CN106248666A - Homocysteine (HCY) detectable that a kind of stability is strong - Google Patents
Homocysteine (HCY) detectable that a kind of stability is strong Download PDFInfo
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- CN106248666A CN106248666A CN201610871111.9A CN201610871111A CN106248666A CN 106248666 A CN106248666 A CN 106248666A CN 201610871111 A CN201610871111 A CN 201610871111A CN 106248666 A CN106248666 A CN 106248666A
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Abstract
The present invention relates to homocysteine (HCY) detectable of the strong double reagent liquid of a kind of stability, main containing buffer, NADH, LDH, serine, preservative, TCEP (trichloroethyl phosphate), protective agent, surfactant, disodiumedetate, substrate stabilizer in reagent R1；Reagent 2 mainly comprises buffer, preservative, protective agent, surfactant, CBS (cystathionine beta-synthase), CBL (cystathionine beta lyases).The buffer that prioritizing selection of the present invention is effective, uses efficient preservative, and uses three kinds of different protective agents in R2; the activity of enzyme in coordinating protection reagent; thus preferably ensure that the stability of reagent, especially heat stability, convenient clinical popularization and use.
Description
Technical field
The present invention relates to a kind of stability strong homocysteine (HCY) detectable, belong to clinical vitro detection skill
Art field.
Background technology
Homocysteine (homocysteine, HCY) also known as homocysteine, is formed after methionine demethyl
Plant sulfur-containing amino acid, belong to the intermediate product of methionine circulation；Be intracellular methionine transmethylated during produce one
Planting the aminoacid containing sulfydryl, Hcy is in circulation is transported to serum, and the Hcy in serum is most with oxidised form and serum
In albumin combined by the form of disulfide bond and form Hcy-albumen composition, the most a small amount of with sequestered and disulfide bond even
Presented in the Hcy-SS-Hcy connect；Homocysteine forms cysteine or methionine in metabolic process, raw in dimension
Element B6 turns in sulfenyl approach, and Hcy is irreversibly decomposed into cysteine, and most of Hcy is in folic acid and vitamin B12
Methionine is re-formed under the methionine synthase effect relied on；Homocysteine is that the one that Methionine metabolism produces contains
Sulfur aminoacid；Hcy level is closely related with cardiovascular disease；The Hcy increased in blood is because stimulating blood vessel wall to cause arterial blood
The damage of pipe, finally causes cardiac flow to be obstructed；Homocysteine patient with urine disease, owing to serious genetic defect affects Hcy
Metabolism, causes high Hcy mass formed by blood stasis；Slight genetic defect or vitamin B group malnutrition can be with moderate or slight Hcy liters
Height, also can increase cardiopathic danger；Hcy raises and neurocele also can be caused abnormal and the birth defect class disease such as congenital malformation.
At present the detection method of blood homocysteine is a lot, and mainly have that Refsum etc. set up in 1985 is same
Position element detection method, this method is highly sensitive, high specificity, due to complex operation and there is radiocontamination, though improved also failing to is promoted
Use；Within 1987, first Stabler reports gas chromatography-mass spectrometry tHCY chromatographic detection, the method measurement accuracy
Great amount of samples analysis can be used for analyzing speed, but relatively costly；Currently used more Immunological Method and enzyme process, both
Method has the advantage that method is simple, veracity and precision is high, and relative to Immunological Method, enzyme process cost is lower, uses more square
Just, can be in conjunction with automatic clinical chemistry analyzer, along with in current enzyme process reagent, enzyme purifies the raising of level, this method detectable
Cost reduces further, and enzyme process detection homocysteine is divided into two kinds of methods, one to be to use S-adenyhomotype
Cysteine (SAH) hydrolytic enzyme (SAHase), is therefore hydrolysis enzyme process, and another kind is to use cystathionie-beta-synthetase (C
BS), being also cystathionie method, the former is relatively costly, and the latter's enzyme cost is relatively low, is more conducive to promote, and its Cleaning Principle is homotype half
Cystine (oxidised form) is reduced into free homocysteine, under the catalysis of cystathionie-beta-synthetase (C BS)
Generating L-cystathionine with serine reaction, L-cystathionine is resolved into homocysteine and acetone by cystathionie-β-lyases
Acid, acetone acid participate in NADH chromogenic reaction, the homocysteine of generation again participate in the first step reaction, so circulate into
OK, but need to add NADH, cystathionie-β-synzyme, Guang owing to cystathionie Enzymatic cycling detects homocysteine
Enzyme or the coenzyme such as thioether-β-lyases, these enzymes are easily decayed in the case of liquid, especially at temperature drift
Under environment, being very easy to decay, mostly be the reagent of lyophilized powder the most on the market, this brings the biggest inconvenience to Clinical practice,
Therefore, it is possible to solve the stability of enzyme, improve the stability of reagent, clinical popularization is had bigger meaning.
Therefore the double reagent liquid that the present invention has invented the strong cystathionie Enzymatic cycling of a kind of stability according to this problem is same
Type cysteine detectable.
Summary of the invention
The present invention relates to double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, and detection
Method.
The present invention is obtained through the following steps:
Double reagent liquid homocysteine (HCY) the detectable reagent constituent that a kind of stability is strong is as follows:
Reagent 1(R1):
TRIS(trishydroxymethylaminomethane)-HCL 100mmol/L
NADH 0.47 mmol/l
LDH 3KU/L
Serine 0.76 mmol
Preservative 0.5-1g/L
TCEP (trichloroethyl phosphate) 2.9mmol/L
Protective agent 2-5ml/L
Surfactant 1-2ml/L
Heavy metal ion chelating agent 1-5mmol/L
Substrate stabilizer 3-5g/L
Reagent 2(R2):
TES (N-tri-(methylol) methyl-2-amino ethyl sulfonic acid) buffer 100mmol/L
Preservative 0.5g/L
Protective agent 1 3-6m/L
Protective agent 2 10-20g/L
Surfactant 1-2ml/L
Protective agent 3 0.5-1g/L
CBS 0.5KU/L
CBL 3 KU/L。
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, buffer in described reagent R1
Being 25 DEG C, pH is the TRIS(trishydroxymethylaminomethane of the 100mmol/L of 7.8)-HCL buffer.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, buffer in described reagent R2
Being 25 DEG C, pH is TES (N-tri-(methylol) methyl-2-amino ethyl sulfonic acid) buffer of the 100mmol/L of 7.8.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, described preservative is MIT(first
Base isothiazolone).
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, protective agent in described reagent R1
For PEG400.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, described surfactant is for telling
Temperature-405.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, heavy metal in described reagent R1
Ion chelating agent is EDTA.NA2.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, in described reagent R1, substrate is steady
Determining agent is CCD (west, Shanghai is precious).
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, protective agent 1 in described reagent R2
For ethylene glycol.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, protective agent 2 in described reagent R2
For beta-schardinger dextrin-.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, protective agent 3 in described reagent R2
For xanthan gum.
Double reagent liquid homocysteine (HCY) detectable that a kind of stability is strong, described homocysteine
Detectable detects the detection method of homocysteine, uses automatic clinical chemistry analyzer to utilize performance rate method to be measured,
Detection dominant wavelength is 340nm.
Described detection method, the ratio of R1 reagent and R2 reagent is 48:13.
Beneficial effects of the present invention:
1) present invention in reagent R1, add protective agent PEG400 and substrate stabilizer can be effectively protected LDH and
NADH；
2) MIT(Methylisothiazolinone is used), can effectively prevent the growth of mushroom, stop the corruption of reagent, and will not
Enzyme in reagent is produced inhibitory action；
3) Tween-40 5 surfactant is used, it is possible to the effective precision improving reagent testing result；
4) in order to improve the stability of two kinds of enzymes of CBS and CBL in reagent R2, with the addition of in reagent ethylene glycol, beta-schardinger dextrin-with
And xanthan gum, the stability of their synergism extraordinary raising enzyme of energy, thus effectively improve the stability of reagent.
Accompanying drawing explanation
Fig. 1 is the dependency graph of embodiment 2 and gas chromatogram-mass spectrography；
Fig. 2 is the dependency graph of embodiment 3 and gas chromatogram-mass spectrography；
Fig. 3 is 15 days corkage STABILITY MONITORING tracing figures；
Fig. 4 is 7 days 37 DEG C of Detection of Stability tracing figures；
Fig. 5 is embodiment 1 reagent test method, wherein calculates: HCY content (umol/L)=(A measures ÷ A standard) × C
Standard；
Fig. 6 is embodiment 2 reagent test method, wherein calculates: HCY content (umol/L)=(A measures ÷ A standard) × C
Standard；
Fig. 7 is embodiment 3 reagent test method, wherein calculates: HCY content (umol/L)=(A measures ÷ A standard) × C
Standard；
Fig. 8 is that table 4 embodiment reagent carbamide that is common with market and that get the nod measures test kit comparison and detection result.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described:
Embodiment 1
A kind of allocation plan of the HCY detectable of the most existing double reagent:
R1:NADH (0.47 mM), LDH (38 KU/L), serine (0.76 mM), Tris buffer (50mM), NaN3
(＜ 1%), TCEP(2.9 mM)；
R2:CBS (0.748 KU/L)；CBL (16.4 KU/L) NaN3(＜ 1%).
The using method of the present embodiment reagent:
The HCY detectable that the present embodiment describes, uses the automatic clinical chemistry analyzer with double reagent function in use, as
Hitachi 7180 fully-automatic analyzers etc., utilize fixed time to be measured.It is right to be placed into according to the ratio of 48:13 by R1 and R2
On the reagent position answered, the correspondence position at specimen disc places distilled water, standard substance and sample, and operation is such as Fig. 5.
Embodiment 2
What the present embodiment described is the HCY detectable of the liquid double reagent that a kind of stability is strong, constituent:
Reagent 1(R1):
TRIS trishydroxymethylaminomethane 100mmol/L
NADH 0.47mmol/l
LDH 3KU/L
Serine 0.76mmol/L
MIT(Methylisothiazolinone) 0.5g/L
TCEP (trichloroethyl phosphate) 2.9mmol/L
PEG400 2ml/L
TritonX-405 1ml/L
Disodiumedetate 1mmol/L
CCD (west, Shanghai is precious) 3g/L
Reagent 2(R2):
TES (N-tri-(methylol) methyl-2-amino ethyl sulfonic acid) buffer 100mmol/L
MIT(Methylisothiazolinone) 0.5g/L
Ethylene glycol 3m/L
Beta-schardinger dextrin-10g/L
TritonX-405 1ml/L
Xanthan gum 0.5g/L
CBS 0.5 KU/L
CBL 3 KU/L。
The using method of the present embodiment reagent:
The HCY detectable that the present embodiment describes, uses the automatic clinical chemistry analyzer with double reagent function in use, as
Hitachi 7180 fully-automatic analyzers etc., utilize fixed time to be measured.It is right to be placed into according to the ratio of 48:13 by R1 and R2
On the reagent position answered, the correspondence position at specimen disc places distilled water, standard substance and sample, and operation is such as Fig. 6.
Embodiment 3
A kind of crucial protective ingredient that the present embodiment describes increase after the examination of the strong liquid double reagent HCY detectable of stability
Agent constituent:
Reagent 1(R1):
TRIS trishydroxymethylaminomethane 100mmol/L
NADH 0.47mmol/l
LDH 3KU/L
Serine 0.76mmol/L
MIT(Methylisothiazolinone) 1g/L
TCEP (trichloroethyl phosphate) 2.9mmol/L
PEG400 5ml/L
TritonX-405 2ml/L
Disodiumedetate 5mmol/L
CCD (west, Shanghai is precious) 5g/L
Reagent 2(R2):
TES (N-tri-(methylol) methyl-2-amino ethyl sulfonic acid) buffer 100mmol/L
MIT(Methylisothiazolinone) 1g/L
Ethylene glycol 6m/L
Beta-schardinger dextrin-20g/L
TritonX-405 2ml/L
Xanthan gum 1g/L
CBS 0.5KU/L
CBL 3KU/L 。
The using method of the present embodiment reagent:
The HCY detectable that the present embodiment describes, uses the automatic clinical chemistry analyzer with double reagent function in use, as
Hitachi 7180 fully-automatic analyzers etc., utilize fixed time to be measured.It is right to be placed into according to the ratio of 48:13 by R1 and R2
On the reagent position answered, the correspondence position at specimen disc places distilled water, standard substance and sample, and operation is such as Fig. 7.
Dependency is tested: utilize the formula reagent preparation in embodiment 2,3, with gas chromatogram-mass spectrography detection serum
Homocysteine, have detected 20 clinical serum samples simultaneously, and testing result is as shown in Figure 8.And obtain two kinds of reagent
Correlation curve (as depicted in figs. 1 and 2), is shown by testing result, embodiment 2, embodiment 3 and gas chromatogram-mass spectrum
The dependency of method testing result is respectively 0.9980 and 0.9985, illustrates embodiment 2, embodiment 3 and gas chromatogram-mass spectrum
Method has great dependency.
Stability compares
Reagent stability checking concrete operation method:
Stability about reagent is verified, 15 days corkage stability and 7 days 37 DEG C of heat stability of being classified into reagent are verified.
First by the detectable in the detectable in the present embodiment and embodiment 1, embodiment 2 and embodiment 3, according to
Recipe configuration is good, takes identical two group respectively, and one group is done 15 days corkage stability tests, reagent is placed on the 2-of instrument
8 DEG C of cold closetes (do not take out) for 15 days, as 15 days corkage Detection of Stability；Another group does 37 DEG C of heat stability testings, closes
Be placed in 37 DEG C of thermostat water baths (every day only detection when take out, after detection, still sealing put back to 37 DEG C of water-baths
In Guo, continuous 7 days), as 37 DEG C of heat stability checkings in 7 days；By reagent simultaneously at Hitachi 7180 automatic clinical chemistry analyzer device
On, detect according to below figure 5 method, and Criterion curve on instrument；Take high and low value sample respectively, the most averagely
Being divided into 15 parts, 4 DEG C of storages, every day, high and low value sample respectively took one, and tracing detection result, and its tracking and monitoring trend is the most attached
Fig. 2 and accompanying drawing 3.
By checking, this reagent is good with gas chromatogram-mass spectrography contrast dependency, and Clinical detection sample results is consistent,
The market application requirement to product can be reached, be a kind of more stable, good HCY detectable.
Claims (9)
1. homocysteine (HCY) the detectable reagent constituent of the double reagent liquid that a stability is strong is as follows:
Reagent 1(R1):
Buffer 100mmol/L
NADH 0.47 mmol/l
LDH 3KU/L
Serine 0.76 mmol
Preservative 0.5-1g/L
TCEP (trichloroethyl phosphate) 2.9mmol/L
Protective agent 2-5ml/L
Surfactant 1-2ml/L
Disodiumedetate 1-5mmol/L
Substrate stabilizer 3-5g/L
Reagent 2(R2):
Buffer 100mmol/L
Preservative 0.5g/L
Protective agent 1 3-6m/L
Protective agent 2 10-20g/L
Surfactant 1-2ml/L
Protective agent 3 0.5-1g/L
CBS (cystathionie-β-synzyme) 0.5KU/L
CBL (cystathionie-β-lyases) 3 KU/L.
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levying and be in reagent R1 that buffer is 25 DEG C, the concentration of PH=7.8 is the TRIS(trishydroxymethylaminomethane of 100mmol/L)-
HCL buffer.
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levying and be in reagent R2 that buffer is 25 DEG C, the concentration of PH=7.8 is TES (N-tri-(methylol) methyl-2-of 100mmol/L
Taurine) buffer.
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levy and be that described preservative is MIT (Methylisothiazolinone).
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levy and be that protective agent described in reagent R1 is PEG400.
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levy and be that described surfactant is TritonX-405.
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levy and be that substrate stabilizer described in reagent R1 is CCD (Xi Bao bio tech ltd, Shanghai).
The homocysteine detectable of the double reagent liquid that a kind of stability the most according to claim 1 is strong, it is special
Levy and be that protective agent 1 described in reagent R2, protective agent 2, protective agent 3 are followed successively by ethylene glycol, beta-schardinger dextrin-, xanthan gum respectively, three
Person has synergistic protective effect.
9. according to the homocysteine detection of the strong double reagent liquid of a kind of stability according to any one of claim 1-8
Reagent, uses automatic clinical chemistry analyzer to utilize performance rate method to be measured, detects homocysteine, it is characterised in that make
Utilizing performance rate method to be measured with automatic clinical chemistry analyzer, detection dominant wavelength is 340nm.
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| CN201610871111.9A CN106248666A (en) | 2016-10-06 | 2016-10-06 | Homocysteine (HCY) detectable that a kind of stability is strong |
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Cited By (9)
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| CN106970230A (en) * | 2017-03-29 | 2017-07-21 | 广州市伊川生物科技有限公司 | A kind of homocysteine detection reagent box |
| CN107153044A (en) * | 2017-07-20 | 2017-09-12 | 青岛浩铂生物科技有限公司 | The homocysteine kit and its detection method of a kind of modified form |
| CN107271691A (en) * | 2017-08-10 | 2017-10-20 | 威特曼生物科技（南京）有限公司 | Homocysteine detection kit and its application method |
| CN107991252A (en) * | 2017-11-23 | 2018-05-04 | 中山市创艺生化工程有限公司 | A kind of stabilizer for α-hydroxybutyrate dehydrogenase assay kit and preparation method thereof |
| CN108562754A (en) * | 2018-01-17 | 2018-09-21 | 河北睿达模生物科技有限公司 | Hepatitis B e antigen individual event quality-control product and its preparation process |
| CN109001462A (en) * | 2018-07-04 | 2018-12-14 | 浙江伊利康生物技术有限公司 | A kind of homocysteine detection kit |
| CN110777190A (en) * | 2019-11-08 | 2020-02-11 | 武汉市长立生物技术有限责任公司 | Kit for detecting homocysteine and application thereof |
| WO2021174873A1 (en) * | 2020-03-05 | 2021-09-10 | 美康生物科技股份有限公司 | Dry homocysteine test card and application thereof |
| CN114689415A (en) * | 2020-12-31 | 2022-07-01 | 桂林优利特医疗电子有限公司 | Homocysteine detection kit with good stability and preparation method thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106970230A (en) * | 2017-03-29 | 2017-07-21 | 广州市伊川生物科技有限公司 | A kind of homocysteine detection reagent box |
| CN106970230B (en) * | 2017-03-29 | 2019-06-11 | 广州市伊川生物科技有限公司 | A kind of homocysteine detection reagent box |
| CN107153044A (en) * | 2017-07-20 | 2017-09-12 | 青岛浩铂生物科技有限公司 | The homocysteine kit and its detection method of a kind of modified form |
| CN107271691A (en) * | 2017-08-10 | 2017-10-20 | 威特曼生物科技（南京）有限公司 | Homocysteine detection kit and its application method |
| CN107991252A (en) * | 2017-11-23 | 2018-05-04 | 中山市创艺生化工程有限公司 | A kind of stabilizer for α-hydroxybutyrate dehydrogenase assay kit and preparation method thereof |
| CN107991252B (en) * | 2017-11-23 | 2021-05-28 | 中山市创艺生化工程有限公司 | Stabilizer for alpha-hydroxybutyrate dehydrogenase determination kit and preparation method thereof |
| CN108562754A (en) * | 2018-01-17 | 2018-09-21 | 河北睿达模生物科技有限公司 | Hepatitis B e antigen individual event quality-control product and its preparation process |
| CN109001462A (en) * | 2018-07-04 | 2018-12-14 | 浙江伊利康生物技术有限公司 | A kind of homocysteine detection kit |
| CN110777190A (en) * | 2019-11-08 | 2020-02-11 | 武汉市长立生物技术有限责任公司 | Kit for detecting homocysteine and application thereof |
| CN110777190B (en) * | 2019-11-08 | 2023-08-22 | 武汉市长立生物技术有限责任公司 | Kit for detecting homocysteine and application thereof |
| WO2021174873A1 (en) * | 2020-03-05 | 2021-09-10 | 美康生物科技股份有限公司 | Dry homocysteine test card and application thereof |
| CN114689415A (en) * | 2020-12-31 | 2022-07-01 | 桂林优利特医疗电子有限公司 | Homocysteine detection kit with good stability and preparation method thereof |
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Application publication date: 20161221 |