbon monosaccharide, is distributed mainly in the mammalian body, most initial
Now in salivary gland mucoprotein, especially vascular endothelial cell content is most abundant.Sialic acid in serum is Total silicic acid, and content exists
1.5~2.5mmol/L, including free sialic acid and bound sialic acid, the title mating type saliva in conjunction with glycoprotein and lipoprotein
Acid is the main ingredient of serum sialic acid, and identification, differentiation with cell, and adherency are related, participates in receptor composition, and induce
Cell Proliferation, tune are died.
Sialic acid is epicyte protein main ingredient, there is many biological functions, especially in most cases with it is pernicious
Tumour is related, and when gland cancer occurs, malignant change of cell, infiltration, transfer, the generation of tumour antigen can generate a large amount of sialic acid,
And be discharged into blood, it can be used as the Subsidiary Index and parameters for observation on effect of diagnosing tumor.It can be used for early detection tumour
Transfer and recurrence.
There are many method for detecting serum sialic acid, typically detection Total silicic acid, here just few narration.Most often at present
Method is enzyme uv detection method, and principle is as follows:
The fall off rate of absorbance is detected, at wavelength 340nm to calculate the content of sialic acid.This method specificity
Height, it is simple and efficient to handle, but there are three disadvantages: first is that sensitive not high, since sialic acid content is very low, this method is being detected
Poor repeatability when low value serum, the coefficient of variation are bigger；It is oxidizable second is that use Reducing Coenzyme I as color developing agent, reagent it is steady
It is qualitative poor, third is that the interference by pyruvic acid in sample is obvious.
In addition, also someone has used Trinder reaction, principle is as follows:
This method stable reagent, high sensitivity repeat when especially detecting low value also not by the interference of pyruvic acid in sample
Property it is good, do critically important when indoor, room interstitial is commented.But specific poor, some amino acid in sample of Ketoamine oxidase
It is reacted with ketone ammonia oxidase, and Ketoamine oxidase is somewhat expensive, increases the cost of reagent.
Summary of the invention
The present invention is reacted using the Trinder of high sensitivity, is detected the pyruvic acid generated in reaction process, is overcome
Above deficiency increases the stability of reagent, improves the sensitivity of detection low value, reproducible, the coefficient of variation is small.Especially
It is pyruvate oxidase to be placed in reagent one, initial oxidation falls original pyruvic acid in sample, eliminates interference.Reaction principle is such as
Under:
The reaction of reagent one:
To remove original pyruvic acid in sample.
Reaction after reagent two is added:
Agent prescription structure
Reagent one:
Reagent two:
By the composition of reagent, it can be made into reagent one: two=1: 1~4 ︰ 1 of reagent different concentration ratios, preferred reagent one: examination
Two=2: 1~4 ︰ 1 of agent.Sample: reagent=1: 20~1: 80, preferably 1 ︰ 50.
Buffer is glycinate, phosphate, trishydroxymethylaminomethane, 2-morpholine ethane sulfonic acid, 3- (N- morpholinyl) -2-
One or both of hydroxy-propanesulfonic acid, 4- hydroxyethyl piperazineethanesulfonic acid, 4- hydroxyethyl piperazine propane sulfonic acid etc..It is preferred that using phosphate
And trishydroxymethylaminomethane, dosage are 20~120mmol/L.Reagent one, reagent two pH of buffer be 6.0~8.0.
Preservative is one of sodium azide, ProClin-300, P-hydroxybenzoic acid and esters etc. or a variety of, still,
Be with sodium azide it is preferred, on reaction system without what influence, price is also low, and amount ranges are in 0.2~10mmol/L.
Stabilizer is Bovine serum albumin, amino acids, carbohydrate, and polyalcohols etc. can be applied alone, can also be two kinds or more
Kind or more combination, preferred Bovine serum albumin.Mannitol, trehalose, polyalcohol can be selected in flavin adenine dinucleotide (FAD), carbohydrate
Optional spent glycol, PEG6000 etc..
The surfactant be Tweens, polyethylene glycol ethers or polyethenoxy ether class nonionic surfactant, it is excellent
Select TritonX-100.
It can be suitably added surfactant in reaction system, reaction can be accelerated, increase the stability of enzyme, including Tweens,
The nonionic surfactants such as polyethenoxy ether class, particularly preferably polyethenoxy ether class.
Zymoexciter is Ca2+Or Mg2+.Dosage in 0.05~10mmol/L,
Color developing agent is 4-AA, phenol, phenyl amines.Phenol includes: phenol, 2,4- Dichlorophenol, 4- chlorine
Phenol, P-hydroxybenzoic acid, tribromo hydroxybenzoic acid, 3, the chloro- 2- sodium hydroxybenzenesulfonate of 5- bis- etc., Detection wavelength 500~
520nm；Phenyl amines includes: N- ethyl-N- (2- hydroxyl -3- sulfopropyl) -3,5- dimethoxyaniline sodium salt, N- (2- hydroxyl -3-
Sulfopropyl) -3,5- dimethoxyaniline sodium salt, N- ethyl-N- (the third sulfo group of 2- hydroxyl -3-) meta-aminotoluene, N- ethyl-N- (3-
Sulfopropyl) -3- aminoanisole sodium salt etc., very much, just seldom describe.Detection wavelength is sensitive between 540~600nm
Degree is higher than phenol, it is therefore preferable that the use of phenyl amines color developing agent being common in current biochemistry detection reagent.Use difference
Color developing agent just have corresponding wavelength.
Detailed description of the invention
Fig. 1 is detection kit testing result linear graph of the present invention；
Fig. 2 is that detection kit of the present invention and outsourcing Application of Sialic Acid Measurement kit detect Experimental Comparison figure.
Specific embodiment
Embodiment one
Reagent one:
Reagent two:
TOOS:N- ethyl-N- (the third sulfo group of 2- hydroxyl -3-) meta-aminotoluene
Reagent Yi ︰ reagent two is 2 ︰ 1, and performance rate method, positive reaction, wavelength: 546nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
6.0 ︰, 180 ︰ 90,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the suction of detection 2 minutes
Light varience rate.
Embodiment two
Reagent one
Reagent two:
Reagent Yi ︰ reagent two is 3 ︰ 1, and performance rate method, positive reaction, wavelength: 510nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
6.0 ︰, 210 ︰ 70,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the suction of detection 2 minutes
Light varience rate.
Embodiment three
Reagent one:
Reagent two:
ADPS:N- ethyl-N- (3- sulfopropyl) -3- aminoanisole sodium salt.
Reagent Yi ︰ reagent two is 4 ︰ 1, and performance rate method, positive reaction, wavelength: 546nm, detected sample Pin ︰ reagent Yi ︰ reagent two is
7 ︰, 240 ︰ 60,37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, and postpones 1 minute, the extinction of detection 2 minutes
Spend change rate.
By taking embodiment three as an example, detection data is as follows:
The linear result of detection kit is
| Theoretical value mmol/L | 0.000 | 0.625 | 1.250 | 1.875 | 2.500 | 3.125 | 3.750 | 4.375 | 5.000 |
| Measured value mmol/L | 0.013 | 0.632 | 1.256 | 1.881 | 2.515 | 3.129 | 3.682 | 4.268 | 4.706 |
Linear equation: y=0.955x+0.066, R2=0.998.See Figure of description 1.
It detects outsourcing quality-control product (1.68~2.10~2.52mmol/L), repeats detection 20 times, as a result as follows:
SD=0.0465, CV=2.247%.
50 parts of clinical blood are detected with the detection kit (Trinder method) and outsourcing Application of Sialic Acid Measurement kit (ultraviolet method)
Comparative test is done clearly, comparative test result is as follows:
The comparative test of 50 clinical samples
The resulting results relevance of two methods is good, r2=0.996.See Figure of description 2.
Detection of Stability: it detects outsourcing quality-control product (1.68~2.10~2.52mmol/L), monthly standard items, quality-control product weight
New to redissolve, reagent opens again detect once, is repeated detection 10 times every time, is averaged, and 13 totally months.As a result as follows:
Quality-control product definite value are as follows: 1.68~2.10~2.52mmol/L, the reagent validity period formulated according to national regulation, enterprise
Be 12 months, should be more than one month validity period in it is still stable, just meet the requirements.
The above is only a preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that for the art
Those of ordinary skill for, several improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of method for measuring sialic acid, steps are as follows:
1), the reaction of reagent one:
To remove original pyruvic acid in sample；
2) after reagent two, is added, the neuraminidase in reagent two starts to react, and it is sub- that oxidized color developing agent generates red quinone
Amine detects at a particular wavelength, to calculate the content of sialic acid；
3), detection end reaction object calculates the content of sialic acid in wavelength 500~600nm absorbance.
2. measuring the method for sialic acid according to claim 1, it is characterised in that: the control of the ratio of sample and reagent exists
1: 20 to 1: 80.
3. a kind of testing agent for sialic acid box, it is characterised in that: including reagent one and reagent two；
Reagent one:
Reagent two:
The ratio of the reagent one and reagent two is 1/1 to 8/1.
4. testing agent for sialic acid box according to claim 3, it is characterised in that: the buffer is phosphate, three hydroxyl first
Base aminomethane, 2-morpholine ethane sulfonic acid, 3- (N- morpholinyl) -2- hydroxy-propanesulfonic acid, 4- hydroxyethyl piperazineethanesulfonic acid, 4- ethoxy
One of piperazine propane sulfonic acid is a variety of.
5. testing agent for sialic acid box according to claim 3, it is characterised in that: the pH of buffer is 6.5~8.5.
6. testing agent for sialic acid box according to claim 3, it is characterised in that: the preservative be sodium azide,
One of ProClin-300, P-hydroxybenzoic acid and esters are a variety of.
7. testing agent for sialic acid box according to claim 3, it is characterised in that: the stabilizer is the white egg of calf serum
It is white, amino acid, one of carbohydrate derivative, polyalcohol, flavin adenine dinucleotide (FAD) or a variety of.
8. testing agent for sialic acid box according to claim 3, it is characterised in that: the surfactant is Tweens, gathers
Gylcol ether or polyethenoxy ether class nonionic surfactant.
9. testing agent for sialic acid box according to claim 3, it is characterised in that: the zymoexciter is Ca2+Or Mg2+。
10. testing agent for sialic acid box according to claim 3, it is characterised in that: the color developing agent be 4- amino peace for than
Woods, phenol, phenyl amines.
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Effective date of registration: 20210513 Address after: 312300 Baiguan Street Chengdong Industrial Zone, Shangyu District, Shaoxing City, Zhejiang Province Applicant after: Zhejiang manweini Biotechnology Co.,Ltd. Address before: 312300 Baiguan Street Chengdong Industrial Zone, Shangyu District, Shaoxing City, Zhejiang Province Applicant before: ZHEJIANG LITACK TRANSMISSION TECHNOLOGY Co.,Ltd. |
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Application publication date: 20191018 |