CN100564539C - The mensuration reagent and the preparation method of cholesterol in the high-density lipoprotein (HDL) - Google Patents
The mensuration reagent and the preparation method of cholesterol in the high-density lipoprotein (HDL) Download PDFInfo
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- CN100564539C CN100564539C CNB2004100993877A CN200410099387A CN100564539C CN 100564539 C CN100564539 C CN 100564539C CN B2004100993877 A CNB2004100993877 A CN B2004100993877A CN 200410099387 A CN200410099387 A CN 200410099387A CN 100564539 C CN100564539 C CN 100564539C
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Abstract
The present invention relates to cholesterol reagent and preparation method in a kind of high-density lipoprotein (HDL) of measuring in the serum.Technical problem to be solved by this invention provides a kind of the measure preparation method of the reagent of cholesterol in the high-density lipoprotein (HDL) and the improvement of reagent, with the prepared reagent of this method answer specificity good, detect easy, test accuracy rate height, and can on the automatic clinical chemistry analyzer of various models, detect.Technical scheme is: the composition of reagent is as follows: A. high affinity enzyme compound, and by the reaction of enzyme and compound and get, the mole proportioning when two kinds of compositions react is 1: 1-20: 1; B. tensio-active agent, its blending ratio is the 0.003-30 of the weight percent of A composition; C. an amount of sanitas, stablizer, chromogen.Also add proper catalyst and anti-interference material in this reagent.Described preparation method may further comprise the steps: one, compound activating; Two, enzyme dissolving; Three, hybrid reaction; Four, configuration.
Description
Technical field
The present invention relates to a kind of preparation method and reagent of measuring serum composition reagent, particularly measure the preparation method and the reagent of cholesterol reagent in the high-density lipoprotein (HDL) in the serum, can be widely used in medical science and technological field of biochemistry.
Background technology
The incidence of cardiovascular and cerebrovascular diseases and serum high-density LP cholesterol (HDL-C) are negative correlation epidemiology and relevant clinical confirmation, detect the clinical diagnosis and treatment that high density lipoprotein cholesterol also can be widely used in being correlated with, and as one of unusual dangerous index of lipid metabolism.Therefore the accurate mensuration of high density lipoprotein cholesterol is most important, particularly at the high density lipoprotein cholesterol lower limit, minimum error may cause relatively large influence, and the result of high density lipoprotein cholesterol directly influences the calculated value of low density lipoprotein cholesterol (LDL-C).At present, the 279 pages of precipitator method of being recommended of " national Clinical Laboratory working specification " second edition that the measuring method of high density lipoprotein cholesterol had known ultracentrifugation and electrophoretic method and the establishment of Department of Medical Administration of Ministry of Health of the People's Republic of China.Ultracentrifugation is the proportion difference according to lipoprotein, with the ultracentrifugation device lipoprotein is classified, and cholesterol is measured again.This procedure is divided ultracentrifugation and two steps of cholesterol detection, expense height, consuming time big.Electrophoretic method need separate as carrier as sepharose etc. by some, operates extremely numerous and diverse.The precipitator method are to use polyanion and divalent-metal ion reagent to precipitate oarse-grained lipoprotein, and are centrifugal then, measure its content with its supernatant liquor.Though this method is easy, but be not suitable for pattern detection full-automatic, in enormous quantities, and, can make the high density lipoprotein cholesterol result higher to the hypertriglyceridemia patient, when can making the Friedwald formula that calculates LDL-C during greater than 5mmol/L, high triglyceride is limited.Above-mentioned these known methods are owing to all can not directly detect on full-automatic, in recent years, directly on full-automatic, detect the also appearance in succession of HDL-C reagent, they utilize LDL in polyanion mixture and the serum (low-density lipoprotein), VLDL (vldl), CM (chylomicron) reaction as Chinese patent (application number 99116565.9), generate water-fast mixture, and HDL-C still exists with solution state, in the presence of cholesterol reagent, carry out its mensuration, having added Sodium cholic acid in present method, may be in order to improve specificity.Not only HDL reacts in the present invention, and LDL, VLDL etc. also reacts lentamente, so will expect that the reaction end of HDL is impossible completely.And specificity also differs and reserves.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned the deficiencies in the prior art, a kind of the measure preparation method of the reagent of cholesterol in the high-density lipoprotein (HDL) and the improvement of reagent are provided, with the prepared reagent of this method answer specificity good, detect easy, test accuracy rate height, and can be on the automatic clinical chemistry analyzer of various models to high-density lipoprotein (HDL) in cholesterol detect.
The present invention has adopted following technical scheme:
The mensuration reagent of cholesterol comprises an amount of sanitas, stablizer and chromogen in the high-density lipoprotein (HDL), and this reagent also comprises following composition:
A, high affinity enzyme compound, this enzyme compound, by the reaction of enzyme and compound and get, wherein:
Compound be a kind of or arbitrary proportion blended in the compounds such as steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotic multiple with activator mix, the activation after activator, compound concentrations is the 1-50 mg/ml;
Enzyme is 1, Sterol esterase and rCO, the two ratio 10: 1-1: 10,
2, lipoprotein lipase and rCO, the two ratio 10: 1-1: 10
In the arbitrary group of solute after mixing, dissolve with damping fluid, the pH value of damping fluid is 5-9, the mole proportioning of damping fluid is 50-200mmol/L, and described damping fluid is GOOD ' S damping fluid or phosphate buffered saline buffer or tris buffer or citrate buffer solution or borate buffer solution;
Mole proportioning during two kinds of composition reactions of above-claimed cpd and enzyme is 1: 1-20: 1;
B, tensio-active agent, the weight percent of its blending ratio and A composition is 0.003-30, and described tensio-active agent is that a kind of or arbitrary proportion blended in cats product, anion surfactant, zwitterionics and the nonionogenic tenside is multiple.
Described enzyme is preferentially selected Sterol esterase and rCO.
This reagent also comprises proper catalyst.
The blending ratio of described tensio-active agent is the 0.01-10 weight percent.
Also has an amount of anti-interference material in this reagent.
The preparation method of the mensuration reagent of cholesterol may further comprise the steps in the described high-density lipoprotein (HDL):
One, compound activating
Select a kind of in the compounds such as described steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotic or the arbitrary proportion blended is multiple, activate with known azide method or carbodiimide method or succimide method or periodate oxidation method in water medium, activation condition is:
The mol ratio of compound and activator is 1: 1-1: 10, temperature 0-25 ℃, PH in the 4-11 scope, time 0.1-24 hour.
Two, enzyme dissolving
Select described Sterol esterase and courage week alcohol oxidase, or in lipoprotein lipase and the rCO arbitrary group, be dissolved in the damping fluid;
Three, hybrid reaction
Compound after the activation with the dissolving after enzyme mix, this solution reacted 18-24 hour under 0-25 ℃ temperature;
Four, configuration:
Reacted enzyme compound mixes with tensio-active agent, and adds sanitas, stablizer, the chromogen that is used for chromogenic assay and catalyzer.
Described steroid glycoside, triterpene glucoside or polyene antibiotic place following ORGANIC SOLVENT MIXTURES to dissolve in advance: methyl-sulphoxide or formyl are pressed 2: 1 blended mixtures for dimethylamine or pregnancy alcohol with toluene, perhaps, formyl is pressed 2: 1 blended mixtures for dimethylamine and ethanol; The part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30-1: 0.01.
Also add the anti-interference agent material in this reagent.
The preparation method of cholesterol determination reagent and reagent in the high-density lipoprotein (HDL) provided by the present invention, can be directly on the automatic clinical chemistry analyzer of various models to high-density lipoprotein (HDL) in cholesterol detect, save numerous and diverse steps such as sample is centrifugal, precipitation, alleviated the testing amount significantly, reduced detection time, reduced the detection cost; And the specificity of reagent is good, and the accuracy rate of detection is higher, the detection effect of the precipitator method of recommending with country relatively, dependency reaches more than 0.98.
Description of drawings
Fig. 1 to Fig. 3 is respectively the dependency synoptic diagram of the measured value of the measured value of embodiments of the invention 1 to embodiment 3 and the precipitator method (" national Clinical Laboratory working specification " second edition 279 pages recommend).
Embodiment
Find after deliberation: use through the high affinity cholesteryl ester enzyme compound of preparation and high affinity rCO compound and cholesterol has the tensio-active agent preparation of pretending usefulness in to high-density lipoprotein (HDL) reagent when being used for measuring through the isolated HDL of ultracentrifugation, LDL, VLDL and CM each component lipoprotein, the reactivity of above-mentioned each component lipoprotein cholesterol demonstrates difference, and the selectivity of different components is HDL＞LDL＞VLDL＞CM with reactivity.When surfactant concentration reaches one regularly, LDL, VLDL, CM do not participate in the reaction of measuring HDL in for a long time.
Thus, reagent provided by the invention and preparation method thereof is:
One, select the high affinity enzyme compound for use:
This enzyme compound, by enzyme and compound and get, wherein:
1, compound is selected from steroid glycoside, and this steroid glycoside has in its structure as the furostan alcohol of aglucon or the cyclopentanoperhydro-phenanthrene enzyme of spirostane alcohol, and has the oligosaccharidase that contains 3-10 wire or branched structure monose.Perhaps be selected from triterpene glucoside, this triterpene glucoside has α-or the aglucon of β-copane, lanostane etc. in its structure, and has an oligose that constitutes by 2-8 branch or linear structure base, perhaps be selected from the hydrophobic protein that can form mixture selectively with cholesterol, perhaps be selected from the archon that can form mixture selectively with cholesterol, perhaps be selected from the polyene antibiotic that can form mixture selectively with cholesterol.Above-claimed cpd can use separately also can two or more mix use, have no particular limits, as long as enzyme compound that makes and the reagent that cholesterol in the high-density lipoprotein (HDL) had a tensio-active agent preparation of pretending usefulness are to by force reactive than other lipoprotein cholesterol of the reaction of cholesterol in the high-density lipoprotein (HDL).
The steroid glycoside example that uses among the present invention has: desugar digitonin, Radix Asparagi glycosides, imperial tongue glycosides, wool wax glycosides or the like.
The example of triterpene glucoside has: escin, theasaponin etc.
The hydrophobic protein example has: the dehydrated protein matter of lipoprotein, lysosomal protein or the like.
Archon can be had as an example by acquisitions such as bacterium, marine microorganisms: streptolysin O, brain ketone cytolysin etc.
The polyene antibiotic example has: Philippines bacterium etc.
2, enzyme is selected Sterol esterase and rCO for use, or in lipoprotein lipase and the rCO any one group.Can certainly select other enzyme for use, as long as the high affinity enzyme compound that makes and cholesterol in the high-density lipoprotein (HDL) had reagent that the tensio-active agent of pretending usefulness is made into to by force reactive than other lipoprotein cholesterol of the reaction of cholesterol in the high-density lipoprotein (HDL).In order to improve the specificity of detection, preferentially use Sterol esterase and rCO among the present invention, concrete restriction is not done in the source of Sterol esterase and rCO.
Two, the preparation of high affinity cholesterol enzyme compound:
1, one or more mixtures in the above-claimed cpd is activated in water medium
One or more mixtures in the above-claimed cpd are activated with known azide method, carbodiimide method, succimide method or periodate oxidation method in water medium, and the method for selecting for use preferably can keep the activity of the enzyme compound for preparing the biglyyest.Activation condition is: the mol ratio of compound and activator is 1: 1-1: 10, compound concentrations is the 1-50 mg/ml, temperature 0-25 ℃, PH in the 4-11 scope, time 0.1-24 hour.As everyone knows, activator wherein is respectively trinitride in azide method, carbodiimide method, succimide method or periodate oxidation method, to cyclohexyl-2-(4-morpholine) ethyl-carbodiimide-methyl-tosilate, N-hydroxy-succinamide and sodium periodate.Some compound described above, be insoluble in water as steroid glycoside, triterpene glucoside or polyene antibiotic, can place the following polar organic solvent mixture that does not provide proton to dissolve in advance for they are dissolved fully: methyl-sulphoxide or formyl replace dimethylamine and ethanol by 2: 1 blended mixture with toluene by 2: 1 blended mixtures, formyls for dimethylamine or pregnancy alcohol.The part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30-1: 0.01.
2, described Sterol esterase or rCO are dissolved in the damping fluid that PH is the 5-9 scope
Sterol esterase or rCO are dissolved in the damping fluid that PH is the 5-9 scope.In order to improve the affinity of cholesterol enzyme compound, also can in solution, add the high molecular polymer that can change the amino in Sterol esterase or the rCO compound.Detecting its active back directly uses.
3, hybrid reaction
Enzyme after compound after the activation and the dissolving is by 1: 1-20: the mixed of 1 (mol ratio), this solution were reacted 18-24 hour under 0-25 ℃ temperature;
Three, configuration:
Add tensio-active agent in the enzyme compound that makes, the blending ratio of tensio-active agent is the 0.003-30 of aforementioned composition weight percent, and preferential blending ratio is 0.01-10; Also can add an amount of sanitas, stablizer and be used for the chromogen of chromogenic assay, also can consider to add proper catalyst and anti-interference material.
Use among the present invention high density lipoprotein cholesterol is had the tensio-active agent of pretending usefulness can be cats product, anion surfactant, zwitterionics or non-ionic surface agent, as long as high density lipoprotein cholesterol had pretend usefulness, have as the example of cats product: lauroyl amido tetrahydroglyoxaline, palmityl amido tetrahydroglyoxaline, polyethylene oxide n-Laurylamine, polyoxyethylene cetylamine; Example as anion surfactant has: 12 (alkane) polyoxyethylenated alcohol phosphoric acid mixing fat, PAM multipolymer, disodium 4-dodecyl-2,4 '-oxydibenzenesulfonate, palmityl methyl thiosulfonic acid sodium; Example as nonionogenic tenside has: sulfo group imidazolinium betaine, alkyl polyoxyethylene trimethyl-glycine; Example as nonionogenic tenside has: Voranol EP 2001, polyoxyethylene phenyl ether, polyoxyethylene alkyl phenyl ether, dibenzyl xenyl Soxylat A 25-7, anhydrous sorbitol polyoxyethylene, polyoxyethylene-polyoxypropylene polyethers, tribenzyl phenol polyethenoxy ether etc.Here preferred nonionic surfactants (as: Tween-80, Triton X-100, GenapolX-081, Thesit etc.). tensio-active agent can use separately also can mix use, and its consumption is as previously mentioned.
In the present invention in order to keep the stability of enzyme compound and reagent, can add stablizer, sanitas or mould inhibitor or other alcohols, as sodium ethylene diamine tetracetate (EDTA.2Na), bovine serum albumin (BSA), polyoxyethylene glycol (PEG), divalent-metal ion.The kind of sanitas or mould inhibitor and consumption are the amount doesn't matter not to be limited, general use range 0.1-20mmol/L.
The chromogen that is used for chromogenic assay in the present invention has: 2.4.6-three bromo-3-hydroxy-benzoic acids (TBHBA), 3.5-two chloro-2 hydroxybenzoic acids (DHBS), N-ethyl-N-(3-sulfopropyl)-3-monomethylaniline sodium salt (TOPS), N-(2-hydroxyl-3-sulfopropyl)-3-5-2 anisidine (HDAOS), TODP, TOOS etc.Chromogenic kind and consumption are also the amount doesn't matter not to be limited, general use range 0.01-10mmol/L.
Damping fluid used in the present invention, can be GOOD ' S damping fluid, phosphate buffered saline buffer, tris buffer, citrate buffer solution, borate buffer solution etc., the kind of damping fluid and concentration are decided because of concrete composition, here do not limit, as long as in PH is the scope of 5-9, guarantee shock absorption and a kind of or its mixture of inhibitory enzyme activity not.Preferentially select GOOD ' the S damping fluid of 50-200mmol/L for use.
The kind of catalyzer and consumption are also the amount doesn't matter not to be limited, and general use range also is 10U/L-10000U/L.
In order to prevent the interference of other non-measurement of species, can add anti-interference materials such as Vitamin C oxidase among the present invention.The kind of this material and consumption also kind and consumption is the amount doesn't matter does not limit, general use range 10U/L-10000U/L.
The equal buyable of all biochemical articles for use of the present invention obtains.
The reagent of cholesterol in the high-density provided by the present invention, the significant parameter when test on the automatic clinical chemistry analyzer is as follows:
| Test | HDL-C |
| Assay code | 2point |
| Assay point | 15-34 |
| Wave(sub/main) | 700nm/546nm |
| Sample volume | 3μl |
| R1 volume | 225μl |
| R2 volume | 75μl |
| Calib.type | Linear |
| Calib/ |
2/2 |
Embodiment 1: high affinity enzyme cholesteryl ester enzyme compound in the present embodiment and high affinity enzyme rCO compound are by imperial tongue glycosides steroid glycoside in the steroid glycoside compound and enzyme preparation; Distributing style as previously mentioned, wherein:
The activation condition of compound activating is:
The mol ratio of compound and activator is 1: 1,15 ℃ of temperature, and pH value is 8,10 hours time.
The mole proportioning of damping fluid is 100mmol/L, and the pH value of damping fluid is 7.
Compound after the activation with the dissolving after enzyme mix, this solution reacted 20 hours under 15 ℃ temperature.
Described imperial tongue glycosides places following ORGANIC SOLVENT MIXTURES to dissolve in advance: formyl is pressed 2: 1 blended mixtures for dimethylamine and toluene, and the part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 0.01.
Reagent I
High affinity enzyme cholesteryl ester enzyme compound 500U/L
High affinity enzyme rCO compound 5000U/L
Damping fluid GOOD ' S (PH:6.7) 100mmol/L
Stablizer BSA 1g/L
Stablizer polyoxyethylene glycol 10g/L
Stablizer EDTA.2Na 2mmol/L
Chromogen TOPS 2mmol/L
Sanitas NaN3 7.5mmol/L
Catalyzer horseradish peroxidase 950U/L
Anti-interference material Vitamin C oxidase 1000U/L
Reagent II
Surfactant polyoxyethylene alkyl phenyl ether 500g/L
Damping fluid GOOD ' S (PH:6.7) 100mmol/L
Sanitas NaN3 7.5mmol/L
Chromogen 4-AAP 0.25mmol/L
Stablizer BSA 1g/L
Stablizer EDTA.2Na 2mmol/L
Reagent I, reagent II mixed simultaneously and add test when last machine was tested.
Table 1 is the last machine test value of present embodiment 1 and the measured value synopsis of the precipitator method.
Fig. 1 makes according to table 1 data, and the dependency r of the measured value of present embodiment 1 measured value and the precipitator method (" national Clinical Laboratory working specification " second edition 279 pages recommend) is 0.992, illustrates that dependency is good.
Use cholesteryl ester enzyme compound, rCO compound by theasaponin in the triterpene glucoside compounds and enzyme preparation in the present embodiment; Distributing style as previously mentioned, wherein:
The activation condition of compound activating is:
The mol ratio of compound and activator is 1: 5,0 ℃ of temperature, and pH value is 4,24 hours time.
The mole proportioning of damping fluid is 50mmol/L, and the pH value of damping fluid is 9.
Compound after the activation with the dissolving after enzyme mix, this solution reacted 24 hours under 0 ℃ temperature;
Described theasaponin places following ORGANIC SOLVENT MIXTURES to dissolve in advance: formyl is pressed 2: 1 blended mixtures for dimethylamine and ethanol; The part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 15.
Reagent I
The Sterol esterase compound amount is 5000U/L
The rCO compound amount is 4000U/L
Damping fluid GOOD ' S (PH:5) 50mmol/L
Stablizer BSA 1g/L
Stablizer Mgcl
25mmol/L
Stablizer EDTA.2Na 2mmol/L
Chromogen TOOS 2.2mmol/L
Sanitas NaN3 7.5mmol/L
Catalyzer horseradish peroxidase 950U/L
Anti-interference material Vitamin C oxidase 1000U/L
Reagent II
Surfactant polyoxyethylene-polyoxypropylene polyethers 600g/L
Damping fluid GOOD ' S (PH:6.7) 50mmol/L
Sanitas NaN3 7.5mmol/L
Chromogen 4-AAP 0.20mmol/L
Stablizer BSA 1g/L
Stablizer EDTA.2Na 2mmo1/L
Reagent I, reagent II mixed simultaneously and add test when last machine was tested.
Table 2 is the last machine test value of present embodiment 2 and the measured value synopsis of the precipitator method.
Fig. 2 makes according to table 2 data, and the dependency r of the measured value of present embodiment 2 measured values and the precipitator method (" national Clinical Laboratory working specification " second edition 279 pages recommend) is 0.994, illustrates that dependency is good.
Embodiment 3
Use cholesteryl ester enzyme compound, rCO compound by preparing in the present embodiment by theasaponin in the triterpene glucoside compounds and enzyme; Distributing style as previously mentioned.
The activation condition of compound activating is:
The mol ratio of compound and activator is 1: 10,25 ℃ of temperature, pH value 11,0.1 hour time.
The mole proportioning of damping fluid is 200mmol/L, and the pH value of damping fluid is 5.
Compound after the activation with the dissolving after enzyme mix, this solution reacted 18 hours under 25 ℃ temperature;
Described theasaponin places following ORGANIC SOLVENT MIXTURES to dissolve in advance: pregnancy alcohol is pressed 2: 1 blended mixtures with toluene, and the part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30.
Reagent I
The Sterol esterase compound amount is 6000U/L
The rCO compound amount is 600U/L
Damping fluid GOOD ' S (PH:9) 200mmol/L
Stablizer BSA 1g/L
Stablizer Mgcl
25mmol/L
Stablizer EDTA.2Na 2mmol/L
Chromogen HDAOS 3mmol/L
Sanitas NaN3 7.5mmol/L
Catalyzer horseradish peroxidase 950U/L
Anti-interference material Vitamin C oxidase 1000U/L
Reagent II
Tensio-active agent tribenzyl phenol polyethenoxy ether 12g/L
Damping fluid GOOD ' S (PH:6.7) 200mmol/L
Sanitas NaN3 7.5mmol/L
Chromogen 4-AAP 0.15mmol/L
Stablizer BSA 1g/L
Stablizer EDTA.2Na 2mmol/L
Reagent I, reagent II mixed simultaneously and add test when last machine was tested.
Table 3 is the last machine test value of present embodiment 3 and the measured value synopsis of the precipitator method.
Fig. 3 makes according to table 3 data, and the dependency r of the measured value of present embodiment 3 measured values and the precipitator method (" national Clinical Laboratory working specification " second edition 279 pages recommend) is 0.989, illustrates that dependency is good.
Table 1
| Precipitator method measured value (mmol/L) | The inventive method (embodiment 1) measured value (mmol/L) |
| 1.2 | 1.2 |
| 1.61 | 1.56 |
| 1.1 | 1.12 |
| 1.62 | 1.63 |
| 0.62 | 0.61 |
| 1.45 | 1.5 |
| 1.55 | 1.54 |
| 1.77 | 1.84 |
| 1.04 | 1.01 |
| 1.57 | 1.54 |
| 1.12 | 1.04 |
| 1.06 | 0.95 |
| 1.68 | 1.63 |
| 1.21 | 1.22 |
| 1.46 | 1.46 |
| 1.11 | 1.04 |
| 1.72 | 1.7 |
| 1.38 | 1.35 |
| 1.27 | 1.25 |
| 0.85 | 0.77 |
| 0.91 | 0.96 |
| 1.49 | 1.5 |
| 0.87 | 0.91 |
| 1.0 | 0.98 |
| 1.79 | 1.75 |
| 1.06 | 1.1 |
| 0.83 | 0.8 |
| 0.99 | 0.98 |
| 0.96 | 0.95 |
| 1.31 | 1.3 |
| 1.43 | 1.4 |
| 1.25 | 1.23 |
| 0.87 | 0.86 |
Table 2
| Precipitator method measured value (mmol/L) | The inventive method (embodiment 2) measured values (mmol/L) |
| 0.94 | 0.92 |
| 1.08 | 1.04 |
| 0.97 | 1.01 |
| 0.86 | 0.89 |
| 0.94 | 0.96 |
| 1.41 | 1.28 |
| 1.17 | 1.21 |
| 0.89 | 0.85 |
| 1.68 | 1.64 |
| 0.89 | 0.94 |
| 0.83 | 0.82 |
| 1.13 | 1.09 |
| 1.49 | 1.5 |
| 1.4 | 1.4 |
| 0.84 | 0.9 |
| 0.79 | 0.78 |
| 1.12 | 1.1 |
| 1.12 | 1.2 |
| 0.91 | 0.9 |
| 1.03 | 1.0 |
| 1.02 | 1.0 |
| 1.29 | 1.27 |
| 0.88 | 0.88 |
| 1.59 | 1.54 |
| 1.06 | 1.09 |
| 1.23 | 1.2 |
| 1.5 | 1.48 |
| 0.38 | 0.36 |
| 1.22 | 1.18 |
| 2.13 | 2.1 |
| 0.43 | 0.41 |
| 1.52 | 1.5 |
| 1.36 | 1.36 |
Table 3
| Precipitator method measured value (mmol/L) | The inventive method (embodiment 3) measured values (mmol/L) |
| 1.5 | 1.43 |
| 1.48 | 1.31 |
| 1.08 | 0.96 |
| 1.12 | 0.99 |
| 0.88 | 0.83 |
| 1.27 | 1.06 |
| 2.0 | 1.79 |
| 1.11 | 1.0 |
| 1.04 | 0.87 |
| 1.72 | 1.49 |
| 1.1 | 0.91 |
| 0.88 | 0.85 |
| 1.42 | 1.27 |
| 1.54 | 1.38 |
| 1.94 | 1.72 |
| 1.18 | 1.11 |
| 1.66 | 1.46 |
| 1.39 | 1.21 |
| 1.86 | 1.68 |
| 1.08 | 1.06 |
| 1.19 | 1.12 |
| 1.75 | 1.57 |
| 1.15 | 1.04 |
| 2.1 | 1.77 |
| 1.75 | 1.55 |
| 1.72 | 1.45 |
| 1.86 | 1.62 |
| 1.28 | 1.1 |
| 1.78 | 1.61 |
| 1.37 | 1.2 |
Claims (8)
1, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) comprises an amount of sanitas, stablizer and chromogen, it is characterized in that this reagent also comprises following composition:
A, high affinity enzyme compound, this enzyme compound is reacted by enzyme and compound and gets, wherein, compound is the activator after two kinds of a kind of or arbitrary proportion blended and activator mix, the activation in imperial tongue glycosides steroid glycoside, the theasaponin, and compound concentrations is the 1-50 mg/ml;
Enzyme is the solute after Sterol esterase and rCO mix, dissolve with damping fluid, the two ratio of described Sterol esterase and rCO is 10: 1-1: 10, the pH value of damping fluid is 5-9, the mole proportioning of damping fluid is 50-200mmol/L, and described damping fluid is GOOD ' S damping fluid or phosphate buffered saline buffer or tris buffer or citrate buffer solution or borate buffer solution; Mole proportioning during two kinds of composition reactions of above-claimed cpd and enzyme is 1: 1-20: 1;
B, tensio-active agent, its blending ratio is the 0.003-30 of the weight percent of A composition, and described tensio-active agent is a kind of in polyoxyethylene alkyl phenyl ether, polyoxyethylene-polyoxypropylene polyethers, the tribenzyl phenol polyethenoxy ether or the arbitrary proportion blended is multiple.
2, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1 is characterized in that this reagent also comprises proper catalyst.
3, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1 and 2, the described blending ratio that it is characterized in that tensio-active agent is the 0.01-10 weight percent.
4, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 3 is characterized in that also having an amount of anti-interference material in this reagent.
5, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1,
It is characterized in that this method may further comprise the steps:
One, compound activating
Select two kinds of a kind of or arbitrary proportion blended in described imperial tongue glycosides steroid glycoside, the theasaponin, in water medium, activate with known azide method or carbodiimide method or succimide method or periodate oxidation method, activation condition is: the mol ratio of compound and activator is 1: 1-1: 10, temperature 0-25 ℃, PH in the 4-11 scope, time 0.1-24 hour;
Two, enzyme dissolving
Described Sterol esterase and rCO are dissolved in the damping fluid;
Three, hybrid reaction
Compound after the activation with the dissolving after enzyme mix, this solution reacted 18-24 hour under 0-25 ℃ temperature;
Four, configuration:
Reacted enzyme compound mixes with tensio-active agent, and adds sanitas, stablizer, is used for the chromogen of chromogenic assay.
6, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 5, it is characterized in that described imperial tongue glycosides steroid glycoside or theasaponin place following ORGANIC SOLVENT MIXTURES to dissolve in advance: methyl-sulphoxide or formyl are pressed 2: 1 blended mixtures for dimethylamine or pregnancy alcohol with toluene, perhaps, formyl is pressed 2: 1 blended mixtures for dimethylamine and ethanol, and the part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30-1: 0.01.
7, according to the preparation method of the mensuration reagent of cholesterol in claim 5 or the 6 described high-density lipoprotein (HDL), it is characterized in that also adding in this reagent catalyzer.
8, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 7 is characterized in that also adding in this reagent anti-interference material.
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| CNB2004100993877A CN100564539C (en) | 2004-12-31 | 2004-12-31 | The mensuration reagent and the preparation method of cholesterol in the high-density lipoprotein (HDL) |
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| US9151768B2 (en) * | 2010-07-23 | 2015-10-06 | Denka Seiken Co., Ltd. | Method for quantifying the amount of cholesterol in high-density lipoprotein 3 |
| CN102252987B (en) * | 2011-06-27 | 2013-01-02 | 南京师范大学 | Method for detecting protein content |
| EP2891718B1 (en) * | 2012-08-31 | 2019-01-30 | Kyowa Medex Co., Ltd. | Method for measuring cholesterol in high-density lipoprotein |
| CN105203472B (en) * | 2015-06-30 | 2019-03-08 | 北京万泰德瑞诊断技术有限公司 | A kind of stable free fatty acid determination reagent kit |
| CN105137098B (en) * | 2015-08-24 | 2017-01-25 | 巩晓东 | Determination reagent of cholesterols in serum high density lipoprotein |
| CN108333175B (en) * | 2018-01-18 | 2021-06-29 | 青岛汉唐生物科技有限公司 | Total cholesterol detection method |
| CN112695071B (en) * | 2020-12-16 | 2022-12-30 | 浙江伊利康生物技术有限公司 | High-density lipoprotein 3 determination reagent, method and kit |
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