CN102706818A - Enzymatic triglyceride measuring method and measuring reagent - Google Patents
Enzymatic triglyceride measuring method and measuring reagent Download PDFInfo
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- CN102706818A CN102706818A CN2012101878334A CN201210187833A CN102706818A CN 102706818 A CN102706818 A CN 102706818A CN 2012101878334 A CN2012101878334 A CN 2012101878334A CN 201210187833 A CN201210187833 A CN 201210187833A CN 102706818 A CN102706818 A CN 102706818A
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Abstract
The invention discloses a method for measuring triglyceride content of a sample by eliminating H2O2 produced by free glycerol through catalase in a reagent 1 of an enzymatic triglyceride measuring reagent and inhibiting the catalase in the reagent 1 through a catalase inhibitor in a reagent 2. According to the method, the H2O2 produced by the free glycerol is eliminated by adopting the catalase, and produced water and oxygen do not increase the cost; the catalase in the reagent is completely inhibited by the inhibitor during detection reaction, so that influence on the reaction is avoided; the method can solve the problems of increased instable background and low detection result accuracy and precision when the H2O2 is oxidized by peroxidase; and the method has the advantages of both high accuracy and high precision.
Description
Technical field
The present invention relates to the medical test technical field, be specifically related to a kind of enzyme process triglyceride determination method and measure reagent.
Background technology
Triglyceride (Triglyceride; TG) being the fat molecule that LCFA and glycerine form, is the maximum lipid of people's in-vivo content, and most tissues all can be utilized TG decomposition product energize; Tissues such as liver, fat can also carry out the synthetic of TG simultaneously, in adipose tissue, store.
Check branch of Chinese Medical Association recommends two step enzyme methods as the serum TG conventional determining method in nineteen ninety-five, and this method generates glycerine and fatty acid with lipoprotein lipase (LPL) hydrolysis TG; Glycerine after the hydrolysis and atriphos (ATP) generate H under the catalysis of glycerokinase (GK) and phosphoglycerol oxidase (glycerol-3-phosphate oxidase GPO)
2O
2, under peroxidase (POD) catalysis, H
2O
2, 4 amino-antipyrines (4-AAP) and 2,4-two chlorophenols (also have the derivant, aniline salt and the benzene sulfonate that use 4-chlorophenol, phenol; Below just with 2; 4-two chlorophenols are representative narration, and other chromogens no longer repeat to mention) form coloured dyestuff (often being quinone imides); The growing amount of these materials is directly proportional with TG content in the sample, calculates corresponding TG concentration through AAS at last.Chemical equation 1 ~ 4 is seen in concrete reaction.
This method has easy and simple to handle quick, and precision is high, and high specificity reacts linear wide ranges, is prone to reach terminal point, can eliminate advantages such as piarhemia, haemolysis, jaundice interference, can be used as double reagent and uses, and also can two kinds of reagent be lumped together as single reagent and use.But; Because original dissociative glycerin has all been participated in reaction in glycerine that produces after the TG hydrolysis in the serum and the serum; So with single reagent mensuration is that the total glyceride of serum (is defined as TG and dissociative glycerin and a small amount of diglyceride, monoglyceride sum; Custom is referred to as TG), its reaction result can not actual response serum in the content of TG.
Disturb in order to remove dissociative glycerin, the scheme that addresses this problem employing in the prior art has: (1) is reagent 2 with 4-AAP and LPL component, and all the other compositions all add in the reagent 1.This method is through adding the first step preincubate phase behind the reagent 1, dissociative glycerin is converted into colourless 2, the dimer of 4-two chlorophenols and benzene oxygen radical.After adding reagent 2, the reaction of after chemical reaction formula (1)-(4) under the catalysis of lipoprotein lipase of the TG in the serum finally is converted into red quinone imines, but because 2; The dimer of 4-two chlorophenols is unstable, adds reagent, after 2; Under the situation that 4-AAP exists; 2, the dimer and the benzene oxygen radical of 4-two chlorophenols also are converted into red quinone imines, and the quinone imines of being surveyed is actual still to be the quinone imines sum that dissociative glycerin and TG produce.(2) during double-colored former material coexisted reagent 1, reagent 2 only contained LPL effective constituent.The glycerine that free glycerine and TG hydrolysis produce in this scheme is colour generation successively; Instrument is a blank with the quinone imines that first step reaction produces, and the quinone imines that only produces with second step calculates TG content, removes the influence of dissociative glycerin with this; But because the absorption signal that dissociative glycerin produces is lower and unstable; Therefore this method reagent accuracy, precision are relatively poor, and clinical effectiveness repeatability is bad, can't satisfy the conventional sense requirement.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, propose a kind of can avoiding originally with superoxide oxydasis H
2O
2Cause the appearance of that background astatically raises, accuracy and the relatively poor phenomenon of precision, but the H that can produce through the reaction of hydrogen peroxide oxydasis dissociative glycerin
2O
2, generate colourless water and oxygen, and suppress hydrogen peroxidase by the suppressant in the reagent, make testing result accuracy, the enzyme process triglyceride determination method that precision is good.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of enzyme process triglyceride determination method, and utilize the hydrogen peroxidase in the reagent 1 of enzyme process triglyceride determination reagent to eliminate the H that dissociative glycerin produces
2O
2, and then by the hydrogen peroxidase in the inhibition of the hydrogen peroxide enzyme inhibitor in the reagent 2 reagent 1, thereby the method for TG content in the mensuration sample, course of reaction is following:
First step reaction
The reaction of second step
Suppressant suppresses catalase activity (4)
The present invention also provides a kind of mensuration reagent of above-mentioned enzyme process triglyceride determination method, and this reagent is made up of reagent 1 and reagent 2, and wherein each component and the component concentration ranges in reagent 1, the reagent 2 is:
Reagent 1:
Reagent 2:
The above-mentioned reagent of the present invention can be dry powder, and use the back that is dissolved in water before use; Also can process liquid reagent, directly use; The industry routine techniques is all adopted in the configuration of mentioned reagent.
Wherein said damping fluid can be the amino damping fluid of phosphate buffer, trihydroxy methyl, glycocoll NaOH damping fluid, N-2-hydroxyethyl piperazine-N'-2-ethyl sulfonic acid damping fluid, N-three (methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-three (methylol) methyl-2-tarine damping fluid, piperazine-N, one or more in two (2-hydroxyethanesulfonic acid) damping fluids of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morphine quinoline) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonic acid damping fluid, two (2-hydroxyethyl) amino of 3--2-hydroxy-propanesulfonic acid damping fluid, 3-(encircling amine)-2-hydroxyl-1-propane sulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-(encircling amine)-1-propane sulfonic acid damping fluid, 3-morpholine propane sulfonic acid damping fluid, N-three (methylol) the methyl-3-aminopropanesulfonicacid acid damping fluid.
Said chromogen comprise phenolic compound (like the 2-chlorophenol, 2,4-chlorophenesic acid, 4-chlorophenol; 2, the 6-chlorophenesic acid), and/or the aniline analog is (like N-ethyl-N-(2-hydroxyl-3-third sulfo group) meta-aminotoluene, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt; N-ethyl-N-(2-hydroxyl-3-sulfopropyl l)-3-aminoanisole sodium salt (dihydrate), N-ethyl-N-(3-sulfopropyl) aniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-aminoanisole sodium salt; N-ethyl-N-(3-sulfopropyl) aniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt; N-(2-hydroxyl-3-sulfopropyl)-35-dimethoxyaniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-xylidin sodium salt); 3-hydroxyl-2,4,6-Triiodobenzoic acid a kind of; Preferred 3-hydroxyl-2,4, the 6-Triiodobenzoic acid.
Said suppressant is an azide, azanol, fluoride, acetate, formates, ethanol, methyl alcohol.Preferred azide.
Said antiseptic is selected from one or more in potassium sorbate, Sodium Benzoate, sodium nitrite, the proclin series antiseptic (like Proclin300).
Said stabilizing agent is selected from one or several among polyglycol, glycerine, propylene glycol, sucrose, trehalose, sorbierite, the BSA (bovine serum albumin(BSA)).
Described surfactant; Preferably; Said surfactant is a non-ionic surfactant, and more preferably, said non-ionic surfactant is selected from a kind of in TWEEN series (like Tween20), SPAN series (like Span-80), the TRITON series (like TritonX-100).
Advantage of the present invention and beneficial effect:
The present invention adopts hydrogen peroxidase to eliminate the H that dissociative glycerin produces
2O
2, the water of generation and oxygen can not cause background to raise, and the hydrogen peroxidase in the reagent is suppressed agent when detection reaction suppresses fully, can not impact reaction; Therefore the present invention can evade with superoxide oxydasis H
2O
2Causing astatically, background raises, cause testing result accuracy, the bad problem of precision; Therefore, the present invention has all advantages preferably of accuracy, precision.
Embodiment
To further specify the present invention through following non-limiting example below, as well known to those skilled in the art, under the situation that does not deviate from spirit of the present invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.
The collocation method of following reagent is conventional method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.
Embodiment
Reagent 1:
Reagent 2:
The compound method of reagent 1 and reagent 2 is a conventional method, and promptly reagent 1 and reagent 2 said components add respectively to mix to stir separately behind the distilled water and get final product.
Embodiment 2
Reagent 1:
Reagent 2:
The preparation method of embodiment 2 reagent is with embodiment 1.
Embodiment 3
Reagent 1:
Reagent 2:
The preparation method of embodiment 3 reagent is with embodiment 1.
The test condition that reagent of the present invention is measured TG in the sample is following: temperature: 37 ℃; The cuvette optical path is 1.0cm.Detect predominant wavelength 546nm, commplementary wave length 700nm.
Using TG of the present invention measures reagent to measure the method for TG in the sample following: sample (calibration tube is made sample with calibration object) adds the R1 mixing, and 37 ℃ add the R2 mixing after hatching 5m in, record absorbance A 1, and behind 37 ℃ of reaction 5m in, record absorbance A 2.Sample consumption 3 μ l wherein, reagent 1 consumption 200 μ l, reagent 2 consumptions 100 μ l.
TG content calculates by following formula in the reagent mensuration sample of the present invention:
Obtain TG of the present invention and measure the concentration that reagent is measured TG in the sample.
Reference examples 4
Reagent 1:
Reagent 2:
The test condition that reagent is measured TG in the sample is following: temperature: 37 ℃; The cuvette optical path is 1.0cm.Detect predominant wavelength 546nm, commplementary wave length 700nm.
Using TG of the present invention measures reagent to measure the method for TG in the sample following: sample (calibration tube is made sample with calibration object) adds the R1 mixing, hatches record absorbance A 1 behind the 5min for 37 ℃, adds the R2 mixing, behind 37 ℃ of reaction 5min, and record absorbance A 2.Sample consumption 3 μ l wherein, reagent 1 consumption 200 μ l, reagent 2 consumptions 100 μ l.
To the target value be the control liquid I of 1.18 (1.00-1.36) mmol/L and control liquid II that the target value is 2.51 (2.12-2.90) mmol/L under the same conditions; Adopt reagent to the TG concentration continuous detecting of same control liquid 20 times; The mean value and the target value scope of testing result are compared,, compare the coefficient of variation of each mensuration simultaneously to detect the accuracy of described reagent; To detect the precision of said embodiment reagent, the result is as shown in table 1:
Table 1:
The result of table 1 shows: the accuracy of reagent of the present invention, precision are all better.
Claims (10)
1. enzyme process triglyceride determination method is characterized in that: utilize the hydrogen peroxidase in the reagent 1 of enzyme process triglyceride determination reagent to eliminate the H that dissociative glycerin produces
2O
2, and then by the hydrogen peroxidase in the inhibition of the hydrogen peroxide enzyme inhibitor in the reagent 2 reagent 1, thereby the method for content of triglyceride in the mensuration sample.
2. the mensuration reagent of the described enzyme process triglyceride determination of claim 1 method is characterized in that: this reagent is made up of reagent 1 and reagent 2, and wherein each component and the component concentration ranges in reagent 1, the reagent 2 is:
Reagent 1:
Reagent 2:
3. the mensuration reagent of enzyme process triglyceride determination method according to claim 2; It is characterized in that: said damping fluid can be the amino damping fluid of phosphate buffer, trihydroxy methyl, glycocoll-NaOH damping fluid, N--2 hydroxyethyl piperazine-N'-2-ethyl sulfonic acid damping fluid, N-three (methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, N-three (methylol) methyl-2-tarine damping fluid, piperazine-N, one or more in two (2-hydroxyethanesulfonic acid) damping fluids of N-, 3-morpholine-2-hydroxypropionate sodium damping fluid, 3-(N-morphine quinoline) ethyl sulfonic acid sodium damping fluid, 4-(2-hydroxyethyl) piperazine-1-2-hydroxy-propanesulfonic acid damping fluid, N-(2-hydroxyethyl) piperazine-N'-4-fourth sulfonic acid damping fluid, two (2-hydroxyethyl) amino of 3--2-hydroxy-propanesulfonic acid damping fluid, 3-(encircling amine)-2-hydroxyl-1-propane sulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-(encircling amine)-1-propane sulfonic acid damping fluid, 3-morpholine propane sulfonic acid damping fluid, N-three (methylol) the methyl-3-aminopropanesulfonicacid acid damping fluid.
4. the mensuration reagent of enzyme process triglyceride determination method according to claim 2 is characterized in that: said chromogen comprises phenolic compound, and/or the aniline analog, 3-hydroxyl-2,4,6-Triiodobenzoic acid a kind of.
5. the mensuration reagent of enzyme process triglyceride determination method according to claim 4 is characterized in that: described phenolic compound is the 2-chlorophenol, 2, and 4-chlorophenesic acid, 4-chlorophenol, 2,6-chlorophenesic acid; Described aniline analog is N-ethyl-N-(2-hydroxyl-3-third sulfo group) meta-aminotoluene, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl l)-3-aminoanisole sodium salt (dihydrate); N-ethyl-N-(3-sulfopropyl) aniline sodium salt; N-ethyl-N-(3-sulfopropyl)-3-aminoanisole sodium salt, N-ethyl-N-(3-sulfopropyl) aniline sodium salt, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3; 5-dimethoxyaniline sodium salt; N-(2-hydroxyl-3-sulfopropyl)-3 5-dimethoxyaniline sodium salts, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-xylidin sodium salt.
6. the mensuration reagent of enzyme process triglyceride determination method according to claim 2 is characterized in that: described suppressant is an azide, azanol, fluoride, acetate, formates, a kind of in ethanol, the methyl alcohol.
7. the mensuration reagent of enzyme process triglyceride determination method according to claim 2 is characterized in that: described antiseptic is one or more in potassium sorbate, Sodium Benzoate, sodium nitrite, proclin series antiseptic.
8. the mensuration reagent of enzyme process triglyceride determination method according to claim 2 is characterized in that: described stabilizing agent is one or several in polyglycol, glycerine, propylene glycol, sucrose, trehalose, sorbierite, the bovine serum albumin(BSA).
9. the mensuration reagent of enzyme process triglyceride determination method according to claim 2 is characterized in that: described surfactant is a non-ionic surfactant.
10. the mensuration reagent of enzyme process triglyceride determination method according to claim 9 is characterized in that: described surfactant is a kind of in serial of, TRITON serial for TWEEN series, SPAN.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101878334A CN102706818A (en) | 2012-06-05 | 2012-06-05 | Enzymatic triglyceride measuring method and measuring reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101878334A CN102706818A (en) | 2012-06-05 | 2012-06-05 | Enzymatic triglyceride measuring method and measuring reagent |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498586A (en) * | 2014-11-28 | 2015-04-08 | 山东博科生物产业有限公司 | Single reagent serum triglyceride detection reagent with strong stability |
| CN106399460A (en) * | 2016-09-29 | 2017-02-15 | 四川迈克生物科技股份有限公司 | Kit and method for determining triglyceride |
| CN107064123A (en) * | 2017-01-03 | 2017-08-18 | 长沙中生众捷生物技术有限公司 | The detection reagent of triglycerides and the Test paper of triglycerides |
| 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 |
| CN108949903A (en) * | 2017-05-17 | 2018-12-07 | 广州市伊川生物科技有限公司 | A kind of triglyceride determination kit and its measuring method |
| CN111808920A (en) * | 2020-06-11 | 2020-10-23 | 武汉生之源生物科技股份有限公司 | Method for removing interference on TBA detection, TC kit and TG kit |
| CN114854705A (en) * | 2022-05-24 | 2022-08-05 | 武汉瀚海新酶生物科技有限公司 | Chemically modified glycerol-3-phosphate oxidase and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498586A (en) * | 2014-11-28 | 2015-04-08 | 山东博科生物产业有限公司 | Single reagent serum triglyceride detection reagent with strong stability |
| CN104498586B (en) * | 2014-11-28 | 2016-06-22 | 山东博科生物产业有限公司 | The single reagent serum triglycerides detectable that a kind of stability is strong |
| CN106399460A (en) * | 2016-09-29 | 2017-02-15 | 四川迈克生物科技股份有限公司 | Kit and method for determining triglyceride |
| CN107064123A (en) * | 2017-01-03 | 2017-08-18 | 长沙中生众捷生物技术有限公司 | The detection reagent of triglycerides and the Test paper of triglycerides |
| CN108949903A (en) * | 2017-05-17 | 2018-12-07 | 广州市伊川生物科技有限公司 | A kind of triglyceride determination kit and its measuring method |
| 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 |
| CN111808920A (en) * | 2020-06-11 | 2020-10-23 | 武汉生之源生物科技股份有限公司 | Method for removing interference on TBA detection, TC kit and TG kit |
| CN114854705A (en) * | 2022-05-24 | 2022-08-05 | 武汉瀚海新酶生物科技有限公司 | Chemically modified glycerol-3-phosphate oxidase and application thereof |
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Application publication date: 20121003 |