CN104215632A - Stable lipase kit - Google Patents
Stable lipase kit Download PDFInfo
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- CN104215632A CN104215632A CN201410433105.6A CN201410433105A CN104215632A CN 104215632 A CN104215632 A CN 104215632A CN 201410433105 A CN201410433105 A CN 201410433105A CN 104215632 A CN104215632 A CN 104215632A
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- reagent
- damping fluid
- stable lipase
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Abstract
The invention discloses a stable lipase kit which comprises a reagent R1 and a reagent R2, wherein the reagent R1 is prepared from 10-200mmol/L of buffer solution, 0.05-5g/L of sodium deoxycholate, 0.05-10g/L of sodium deoxycholate, 0.1-5mg/L of colipase, 1-20mmol/L of calcium chloride, 0.01-1% of preservative and 0.01-1% of surfactant; the reagent R2 is prepared from 10-200mmol/L of tartaric acid, 0.1-10g/L of taurodeoxycholic acid sodium salt, 0.01-1% of preservative, 0.01-2% of cosolvent, 0.01-2% of emulsifying agent, 0.01-2% of stabilizer and 0.1-0.5mmol/L of 6-methyl resorufin. The kit is high in sensitivity, small in error, stable in quality and convenient to store and has a strong function of protecting a substrate so as to improve the stability of the reagents to obtain a good detection effect. Thus, the kit has a high clinical application value.
Description
Technical field
The present invention relates to a kind of biological detection reagent, particularly a kind of stable lipase kit.
Illustrate the implication of partial symbols in the application:
Antiseptic 0.01-1% represents in every 100 ml solns and adds antiseptic 0.01-1g.
Similar part in the application, all represents this type of implication, except being otherwise noted.
Background technology
Lipase (Lipase EC3.1.1.3., LPS, GEH) is the enzyme of a class hydrolysis grease.The hydrolysis substrate of lipase is generally natural oil, and its hydrolysis position is the ester bond that in grease, fatty acid is connected with glycerine.It is different from other hydrolytic enzyme, lipase-catalyzed action system is a kind of heterogeneous system, water miscible enzyme catalysis occurs on the interface of water insoluble substrates and water, catalytic action mechanism on this interface is very unclear, can not simply with the reaction mechanism between Michaelis2Menten theory explain enzyme-to-substrate.Someone proposes the hypothesis that lipase is located on oil-water interface, proposes the model of " super substrate ", and this model can explain some behaviors of lipase, but also needs further experiment prove and revise.In addition, lipase is with reverse catalyzing glycerol and free fatty acid synthetic glycerine fat activity.Lipase is in clinical meaning: normal human blood LPS content is few, but when acute pancreatitis, 2 ~ 12h blood LPS significantly raises, 24h, to peak value, can reach 10 times of Upper Limit of Normal Value, even 50 times, may recover normal to 48 ~ 72h, but sustainable rising 8 ~ 15 days again subsequently.Due to blood LPS, early the amplitude of rising large, the time continued is long, therefore its diagnostic value is better than diastase the active time raised when acute pancreatitis.Clinical observation finds, the case that all blood AMY raise, and its LPS all raises; And LPS rising person AMY not necessarily raises, about have the normal pancreatitis patient of 2/3AMY, its LPS is normal; Non-pancreatitic acute abdomen has blood AMY rising and LPS does not raise.The blood LPS such as excessive drinking, alcohol repellency pancreatitis, chronic pancreatitis, cancer of pancreas, hepatobiliary disease can have rising in various degree.The method measuring LPS so far can be divided into 3 classes: the increase (as titrimetry, colourimetry, spectrophotometric method, fluorescence method and pH electrode method etc.) 1. measuring product (free fatty acid); 2. the reduction (as turbidimetry, diffusion method etc.) of substrate is measured; 3. the actual mass (double-antibody sandwich immunoassay method, latex agglutination) of LPS is measured.Current most laboratory is at home mainly based on titrimetry, turbidimetry and spectrophotometric method.Spectrophotometric method has two classes more conventional at present: 1. enzyme coupling colour developing colourimetry, multiplex 1,2-diglyceride is substrate, under the catalysis of LPS and monoglyceride lipase, hydrolysis generates glycerine and fatty acid, glycerine generates glycerol 3-phosphate by glycerokinase effect, then produces aubergine by GPO/peroxidase system and 4-AAP chromogen system.The change of monitoring absorbance in 550nm wavelength continuously can calculate LPS activity.This class methods specificity is high, also substantially can be solved the interference problem of endogenous glycerine by double reagent.The continuous monitoring method of 2. 1,2-o-bis-bay racemic glycerine-3-valeric acid-(6-methyl resorufin) ester design.This substrate, in alkaline environment, under LPS and colipase effect, is hydrolyzed generation 1,2-0-dilaurylglycerol and glutaric acid-6 '-methyl resorufin.The latter is unstable, can generate glutaric acid and methyl resorufin by Auto-decomposition.Methyl resorufin has absorption peak at 581nm wavelength place, and the change of its absorbance of monitoring can quantitative measurement LPS activity continuously.This method has the features such as easy, quick, sensitive, stable and antijamming capability is strong.But enzyme coupling development process, toolenzyme expensive, and also enzyme source is few and unstable, seldom has producer in this way.Substrate hydrolysis method, substrate extremely unstable, what the background of reagent raised is exceedingly fast, and causes reagent to calibrate every day.Waste reagent also wastes calibration object.And the inaccurate of result can be caused.Therefore better detection method is urgently sought.And existing reagent is not easy to preserve, and is very easily hydrolyzed, breakdown of emulsion, and long-term placement can precipitate, and quality is unstable, is not easy to preserve, with high costs.
Summary of the invention
For solving the problem, the invention discloses a kind of stable lipase kit, efficiently solving reagent stability, reduce the use cost that lipase detects reagent, improve ease of use, improve detection efficiency and accuracy of detection.
Stable lipase kit disclosed by the invention comprises
Preferred as one, damping fluid is the one in tris damping fluid, Good ' s damping fluid, mops damping fluid, hepes damping fluid.
Preferred as one, antiseptic is the one in Sodium azide, proclin300, mit.
Preferred as one, surfactant is the one in TritonX-100, Tween 80, brij-35, two bay sulfuric ester of glycerols.
Preferred as one, cosolvent is the one in acetone, methyl alcohol, ethanol, propyl alcohol, butanols.
Preferred as one, emulsifying agent is the one in two bay sulfuric ester of glycerols, TritonX-100, Triton-114, Tween 80, brij-35.
Preferred as one, stabilizing agent is the one in DMSO, sweet mellow wine, mercaptoacetic acid.
Preferred as one, reagent R2 adopts and configures with the following method, successively tartrate, cow-bezoar NaTDC, antiseptic, emulsifying agent, stabilizing agent is added to the water dissolving, is heated to 37 DEG C, and wherein water is deionized water or redistilled water.
Kit of the present invention detects mechanism:
The present invention, through research experiment, adds emulsifying agent two bay sulfuric ester of glycerol and stabilizing agent DMSO in kit, and under the environment of 37 DEG C, adds substrate.Such compound method, has no bibliographical information.The solution obtained is clarified and is stablized, and correlativity is also fabulous.
Accompanying drawing explanation
Embodiment 1 and the correlativity figure with Roche reagent of Fig. 1, stable lipase kit disclosed by the invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, illustrate the present invention further, following embodiment should be understood and be only not used in for illustration of the present invention and limit the scope of the invention.
Stable lipase kit disclosed by the invention comprises
Preferred as one, damping fluid is the one in tris damping fluid, Good ' s damping fluid, mops damping fluid, hepes damping fluid.
Preferred as one, antiseptic is the one in Sodium azide, proclin300, mit.
Preferred as one, surfactant is the one in TritonX-100, Tween 80, brij-35, two bay sulfuric ester of glycerols.
Preferred as one, cosolvent is the one in acetone, methyl alcohol, ethanol, propyl alcohol, butanols.
Preferred as one, emulsifying agent is the one in two bay sulfuric ester of glycerols, TritonX-100, Triton-114, Tween 80, brij-35.
Preferred as one, stabilizing agent is the one in DMSO, sweet mellow wine, mercaptoacetic acid.
Preferred as one, reagent R2 adopts and configures with the following method, successively tartrate, cow-bezoar NaTDC, antiseptic, emulsifying agent, stabilizing agent is added to the water dissolving, is heated to 37 DEG C.Substrate joins in above-mentioned solution, and stirs 30 minutes after dissolving with cosolvent.The object of heating is stable in order to obtain, the emulsion of clarification.
Embodiment
Test condition and method
| Wavelength | 580nm | Correct type | Linearly |
| Addition (sample/R1/R2, μ 1) | 4/200/50 | Serum+R1 the time | 3～5min |
| Method | Rate method | Add the reaction time after R2 | 4min |
| Bearing calibration | Two-point calibration | The Direction of Reaction | Upwards |
The present invention all adopts following steps about the configuration of reagent R2:
Successively tartrate, cow-bezoar NaTDC, antiseptic, emulsifying agent, stabilizing agent are added to the water dissolving, are heated to 37 DEG C, substrate joins in above-mentioned solution, and stirs 30 minutes after dissolving with cosolvent.Applicable equally in all embodiments below.
Embodiment 1
In LPS test, the embodiment of the present invention 1 is tested with the correlativity of Roche reagent test:
| Sample number | Roche reagent | Embodiment 1 |
| 1 | 9.8 | 9.6 |
| 2 | 32.6 | 31.6 |
| 3 | 17.5 | 17.6 |
| 4 | 34.1 | 35.1 |
| 5 | 63.1 | 63 |
| 6 | 43.4 | 42.9 |
| 7 | 33.8 | 33.8 |
| 8 | 49.8 | 49.5 |
| 9 | 36.4 | 35.7 |
| 10 | 25.5 | 26.1 |
| 11 | 19.6 | 19.4 |
| 12 | 85.6 | 87.1 |
| 13 | 22.5 | 22.9 |
| 14 | 47.9 | 46.1 |
| 15 | 28.7 | 28.5 |
| 16 | 77.4 | 76.9 |
| 17 | 44.6 | 43.8 |
| 18 | 16.9 | 16.7 |
| 19 | 55.4 | 55.2 |
| 20 | 46.8 | 45.2 |
| 21 | 44.5 | 44.1 |
| 22 | 44.9 | 44.5 |
| 23 | 51.3 | 51.1 |
| 24 | 36.4 | 36.1 |
| 25 | 31.2 | 31 |
| 26 | 42.1 | 41.1 |
| 27 | 33.3 | 31 |
| 28 | 45.6 | 44.2 |
| 29 | 28.9 | 27.9 |
| 30 | 37.4 | 36.5 |
Stability precipitation status: the embodiment of the present invention 1 reagent and contrast agents (commercially available as Roche reagent) when storing 12 months for 2-8 DEG C, to levels of precipitate observations in two kinds of reagent.
| 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 embodiment of the present invention 1 reagent and contrast agents (commercially available as Roche reagent), when storing 12 months for 2-8 DEG C, are observed the sensitivity of two kinds of reagent.
| 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 sum, the present embodiment reagent, measures response accurately to LPS, highly sensitive, there is higher response accuracy and correlativity compared with commercial reagent, and there is good stability, in long-term preservation, not easily produce precipitation, effectively inhibit the decay of active component in storage in reagent simultaneously, thus effectively ensure that the stability in use of reagent, extend serviceable life and the term of validity of reagent, make use flexibly more convenient, reduce LPS testing cost, be convenient to promote.
Embodiment 2
Embodiment 3
Embodiment 4
Embodiment 5
Embodiment 6
Embodiment 7
Embodiment 8
Embodiment 9
Unique difference of embodiment 10-18 and embodiment 1-9 is only: damping fluid is Good ' s damping fluid.
Unique difference of embodiment 19-27 and embodiment 1-9 is only: damping fluid is mops damping fluid.
Unique difference instrument of embodiment 28-36 and embodiment 1-9 is: damping fluid is hepes damping fluid.
Unique difference of embodiment 37-45 and embodiment 19 is only: antiseptic is proclin300.
Unique difference of embodiment 46-54 and embodiment 1-9 is only: antiseptic is mit (methylisothiazolinone).
Unique difference of embodiment 55-63 and embodiment 19 is only: surfactant is Tween 80.
Unique difference of embodiment 64-72 and embodiment 1-9 is only: surfactant is brij-35.
Unique difference of embodiment 73-81 and embodiment 19 is only: surfactant is two bay sulfuric ester of glycerols.
Unique difference of embodiment 81-90 and embodiment 1-9 is only: cosolvent is acetone.
Unique difference of embodiment 91-99 and embodiment 1-9 is only: cosolvent is ethanol.
Unique difference of embodiment 100-108 and embodiment 19 is only: cosolvent is propyl alcohol.
Unique difference of embodiment 109-117 and embodiment 1-9 is only: cosolvent is butanols.
Unique difference of embodiment 118-126 and embodiment 1-9 is only: emulsifying agent is TritonX-100.
Unique difference of embodiment 127-135 and embodiment 1-9 is only: emulsifying agent is Triton-114.
Unique difference instrument of embodiment 136-144 and embodiment 1-9 is: emulsifying agent is Tween 80.
Unique difference of embodiment 145-153 and embodiment 1-9 is only: emulsifying agent is brij-35.
Unique difference of embodiment 154-162 and embodiment 1-9 is only: stabilizing agent is sweet mellow wine.
Unique difference of embodiment 163-171 and embodiment 1-9 is only: stabilizing agent is mercaptoacetic acid.
The non-limit part of technical scope midrange that this place embodiment is protected application claims, equally all in the scope of protection of present invention.
In view of the present invention program's embodiment is numerous, each embodiment experimental data is huge numerous, be not suitable for particularize explanation herein, but the content of the required checking of each embodiment is all close with the final conclusion obtained, so do not illustrate one by one the checking content of each embodiment, only with embodiment 1, the excellent part of the present patent application is representatively described herein.All can prove that the present invention is all by reagent test enumerate or do not enumerate embodiment, existing technology is all being better than conveniently to LPS measuring accuracy, response efficiency, response correlativity, measurement range, all producing without precipitation after preserving for a long time simultaneously, effective active composition decay is simultaneously few, and the kit of technical solution of the present invention all has 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, also comprises the technical scheme be made up of above technical characteristic combination in any.The above is the specific embodiment of the present invention; it should be pointed out that 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 (8)
1. a stable lipase kit, is characterized in that: described stable lipase kit comprises
2. stable lipase kit according to claim 1, is characterized in that: described damping fluid is the one in tris damping fluid, Good ' s damping fluid, mops damping fluid, hepes damping fluid.
3. stable lipase kit according to claim 1, is characterized in that: described antiseptic is the one in Sodium azide, proclin300, mit.
4. stable lipase kit according to claim 1, is characterized in that: described surfactant is the one in TritonX-100, Tween 80, brij-35, two bay sulfuric ester of glycerols.
5. stable lipase kit according to claim 1, is characterized in that: described cosolvent is the one in acetone, methyl alcohol, ethanol, propyl alcohol, butanols.
6. stable lipase kit according to claim 1, is characterized in that: described emulsifying agent is the one in two bay sulfuric ester of glycerols, TritonX-100, Triton-114, Tween 80, brij-35.
7. stable lipase kit according to claim 1, is characterized in that: described stabilizing agent is the one in DMSO, sweet mellow wine, mercaptoacetic acid.
8. stable lipase kit according to claim 1; it is characterized in that: described reagent R2 adopts and configures with the following method; successively tartrate, cow-bezoar NaTDC, antiseptic, emulsifying agent, stabilizing agent are added to the water dissolving, are heated to 37 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410433105.6A CN104215632B (en) | 2014-08-28 | 2014-08-28 | A kind of fatty enzyme reagent kit of stabilization |
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| CN201410433105.6A CN104215632B (en) | 2014-08-28 | 2014-08-28 | A kind of fatty enzyme reagent kit of stabilization |
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| CN104215632B CN104215632B (en) | 2017-12-19 |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241873A (en) * | 2015-09-14 | 2016-01-13 | 郁东 | Lipase 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 |
| CN107796941A (en) * | 2017-08-25 | 2018-03-13 | 宁波瑞源生物科技有限公司 | The measure kit and its detection method of a kind of platelet-activating factor acetylhydro-lase |
| 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 |
| CN110031631A (en) * | 2019-04-02 | 2019-07-19 | 山东博科生物产业有限公司 | Stablize, the hoptoglobin detection kit of high specificity |
| 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 |
| CN112051354A (en) * | 2020-08-05 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Lipase determination kit and preparation method thereof |
Families Citing this family (1)
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| 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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| 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 |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241873A (en) * | 2015-09-14 | 2016-01-13 | 郁东 | Lipase detection kit |
| 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 |
| CN107796941A (en) * | 2017-08-25 | 2018-03-13 | 宁波瑞源生物科技有限公司 | The measure kit and its detection method of a kind of platelet-activating factor acetylhydro-lase |
| 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 |
| CN110031631A (en) * | 2019-04-02 | 2019-07-19 | 山东博科生物产业有限公司 | Stablize, the hoptoglobin detection kit of high specificity |
| 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 |
| CN112051354A (en) * | 2020-08-05 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Lipase determination kit and preparation method thereof |
| 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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