CN101762577B - Two-step enzyme testing method of triglycercide in blood serum - Google Patents
Two-step enzyme testing method of triglycercide in blood serum Download PDFInfo
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- 238000002835 absorbance Methods 0.000 claims description 8
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Abstract
The invention discloses a two-step enzyme testing method of triglycercide in blood serum, belonging to a method for testing material according to the color change of the result of test reaction with visible light. The method has the technical scheme as follows: double-color raw materials are in reagent I, and reagent II only has active constituent of lipoprotein lipase. The test method comprises the steps of: bathing the blood serum and the reagent I under the temperature of 37 DEG C for 3-5 minutes; reaching free glycerin in the blood serum with the reagent I to generate quinone imide; bathing under the temperature of 37 DEG C for 4-7 minutes after adding the reagent II; and hydrolyzing the triglycercide and reacting to generate the red quinone imide. An instrument is used for detecting at the position with the wave length of 500-520nm, the quinone imide generated by the reaction of the reagent I is taken as blank, and the content of the triglycercide is computed through the quinone imide generated by the reaction of the reagent II. The method is not influenced by endogenic glycerine, has the same steps and range as the existing two-step method, does not increase the cost of the reagent, and is economic and convenient, thereby being a triglycercide testing method with higher accuracy.
Description
Technical field
The invention belongs to a kind of assay method that comprises enzyme; Or utilizing visible light, the result by test reaction produces the method that change color is come test material, particularly relates to a kind of two-step enzyme testing method that detects triglyceride in the serum with Biochemical Analyzer.
Background technology
The assay method of triglyceride in the serum (TG) generally can be divided into chemical method, enzyme process and chromatography 3 big classes.Early stage assay method is the difference estimation with TL and cholesterol and phosphatide.Chemical method is with the TG in the organic solvent extracting sample, remove in the extract chaff interference such as phosphatide after, with basic hydrolysis (saponification) TG, generate formaldehyde with periodate oxidation glycerine, survey formaldehyde with chromogenic reaction then.Be methylene chloride-silicic acid-variable color acid system (Van Handel-Caslson method) more accurately, this method extracting fully, can remove phosphatide and glycerine interferences, chromotropic acid developing sensitivity height, color stability, still be the internal reference method of U.S. CDC (CDC) so far.But high being unsuitable for of, technical requirement various because of operation steps used in the routine clinical testing.Chromatography comprises methods (ID/GC/MS) such as nucleic dilution/gas chromatography/mass spectrometry, mainly as the foundation of decisive method in the frame of reference and the preparation and the definite value of reference material, and this method expense costliness, the sample preparation complexity is difficult to apply.Use the triglyceride in the high performance liquid chromatograph mensuration serum in addition, detect the cost height, be difficult at clinical expansion.
The serum TG level all detects with enzyme process in domestic nearly all clinical labororatory at present, the method is divided into single stage method and two-step approach, though method is different, generally all comprise following fundamental reaction step: generate glycerine and fatty acid with only lipoprotein lipase (LPL) hydrolysis TG; Glycerine after the hydrolysis and atriphos (ATP) generate H under the catalysis of glycerokinase (GK) and phosphoglycerol oxidase (GPO)
2O
2, under the catalysis of peroxidase (POD), H
2O
24-amino-antipyrine (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 the representative narration, other chromogens no longer repeat to mention) form coloured dyestuff (often for quinone imides), the growing amount of these materials is directly proportional with content of triglyceride in the sample.Calculate corresponding TG concentration by spectrophotometric method at last.Chemical equation 1～4 is seen in concrete reaction.
What TG adopted in the Biochemical Analyzer detection serum is enzyme process, generally adopts compositions and methods (GPO-PAP method) such as LPL, GPO, POD, 4-amino-antipyrine and phenol.Its single stage method is with LPL, GK, GPO, POD, 4-amino-antipyrine and 2, and compositions such as 4-two chlorophenols, magnesium salts, surfactant and antiseptic are dissolved in the Tris-HCl damping fluid and are mixed with single agents.Prepare by Chinese Medical Association's recommendation method: every liter of principal ingredient that contains of Tris-HCl damping fluid has: Tris 0.15mmol (pH7.6), 2,4-chlorophenesic acid 2.5mmol, MgSO
410mmol, sodium taurocholate 3.5mmol, Triton X-1000.1g, ATP1.0mmol.GK 500U, GPO 3000U, POD1000U, LPL 3000U, 4-amino-antipyrine 1.2mmol.This method have easy fast, trace, advantage that precision is high, and high specificity reacts linear wide ranges, easily reaches terminal point.But because original dissociative glycerin has all participated in reaction in glycerine that produces after the TG hydrolysis in the serum and the serum, so with single stage method 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), make reaction result can not truly reflect the content of TG in the serum.Check branch of Chinese Medical Association recommends two step enzyme methods of GPO-PAP method as the serum TG conventional determining method in nineteen ninety-five.This method is that former single reaction reagent is divided into double reagent, and wherein LPL and 4-amino-antipyrine are formed the reagent II, and all the other one-tenth are grouped into the reagent I, and hope can reduce the interference of dissociative glycerin.By Chinese Medical Association's recommendation method preparation, every liter of Tris-HCl damping fluid of its reagent II contains principal ingredient to be had: Tris 0.15mmol (pH7.6), LPL 30000U, 4-amino-antipyrine 1.0mmol.Reagent I: every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), 2,4-two chlorophenol 3.0mmol, MgSO
412mmol, sodium taurocholate 4.2mmol, Triton X-1000.12g, ATP1.2mmol, GK 600U, GPO 3600U, POD 1200U.
Domestic most of clinical labororatory adopts a step enzyme method and two step enzyme methods to detect triglyceride.The kit of more domestic reagent manufacturer production two step enzyme methods is with GK, GPO, POD, 2, and 4-two chlorophenols are reagent I, with LPL, 4-amino-antipyrine as the reagent II.The producer that also has is with GK, GPO, 2, and 4-two chlorophenols are reagent I, with LPL, 4-amino-antipyrine and POD as the reagent II.These kits both can be used as double reagent and had used, and also two kinds of reagent can be lumped together as single reagent and use.The two step enzyme methods that Chinese Medical Association is recommended can be eliminated the advantages such as interference of piarhemia, haemolysis, yellow subcutaneous ulcer, but also have following problem in actual applications:
Now institute is in the two step enzyme methods of reagent II in order to 4-amino-antipyrine and lipoprotein lipase component, and the first step preincubate phase behind adding reagent I, it is colourless 2 that dissociative glycerin is converted into, and the dimer of 4-two chlorophenols and benzene oxygen radical are seen reaction equation 5.After adding the reagent II, the reaction of after chemical reaction formula (1)～(4) under the catalysis of lipoprotein lipase of the triglyceride in the serum finally is converted into red quinone imines.But because 2, the dimer instability of 4-two chlorophenols is after the adding reagent II, under the situation that the 4-amino-antipyrine exists, 2, the dimer and the benzene oxygen radical of 4-two chlorophenols also are converted into red quinone imines, the actual quinone imines sum for dissociative glycerin and triglyceride generation of the quinone imines of being surveyed.Existing two step enzyme methods can not be eliminated the interference of the dissociative glycerin in the serum, make detected value higher, bring misleading to clinical diagnosis.
Summary of the invention:
In order to solve in the prior art problem that the assay method of TG exists endogenous glycerine to disturb in the serum, it is convenient and easy to the invention provides a kind of economy, the two-step enzyme testing method that accuracy is higher, can eliminate the serum triglyceride of endogenous glycerine influence.
The technical scheme that solves this technical problem employing is: double-colored former material coexists among the reagent I, and reagent II only contains lipoprotein lipase effective constituent; Its assay method is: serum was bathed 3～5 minutes in 37 ℃ of temperature with the reagent I earlier, and free glycerine and the reaction of reagent I generate quinone imines in the serum, add the reagent II and bathe 4～7 minutes in 37 ℃ of temperature, and triglyceride hydrolysis is after series reaction generates red quinone imines.Instrument detects at 500nm wavelength place, and the quinone imines that reacts with the reagent I is a blank, is calculated the content of triglyceride by the quinone imines of reagent II reaction generation.
Every liter of Tris-HCl damping fluid of mentioned reagent I includes Tris 0.10mmol～0.20mmol, 2,4-two chlorophenol 2.0mmol～4.0mmol, MgSO
49mmol～15mmol, sodium taurocholate 3.0mmol～5.5mmol, Triton X-1000.08g～0.15g, ATP 0.8mmol～2.0mmol, GK 400U～800U, GPO3000U～4200U, POD 800U～1600U, 4-amino-antipyrine 0.8mmol～2.4mmol, Procl in300 antiseptic 100 μ l～300 μ l; Every liter of Tri s-HCl of reagent II damping fluid includes Tris 0.10mmol～0.20mmol, LPL 16000U～32000U, Proclin300 antiseptic 100 μ l～300 μ l.
One of double-colored former material is the 4-amino-antipyrine in the mentioned reagent I, and another is selected from 2, the derivant of 4-two chlorophenols or phenol.
The pH value of Tri s-HCl damping fluid is 7.6 ± 0.2 in mentioned reagent I and the reagent II.
The volume ratio of reactant is in the said determination: sample: reagent I: reagent II=1: 75～80: 25～20.
Though the reagent of this method is identical with original two step enzyme method reagent compositions, just the 4-amino-antipyrine is changed into and 2, the 4-two chlorophenols reagent I that coexists, but its reaction product that adds after reagent I is different with original adding reagent I afterreaction product, measures obviously difference of effect.
Contain GK, GPO, POD, 4-amino-antipyrine and 2 in the reagent I of the present invention, components such as the double-colored former material of 4-two chlorophenols, the reagent II only contains the lipoprotein lipase active principle.Serum is bathed with reagent I temperature earlier, owing to include GK, GPO, POD, 4-amino-antipyrine and 2 in the reagent I, double-colored former materials such as 4-two chlorophenols, therefore free glycerine generates quinone imines in the reaction of reagent I, after adding the reagent II, triglyceride generates glycerine by the lipoprotein lipase hydrolysis, through GK, GPO, POD, 4-amino-antipyrine and 2, the double-colored former material effect of 4-two chlorophenols makes also to generate red quinone imines after the TG hydrolysis.By the Instrument measuring parameter is set, instrument is made as blank with the quinone imines that the reaction of reagent I generates, and reacts the content that the quinone imines that generates calculates TG by the reagent II, and measurement result has been eliminated the influence that original dissociative glycerin disturbs like this.
Among the present invention 2,4-two chlorophenols also can select for use derivant, aniline salt and the benzene sulfonate of 4-chlorophenol, phenol to substitute.
The present invention compared with prior art has following advantage: two of triglyceride steps were not influenced by endogenous glycerine in the serum of the present invention when enzyme method detects, and the range of linearity can reach 11.3mmol/L.Its using method is identical with original two step enzyme methods with scope, can not increase experimenter's burden, does not increase reagent cost, and is economical convenient and easy, is the higher TG detection method of a kind of accuracy.
Description of drawings
Accompanying drawing is the response curve figure that the present invention measures healthy human body TG.
Embodiment:
Below by embodiment and accompanying drawing the present invention is described in further details.
Embodiment 1
The composition of reagent:
A. reagent I:
Every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), 2,4-two chlorophenol 3.0mmol, MgSO
412mmol, sodium taurocholate 4.2mmol, Triton X-1000.12g, ATP 1.2mmol, GK 600U, GPO 3600U, POD 1200U, 4-amino-antipyrine 1.6mmol, Proclin-300 antiseptic 200 μ l.
B. reagent II:
Every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), LPL 24000U, Proclin-300 antiseptic 200 μ l.
C. titer: 2mmol/L trioleate aqueous solution.
Wherein, MgSO
4Be the activator of glycerokinase, Triton X-100 is a surfactant, and Proclin-300 is the efficient liquid antiseptic, and sodium taurocholate is a bacteriostatic agent.
Embodiment 2.
With 2,4-two chlorophenols change the 4-chlorophenol in the reagent I.Content is constant.Other compositions are all constant, and the reagent II is constant.
Embodiment 3
The mensuration program
Double reagent method: on the full-automatic Biochemical Analyzer of Japanese OLYMPUS AU2700, instrument joins 2 μ l samples mixing in the 150 μ l reagent I automatically, hatches 3 minutes for 37 ℃, adds 50 μ l reagent II mixings, hatched 5.1 minutes for 37 ℃, fully-automatic analyzer detects at 500nm wavelength place.Instrument calculates TG result automatically.Specifically see Table 1
Table 1. robotization Biochemical Analyzer of the present invention test condition
Reaction OD
TGCalculated value=OD
2-OD
1* [(SV+R
1V
1)/(SV+R
1V
1+ R
2V
2)]
Triglyceride concentration=F * OD
TG
OD wherein
TGIt is the absorbance that triglyceride produces.OD
1Be that sample adds the absorbance that records after the reaction of reagent I, OD
2Be that sample adds the absorbance that records after the reaction of reagent II, SV is the volume of blood serum sample, R
1V
1Be the volume of reagent I, R
2V
2It is the volume of reagent II.F is a correction factor.
OD behind the adding reagent II
1Extension rate be [(SV+R
1V
1)/(SV+R
1V
1+ R
2V
2)], the absorbance behind the adding reagent II is OD
1* [(SV+R
1V
1)/(SV+R
1V
1+ R
2V
2)].So the absorbance of the quinone imines that is produced by triglyceride is: OD
TG=OD
2-OD
1* [(SV+R
1V
1)/(SV+R
1V
1+ R
2V
2)], i.e. OD
TG=OD
2-(2+150)/(2+150+50) * OD
1As long as measure OD
1And OD
2Can calculate the concentration of triglyceride.
Below by adopting three kinds of glycerine recovery in the different detectable detection serum, further specify good effect of the present invention.The glycerine recovery is the percent of ratio of the glycerine numerical value of the glycerine numerical value measured of reaction back and adding.Glycerine is the geometric ratio product of triglyceride in this reaction.It is best that the glycerine mensuration recovery does not influence triglyceride determination with the glycerine that adds.Be that the glycerine recovery is low more, illustrate that glycerine is low more to the triglyceride determination influence.Glycerine does not count in the triglyceride determination value.
1. detected object: health examination personnel 86 people, the male sex 52 people wherein, 42.5 years old mean age; Women 34 people, 39.5 years old mean age, blood sampling on an empty stomach.
2. employing method and reagent
2.1 reagent:
(1) reagent A: single agents, prepare by Chinese Medical Association's recommendation method: the Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), 2 for every liter, 4-chlorophenesic acid 2.5mmol, MgSO
410mmol, sodium taurocholate 3.5mmol, Triton X-1000.1g, ATP 1.0mmol.GK 500U, GPO 3000U, POD 1000U, LPL 3000U, 4-amino-antipyrine 1.2mmol.
(2) reagent B: by Chinese Medical Association's recommendation method preparation, mentioned reagent is divided into reagent I and reagent II, wherein reagent II: every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), LPL 30000U, 4-amino-antipyrine 1.0mmol.Reagent I: every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), 2,4-two chlorophenol 3.0mmol, MgSO
412mmol, sodium taurocholate 4.2mmol, TritonX-1000.12g, ATP 1.2mmol, GK 600U, GPO 3600U, POD 1200U.
(3) reagent C: be mixed with double reagent, the amino antipyrine and 2 of 4-, 4-two chlorophenols coexist among the reagent I, and reagent II only has lipoprotein lipase effective constituent.Reagent II: every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), LPL 24000U, Proclin-300 antiseptic 200 μ l.Reagent I: every liter of Tris-HCl damping fluid includes Tris 0.15mmol (pH7.6), 2,4-two chlorophenol 3.0mmol, MgSO
412mmol, sodium taurocholate 4.2mmol, Triton X-1000.12g, ATP 1.2mmol, GK 600U, GPO 3600U, POD 1200U, 4-amino-antipyrine 1.6mmol, Proclin-300 antiseptic 200 μ l.
(4) glycerine (analyze pure, sigma company, molecular weight 92.09, density is 1.2613mg/dl, 25 ℃)
2.2. instrument: Japanese Olympus AU2700 type automatic clinical chemistry analyzer
2.3. method
2.3.1 measure 86 parts of healthy population triglyceride levels with reagent A, three kinds of reagent of B, C respectively, and carry out the result relatively.
2.3.2. above-mentioned serum is hybridly prepared into pooled serum, add 0,0.85,1.70 respectively, 3.40mmol/L glycerine, measure with reagent A, three kinds of reagent of B, C respectively, relatively the recovery tests of three kinds of methods.
2.3.3. the assay method of three kinds of reagent:
Reagent A: sample: reagent=1: 100.37 ℃, in 8.1 minutes reaction time, 500nm wavelength place end-point method is measured.
Reagent B: sample: reagent I: reagent II=1: 75: 25.37 ℃, sample and reagent I temperature were bathed 3 minutes, added reagent II, reacted 5.1 minutes, and 500nm wavelength place end-point method is measured.
Reagent C: sample: reagent I: reagent II=1: 75: 25.37 ℃, sample and reagent I temperature were bathed 3 minutes, added reagent II, reacted 5.1 minutes, and 500nm wavelength place end-point method is measured.
3. three kinds of methods are measured TG relatively:
Pass through statistical analysis: adopt reagent A and two kinds of assay method measurement results of reagent B there was no significant difference, t=1.04, P=0.3338, correlativity is good, r=0.984, Y
Reagent A=0.9307+0.931X
Reagent B, two kinds of assay methods of reagent A and reagent B all can not be eliminated the dissociative glycerin influence and see Table 2, adopt the assay method measurement result of reagent C and reagent B significant difference to be arranged, t=7.03, P=0.0026.Reagent C has been eliminated the dissociative glycerin interference and has been seen Table 2.
Table 2. reclaims experimental data table (n=34)
In the reagent A single agents, dissociative glycerin and TG react the generation quinone imines simultaneously, and result of calculation is dissociative glycerin and TG sum.Among the recommend method reagent B, after serum added reagent I, in the first step preincubate phase, dissociative glycerin was converted into unsettled colourless 2, the dimer of 4-two chlorophenols, after adding reagent II, under the situation that the 4-amino-antipyrine exists, 2, the dimer of 4-two chlorophenols is converted into red quinone imines, finally also be converted into quinone imines after the TG hydrolysis, the quinone imines of surveying is the sum that dissociative glycerin and TG produce, and the influence of dissociative glycerin is not eliminated as yet.In the reagent C of the present invention, in the first step preincubate phase, dissociative glycerin is converted into quinone imines, after adding reagent II, starts the TG hydrolysis reaction, and TG is converted into quinone imines, and response curve is seen accompanying drawing.The red quinone imines that instrument produces first step reaction is blank, goes on foot with second and reacts the content that the quinone imines that produces calculates TG, and be real TG content.
Can find out through above comparison, though the present invention forms identical with original formulation, but because of the amino antipyrine and 2 of 4-, 4-two chlorophenols coexist among the reagent I, reagent II only has lipoprotein lipase, so it is different with the reaction behind the existing adding reagent I that serum adds the reaction that takes place behind the reagent I, actual effect is also different fully.Therefore, the present invention can guarantee the accuracy that triglyceride detects in the serum by the component that changes reagent I, II.
Claims (5)
1. the two-step enzyme testing method of triglyceride in the serum is characterized in that double-colored former material coexists among the reagent I, and reagent II only contains lipoprotein lipase effective constituent; Its assay method is: serum elder generation and reagent I were bathed 3～5 minutes in 37 ℃ of temperature, and the reaction generation quinone imines of free glycerine and reagent I was bathed 4～7 minutes in 37 ℃ of temperature after the adding reagent II in the serum, and triglyceride hydrolysis is after reaction also generates quinone imines; Instrument detects at 500-520nm wavelength place, and the quinone imines that produces with the reaction of reagent I is a blank, reacts the content that the quinone imines that produces calculates triglyceride by the reagent II, and computing formula is:
OD
TG＝OD
2-OD
1×[(SV+R
1V
1)/(SV+R
1V
1+R
2V
2)]
Triglyceride concentration=F * OD
TG
OD wherein
TGBe the absorbance that triglyceride produces, OD
1Be that sample adds the absorbance that records after the reaction of reagent I, OD
2Be that sample adds the absorbance that records after the reaction of reagent II, SV is the volume of blood serum sample, R
1V
1Be the volume of reagent I, R
2V
2Be the volume of reagent II, F is a correction factor.
2. the two-step enzyme testing method of triglyceride in the serum according to claim 1 is characterized in that one of double-colored former material is the 4-amino-antipyrine in the reagent I, and another is selected from 2, the derivant of 4-two chlorophenols or phenol.
3. the two-step enzyme testing method of triglyceride in the serum according to claim 1 is characterized in that every liter of Tris-HCl damping fluid of reagent I includes Tris 0.10mmol～0.20mmol, 2,4-two chlorophenol 2.0mmol～4.0mmol, MgSO
49mmol～15mmol, sodium taurocholate 3.0mmol～5.5mmol, TritonX-1000.08g～0.15g, ATP 0.8mmol～2.0mmol, glycerokinase 400U～800U, phosphoglycerol oxidase 3000U～4200U, peroxidase 800U～1600U, 4-amino-antipyrine 0.8mmol～2.4mmol, Proclin-300 antiseptic 100 μ l～300 μ l; Every liter of Tris-HCl damping fluid of reagent II includes Tris 0.10mmol～0.20mmol, lipoprotein lipase 16000U～32000U, Proclin-300 antiseptic 100 μ l～300 μ l.
4. the two-step enzyme testing method of triglyceride in the serum according to claim 1, the pH value that it is characterized in that Tris-HCl damping fluid in reagent I and the reagent II is 7.6 ± 0.2.
5. the two-step enzyme testing method of triglyceride in the serum according to claim 1 is characterized in that the volume ratio of measuring is: sample: reagent I: reagent II=1: 75～80: 25～20.
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| CN101975753B (en) * | 2010-09-17 | 2012-10-31 | 李立和 | Two-step enzymatic determination method for cholesterol ester in serum |
| CN103602718A (en) * | 2013-11-20 | 2014-02-26 | 天津市宝坻区人民医院 | Method for testing triglyceride in serum by using glycerol dehydrogenase |
| 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 |
| CN110146499B (en) * | 2019-06-21 | 2022-03-22 | 长春汇力生物技术有限公司 | Dry sheet detection reagent card for quantitatively detecting hydrogen peroxide concentration and preparation method thereof |
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