CN106501201B - A kind of enzyme round-robin method for being used to detect homocysteine - Google Patents
A kind of enzyme round-robin method for being used to detect homocysteine Download PDFInfo
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- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 title claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 36
- 238000007616 round robin method Methods 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 89
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 66
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical group N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 claims abstract description 45
- 230000004044 response Effects 0.000 claims abstract description 38
- 239000004471 Glycine Substances 0.000 claims abstract description 36
- 239000000758 substrate Substances 0.000 claims abstract description 21
- FFFHZYDWPBMWHY-UHFFFAOYSA-N L-Homocysteine Natural products OC(=O)C(N)CCS FFFHZYDWPBMWHY-UHFFFAOYSA-N 0.000 claims abstract description 18
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) 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-O 0.000 claims abstract description 18
- 239000000376 reactant Substances 0.000 claims abstract description 16
- 238000002835 absorbance Methods 0.000 claims abstract description 15
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 14
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 14
- 235000007635 levomefolic acid Nutrition 0.000 claims abstract description 10
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims abstract description 4
- 238000005259 measurement Methods 0.000 claims abstract description 4
- 230000000630 rising effect Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 50
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims description 19
- 108010075604 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase Proteins 0.000 claims description 15
- 102000011848 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase Human genes 0.000 claims description 15
- 108010058065 Aminomethyltransferase Proteins 0.000 claims description 15
- 102100039338 Aminomethyltransferase, mitochondrial Human genes 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims description 15
- 239000007983 Tris buffer Substances 0.000 claims description 14
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 108010043428 Glycine hydroxymethyltransferase Proteins 0.000 claims description 9
- 102000002667 Glycine hydroxymethyltransferase Human genes 0.000 claims description 9
- JMNIIIQOMSQWJN-ACGFUFEJSA-L calcium;(4s)-4-[[4-[(2-amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound [Ca+2].C1NC=2N=C(N)NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1.C1NC=2N=C(N)NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 JMNIIIQOMSQWJN-ACGFUFEJSA-L 0.000 claims description 9
- ZNOVTXRBGFNYRX-ABLWVSNPSA-N levomefolic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-ABLWVSNPSA-N 0.000 claims description 9
- 239000011578 levomefolic acid Substances 0.000 claims description 9
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 9
- 239000008213 purified water Substances 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- 238000005374 membrane filtration Methods 0.000 claims description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 4
- -1 glycine methylol Chemical class 0.000 claims description 4
- BOPGDPNILDQYTO-NNYOXOHSSA-L NADH(2-) Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-L 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 229910017435 S2 In Inorganic materials 0.000 claims description 2
- 230000008033 biological extinction Effects 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000012956 testing procedure Methods 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 3
- 229910052791 calcium Inorganic materials 0.000 claims 3
- 239000011575 calcium Substances 0.000 claims 3
- QWWVBNODQCWBAZ-WHFBIAKZSA-N (2r)-2-amino-3-[(2r)-2-carboxy-2-(methylamino)ethyl]sulfanylpropanoic acid Chemical compound CN[C@H](C(O)=O)CSC[C@H](N)C(O)=O QWWVBNODQCWBAZ-WHFBIAKZSA-N 0.000 claims 2
- 238000012546 transfer Methods 0.000 claims 2
- FFEARJCKVFRZRR-SCSAIBSYSA-N D-methionine Chemical compound CSCC[C@@H](N)C(O)=O FFEARJCKVFRZRR-SCSAIBSYSA-N 0.000 claims 1
- 150000007513 acids Chemical class 0.000 claims 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 4
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- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- TZBGSHAFWLGWBO-ABLWVSNPSA-N (2s)-2-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-1h-pteridin-6-yl)methylamino]benzoyl]amino]-5-methoxy-5-oxopentanoic acid Chemical compound C1=CC(C(=O)N[C@@H](CCC(=O)OC)C(O)=O)=CC=C1NCC1NC(C(=O)NC(N)=N2)=C2NC1 TZBGSHAFWLGWBO-ABLWVSNPSA-N 0.000 abstract 4
- 150000008546 L-methionines Chemical class 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 description 8
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 6
- 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 6
- 229950006238 nadide Drugs 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 102000004357 Transferases Human genes 0.000 description 4
- 108090000992 Transferases Proteins 0.000 description 4
- 230000003139 buffering effect Effects 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 108010072462 Hydroxymethyl and Formyl Transferases Proteins 0.000 description 2
- 102000006933 Hydroxymethyl and Formyl Transferases Human genes 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- 230000009137 competitive binding Effects 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 1
- 206010003178 Arterial thrombosis Diseases 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 208000033892 Hyperhomocysteinemia Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 108030006431 Methionine synthases Proteins 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- ZJUKTBDSGOFHSH-WFMPWKQPSA-N S-Adenosylhomocysteine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSCC[C@H](N)C(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZJUKTBDSGOFHSH-WFMPWKQPSA-N 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 235000021004 dietary regimen Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000008191 folinic acid Nutrition 0.000 description 1
- 239000011672 folinic acid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003225 hyperhomocysteinemia Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229960001691 leucovorin Drugs 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000007119 pathological manifestation Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
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- 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
-
- 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
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- 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
- G01N1/34—Purifying; Cleaning
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- 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
- 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
- G01N2001/386—Other diluting or mixing processes
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
It is a kind of to include the circular response of two reactants for detecting the enzyme round-robin method of homocysteine, it is tetrahydrofolic acid circular response and 5 respectively, 10 methyl tetrahydrofolate circular responses；After the tetrahydrofolic acid circular response reacts generation tetrahydrofolic acid and L methionines with L homocysteine for 5 methyl tetrahydrofolates, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction 5,10 methyl tetrahydrofolates of generation and NADH；Described 5,10 methyl tetrahydrofolate circular responses are that 5,10 methyl tetrahydrofolates that will be generated in tetrahydrofolic acid circular response are reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；By detecting the absorbance rising degree of NADH at 340nm wavelength, the concentration measurement result of L homocysteine is calculated.This method is that circular response is generated based on tetrahydrofolic acid, generates NADH amplification detection signals by accumulation, is measured in wavelength 340nm, NADH is directly proportional to the homotype semicystinol concentration investigating for participating in reaction.
Description
Technical field
The present invention relates to the detection method technical fields of biological micromolecule homocysteine, are used for more particularly, to one kind
Detect the enzyme round-robin method of homocysteine.
Background technology
Homocysteine (Homocysteine, Hcy) or for homocysteine, is the different of amino acid cysteine
Kind, sulfydryl (- SH) is contained in side chain.Hcy is mainly derived from the methionine of diet regimen, is methionine and cysteine metabolism
An important intermediate product in the process, itself does not participate in the synthesis of protein.Mccully in 1969 is from heredity homotype
It is found in cysteine urine disease death children's postmortem, there are extensive Arterial thrombosis and atherosclerosis in body circulation
(AS) thus pathological manifestations propose that hyperhomocysteinemiainjury (hyperhomocysteinemia, HHCY) can cause to move
The hypothesis of pulse atherosclerosis vascular conditions.Hcy can directly or indirectly lead to vascular endothelial cell damage, and blood vessel is promoted to put down
Sliding muscle cell multiplication, influences the oxidation of low-density lipoprotein, enhances platelet function, promotes thrombosis.
The assay method of homocysteine is in addition to traditional high performance liquid chromatography, enzyme immunoassay (EIA), fluorescence polarization
Method etc. is outer, also occurs the Enzymatic cycling based on methionine and adenosyl homocysteine in recent years (referring to Chinese special
Sharp CN200480026009.4), but since the production technology of critical materials is difficult to grasp and cost is very high, it is difficult to forming market should
With.
The fundamental analysis principle of traditional enzyme immunoassay (EIA) is that the antigen that enzymic-labelled antibody or enzymic-labelled antibody carry out resists
Then precursor reactant generates color reaction by enzyme-to-substrate, for quantitative determining, step is：Enzyme is first used by institute in blood sample
The Hcy for having form is transformed into s- adenosine-L-Hcy, then adds in the monoclonal or Anti-TNF-α of the anti-s- adenosines-L-Hcy of enzyme target
Body, using the principle of competitive binding, by the standard items of different level and enzyme labelled antibody competitive binding, then with colour reagent and end
Only reagent is developed the color and is terminated respectively, produces the standard curve of hcy concentration and coloring intensity.Sample is also by same steps
Reason can find the concentration of its Hcy on standard curve.
Invention content
In order to overcome the shortcomings of in background technology, the invention discloses be different from traditional assay method and existing Enzymatic cycling
Body fluid in homocysteine assay method, this method is that circular response is generated based on tetrahydrofolic acid, passes through accumulation
Reduced Coenzyme I (NADH) amplification detection signal is generated, is measured in wavelength 340nm, NADH and the homotype half for participating in reacting
Cystine concentration is directly proportional.Key enzyme involved in the reaction includes：Methionine synthetase (EC 2.1.1.13), amino methyl
Transferase (EC 2.1.2.10), glycine hydroxymethyl transferase (EC 2.1.2.1) are examined for the cycle of homocysteine
It surveys.The present invention also discloses the reagents of the homocysteine detection based on above-mentioned detection method, which can be extensive
Detection applied to homocysteine in clinic.
In order to realize the goal of the invention, the present invention adopts the following technical scheme that：One kind is used to detect homocysteine
Enzyme round-robin method, include the circular response of two reactants, be tetrahydrofolic acid circular response and 5,10- methyl tetrahydrochysene leaf respectively
Sour circular response；
The tetrahydrofolic acid circular response for 5-methyltetrahydrofolate reacted with L- homocysteine generate tetrahydrofolic acid and
After l-methionine, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl tetrahydrofolates and
NADH；
5, the 10- methyl tetrahydrofolates circular response is 5, the 10- methyl four that will be generated in tetrahydrofolic acid circular response
Hydrogen folic acid is reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；
By detecting the absorbance rising degree of NADH at 340nm wavelength, the concentration of L- homocysteine is calculated
Measurement result.
In order to be further improved technical solution, in tetrahydrofolic acid circular response of the present invention, 5-methyltetrahydrofolate with
In the reaction step of L- homocysteine reaction generation tetrahydrofolic acid and l-methionine, the enzyme used is synthesized for methionine
Enzyme, the reaction equation of the reaction are：5-methyltetrahydrofolate+L- homocysteine → tetrahydrofolic acid+l-methionine.
In order to be further improved technical solution, in tetrahydrofolic acid circular response of the present invention, using tetrahydrofolic acid as reaction
Substrate and glycine and NAD+In the reaction step of reaction generation 5,10- methyl tetrahydrofolates and NADH, the enzyme used is amino
Transmethylase；The reaction equation of the reaction is：Glycine+tetrahydrofolic acid+NAD+→ 5,10- methyl tetrahydrofolate+NH3+CO2+
NADH+H+。
In order to be further improved technical solution, in 5,10- methyl tetrahydrofolates circular response of the present invention, 5,10- first
Base tetrahydrofolic acid is generated with glycine reactant in the reaction step of tetrahydrofolic acid and serine, and the enzyme used is glycine methylol
Transferase；The reaction equation of the reaction is：5,10- methyl tetrahydrofolates+glycine+H2O → tetrahydrofolic acid+serine.
A kind of enzyme round-robin method for being used to detect homocysteine, it is characterized in that：Its testing procedure is：
S1:Reagent preparation box：The kit reagent includes following each component：Buffer solution：Tris, reaction substrate 5- methyl
Calcium leucovorin, reaction substrate glycine, reaction substrate NAD+, enzyme activition ION Mg Cl2, methionine synthetase, amino methyl
Transferase, glycine hydroxymethyltransferase；
S2：The buffer solution Tris in step S1 is taken, is dissolved in purified water, and pH value is adjusted with concentrated hydrochloric acid；
S3：5-methyltetrahydrofolate, glycine, the NAD in step S1 are weighed successively+、MgCl2, and add in the molten of step S2
In liquid, stirring is to being completely dissolved；
S4：Take prepared reagent methionine synthetase, Aminomethyltransferase and glycine methylol in step S1
After transferase and respectively independent dissolving, add in the solution described in step S3, stir and evenly mix；
S5：The pH value of the mixing solution of determination step S4, and pH value is adjusted, purified water constant volume is added in, then filters postposition
It is spare in 2-8 DEG C；
S6：Solution after being filtered in step S5 is placed in after water-bath balance and obtains reaction solution, and compound concentration is 0.05mM's
L-HCY solution is as detectable substance；
S7：By reaction solution：Detectable substance in proportion, sets response parameter on Biochemical Analyzer, and records each read point
Absorbance, for calculating absorbance change rate.
In order to be further improved technical solution, each component is a concentration of in the kit of step S1 of the present invention：Buffering
Liquid Tris：20mM-50mM；Reaction substrate 5-methyltetrahydrofolate calcium：5mM-10mM；Reaction substrate glycine：10mM-20mM；
NAD+1mM-5mM；Enzyme activition ION Mg Cl2：2mM-10mM；Methionine synthetase：5-15kU/L；Aminomethyltransferase：1-
4kU/L；Glycine hydroxymethyltransferase：2-6kU/L.
In order to be further improved technical solution, the pH value after being adjusted in step S2 and step S5 of the present invention is to 8.00
±0.05；Purified water is added in the step S5 and is settled to 500ml, solution after constant volume is passed through into 0.22 μm of membrane filtration.
In order to be further improved technical solution, bath temperature is 37 DEG C in step S6 of the present invention, equilibration time 15-
20min。
In order to be further improved technical solution, reaction solution in step S7 of the present invention：The ratio of detectable substance is 50:1.
In order to be further improved technical solution, reagent Tris of the present invention, 5-methyltetrahydrofolate calcium, glycine, NAD+And MgCl2, it is the reagent that Sigma companies produce.
By adopting the above-described technical solution, the present invention has the advantages that：Detection half Guang of homotype of the present invention
The enzyme round-robin method of propylhomoserin includes the circular response of two reactants, mainly passes through tetrahydrofolic acid and 5,10- methyl tetrahydrochysene leaves
Two-way Cycle reaction between acid, makes NADH (reduced Coenzyme I) constantly accumulate under the startup of L- homocysteine, that is, exists
Absorbance is continuously increased at 340nm wavelength, and detection signal is made constantly to enhance, and letter obtains to participate in the L- homocysteine of reaction
Concentration.Each reaction is as follows：
1) 5-methyltetrahydrofolate+L- homocysteine → tetrahydrofolic acid+l-methionine in the reaction process, uses
Methionine synthetase is as reaction enzymes；
2) glycine+tetrahydrofolic acid+NAD+→ 5,10- methyl tetrahydrofolate+NH3+CO2+NADH+H+, the reaction process
In, using Aminomethyltransferase as reaction enzymes；
3) 5,10- methyl tetrahydrofolates+glycine+H2O → tetrahydrofolic acid+serine in the reaction process, uses sweet ammonia
Sour hydroxymethyl transferases are as reaction enzymes.
Two-way Cycle of the present invention, first circular response：Above-mentioned reaction 1) and reaction 2) in tetrahydrofolic acid follow
Ring reacts, and tetrahydrofolic acid is product in reaction 1), is substrate in reaction 2)；Second circular response：It is above-mentioned reaction 2)
With 5, the 10- methyl tetrahydrofolates cycle in reaction 3), 5,10- methyl tetrahydrofolates are product in reaction 2, in reaction 3
For substrate；When reacting startup, the concentration of tetrahydrofolic acid and 5,10- methyl tetrahydrofolate can maintain certain level, constantly disappear
Consumption and generation, and NADH (final indicant) concentration reacted in 2 then will continue to accumulate, and increase the suction at wavelength 340nm
Luminosity, and it is related to the L- homocysteine concentration in reaction 1, increase the sensitivity of measure L- homocysteine.
When reaction is acyclic reaction, sensitivity is mainly related to initial reactant concentration, can only be in certain model
Enclose interior increase；When reaction is single cycle reaction, sensitivity is mainly related to initial reactant and circular response object concentration,
Relatively acyclic reaction can greatly improve sensitivity；When reaction is Two-way Cycle reaction, reaction signal is obviously improved, is enhanced
Reaction sensitivity, sensitivity is mainly related to initial reactant and circular response object concentration, can with respect to single cycle reaction
Greatly improve sensitivity.
Description of the drawings
Fig. 1 is response curve figure of the embodiment one to embodiment three.
Fig. 2 be embodiment one to embodiment three 22-23 range read points response curve figure.
Specific embodiment
Explanation that can be detailed by the following examples is of the invention, and the open purpose of the present invention is intended to protect model of the present invention
Enclose all interior technological improvements.
Embodiment one
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 20mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 5mM；The glycine of a concentration of 10mM；The NAD of a concentration of 1mM+；A concentration of 2mM's
MgCl2；The methionine synthetase of a concentration of 5kU/L；The Aminomethyltransferase of a concentration of 1kU/L；A concentration of 2kU/L's is sweet
Propylhomoserin hydroxymethyl transferases.
S2：The Tris buffer solutions in 1.211g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 1.244g in step S1, glycine 0.375g, NAD+0.331g and
MgCl20.203g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：Weigh methionine synthetase 2.5kU, Aminomethyltransferase 0.5kU, the glycine methylol in step S1
Transferase 1kU, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2~8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
Embodiment two
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 35mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 7.5mM；The glycine of a concentration of 15mM；The NAD of a concentration of 3mM+；It is a concentration of
The MgCl of 3.5mM2；The methionine synthetase of a concentration of 7.5kU/L；The Aminomethyltransferase of a concentration of 3kU/L；It is a concentration of
The glycine hydroxymethyltransferase of 4kU/L.
S2：The Tris buffer solutions in 2.120g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 1.866g in step S1, glycine 0.563g, NAD+0.994g and
MgCl20.356g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：Weigh methionine synthetase 3.75kU in step S1, Aminomethyltransferase 1.5kU, glycine hydroxyl first
Based transferase 2kU, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2-8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
Embodiment three
S1：Reagent preparation box, the kit reagent include the substance of following each component：The Tris bufferings of a concentration of 50mM
Liquid；The 5-methyltetrahydrofolate calcium of a concentration of 10mM；The glycine of a concentration of 20mM；The NAD of a concentration of 5mM+；A concentration of 5mM
MgCl2；The methionine synthetase of a concentration of 10kU/L；The Aminomethyltransferase of a concentration of 5kU/L；A concentration of 6kU/L
Glycine hydroxymethyltransferase.
S2：The Tris buffer solutions in 3.029g steps S1 are weighed, are dissolved in 300ml purified waters, and adjusted with concentrated hydrochloric acid
PH to 8.00 ± 0.05；
S3：Weigh the 5-methyltetrahydrofolate calcium 2.488g in step S1, glycine 0.751g, NAD+1.656g and
MgCl20.508g is added in the buffer solution of step S2, and stirring is to being completely dissolved；
S4：The methionine synthetase 5kU in step S1, Aminomethyltransferase 2.5kU, glycine methylol is weighed to turn
Enzyme 3kU is moved, and individually after dissolving, add in the solution of step S3, stir to abundant mixing；
S5：Above-mentioned solution ph is measured, and adjusts pH to 8.00 ± 0.05, purified water constant volume is added in using 500ml graduated cylinders
To 500ml.By solution after constant volume by 0.22 μm of membrane filtration, be placed in 2-8 DEG C it is spare.
S6:Solution after being filtered in step S5 is placed in 37 DEG C of water-baths after balancing 15min and obtains reaction solution, and prepare
The L- homocysteine solution of a concentration of 0.05mM is as detectable substance；
S7：By reaction solution：Detectable substance presses 50：1 ratio sets response parameter on Biochemical Analyzer, and records each
The absorbance of read point, for calculating absorbance change rate.
The measurement result of embodiment one to three is as shown in the table：
It is shown by reaction result, three embodiments show preferable linear relationship, are shown in embodiment one
In the concentration range of each reactant, which can implement, and when reactant concentration increases, reactions change rate (Δ A/
Min) can also increase, but when concentration increases to the formula upper limit, Δ A/min is excessively high, can make extinction when measuring high concentration sample
Degree is more than the readable upper limit of instrument.Embodiment three the results show that the reaction later stage change greatly, may when testing high concentration sample
It is that absorbance reaches biochemical instruments detection threshold value, each reactant concentration is more than that the value that embodiment 3 provides may cause absorbance to exceed
Instrument detection threshold value.So as to which test result is not allowed.Therefore in 1 given concentration range of table, linear and reaction sensitivity is equal
It can meet the requirements.
Part not in the detailed description of the invention is the prior art.
Claims (5)
1. it is a kind of for detecting the enzyme round-robin method of homocysteine, it is characterized in that：Include the circular response of two reactants,
It is tetrahydrofolic acid circular response and 5,10- methyl tetrahydrofolate circular response respectively；
The tetrahydrofolic acid circular response reacts generation tetrahydrofolic acid and L- first with L- homocysteine for 5-methyltetrahydrofolate
After methyllanthionine, then using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl tetrahydrofolates and NADH；
In the tetrahydrofolic acid circular response, 5-methyltetrahydrofolate reacts generation tetrahydrofolic acid and L- first with L- homocysteine
In the reaction step of methyllanthionine, the enzyme used is methionine synthetase, and the reaction equation of the reaction is：5-methyltetrahydrofolate
+ L- homocysteine=tetrahydrofolic acid+l-methionine；
In the tetrahydrofolic acid circular response, using tetrahydrofolic acid as reaction substrate and glycine and NAD+Reaction generation 5,10- methyl
In the reaction step of tetrahydrofolic acid and NADH, the enzyme used is Aminomethyltransferase；The reaction equation of the reaction is：Glycine
+ tetrahydrofolic acid+NAD+=5,10- methyl tetrahydrofolate+NH3+CO2+NADH+H+；
5, the 10- methyl tetrahydrofolates circular response is 5, the 10- methyl tetrahydrochysene leaves that will be generated in tetrahydrofolic acid circular response
Acid is reaction substrate, and tetrahydrofolic acid and serine are generated with glycine reactant；
In 5, the 10- methyl tetrahydrofolates circular response, 5,10- methyl tetrahydrofolates generate tetrahydrofolic acid with glycine reactant
In the reaction step of serine, the enzyme used is glycine hydroxymethyltransferase；The reaction equation of the reaction is：5,10- first
Base tetrahydrofolic acid+glycine+H2O=tetrahydrofolic acids+serine；
By detecting the absorbance rising degree of NADH at 340nm wavelength, calculate L- homocysteine concentration and then
Obtain measurement result；
The enzyme round-robin method for being used to detect homocysteine, testing procedure are：
S1:Reagent preparation box：The kit reagent includes following each component：Buffer solution Tris, reaction substrate 5- methyl tetrahydrochysenes
Calcium Folinate-SF, reaction substrate glycine, reaction substrate NAD+, enzyme activition ION Mg Cl2, methionine synthetase, amino methyl transfer
Enzyme, glycine hydroxymethyltransferase；
Each component is a concentration of in the kit of the step S1：Buffer solution Tris：20mM-50mM；Reaction substrate 5- methyl four
Hydrogen Calcium Folinate-SF：5mM-10mM；Reaction substrate glycine：10mM-20mM；NAD+：1mM-5mM；Enzyme activition ION Mg Cl2：2mM-
10mM；Methionine synthetase：5-15kU/L；Aminomethyltransferase：1-4kU/L；Glycine hydroxymethyltransferase：2-6kU/
L；
S2：The buffer solution Tris in step S1 is taken, is dissolved in purified water, and pH value is adjusted with concentrated hydrochloric acid；
S3：5-methyltetrahydrofolate calcium, glycine, the NAD in step S1 are weighed successively+、MgCl2, and add in the solution of step S2
In, stirring is to being completely dissolved；
S4：Take prepared reagent methionine synthetase in step S1, Aminomethyltransferase and the transfer of glycine methylol
After enzyme and respectively independent dissolving, add in the solution described in step S3, stir and evenly mix；
S5：The pH value of the mixing solution of determination step S4, and pH value is adjusted, purified water constant volume is added in, then filtering is placed on 2-8
It is DEG C spare；
S6:Solution after being filtered in step S5 is placed in after water-bath balance and obtains reaction solution, and the L- high that compound concentration is 0.05mM
Cysteine solution is as detectable substance；
S7：By reaction solution：Detectable substance in proportion, sets response parameter on Biochemical Analyzer, and records the extinction of each read point
Degree, for calculating absorbance change rate.
2. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：The step S2 and
PH value after being adjusted in step S5 is to 8.00 ± 0.05；Purified water is added in the step S5 and is settled to 500ml, after constant volume
Solution passes through 0.22 μm of membrane filtration.
3. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：In the step S6
Bath temperature is 37 DEG C, equilibration time 15-20min.
4. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：In the step S7
Reaction solution：The ratio of detectable substance is 50:1.
5. it is as described in claim 1 for detecting the enzyme round-robin method of homocysteine, it is characterized in that：The reagent
Tris, 5-methyltetrahydrofolate calcium, glycine, NAD+And MgCl2, it is the reagent that Sigma companies produce.
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| CN103184269A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting SNP sites related to homocysteine metabolism and amplification method and detection method thereof |
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| CN103184269A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting SNP sites related to homocysteine metabolism and amplification method and detection method thereof |
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