CN104215632B - A kind of fatty enzyme reagent kit of stabilization - Google Patents
A kind of fatty enzyme reagent kit of stabilization Download PDFInfo
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- CN104215632B CN104215632B CN201410433105.6A CN201410433105A CN104215632B CN 104215632 B CN104215632 B CN 104215632B CN 201410433105 A CN201410433105 A CN 201410433105A CN 104215632 B CN104215632 B CN 104215632B
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Abstract
The fatty enzyme reagent kit of stabilization disclosed by the invention, including reagent R1：The 200mmol/L of buffer solution 10；The 5g/L of NaTDC 0.05；The 10g/L of sodium taurodeoxycholate 0.05；The 5mg/L of colipase 0.1；The 20mmol/L of calcium chloride 1；Preservative 0.01 1%；Surfactant 0.01 1%；Reagent R2：The 200mmol/L of tartaric acid 10；The 10g/L of cow-bezoar NaTDC 0.1；Preservative 0.01 1%；Cosolvent 0.01 2%；Emulsifying agent 0.01 2%；Stabilizer 0.01 2%；The 0.5mmol/L of 6 methyl resorufin 0.1.The kit of the present invention, high sensitivity, error is small, steady quality, is easy to preserve, and has very strong protective effect to substrate, improves the stability of reagent, and Detection results are good, there is larger clinical value.
Description
Technical field
The present invention relates to a kind of biological detection reagent, the fatty enzyme reagent kit of particularly a kind of stabilization.
Illustrate the implication of partial symbols in the application：
Preservative 0.01-1% represents to add preservative 0.01-1g in every 100 milliliters of solution.
Similar part, represents such implication, unless otherwise specified in the application.
Background technology
Lipase (Lipase EC3.1.1.3., LPS, glycerol ester hydrolase) is the enzyme of a kind of hydrolysis grease.Lipase
Hydrolysis substrate be usually natural oil, it is aliphatic acid is connected with glycerine in grease ester bond that it, which hydrolyzes position,.It is different from
Other hydrolases, lipase-catalyzed action system are a kind of heterogeneous systems, and water miscible enzyme catalysis occur insoluble in water
Property substrate and water interface on, the catalytic action mechanism on this interface is not very clear, it is impossible to simple to use
Michaelis2Menten theories explain the reaction mechanism between enzyme-to-substrate.It is proposed that lipase is fixed on oil-water interface
The hypothesis of position, it is proposed that the model of " super substrate ", the model can explain some behaviors of lipase, but also need to further reality
Verify bright and amendment.In addition, lipase is with reverse catalyzing glycerol and free fatty synthetic glycerine fat activity.Lipase exists
Clinical meaning：Normal human blood LPS contents are few, but in acute pancreatitis, 2~12h blood LPS is significantly raised, and 24h is extremely
Peak value, up to 10 times of Upper Limit of Normal Value, or even 50 times, normal but subsequent and sustainable rise 8 may be recovered to 48~72h
~15 days.Due to blood LPS, in acute pancreatitis, the activity elevated time is early, big, the duration length of the amplitude of rising,
Therefore its diagnostic value is better than amylase.Clinical observation finds that all elevated cases of blood AMY, its LPS is raised；And LPS is raised
Person AMY is not necessarily raised, and there are about the normal pancreatitis patients of 2/3AMY, and its LPS is normal；The acute abdomen of non-pancreatitis has blood
AMY is raised and LPS is not raised.The blood LPS such as excessive drinking, alcohol repellency pancreatitis, chronic pancreatitis, cancer of pancreas, hepatobiliary disease can have
Different degrees of rise.3 classes can be divided into by determining LPS method so far：1. the increase of product (free fatty) is determined (as titrated
Method, colorimetric method, AAS, fluorescence method and pH electrode methods etc.)；2. determine decrement (such as turbidimetry, diffusion method of substrate
Deng)；3. determine LPS actual mass (double-antibody sandwich immunoassay method, latex agglutination).Current laboratory most at home
Mainly based on titration, turbidimetry and AAS.AAS has two classes more commonly used at present：1. enzyme coupling colour developing
Colorimetric method, use 1,2- diglycerides be substrate, under the catalysis of LPS and monoglyceride lipase, hydrolysis generation glycerine with
Aliphatic acid, glycerine acts on generation glycerol 3-phosphate by glycerokinase, then passes through GPO/peroxidase system
Aubergine is produced with 4-AAP chromogens system.LPS activity can be calculated in the change of 550nm wavelength continuous monitoring absorbances.This
Class method specificity is high, can also solve the interference problem of endogenous glycerine substantially by double reagent.2. 1,2-o- bis- bay-disappear outside
The continuous monitoring method of rotation-glycerine -3- valeric acids-(6- methyl resorufin) ester design.The substrate is in alkaline environment, in LPS and auxiliary
Under fatty enzyme effect, hydrolysis generation 1,2-O- dilaurylglycerols and glutaric acid -6 '-methyl resorufin.The latter is unstable, can be certainly
Hair decomposes generation glutaric acid and methyl resorufin.Methyl resorufin has absworption peak, its absorbance of continuous monitoring at 581nm wavelength
Change can quantitative determine LPS activity.This method has the characteristics that easy, quick, sensitive, stable and strong antijamming capability.But enzyme
Be coupled development process, toolenzyme it is expensive, and enzyme source is few and unstable, and few producers are in this way.Substrate water
Solution, substrate is extremely unstable, and the background of reagent is elevated to be exceedingly fast, and causes reagent to calibrate daily.Waste reagent and also waste calibration
Product.And the inaccuracy of result can be caused.Therefore more preferable detection method is urgently sought.And existing reagent is not easy to protect
Deposit, hydrolysis, demulsification easily occurs, and place can precipitate for a long time, quality is unstable, is not easy to preserve, with high costs.
The content of the invention
To solve the above problems, the invention discloses a kind of fatty enzyme reagent kit of stabilization, stable reagent is efficiently solved
Property, the use cost of lipase detection reagent is reduced, improves ease of use, improves detection efficiency and accuracy of detection.
The fatty enzyme reagent kit of stabilization disclosed by the invention includes
As a preferred embodiment, buffer solution is in tris buffer solutions, Good's buffer solutions, mops buffer solutions, hepes buffer solutions
One kind.
As a preferred embodiment, preservative is one kind in Sodium azide, proclin300, mit.
As a preferred embodiment, surfactant is TritonX-100, Tween 80, brij-35, two bay sulfuric ester of glycerols
In one kind.
As a preferred embodiment, cosolvent is one kind in acetone, methanol, ethanol, propyl alcohol, butanol.
As a preferred embodiment, emulsifying agent be two bay sulfuric ester of glycerols, TritonX-100, Triton-114, Tween 80,
One kind in brij-35.
As a preferred embodiment, stabilizer is one kind in DMSO, mannitol, TGA.
As a preferred embodiment, reagent R2 is configured with the following method, successively by tartaric acid, cow-bezoar NaTDC, anti-corrosion
Agent, emulsifying agent, stabilizer are added to the water dissolving, are heated to 37 DEG C, and its reclaimed water is deionized water or redistilled water.
Kit of the present invention detects mechanism：
The present invention passes through research experiment, and the bay sulfuric ester of glycerol of emulsifying agent two and stabilizer DMSO are added in kit,
And in the environment of 37 DEG C, add substrate.Such compound method, has no document report.Obtained solution clarification and steady
Fixed, correlation is also fabulous.
Brief description of the drawings
Fig. 1, stabilization disclosed by the invention fatty enzyme reagent kit embodiment 1 and dependency graph with Roche reagent.
Embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following embodiments are only
For illustrating the present invention rather than limitation the scope of the present invention.
The fatty enzyme reagent kit of stabilization disclosed by the invention includes
As a preferred embodiment, buffer solution is in tris buffer solutions, Good's buffer solutions, mops buffer solutions, hepes buffer solutions
One kind.
As a preferred embodiment, preservative is one kind in Sodium azide, proclin300, mit.
As a preferred embodiment, surfactant is TritonX-100, Tween 80, brij-35, two bay sulfuric ester of glycerols
In one kind.
As a preferred embodiment, cosolvent is one kind in acetone, methanol, ethanol, propyl alcohol, butanol.
As a preferred embodiment, emulsifying agent be two bay sulfuric ester of glycerols, TritonX-100, Triton-114, Tween 80,
One kind in brij-35.
As a preferred embodiment, stabilizer is one kind in DMSO, mannitol, TGA.
As a preferred embodiment, reagent R2 is configured with the following method, successively by tartaric acid, cow-bezoar NaTDC, anti-corrosion
Agent, emulsifying agent, stabilizer are added to the water dissolving, are heated to 37 DEG C.After substrate is dissolved with cosolvent, it is added in above-mentioned solution,
And stir 30 minutes.The purpose of heating is the emulsion of clarification in order to be stablized.
Embodiment
Test condition and method
| Wavelength | 580nm | Correct type | Linearly |
| Addition (sample/R1/R2, μ l) | 4/200/50 | Serum+R1 the times | 3~5min |
| Method | Performance rate method | Reaction time after addition R2 | 4min |
| Bearing calibration | Two-point calibration | The Direction of Reaction | Upwards |
The present invention uses following steps on reagent R2 configuration：
Tartaric acid, cow-bezoar NaTDC, preservative, emulsifying agent, stabilizer are added to the water dissolving successively, are heated to 37
DEG C, after substrate is dissolved with cosolvent, it is added in above-mentioned solution, and stir 30 minutes.It is same in all examples below
It is applicable.
Embodiment 1
In LPS tests, the embodiment of the present invention 1 and the correlation of Roche reagent test are tested：
Stability precipitation status：The reagent of the embodiment of the present invention 1 is with contrast agents (commercially available such as Roche reagent) in 2-8 DEG C of storage
At 12 months, result is observed to levels of precipitate in two kinds of reagents.
| Time | Contrast agents | The embodiment of the present invention 1 |
| 1 month | Without precipitation | Without precipitation |
| 2 months | Without precipitation | Without precipitation |
| 3 months | Without precipitation | Without precipitation |
| 4 months | There is a small amount of precipitation | Without precipitation |
| 5 months | There is a small amount of precipitation | Without precipitation |
| 6 months | There is a small amount of precipitation | Without precipitation |
| 7 months | There is a small amount of precipitation | Without precipitation |
| 8 months | There is a small amount of precipitation | Without precipitation |
| 9 months | There is a small amount of precipitation | Without precipitation |
| 10 months | More precipitation | Without precipitation |
| 11 months | More precipitation | Without precipitation |
| 12 months | More precipitation | Without precipitation |
Stability change of sensitivity：The reagent of the embodiment of the present invention 1 is with contrast agents (commercially available such as Roche reagent) in 2-8 DEG C of storage
When depositing 12 months, the sensitivity to two kinds of reagents is observed.
| Time | Contrast agents | The embodiment of the present invention 1 |
| 0 month | 480 | 510 |
| 1 month | 480 | 510 |
| 3 months | 465 | 509 |
| 6 months | 451 | 493 |
| 12 months | 420 | 486 |
Visible in summary, the present embodiment reagent is accurate to LPS measure responses, high sensitivity, has compared with commercial reagent
There are higher response accuracy and correlation, and there is good stability, precipitation is not likely to produce in long-term preserve, while effectively
Decay of the active component in storage in reagent is inhibited, so as to be effectively guaranteed the stability in use of reagent, extends examination
The service life and the term of validity of agent so that use is more convenient flexibly, reduces LPS testing costs, is easy to promote.
Embodiment 2
Embodiment 3
Embodiment 4
Embodiment 5
Embodiment 6
Embodiment 7
Embodiment 8
Embodiment 9
The unique of embodiment 10-18 and embodiment 1-9 differs only in：Buffer solution is Good's buffer solutions.
The unique of embodiment 19-27 and embodiment 1-9 differs only in：Buffer solution is mops buffer solutions.
The unique of embodiment 28-36 and embodiment 1-9 differs only in：Buffer solution is hepes buffer solutions.
The unique of embodiment 37-45 and embodiment 1-9 differs only in：Preservative is proclin300.
The unique of embodiment 46-54 and embodiment 1-9 differs only in：Preservative is mit (methylisothiazolinone).
The unique of embodiment 55-63 and embodiment 1-9 differs only in：Surfactant is Tween 80.
The unique of embodiment 64-72 and embodiment 1-9 differs only in：Surfactant is brij-35.
The unique of embodiment 73-81 and embodiment 1-9 differs only in：Surfactant is two bay sulfuric ester of glycerols.
The unique of embodiment 81-90 and embodiment 1-9 differs only in：Cosolvent is acetone.
The unique of embodiment 91-99 and embodiment 1-9 differs only in：Cosolvent is ethanol.
The unique of embodiment 100-108 and embodiment 1-9 differs only in：Cosolvent is propyl alcohol.
The unique of embodiment 109-117 and embodiment 1-9 differs only in：Cosolvent is butanol.
The unique of embodiment 118-126 and embodiment 1-9 differs only in：Emulsifying agent is TritonX-100.
The unique of embodiment 127-135 and embodiment 1-9 differs only in：Emulsifying agent is Triton-114.
The unique of embodiment 136-144 and embodiment 1-9 differs only in：Emulsifying agent is Tween 80.
The unique of embodiment 145-153 and embodiment 1-9 differs only in：Emulsifying agent is brij-35.
The unique of embodiment 154-162 and embodiment 1-9 differs only in：Stabilizer is mannitol.
The unique of embodiment 163-171 and embodiment 1-9 differs only in：Stabilizer is TGA.
, equally all will in the present invention in place of this place embodiment is to the claimed non-limit of technical scope midrange
In the range of asking protection.
In view of the present invention program embodiment is numerous, each embodiment experimental data is huge numerous, is not suitable for arranging one by one herein
Act explanation, but the content of checking required for each embodiment approaches with obtained final conclusion, so herein not to each reality
The checking content for applying example is illustrated one by one, only illustrates the excellent part of the present patent application using embodiment 1 as representative.Pass through reagent
Test can prove that the present invention is all and enumerates or unrequited embodiment, to LPS measuring accuracies, response efficiency, response correlation,
It is convenient that measurement range is superior to existing technology, while is being produced after long-term preserve without precipitation, at the same effective active into
Divide decay few, the kit of technical solution of the present invention is respectively provided with good quality stability and quality guarantee.
Technological means disclosed in the present invention program is not limited only to the technological means disclosed in above-mentioned technological means, in addition to
Formed technical scheme is combined by above technical characteristic.Described above is the embodiment of the present invention, should be referred to
Go out, for those skilled in the art, under the premise without departing from the principles of the invention, can also make some
Improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (1)
- A kind of 1. fatty enzyme reagent kit of stabilization, it is characterised in that：The fatty enzyme reagent kit of the stabilization includesThe reagent R2 is configured with the following method, successively by tartaric acid, cow-bezoar NaTDC, preservative Sodium azide, emulsification The bay sulfuric ester of glycerol of agent two, stabilizer DMSO, which are added to the water, is dissolved into solution, is heated to 37 DEG C, 6- methyl resorufins After substrate is dissolved with methanol, it is added in above-mentioned solution, and stirs 30 minutes.
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Cited By (1)
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|---|---|---|---|---|
| CN109490296A (en) * | 2018-12-29 | 2019-03-19 | 南京澳林生物科技有限公司 | A kind of fat enzyme detection kit and production technology |
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| CN105241873B (en) * | 2015-09-14 | 2018-05-22 | 郁东 | A kind of fat enzyme detection kit |
| CN107782680A (en) * | 2016-08-26 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of antiheparin fat enzyme detection kit of stabilization |
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| CN109490227A (en) * | 2018-10-18 | 2019-03-19 | 闫玮钰 | A kind of highly sensitive fatty enzyme reagent kit |
| CN109580515A (en) * | 2019-01-14 | 2019-04-05 | 中生北控生物科技股份有限公司 | A kind of reagent and the preparation method and application thereof measured for pancreatic lipase in serum or blood plasma |
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| CN110669822A (en) * | 2019-11-07 | 2020-01-10 | 浙江爱康生物科技有限公司 | Lipase kit and preparation method thereof |
| CN111272991A (en) * | 2020-01-21 | 2020-06-12 | 苏州德沃生物技术有限公司 | Antigen stabilizing agent |
| CN112051354B (en) * | 2020-08-05 | 2022-06-14 | 武汉生之源生物科技股份有限公司 | Lipase determination kit and preparation method thereof |
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| CN1205034A (en) * | 1996-09-19 | 1999-01-13 | 曼海姆泊灵格股份公司 | Improved method for determining lipase |
| CN102621138A (en) * | 2012-04-06 | 2012-08-01 | 上海蓝怡科技有限公司 | Preparation method of micro-emulsion kit |
| CN103173518A (en) * | 2011-12-20 | 2013-06-26 | 上海复星医药（集团）股份有限公司 | Kit for detecting lipase by enzyme method and preparation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP3916086B2 (en) * | 2004-06-29 | 2007-05-16 | ソニー株式会社 | Method and detection unit for detecting interaction such as hybridization, substrate for bioassay including the detection unit, apparatus for detecting interaction such as hybridization, and reagent kit |
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| CN1205034A (en) * | 1996-09-19 | 1999-01-13 | 曼海姆泊灵格股份公司 | Improved method for determining lipase |
| CN103173518A (en) * | 2011-12-20 | 2013-06-26 | 上海复星医药（集团）股份有限公司 | Kit for detecting lipase by enzyme method and preparation method |
| CN102621138A (en) * | 2012-04-06 | 2012-08-01 | 上海蓝怡科技有限公司 | Preparation method of micro-emulsion kit |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109490296A (en) * | 2018-12-29 | 2019-03-19 | 南京澳林生物科技有限公司 | A kind of fat enzyme detection kit and production technology |
| CN109490296B (en) * | 2018-12-29 | 2021-07-20 | 南京澳林生物科技有限公司 | Lipase detection kit and production process |
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