CN104498586B - The single reagent serum triglycerides detectable that a kind of stability is strong - Google Patents
The single reagent serum triglycerides detectable that a kind of stability is strong Download PDFInfo
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Abstract
The invention discloses the single reagent serum triglycerides detectable that a kind of stability is strong, belong to clinical vitro detection technical field。This reagent adopts 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)-sodium glutamate buffer system, makes system have good buffer capacity；Reagent also added the materials such as dodecyltriethanolamine sulfate, coenzyme F AD and polyvinyl alcohol AH-26, all effectively raise the stability of reagent。The present invention has more excellent stability compared with conventional single reagent detection Triglyceride Reagent, is more beneficial for clinical expansion and uses。
Description
Technical field
The present invention relates to the single reagent serum triglycerides detectable that a kind of stability is strong, belong to clinical vitro detection technical field。
Background technology
Triglyceride (TG) is the key component of blood fat, is the important energy supply material of body。Triglyceride in food is at intestinal through pancreatic lipase digest and decompose, and after absorption, esterification is triglyceride again, enters blood through lymphsystem。Liver is the main place of synthetic glycerine three ester, and fatty tissue is maximum triglyceride storage vault。Under pathological conditions, the synthesis of various histiocytes and the triglyceride preserved dramatically increase。Content of triglyceride in detection by quantitative blood and histiocyte, is the conventional biochemical indicator of clinic, Clinical Basis, preclinical medicine biological study。
Current serum TG detection method is numerous, generally can be divided into chemical method, enzyme process and the big class of chromatography 3。Chemical method is with the TG in organic solvent extracting specimen, after removing in extract the chaff interferences such as phospholipid, with basic hydrolysis (saponification) TG, generates formaldehyde with periodate oxidation glycerol, then surveys formaldehyde by chromogenic reaction。Being dichloromethane silicic acid off-color acid method (VanHande-Caslson method) more accurately, but operate more complicated, technology requires height, is unsuitable in routine clinical analysis and applies；And chromatography is to the unusual height of the requirement of instrument and operator, and analysis cost is expensive, and therefore chemical method and chromatography are unfavorable for clinical expansion。
Domestic almost all of Clinical detection mainly adopts TG content in enzyme process detection serum, and its main operational principle is as follows: it is glycerol and fatty acid that lipase decomposes Triglycerides in Serum。Under ATP exists, phosphoglycerolization is generated glycerol 3-phosphate by glycerol kinase。The latter is generated dihydroxyacetone phosphate and hydrogen peroxide by GPO oxidation。Hydrogen peroxide and peroxidase, 4-aminophenazone carry out chromogenic reaction, generate coloured benzoquinone imine, and at 480 ~ 550nm place, absorbance is directly proportional to triglyceride concentration。This method has the advantage that simplicity is quick, trace, precision are high and high specificity, it is easy to reaches terminal, range of linearity width, is conducive to clinical expansion。What general comparison was many is the TG detectable of double reagent on the market, and the TG detectable of single reagent is due to its less stable, and reagent is extremely easily subject to the pollution of environment and is not accepted by market。
Summary of the invention
For solving problems of the prior art, the invention provides the single reagent serum levels of triglyceride detectable that a kind of stability is strong。
The present invention is by the following technical solutions:
The single reagent serum levels of triglyceride detectable that a kind of stability is strong, it is characterized in that, described reagent includes: 50-60mmol/L4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer (pH=7.4), 1-5g/L bovine serum albumin, 0.1-1g/L dodecyltriethanolamine sulfate, 0.1-1g/L benzylhydroperoxide sodium, 1-2g/L coenzyme F AD, 0.1-1g/L polyvinyl alcohol AH-26, 10-100mmol/L disodiumedetate, 3.5-5mmol/L sodium cholate, 17.5-20.5mo/Ll bitter salt, 1-3mmol/L4-amino-antipyrine, 3.5-5mmol/LN, accelerine, 0.1-1g/LTritonX-100, 1-3KU/L lipoprotein lipase, 0.25-500KU/L glycerol kinase, 1-3KU/L peroxidase, 1-3KU/L glycerol-3-phosphate oxidase, 0.1-1g/L sodium azide。
One as the present invention is preferred, described reagent includes: 50mmol/L4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer (pH=7.4), 3g/L bovine serum albumin, 0.5g/L dodecyltriethanolamine sulfate, 0.5g/L benzylhydroperoxide sodium, 1.5g/L coenzyme F AD, 0.5g/L polyvinyl alcohol AH-26, 50mmol/L disodiumedetate, 4mmol/L sodium cholate, 18mol/L bitter salt, 2mmol/L4-amino-antipyrine, 4.5mmol/LN, accelerine, 0.5g/LTritonX-100, 2KU/L lipoprotein lipase, 300KU/L glycerol kinase, 2KU/L peroxidase, 2KU/L glycerol-3-phosphate oxidase, 0.5g/L sodium azide。
The preparation method of reagent of the present invention is to be dissolved in each component successively in 1L distilled water having prepared。
The invention has the beneficial effects as follows:
1) present invention have selected 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)-sodium glutamate buffer, and this buffer system is have good buffer capacity within the scope of 6.8-8.2 at pH value, makes whole formula be in a kind of extremely stable state, improves the stability of reagent。
2) adding dodecyltriethanolamine sulfate respectively in reagent of the present invention, it can be effectively protected the stability of lipoprotein lipase；Additionally, reagent adds coenzyme F AD and polyvinyl alcohol AH-26, they can effectively in protected glycerol-3-phosphoric acid oxidase the active group of enzyme make enzyme not easy in inactivation, thus improving the stability of enzyme。
4) choosing stability in the present invention relatively strong, the DMA being not easily decomposed, as chromogen, substitutes original 4-chlorophenol, effectively slows down the disadvantage that reagent blank raises, thus ensure that the stability of reagent。
5) present invention also adds the benzylhydroperoxide sodium with weak oxide character in reagent, it is possible to effectively remove the interference to biochemical reagents reducing substances of the reducing substances such as ascorbic acid, bilirubin, thus improving capacity of resisting disturbance and the stability of reagent。
Accompanying drawing explanation
Fig. 1 is embodiment of the present invention 1-3 and 7 days 37 DEG C of heat stabilization test result figure of matched group；
Fig. 2 is embodiment of the present invention 1-3 and 15 days reagent stability test result figure of matched group；
Fig. 3 is embodiment of the present invention 1-3 and 7 days 37 DEG C of heat stability reagent blank testing result figure of matched group；
Fig. 4 is embodiment of the present invention 1-3 and 15 days reagent stability reagent blank testing result figure of matched group。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail。
Embodiment 1
The single reagent serum levels of triglyceride detectable that a kind of stability is strong, described reagent includes: 50mmol/L4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer (pH=7.4), 1g/L bovine serum albumin, 0.1g/L dodecyltriethanolamine sulfate, 0.1g/L benzylhydroperoxide sodium, 1g/L coenzyme F AD, 0.1g/L polyvinyl alcohol AH-26, 10mmol/L disodiumedetate, 3.5mmol/L sodium cholate, 17.5mo/Ll bitter salt, 1mmol/L4-amino-antipyrine, 3.5mmol/LN, accelerine, 0.1g/LTritonX-100, 1KU/L lipoprotein lipase, 0.25KU/L glycerol kinase, 1KU/L peroxidase, 1KU/L glycerol-3-phosphate oxidase, 0.1g/L sodium azide。
Above-mentioned each component is dissolved in 1L distilled water successively and has prepared。
Embodiment 2
The single reagent serum triglycerides detectable that a kind of stability is strong, described reagent includes: 60mmol/L4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer (pH=7.4), 5g/L bovine serum albumin, 1g/L dodecyltriethanolamine sulfate, 1g/L benzylhydroperoxide sodium, 2g/L coenzyme F AD, 1g/L polyvinyl alcohol AH-26, 100mmol/L disodiumedetate, 5mmol/L sodium cholate, 20.5mo/Ll bitter salt, 3mmol/L4-amino-antipyrine, 5mmol/LN, accelerine, 1g/LTritonX-100, 3KU/L lipoprotein lipase, 500KU/L glycerol kinase, 3KU/L peroxidase, 3KU/L glycerol-3-phosphate oxidase, 1g/L sodium azide。
Above-mentioned each component is dissolved in 1L distilled water successively and has prepared。
Embodiment 3
The single reagent serum triglycerides detectable that a kind of stability is strong, described reagent includes:
50mmol/L4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer (pH=7.4), 3g/L bovine serum albumin, 0.5g/L dodecyltriethanolamine sulfate, 0.5g/L benzylhydroperoxide sodium, 1.5g/L coenzyme F AD, 0.5g/L polyvinyl alcohol AH-26, 50mmol/L disodiumedetate, 4mmol/L sodium cholate, 18mol/L bitter salt, 2mmol/L4-amino-antipyrine, 4.5mmol/LN, accelerine, 0.5g/LTritonX-100, 2KU/L lipoprotein lipase, 300KU/L glycerol kinase, 2KU/L peroxidase, 2KU/L glycerol-3-phosphate oxidase, 0.5g/L sodium azide。
Said components is dissolved in 1L distilled water successively and has prepared。
Reagent obtained by embodiment of the present invention 1-3 is carried out stability test checking。Test is divided into corkage stability checking in 15 days and 37 DEG C of heat stability checking tests in 7 days。
Test calibration object used and quality-control product are respectively as follows:
Calibration object: the content of Landau compound standard (848UN) wherein TG is 1.14mmol/L
Quality-control product: Landau compound high level Quality Control: 620UE (TG target value: 2.86mmol/L, target value scope: 2.41-3.31mmol/L)；Landau compound low value Quality Control: 897UN (TG target value: 1.09mmol/L, target value scope: 0.92-1.26mmol/L)。
The concrete operation method of reagent stability checking test:
Using gained detectable in embodiment of the present invention 1-3 as test group, take the single reagent serum triglycerides detectable of a kind of certain company commercially available production as a control group, identical two part that ask for often organized by test group and matched group reagent, portion does 15 days corkage stability tests, reagent is placed in 2-8 DEG C of cold closet of instrument (within 15 days, not taking out), opened Detection of Stability as 15 days；Another part does 37 DEG C of heat stability test tests, closes and is placed in 37 DEG C of thermostat water baths (every day only takes out when detection, and after detection, still sealing is put back in 37 DEG C of water-baths, continuous 7 days), as 37 DEG C of heat stability checkings in 7 days。By reagent simultaneously on Toshiba-120 automatic clinical chemistry analyzer device, detect according to such as table 1 below method, and on instrument Criterion curve。Taking the high and low value lyophilized powder quality-control product of Landau respectively, after being uniformly dissolved, be each divided into 15 parts ,-20 DEG C of storages, every day, high and low value Quality Control respectively took one, and tracing detection result, its tracking and monitoring trend such as Fig. 1 and Fig. 2。Every day detects the reagent blank of each group reagent, and testing result is Fig. 3 such as, Fig. 4。
Table 1TG (triglyceride) detectable detection method
Calculate TG(triglyceride) content (μm ol/L)=(?A mensuration/min ÷?A standard/min) × C standard。
Can be seen that no matter the TG detectable in embodiment 1-3 is 7 days 37 DEG C of heat stabilization tests or 15 days reagent stability tests by Fig. 1 and Fig. 2, the stability of reagent is all good than comparison group reagent, is conducive to clinical expansion to use。This is stable mainly due to buffer system in reagent of the present invention, adds dodecyltriethanolamine sulfate in reagent respectively, and it can be effectively protected the stability of lipoprotein lipase；Simultaneously reagent is also added into coenzyme F AD and polyvinyl alcohol AH-26, they can effectively in protected glycerol-3-phosphoric acid oxidase the active group of enzyme make enzyme not easy in inactivation, thus improving the stability of enzyme。In addition, choosing stability in the present invention relatively strong, the DMA being not easily decomposed, as chromogen, substitutes original 4-chlorophenol, effectively slows down the disadvantage that reagent blank raises, thus ensure that the stability of reagent。
By Fig. 3 and Fig. 4 it is found that matched group reagent attenuation ratio is very fast, and reagent blank is very easy to raise, and the stability of reagent self is excessively poor, and reagent blank absorbance is when more than 0.08, and reagent just cannot use。And embodiment of the present invention 1-3 gained reagent, its 7 days 37 DEG C of heat stability and 15 days reagent stability, testing result is all very stable, there is not the situation of decay in reagent, although reagent blank also has a degree of rising, but ascensional range is little, it does not have more than 0.08, reagent is still highly stable, it is appreciated that use in clinical expansion。
Claims (2)
1. the single reagent serum triglycerides detectable that a stability is strong, it is characterized in that, described reagent includes: the 4-hydroxyethyl piperazine ethanesulfonic acid-sodium glutamate buffer of 50-60mmol/LpH=7.4, 1-5g/L bovine serum albumin, 0.1-1g/L dodecyltriethanolamine sulfate, 0.1-1g/L benzylhydroperoxide sodium, 1-2g/L coenzyme F AD, 0.1-1g/L polyvinyl alcohol AH-26, 10-100mmol/L disodiumedetate, 3.5-5mmol/L sodium cholate, 17.5-20.5mo/Ll bitter salt, 1-3mmol/L4-amino-antipyrine, 3.5-5mmol/LN, accelerine, 0.1-1g/LTritonX-100, 1-3KU/L lipoprotein lipase, 0.25-500KU/L glycerol kinase, 1-3KU/L peroxidase, 1-3KU/L glycerol-3-phosphate oxidase, 0.1-1g/L sodium azide。
2. single reagent serum triglycerides detectable according to claim 1, it is characterized in that, described reagent includes: the 4-hydroxyethyl piperazine ethanesulfonic acid of 50mmol/LpH=7.4-sodium glutamate buffer (pH=7.4), 3g/L bovine serum albumin, 0.5g/L dodecyltriethanolamine sulfate, 0.5g/L benzylhydroperoxide sodium, 1.5g/L coenzyme F AD, 0.5g/L polyvinyl alcohol AH-26, 50mmol/L disodiumedetate, 4mmol/L sodium cholate, 18mol/L bitter salt, 2mmol/L4-amino-antipyrine, 4.5mmol/LN, accelerine, 0.5g/LTritonX-100, 2KU/L lipoprotein lipase, 300KU/L glycerol kinase, 2KU/L peroxidase, 2KU/L glycerol-3-phosphate oxidase, 0.5g/L sodium azide。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108949903B (en) * | 2017-05-17 | 2021-11-16 | 广州市伊川生物科技有限公司 | Triglyceride determination kit and determination method thereof |
| CN107884401B (en) * | 2017-11-13 | 2020-03-24 | 天津市宝坻区人民医院 | Glucose oxidase determination method for eliminating lipemia interference |
| CN108467882A (en) * | 2018-03-30 | 2018-08-31 | 潍坊市康华生物技术有限公司 | A kind of triglyceride detection kit |
| CN110938607B (en) * | 2019-12-18 | 2023-03-28 | 美康生物科技股份有限公司 | Glycerol-3-phosphate oxidase with good thermal stability and application thereof in kit |
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| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| CN102706818A (en) * | 2012-06-05 | 2012-10-03 | 宁波美康生物科技股份有限公司 | Enzymatic triglyceride measuring method and measuring reagent |
| CN104165992A (en) * | 2014-07-28 | 2014-11-26 | 武汉璟泓万方堂医药科技股份有限公司 | Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| CN102706818A (en) * | 2012-06-05 | 2012-10-03 | 宁波美康生物科技股份有限公司 | Enzymatic triglyceride measuring method and measuring reagent |
| CN104165992A (en) * | 2014-07-28 | 2014-11-26 | 武汉璟泓万方堂医药科技股份有限公司 | Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia |
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