CN100365409C - Reagent for measuring low-density lipoproteincholesterol and preparation method - Google Patents
Reagent for measuring low-density lipoproteincholesterol and preparation method Download PDFInfo
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- CN100365409C CN100365409C CNB2005100499129A CN200510049912A CN100365409C CN 100365409 C CN100365409 C CN 100365409C CN B2005100499129 A CNB2005100499129 A CN B2005100499129A CN 200510049912 A CN200510049912 A CN 200510049912A CN 100365409 C CN100365409 C CN 100365409C
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Abstract
The present invention relates to a reagent for measuring cholesterol in low-density lipoprotein in blood serum and a preparation method thereof. The goal of the present invention is to provide improvements on a reagent for measuring low-density lipoprotein cholesterol and the preparation method thereof. The reagent has the characteristics of simple, convenient and exact detection and stable reagent characters, and adapts to various models of full-automatic biochemical analyzers. The technical scheme provided by the present invention comprises: the reagent for measuring low-density lipoprotein cholesterol comprises a proper amount of buffer solution, stabilizing agent, a colour source and the following components: A, an enzyme compound with high affinity, which is made by an enzyme and an activator reaction; B, a surface active agent; the adding proportion of the surface active agent is 10 to 50 g/l; the reagent also comprises a proper amount of catalysts, a preservative and antijam substances. The preparation method of the reagent for measuring low-density lipoprotein cholesterol comprises the following steps: (1), compound activation; (2), enzyme dissolution; (3), a mixing reaction; (4), configuration. The preservative and the antijam substances are also added in the reagent.
Description
Technical field
The present invention relates to a kind of reagent and preparation method who measures serum composition, particularly measure the reagent and the preparation method of cholesterol in the low-density lipoprotein in the serum, can be widely used in medical science and technological field of biochemistry.
Background technology
The incidence of serum (slurry) LDL-C (LDL-C) and cardiovascular and cerebrovascular disease is proportionate, and epidemiology and relevant clinical confirm, detect the clinical diagnosis and treatment that LDL-C also can be widely used in being correlated with, and as one of unusual dangerous index of lipid metabolism.
The Friedewald equation and the polyvinyl sulfuric acid precipitation method that have " national clinical examination working specification " (281 pages of second editions) of known supercentrifugation and electrophoresis and the establishment of Department of Medical Administration of Ministry of Health of the People's Republic of China to be recommended to the assay method of LDL-C.Though the Friedewald equation is easy, if but have one to measure inaccurate or when triglyceride during greater than 4.52mmol/L, it is inaccurate adopting the result of this equation calculating LDL-C in T-CHOL (TC), triglyceride (TG) and the HDL-C (HDL-C).The polyvinyl sulfuric acid precipitation method are that the cholesterol of measuring in the supernatant is represented HDL-C and VLDL-C sum, deducts the value that the supernatant cholesterol promptly gets LDL-C with TC with the LDL-C in polyvinyl sulfuric acid (PVP) the selective precipitation serum.Above-mentioned these known methods are owing to all can not directly detect on automatic clinical chemistry analyzer, in recent years, directly the LDL-C reagent that detects on automatic clinical chemistry analyzer also occurs in succession, the method of cholesterol in the specific determination target lipoprotein is disclosed as Jap.P. 6-242110, this method is to make the lipoprotein aggegation in addition of target lipoprotein, control reaction with enzyme, thereby measure target lipoprotein (LDL), this method is realizing that aspect the robotization be that practicality is arranged very much, but LDL that can only fractionated goes out from the lipoprotein except that HDL, and can not be from the potpourri of VLDL (very low density lipoprotein (VLDL)) and CM (chylomicron) quantitatively and fractionation testing LDL, so still must measure the LDL cholesterol by separation means.Utilize polyanion and divalent metal salt composite and non-measurement lipoprotein to form cotton-shaped compound at present in addition, thereby measure the mensuration of lipoprotein, but after these divalent metal salt composites detect and finish, easy and other alkaline reagent waste liquids reaction formation precipitations in the Drainage pipe of automatic clinical chemistry analyzer, blocking pipe causes harmful effect to automatic clinical chemistry analyzer.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned background technology, a kind of the measure reagent of LDL-C and preparation method's improvement are provided, this reagent should have detect easy accurately, the reagent stable in properties, be suitable for the characteristics of the automatic clinical chemistry analyzer of various models.
Technical scheme provided by the invention is: a kind of reagent of measuring LDL-C, comprise appropriate amount of buffer solution, stabilizing agent and chromogen, and this reagent also comprises following composition:
A, high affinity enzyme compound, this enzyme compound, by the reaction of enzyme and activator and get, wherein:
Activator is that multiple and activator mix, the activation back that a kind of or arbitrary proportion in the compounds such as steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotics mixes obtains, compound concentrations is the 1-100 mg/ml, and the mol ratio of compound and activator is 1: 1-1: 20;
The enzyme that is adopted is cholesterol esterase and cholesterol oxidase, the two mol ratio 10: 1-1 of cholesterol esterase and cholesterol oxidase: 10;
Mole proportioning when above-mentioned activator and enzyme reaction is 1: 1-20: 1;
B, surfactant, its additional proportion is every liter of reagent 10-50 gram, described surfactant be a kind of or arbitrary proportion in polyoxyethylene lauryl ether, many alkylphenol-polyethenoxies lauryl amine, alkyl polyoxyethylene ether hydroxypropyl azochlorosulfonate acid sodium, the senior alcohol ether of polyoxyethylene, the NONIN HS 240 mix multiple.
Described damping fluid is GOOD ' S damping fluid or phosphate buffer or TRIS buffer or citrate buffer solution or borate buffer solution, and the pH value of damping fluid is 5-9, and the concentration of damping fluid is 50-200mmol/L.
Also have proper catalyst, antiseptic and anti-interference material in this reagent.
The preparation method that described LDL-C is measured reagent may further comprise the steps:
One, compound activating
That selects that a kind of or arbitrary proportion in the compounds such as described steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotics mixes is multiple, activate with known azide method or carbodiimide method or succimide method or periodate oxidation method in aqueous medium, activation condition is:
The mol ratio of compound and activator is 1: 1-1: 20, temperature 0-25 ℃, PH in the 3-11 scope, time 0.1-24 hour;
Two, enzyme dissolving
Select described cholesterol esterase and cholesterol oxidase, be dissolved in the damping fluid;
Three, hybrid reaction
Activator after the activation with the dissolving after enzyme solutions mix, this solution reacted 18-24 hour under 0-25 ℃ temperature;
Four, configuration:
Reacted enzyme compound mixes with surfactant, and adds damping fluid, stabilizing agent, 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: dimethyl sulfoxide or formyl for dimethylamine or pregnancy alcohol and toluene by the potpourri that mixes at 2: 1, perhaps, formyl for dimethylamine and ethanol by the potpourri that mixes at 2: 1; The part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30-1: 0.01.
Also add antiseptic and anti-interference material in this reagent.
LDL-C provided by the present invention is measured the preparation method and the reagent of reagent, can directly on the automatic clinical chemistry analyzer of various models, detect LDL-C, need not numerous and diverse steps such as sample is centrifugal, precipitation, alleviated the testing amount significantly, reduced detection time, reduced the detection cost; The stable in properties of reagent: can stablize more than 12 months at 2-8 ℃; And specificity is good, and the accuracy rate of detection is higher, and the Friedewald equation testing result of recommending with country compares, and correlativity reaches more than 0.98.
Description of drawings
Fig. 1 to Fig. 3 is respectively the correlativity synoptic diagram of the measured value of embodiments of the invention 1 to embodiment 3 and the Friedewald equation calculating income value that " national clinical examination working specification " (279 pages of second editions) recommended.Wherein, the ordinate among each figure is a Friedewald formula calculated value, and horizontal ordinate is the measured value of each embodiment.
Embodiment
Find after deliberation: use through the high affinity cholesteryl ester enzyme compound of preparation and high affinity cholesterol oxidase compound and when only selectivity reagent that LDL-C is had a surfactant preparation of pretending usefulness was 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 demonstrated difference.Its reactivity shows as LDL＞VLDL＞CM＞HDL.
When surfactant concentrations reaches one regularly, HDL, VLDL, CM lipoprotein component are not participated in the reaction of measuring LDL in for a long time.Just in for a long time, have only the reaction of LDL lipoprotein component, so the present invention is finished.
Thus, the invention provides a kind of assay method of LDL-C, it is characterized in that in the sample LDL-C high affinity cholesterol esterase and cholesterol oxidase and only selectivity generate 4-courage steroid-3-ketenes and hydrogen peroxide (H under to the effect of the surfactant of LDL-C effect
2O
2), H
2O
2Under the peroxidase effect,, generate coloured quinones, utilize the colorimetric analysis principle can on automatic clinical chemistry analyzer, draw the content of LDL-C in the sample with 4-amino-antipyrine (4-APP), phenols or amino benzenes derivates reaction.
For this reason, reagent provided by the invention and preparation method thereof is:
One, the high affinity cholesterol enzyme compound of preparation
(1) selects enzyme and compound
This enzyme compound, by the preparation of enzyme and activator and get, wherein: the compound of preparation activator 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 spirostan 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 the compound sugar that is made of 2-8 branch or linear structure base; Perhaps be selected from the hydrophobic protein that can form compound selectively with cholesterol; Perhaps be selected from the archon that can form compound selectively with cholesterol; Perhaps be selected from the polyene antibiotics that can form compound selectively with cholesterol, above-claimed cpd can use separately also can two or more mix use, have no particular limits, as long as the enzyme compound that makes must be stronger than the reactivity of other lipoprotein cholesterol to the reaction of LDL-C.
The steroid glycoside example that uses among the present invention has: lucid asparagus glycosides, digitonin, imperial tongue glycosides, lanocerin glycosides, desugar digitonin or the like.
The example of triterpene glucoside has: aescin, tea saponin 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 antibiotics example has: amphotericin B, Philippine bacterium etc.
In order to improve the selectivity of detection, the enzyme that adopts among the present invention is cholesterol esterase and cholesterol oxidase, can certainly select other enzyme for use, as long as the enzyme compound that makes must be stronger than the reactivity of other lipoprotein cholesterol to the reaction of cholesterol in the low-density lipoprotein.
(2) high affinity cholesteryl ester enzyme compound produces
One or more potpourris in the aforesaid compound are activated with known azide method, carbodiimide method, succimide method or periodate oxidation method in aqueous medium, and the method for selecting for use preferably can keep the activity of the enzyme compound for preparing to greatest extent.As everyone knows, activator wherein is respectively azide in azide method, carbodiimide method, succimide method or periodate oxidation method, to cyclohexyl-2-(4-morpholine) ethyl-carbodiimides-methyl-tosilate, N-hydroxy-succinamide and sodium metaperiodate.Compound concentrations is the 1-100 mg/ml.Activation condition is: the mol ratio of compound and activator is 1: 1-1: 20, and temperature 0-25 ℃, PH3-11, time 0.1-24 hour;
Cholesterol esterase is dissolved in the damping fluid of PH=5-9, and the unqualified requirement of the proportioning of cholesterol esterase and damping fluid is as long as the cholesterol esterase dissolving fully;
The proportioning of activator and cholesterol esterase is 1: 1-20: 1 (mol ratio); Activator after the activation mixes with the damping fluid that is dissolved with cholesterol esterase, this solution reacted 18-24 hour under 0-25 ℃ temperature, in order to improve the high affinity of cholesteryl ester enzyme compound, also can in solution, add the high molecular polymer that can change the amino in the cholesteryl ester enzyme compound.The enzyme compound of preparation does not need purifying, detects its active back and directly uses.
(3) high affinity cholesterol oxidase compound produces
Cholesterol oxidase is dissolved in the damping fluid of PH=5-9, and the unqualified requirement of the proportioning of cholesterol oxidase and damping fluid is as long as the cholesterol oxidase dissolving fully;
One or more potpourris in the aforesaid compound are activated with known azide method, carbodiimide method, succimide method or periodate oxidation method in aqueous medium, and the method for selecting for use preferably can keep the activity of the enzyme compound for preparing to greatest extent.As everyone knows, activator wherein is respectively azide in azide method, carbodiimide method, succimide method or periodate oxidation method, to cyclohexyl-2-(4-morpholine) ethyl-carbodiimides-methyl-tosilate, N-hydroxy-succinamide and sodium metaperiodate.Compound concentrations is the 1-100 mg/ml.Activation condition is: the mol ratio of compound and activator is 1: 1-1: 20, and temperature 0-25 ℃, PH3-11, time 0.1-24 hour;
The proportioning of activator and cholesterol oxidase is 1: 1-20: 1 (mol ratio), activator after the activation mixes with the damping fluid that is dissolved with cholesterol oxidase, this solution carried out under 0-25 ℃ temperature 18-24 hour, in order to improve the high affinity of cholesterol oxidase compound, also can in solution, add the high molecular polymer that can change the amino in the cholesterol oxidase compound.The enzyme compound of preparation does not need purifying, detects its active back and directly uses.
Some compound recited above, few or water insoluble as steroid glycoside, triterpene glucoside or polyene antibiotic, for they are dissolved fully, can in advance they be dissolved in the following polar organic solvent that does not provide proton: dimethyl sulfoxide, formyl replace dimethylamine and ethanol by the potpourri that 2: 1 mix for dimethylamine and toluene by the potpourri, the formyl that mix at 2: 1 for dimethylamine, pregnancy alcohol, formyl.
Above high affinity cholesteryl ester enzyme compound and high affinity cholesterol oxidase compound are to prepare respectively, also can be earlier cholesterol esterase and cholesterol oxidase be made enzyme compound after mixed again.
Two, select surfactant for use
Used in the present invention have the surfactant of pretending usefulness to LDL-C, can be polyoxyethylene lauryl ether, many alkylphenol-polyethenoxies lauryl amine, alkyl polyoxyethylene ether hydroxypropyl azochlorosulfonate acid sodium, the senior alcohol ether of polyoxyethylene, NONIN HS 240 etc.Surfactant can use separately also can mix use, and its consumption is decided because of concrete composition, and usable range is every liter of reagent 10-50 gram usually.
Three, configuration
Reacted enzyme compound mixes with surfactant, and can add damping fluid, stabilizing agent, the chromogen that is used for chromogenic assay and catalyzer as one sees fit.
In the present invention in order to keep the stability of enzyme compound and reagent, can add antiseptic, mildewproof agent or other alcohols, macromolecular compound, as disodium ethylene diamine tetraacetate (EDTA.2Na), seralbumin (BSA), polyglycol (PEG), bivalent metal ion, ethylene glycol etc.
The chromogen that is used for chromogenic assay in the present invention can be: N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-(2-hydroxyl-3-sulfopropyl)-3-5-2 aminoanisole (HDAOS), TODP, TOOS etc.
Damping fluid used in the present invention, can be GOOD ' S damping fluid, phosphate 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 bigger buffer action and one or more of inhibitory enzyme activity not.General concentration can be at 50-200mmol/L, GOOD ' the S damping fluid of the preferred 100mmol/L of the present invention.
In order to prevent the interference of other non-measurement of species, can add anti-interference materials such as ascorbic acid oxidase among the present invention.
The quantity of damping fluid, antiseptic, stabilizing agent, chromogen and catalyzer that the present invention is added when configuration is determined unqualified requirement as required.
Above-described all biochemical reagents, the equal buyable of raw material obtain.
Main setting parameter when reagent provided by the present invention is tested on automatic clinical chemistry analyzer is as follows:
| Test | LDL-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: use the high affinity enzyme compound of digitonin compound and enzyme preparation and the reagent of surfactant-NPE sulfuric acid amine sodium preparation to measure the LDL cholesterol
Reagent I (R1)
Damping fluid MES (PH:7.0) 100mmol/L
Stabilizing agent MgCL.6H
2O 2mmol/L
Chromogen 4-AAP 0.25mmol/L
Antiseptic NaN3 5mmol/L
Stabilizing agent BSA 0.5g/L
Catalyzer sodium taurocholate 20mmol/L
High affinity cholesteryl ester enzyme compound 1500U/L
High affinity cholesterol oxidase compound 3000U/L
Anti-interference material ascorbic acid oxidase 1000U/L
Surfactant polyoxyethylene lauryl ether 13g/L (content is 10g/L when reagent I mixes with reagent II)
Reagent II (R2)
Damping fluid MES (PH:7.0) 100mmol/L
Stabilizing agent BSA 0.5g/L
Antiseptic NaN3 5mmol/L
Stabilizing agent MgCL.6H2O 2mmol/L
Catalyzer horseradish peroxidase 950U/L
Chromogen TOPS 2mmol/L
(when wherein preparing high affinity cholesteryl ester enzyme compound and high affinity cholesterol oxidase compound: the digitonin compound concentrations is 50 mg/ml.Activation condition is: the mol ratio of compound and activator is 1: 10,25 ℃ of temperature, PH3,24 hours time; Mole proportioning when activator and enzyme react respectively is 1: 1)
Table 1 is the measured value table of comparisons of the embodiment of the invention 1 and Friedewald equation.
Fig. 1 makes according to table 1 data, the correlativity r of present embodiment 1 measured value and Friedewald equation measured value
2Be 0.9901, illustrate that correlativity is good.
Embodiment 2: use the high affinity enzyme compound of aescin and enzyme preparation and the reagent of surfactant-alkyl polyoxyethylene ether hydroxypropyl azochlorosulfonate acid sodium preparation to measure the LDL cholesterol
Reagent I (R1)
Damping fluid MOPS (PH:6.7) 50mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Chromogen 4-AAP 0.20mmol/L
Antiseptic NaN3 5mmol/L
Stabilizing agent BSA 0.5g/L
Catalyzer sodium taurocholate 15mmol/L
High affinity cholesteryl ester enzyme compound 350U/L
High affinity cholesterol oxidase compound 3500U/L
Anti-interference material ascorbic acid oxidase 1000U/L
(content is surfactant alkyl polyoxyethylene ether hydroxypropyl azochlorosulfonate acid sodium 30g/L when reagent I mixes with reagent II
22.5g/L)
Reagent II (R2)
Damping fluid MOPS damping fluid (PH:6.7) 50mmol/L
Stabilizing agent BSA 0.5g/L
Antiseptic NaN3 5mmol/L
Stabilizing agent MgCL.6H2O 2mmol/L
Catalyzer horseradish peroxidase 850U/L
Chromogen TOPS 3mmol/L
(when wherein preparing high affinity cholesteryl ester enzyme compound and high affinity cholesterol oxidase compound: the aescin compound concentrations is 1 mg/ml.Activation condition is: the mol ratio of compound and activator is 1: 20,10 ℃ of temperature, PH6,10 hours time; Mole proportioning when activator and enzyme react respectively is 20: 1)
Table 2 is the measured value table of comparisons of the embodiment of the invention 2 and Friedewald equation.
Fig. 2 makes according to table 2 data, the correlativity r of present embodiment 2 measured values and Friedewald equation measured value
2Be 0.9889, illustrate that correlativity is good.
Embodiment 3: use the dehydrated protein matter of lipoprotein and the high affinity enzyme compound of enzyme preparation and the reagent of the senior alcohol ether preparation of surfactant-polyoxyethylene to measure the LDL cholesterol.
Reagent I (R1)
Damping fluid PIPES (PH:6.7) 200mmol/L
Stabilizing agent MgCL.6H2O 2mmol/L
Chromogen 4-AAP 0.25mmol/L
Antiseptic NaN3 5mmol/L
Stabilizing agent BSA 0.5g/L
Catalytic materials sodium taurocholate 20mmol/L
High affinity cholesteryl ester enzyme compound 2600U/L
High affinity cholesterol oxidase compound 260U/L
Anti-interference material ascorbic acid oxidase 1000U/L
The senior alcohol ether 66g/L of surfactant polyoxyethylene (content is 50g/L when reagent I mixes with reagent II)
Reagent II (R2)
Damping fluid PIPES (PH:6.7) 200mmol/L
Stabilizing agent BSA 0.5g/L
Antiseptic NaN3 5mmol/L
Stabilizing agent MgCL.6H2O 2mmol/L
Catalyzer horseradish peroxidase 1000U/L
Chromogen TOPS 2mmol/L
(when wherein preparing high affinity cholesteryl ester enzyme compound and high affinity cholesterol oxidase compound: dehydrated protein matter compound concentrations is 100 mg/ml.Activation condition is: the mol ratio of compound and activator is 1: 1,0 ℃ of temperature, PH11,0.1 hour time; Mole proportioning when activator and enzyme react respectively is 10: 1)
Table 3 is the measured value table of comparisons of the embodiment of the invention 3 and Friedewald equation.
Fig. 3 makes according to table 3 data, the correlativity r of present embodiment 3 measured values and Friedewald equation measured value
2Be 0.9814, illustrate that correlativity is good.
Table 1 (the inventive method embodiment 1 measured value and Friedewald computing method of formula are relatively)
| Friedewald equation value (mmol/L) | The inventive method (embodiment 1) measured value (mmol/L) |
| 2.66 | 2.63 |
| 1.92 | 1.76 |
| 2.97 | 2.85 |
| 3.31 | 3.29 |
| 2.76 | 2.58 |
| 1.42 | 1.37 |
| 2.78 | 2.49 |
| 2.33 | 2.24 |
| 2.09 | 2.03 |
| 4.30 | 4.03 |
| 2.34 | 2.18 |
| 3.82 | 3.67 |
| 2.51 | 2.39 |
| 2.70 | 2.60 |
| 3.69 | 3.58 |
| 2.23 | 2.17 |
| 2.82 | 2.73 |
| 1.73 | 1.60 |
| 3.52 | 3.49 |
| 2.89 | 2.76 |
| 5.09 | 5.01 |
| 2.11 | 2.18 |
| 2.72 | 2.39 |
| 2.27 | 2.18 |
| 2.49 | 2.26 |
| 3.31 | 3.26 |
| 4.76 | 4.71 |
| 2.73 | 2.54 |
| 1.29 | 1.20 |
| 2.66 | 2.57 |
| 3.07 | 3.04 |
| 2.69 | 2.58 |
| 3.19 | 3.17 |
Table 2 (the inventive method embodiment 2 measured values and Friedewald computing method of formula are relatively)
| Friedewald equation value (mmol/L) | The inventive method (embodiment 2) measured values (mmol/L) |
| 2.55 | 2.27 |
| 3.77 | 3.73 |
| 2.45 | 2.26 |
| 2.82 | 2.43 |
| 3.62 | 3.33 |
| 2.3 | 2.0 |
| 2.67 | 2.41 |
| 1.13 | 1.01 |
| 2.84 | 2.45 |
| 4.36 | 4. 28 |
| 2.57 | 2.26 |
| 3.12 | 3.09 |
| 2.96 | 2.59 |
| 3.68 | 3.42 |
| 2.71 | 2.24 |
| 2.46 | 2.43 |
| 3.26 | 3.10 |
| 2.72 | 2.49 |
| 3.38 | 3.28 |
| 2.27 | 2.18 |
| 2.49 | 2.36 |
| 3.31 | 3.26 |
| 1.45 | 1.38 |
| 2.08 | 2.04 |
| 1.73 | 1.66 |
| 1.29 | 1.14 |
| 3.43 | 3.27 |
| 3.52 | 3.29 |
| 3.22 | 3.09 |
| 5.13 | 5.10 |
| 7.26 | 7.07 |
| 4.07 | 4.0 |
| 3.19 | 3.07 |
Table 3 (the inventive method embodiment 3 measured values and Friedewald computing method of formula are relatively)
| Friedewald equation value (mmol/L) | The inventive method (embodiment 3) measured values (mmol/L) |
| 2.73 | 2.64 |
| 3.4 | 3.29 |
| 2.71 | 2.61 |
| 3.66 | 3.54 |
| 2.89 | 2.64 |
| 3.1 | 3.4 |
| 3.73 | 3.59 |
| 2.9 | 2.85 |
| 3.59 | 3.4 |
| 2.12 | 2.20 |
| 2.93 | 3.0 |
| 2.14 | 2.2 |
| 2.53 | 2.52 |
| 2.95 | 2.93 |
| 2.25 | 2.43 |
| 3.57 | 3.38 |
| 1.87 | 1.9 |
| 3.71 | 3.84 |
| 1.46 | 1.71 |
| 2.28 | 2.21 |
| 4.89 | 4.92 |
| 3.62 | 3.02 |
| 2.37 | 2.53 |
| 2.8 | 2.73 |
| 0.95 | 1.0 |
| 2.14 | 2.38 |
| 2.88 | 3.01 |
| 2.51 | 2.41 |
| 2.17 | 2.09 |
| 2.16 | 2.16 |
| 4.29 | 4.21 |
| 1.92 | 2.20 |
| 3.74 | 3.465 |
Claims (6)
1. a reagent of measuring LDL-C comprises appropriate amount of buffer solution, stabilizing agent and chromogen, it is characterized in that this reagent also comprises following composition:
A, high affinity enzyme compound, this enzyme compound, by the reaction of enzyme and activator and get, wherein:
Activator is that multiple and activator mix, the activation back that a kind of or arbitrary proportion in digitonin compound, aescin compound and the dehydrated protein materialization compound mixes obtains, compound concentrations is the 1-100 mg/ml, and the mol ratio of compound and activator is 1: 1-1: 20;
The enzyme that is adopted is cholesterol esterase and cholesterol oxidase, and the two mol ratio of cholesterol esterase and cholesterol oxidase is 10: 1-1: 10;
Mole proportioning when above-mentioned activator and enzyme reaction is 1: 1-20: 1;
B, surfactant, its additional proportion are every liter of reagent 10-50 gram, described surfactant be a kind of or arbitrary proportion in polyoxyethylene ether lauryl ether, alkyl polyoxyethylene ether hydroxypropyl azochlorosulfonate acid sodium, the senior alcohol ether of polyoxyethylene mix multiple.
2. a kind of reagent of measuring LDL-C according to claim 1, it is characterized in that described damping fluid is GOOD ' S damping fluid or phosphate buffer or TRIS buffer or citrate buffer solution or borate buffer solution, the pH value of damping fluid is 5-9, and the concentration of damping fluid is 50-200mmol/L.
3. a kind of reagent of measuring LDL-C according to claim 1 and 2 is characterized in that also having proper catalyst, antiseptic and anti-interference material in this reagent.
4. the described a kind of preparation method who measures LDL-C reagent of claim 1 is characterized in that described LDL-C measures the preparation method of reagent and may further comprise the steps:
1), compound activating
That selects that a kind of or arbitrary proportion in described digitonin compound, aescin compound and the dehydrated protein materialization compound mixes is multiple, activate with known azide method or carbodiimide method or succimide method or periodate oxidation method in aqueous medium, activation condition is:
The mol ratio of compound and activator is 1: 1-1: 20, temperature 0-25 ℃, PH in the 3-11 scope, time 0.1-24 hour;
2), enzyme dissolving
Select described cholesterol esterase and cholesterol oxidase, be dissolved in the damping fluid; The two mol ratio of cholesterol esterase and cholesterol oxidase is 10: 1-1: 10;
3), hybrid reaction
Activator after the activation with the dissolving after enzyme solutions mix, this solution reacted 18-24 hour under 0-25 ℃ temperature; Mole proportioning when activator and enzyme reaction is 1: 1-20: 1;
4), configuration:
Reacted enzyme compound mixes with surfactant, and adds damping fluid, stabilizing agent, the chromogen that is used for chromogenic assay and catalyzer.
5. a kind of preparation method who measures LDL-C reagent according to claim 4, it is characterized in that described digitonin compound, aescin compound and dehydrated protein materialization compound place following ORGANIC SOLVENT MIXTURES to dissolve in advance: dimethyl sulfoxide or formyl for dimethylamine or pregnancy alcohol and toluene by the potpourri that mixes at 2: 1, perhaps, formyl for dimethylamine and ethanol by the potpourri that mixes at 2: 1; The part by weight of compound and ORGANIC SOLVENT MIXTURES is 1: 30-1: 0.01.
6. according to claim 4 or 5 described a kind of preparation methods that measure LDL-C reagent, it is characterized in that also adding in this reagent antiseptic and anti-interference material.
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| CN102252987B (en) * | 2011-06-27 | 2013-01-02 | 南京师范大学 | Method for detecting protein content |
| 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 |
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| CN106645763B (en) * | 2017-01-03 | 2019-03-22 | 长沙中生众捷生物技术有限公司 | The Test paper of total cholesterol |
| CN106841189A (en) * | 2017-03-01 | 2017-06-13 | 扬州市农产品质量监督检测中心 | The enzymic colorimetric of cholesterol level in a kind of detection birds, beasts and eggs |
| CN110029145A (en) * | 2018-01-11 | 2019-07-19 | 北京瑞正善达生物工程技术有限公司 | Low density lipoprotein cholesterol measures reagent, preparation method and its application method |
| CN108333175B (en) * | 2018-01-18 | 2021-06-29 | 青岛汉唐生物科技有限公司 | Total cholesterol detection method |
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