CN103173518A - Kit for detecting lipase by enzyme method and preparation method - Google Patents
Kit for detecting lipase by enzyme method and preparation method Download PDFInfo
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- CN103173518A CN103173518A CN 201110430794 CN201110430794A CN103173518A CN 103173518 A CN103173518 A CN 103173518A CN 201110430794 CN201110430794 CN 201110430794 CN 201110430794 A CN201110430794 A CN 201110430794A CN 103173518 A CN103173518 A CN 103173518A
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Abstract
The invention provides a kit for detecting lipase by an enzyme method, and is composed of a reagent 1 and a reagent 2. The reagent 1 is composed of 10-200mmol/L of 2L buffer, 0.01-20ml/L of sodium deoxycholate, 0.1-2mg/L of colipase, 5-20mmol/L of calcium chloride, 0.5-2g/L of sodium azide, and the balance of deionized water to 2L; and the reagent 2 comprises the following components by weight: 10-200mmol/L of 1L tartrate, 0.1-10mmol/L of bezoar sodium deoxycholate, 10-200mmol/L of mannitol, 0.01-0.5ml/L of proclin300, 10-200mmol/L of mercaptoacetic acid, 0.1-0.5mmol/L of 6-methyl resorufin, and the balance of deionized water to 1L. The kit has strong protection effect on a substrate, and the stability of the reagent is increased, the kit has good detection effect, and has large clinical application value.
Description
Technical field
The present invention relates to biological reagent, be specifically related to a kind of detection kit, relate in particular to a kind of enzyme process and detect lipase test kit and preparation method thereof.
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 fats and oils, and it is hydrolyzed the position is the ester bond that in grease, lipid acid is connected with glycerine.It is different from other lytic enzyme, lipase-catalyzed action system is a kind of nonhomogeneous system, water miscible enzyme catalysis occurs on the interface of water insoluble substrates and water, katalysis mechanism on this interface is very unclear, can not be simply with the reaction mechanism between Michaelis2Menten theory explain enzyme-to-substrate.Someone has proposed the hypothesis that lipase is located on oil-water interface, proposed the model of " super substrate ", and this model can be explained some behaviors of lipase, but also needs further experimental results show that and revise.In addition, lipase is active with reverse catalyzing glycerol and free fatty acids synthetic glycerine fat.Lipase is in clinical meaning: normal human blood LPS content is few, but when acute pancreatitis, 2～12h blood LPS significantly raises, 24h can reach 10 times of Upper Limit of Normal Value to peak value, even 50 times, may recover normally to 48～72h, but sustainable rising 8～15 days again subsequently.Due to blood LPS when the acute pancreatitis the active time that raises early, the amplitude of rising large, the time that continues is long, therefore its diagnostic value is better than amylase.Clinical observation is found, the case that all blood AMY raise, and its LPS all raises; And LPS rising person AMY not necessarily raises, and the normal pancreatitis patient of 2/3AMY is approximately arranged, and its LPS is normal; Non-pancreatitic acute abdomen has blood AMY to raise and LPS does not raise.The blood LPS such as excessive drinking, ethanol pancreatitis, chronic pancreatitis, carcinoma of the pancreas, hepatobiliary disease can have rising in various degree.The method of measuring so far LPS can be divided into 3 classes: 1. measure the increase (as volumetry, colorimetry, spectrophotometry, fluorescent method and pH electrode method etc.) of product (free fatty acids); 2. measure the reduction (as turbidimetry, diffusion process etc.) of substrate; 3. measure the actual mass (double-antibody sandwich immunoassay method, latex agglutination) of LPS.At present at home mostly the laboratory mainly take volumetry, turbidimetry and spectrophotometry as main.Spectrophotometry has two classes more commonly used at present: 1. enzyme coupling colour developing colorimetry, multiplex 1, the 2-triglyceride is substrate, under the catalysis of LPS and monoglyceride lipase, hydrolysis generates glycerine and lipid acid, glycerine generates glycerol 3-phosphate by the glycerol kinase effect, then produces red-purple by GPO/peroxidase system and 4-AAP chromogen system.Variation in 550nm wavelength continuous monitoring absorbancy can be calculated the LPS activity.This class methods specificity is high, also substantially can solve the interference problem of endogenous glycerine by double reagent.2. 1, the continuous monitoring method of 2-o-two bays-racemize-glycerine-3-valeric acid-(6-methyl resorufin) ester design.This substrate is in alkaline environment, and under LPS and colipase effect, hydrolysis generates 1,2-O-dilauryl glycerine and pentanedioic acid-6 '-methyl resorufin.The latter is unstable, but Auto-decomposition generates pentanedioic acid and methyl resorufin.The methyl resorufin has absorption peak at 581nm wavelength place, but that its absorbancy of continuous monitoring changes quantitative assay LPS is active.This method has easy, quick, sensitive, stable and the characteristics such as immunity from interference is strong.But enzyme coupling development process, toolenzyme expensive, and also enzyme source is few and unstable, producer is seldom arranged in this way.Substrate hydrolysis method, substrate are extremely unstable, and what the background of reagent raise is exceedingly fast, and causes reagent to calibrate every day.Waste reagent is also wasted calibration object.And can cause the inaccurate of result.Therefore demand seeking better detection method urgently.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and the synthetic substrate of research and design stabilised fat enzyme improves reagent stability, for detection of the reagent of lipase.
The invention provides a kind of enzyme process and detect the lipase test kit, this test kit is by following reagent 1 and reagent 2, in 1 part: the ratio of 1 part forms:
Reagent 1:(2L) (reagent volume)
Reagent 2:(1L) (reagent volume)
It is 1L that deionized water adds to cumulative volume.
Described reagent 1 damping fluid is selected from TRIS (Tutofusin tris tris (hydroxymethyl) aminomethane), MOPS (3-(N-morpholine) propanesulfonic acid 3-(N-morpholino) propanesulfonic acid), BICINE (N, N-two (2-hydroxyethyl glycine) or HEPES (4-hydroxyethyl piperazine ethanesulfonic acid 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
The present invention detects the lipase test kit, and in use, reagent 1 adds reagent 2 after adding the detection sample again, can detect.
Another object of the present invention has been to provide the preparation method of lipase test kit: the method comprises the following steps:
(1) preparation reagent 1:(2L) (reagent volume)
(1) first be incorporated as the deionized water of total amount 80% in the container;
(2) add successively damping fluid, Sodium desoxycholate, calcium chloride, sodium azide;
(3) add colipase;
(4) adding at last deionized water to the cumulative volume of residual content is that 2L mixes, and get final product;
(2) preparation reagent 2:(1L) (reagent volume)
Add successively cow-bezoar Sodium desoxycholate, N.F,USP MANNITOL, proclin300 (biological preservative), Thiovanic acid, 6-methyl resorufin in container, it is that 1L mixes that deionized water adds to cumulative volume, and get final product.
The inventor is through research trial, added Substrate Protection agent N.F,USP MANNITOL and Thiovanic acid in test kit, wherein agent has no bibliographical information to Thiovanic acid as Substrate Protection, although, N.F,USP MANNITOL has the removal Free Radical, can reach the protective material effect but must use just together with Thiovanic acid; On the other hand, if N.F,USP MANNITOL does not add, Thiovanic acid self just is easy to oxidation and hydrolysis.After the present invention has added Substrate Protection agent N.F,USP MANNITOL and Thiovanic acid, guaranteed the stability of reagent.
Test kit of the present invention detects effective, and the substrate of the synthetic in reagent is had very strong provide protection, and is As time goes on more obvious to the provide protection meeting of substrate, and larger clinical value is arranged.
Embodiment
Following examples agents useful for same raw material all obtains by commercially available.
Embodiment 1 preparation lipase detection kit
Form:
Reagent 1:(2L) (reagent volume)
It is 2L that deionized water adds to cumulative volume;
Reagent 2:(1L) (reagent volume)
It is 1L that deionized water adds to cumulative volume.
Preparation:
(1) preparation reagent 1:(2L)
(1) first add the deionized water of 1.6L in the container;
(2) add successively TRIS damping fluid, Sodium desoxycholate, calcium chloride, sodium azide;
(3) add enzyme: colipase
(4) adding at last deionized water to cumulative volume is that 2L mixes, and get final product;
(2) preparation reagent 2:(1L)
Adding successively tartrate, cow-bezoar Septochol, N.F,USP MANNITOL proclin300 (biological preservative), Thiovanic acid, 6-methyl resorufin, deionized water to add to cumulative volume in the container is 2L, mixes.
Embodiment 2
Reagent 1:(2L)
It is 2L that deionized water adds to cumulative volume.
Reagent 2:(1L)
It is 1L that deionized water adds to cumulative volume.
Embodiment 3
Reagent 1:(2L)
It is 2L that deionized water adds to cumulative volume.
Reagent 2:(1L)
It is 1L that deionized water adds to cumulative volume.
The preparation method is with embodiment 1
Embodiment 4
The test kit of the embodiment of the present invention 1 detects effect test:
Measuring method:
Step 1: input parameter is in automatic biochemistry analyzer: 37 ℃ of temperature, and predominant wavelength 583nm, commplementary wave length 800nm,
R1 300ul (embodiment 1 reagent 1) .R2150ul (embodiment 1 reagent 2).
Contrast agents: Switzerland Luo Shi lipase test kit (reagent 1300ul; Reagent 2150ul does not wherein add N.F,USP MANNITOL, Thiovanic acid) manufacturer (Switzerland Roche Holding Ag);
Detect sample 4ul (serum sample)
Reading point: 16-24
Step 2: before measuring with R1 and R2 balance to room temperature., then put into agent bin
Step 3 input scaling ratio: 2218
In use, reagent 1 adds reagent 2 after adding the detection sample again, can detect.
(1) embodiment 1 reagent with N.F,USP MANNITOL, Thiovanic acid as the impact on detection reaction of protective material and sample reagent
(2) embodiment 1 reagent and the sample reagent provide protection to substrate
Above-mentioned detected result shows that test kit of the present invention adds N.F,USP MANNITOL and Thiovanic acid to the substrate in reagent, very strong provide protection to be arranged; As time goes on; can be more obvious to Protection of Substrates; add protectant reagent after 12 months and do not add protectant reagent and compare; the reagent background has only raise 10%, and the reagent background of sample reagent box has raise 10 times.
The clinical correlation comparison test of embodiment 5 and Luo Shi lipase test kit
The present embodiment 1 test kit and Luo Shi lipase test kit have been made clinical correlation relatively:
Get the clinical sample and parallel detection of the commercially available Luo Shi test kit that obtains (reagent 1:2*80ml reagent 2:16*10ml) manufacturer (Switzerland Roche Holding Ag) of 30 routine different concns
Use instrument: Olympus 400
Measuring method
Step 1: input parameter is in automatic biochemistry analyzer: 37 ℃ of temperature, and predominant wavelength 583nm, auxiliary wavelength 800nm,
In use, reagent 1 adds reagent 2 after adding the detection sample again, can detect.
Contrast agents: Switzerland Luo Shi lipase test kit (reagent 1300ul; Reagent 2150ul does not wherein add N.F,USP MANNITOL, Thiovanic acid) manufacturer (Switzerland Roche Holding Ag);
Detect sample 4ul (serum sample)
Reading point: 16-24
Step 2: before measuring with R1 and R2 balance to room temperature., then put into agent bin
Step 3 input scaling ratio: 2218
The above parameter of input on Olympus 400, in use, reagent 1 adds reagent 2 after adding the detection sample again, can detect.
Test-results:
According to top clinical comparison data, this reagent is good with the clinical correlation of Luo Shi reagent, there is no difference.Can for clinical, reduce medical expense.
Embodiment 6
Reagent 1:(2L)
It is 2L that deionized water adds to cumulative volume.
Reagent 2:(1L)
It is 1L that deionized water adds to cumulative volume.
The preparation method is with embodiment 1.
Embodiment 7
Reagent 1:(2L)
It is 2L that deionized water adds to cumulative volume.
Reagent 2:(1L)
It is 1L that deionized water adds to cumulative volume.
The preparation method is with embodiment 1.
Claims (3)
1. an enzyme process detects the lipase test kit, it is characterized in that, described test kit by following reagent 1 and reagent 2 in 1 part: the ratio of 1 part forms:
Reagent 1:2L
It is 2L that deionized water adds to cumulative volume
；
Reagent 2:1L
It is 1L that deionized water adds to cumulative volume.
2. enzyme process detects the lipase test kit according to claim 1, it is characterized in that, the damping fluid of described reagent 1 is selected from TRIS, MOPS or HEPES.
One kind as claimed in claim 1 enzyme process detect the preparation method of lipase test kit, it is characterized in that, described test kit by following reagent 1 and reagent 2 in 1 part: the ratio of 1 part forms:
Reagent 1:2L
It is 2L that deionized water adds to cumulative volume
；
Reagent 2:1L
It is 1L that deionized water adds to cumulative volume
；
Described preparation method comprises the following steps:
(1) preparation reagent 1:2L
(1) first be incorporated as the deionized water of total amount 80% in the container;
(2) add successively damping fluid, Sodium desoxycholate, calcium chloride, sodium azide;
(3) add colipase;
(4) adding at last deionized water to cumulative volume is 2L, mixes, and get final product;
(2) preparation reagent 2:1L
Add successively cow-bezoar Sodium desoxycholate, N.F,USP MANNITOL, proclin300, Thiovanic acid, 6-methyl resorufin in container, it is 1L that deionized water adds to cumulative volume,
Mix, and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110430794 CN103173518A (en) | 2011-12-20 | 2011-12-20 | Kit for detecting lipase by enzyme method and preparation method |
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|---|---|---|---|
| CN 201110430794 CN103173518A (en) | 2011-12-20 | 2011-12-20 | Kit for detecting lipase by enzyme method and preparation method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215632A (en) * | 2014-08-28 | 2014-12-17 | 宁波瑞源生物科技有限公司 | Stable lipase kit |
| CN105755103A (en) * | 2014-12-16 | 2016-07-13 | 上海复星长征医学科学有限公司 | Reagent for measuring lipase activity by stable enzymic method |
| 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 |
| 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 |
| 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 |
| CN110923292A (en) * | 2019-11-15 | 2020-03-27 | 中山市创艺生化工程有限公司 | Serum lipase detection kit and preparation method and application thereof |
| 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 |
-
2011
- 2011-12-20 CN CN 201110430794 patent/CN103173518A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215632A (en) * | 2014-08-28 | 2014-12-17 | 宁波瑞源生物科技有限公司 | Stable lipase kit |
| CN104215632B (en) * | 2014-08-28 | 2017-12-19 | 宁波瑞源生物科技有限公司 | A kind of fatty enzyme reagent kit of stabilization |
| CN105755103A (en) * | 2014-12-16 | 2016-07-13 | 上海复星长征医学科学有限公司 | Reagent for measuring lipase activity by stable enzymic method |
| 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 |
| 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 |
| 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 |
| CN110923292A (en) * | 2019-11-15 | 2020-03-27 | 中山市创艺生化工程有限公司 | Serum lipase detection kit and preparation method and application thereof |
| CN110923292B (en) * | 2019-11-15 | 2024-03-29 | 中山市创艺生化工程有限公司 | Serum lipase detection kit and preparation method and application thereof |
| 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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Application publication date: 20130626 |