CN102323225B - Method and reagent kit used for detecting low-density lipoprotein cholesterin - Google Patents
Method and reagent kit used for detecting low-density lipoprotein cholesterin Download PDFInfo
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Abstract
The invention relates to the filed of biochemistry, and discloses a method and a reagent kit used for detecting low-density lipoprotein cholesterin. The method comprises the steps of: combining phosphotungstic acid and agaropectin into a mixed liquid with the pH value of 6.1-6.2; mixing a sample to be tested with the mixed liquid and polyethylene glycol 20000 under the action of magnesium ions to obtain a suspension turbid liquid; then detecting light absorbance under a wavelength of 630nm; and calculating the concentration of low-density lipoprotein cholesterin of the sample to be tested through turbidimetry with a standard solution subjected to the same treatment abovementioned. The reagent kit disclosed by the invention comprises 2-4g/L magnesium chloride, 6-10g/L polyethylene glycol 20000, 4-5g/L phosphotungstic acid and 0.2-0.4g/L agaropectin. According to the method disclosed by the invention, the specific reaction with low-density lipoprotein without removing impure proteins can be realized, so that the detection of the low-density lipoprotein cholesterin is simple, quick and accurate, and the method can be widely applied to the detection of low-density lipoprotein cholesterin.
Description
Technical field
The present invention relates to biochemical field, be specifically related to a kind of method and kit that detects LDL-C.
Background technology
Cholesterol is the indispensable important substance of human body, and it not only participates in forming plasma membrane, but also is synthetic bile acid, the raw material of vitamin D and steroid hormone.In normal blood, cholesterol is main to turn round in blood with the lipoprotein form with low-density lipoprotein (LDL) combination, the cholesterol of institute's combination is called LDL-C (LDL-C), in the time of need to utilizing the materials such as cholesterol biosynthesis plasma membrane when cell, cell produces ldl receptor protein, and it is installed on plasma membrane.LDL is combined rear with cell surface receptor and lysosome merges, and the cholesterol on LDL is hydrolyzed, and is synthetic for materials such as plasma membranes.
Along with growth in the living standard, people's diet is tending towards sophistication, usually absorb unconsciously excessive, higher than the cholesterol of human body cell demand, this makes the concentration of LDL in blood constantly raise.And the LDL of high concentration easily is oxidized to ox-LDL, then by macrophage phagocytic, owing to being combined with cholesterol on LDL, is deposited on arterial wall so can form foam cells, causes atherosclerotic, finally causes cardiovascular and cerebrovascular disease.There are some researches show, the every rising 1% of LDL-C concentration in blood, the risk of suffering from cardiovascular and cerebrovascular disease increases 2-3%.Therefore, in detection blood, LDL-C concentration is significant to prevention and treatment cardiovascular and cerebrovascular disease.
At present, in order to be adapted to the generally application of automatic clinical chemistry analyzer, the method for many detection LDL-C has appearred.As suppress method, its reaction principle is to adopt a kind of surfactant, HDL-C, C-VLDL and chylomicron are suppressed, the cholesterol of its combination is not hydrolyzed, and the cholesterol of LDL-C combination is hydrolyzed, then generate the quinoid colored compound by cholesterol oxidase, peroxidase, then detect the concentration that absorbance is calculated LDL-C.also having a kind of is to utilize two kinds of surfactants to HDL-C, LDL-C, the null method of the different foundation of the affinity of C-VLDL and chylomicron, its reaction principle is that polyanion is combined with LDL-C and surfactant I, when lacking coupling agent not with lentochol reaction, and HDL-C, C-VLDL and chylomicron reaction Hydrogen Peroxide are eliminated, add surfactant II to make LDL-C hydrolysis cholesterol, at this moment in whole reaction system, product is LDL-C, by the cholesterol level determinations LDL-C content that develops the color.But surfactant mentioned in said method is all unexposed, and needs simultaneously accurate full automatic biochemical apparatus strictly to operate, and this just makes it can not be widely used in detecting LDL-C.
In order to make the detection method widespread use, particularly can use at some different medical units, prior art utilizes precipitation agent and low-density lipoprotein reaction to generate suspension, then detect absorbance, because the concentration of the suspension absorbance of reacting generation and LDL-C is proportional, can obtain testing result so just calculate by turbidimetry.Yet this precipitation agent method need to be removed the materials such as the globulin of Interference Detection in blood sample, high-density lipoprotein (HDL), makes detecting step loaded down with trivial details, complicated, is unfavorable for fast detecting.Simultaneously, the precipitation agent method in accuracy a little less than inhibition method and null method.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method and kit that detects LDL-C, make the method and kit can remove in blood sample interfering material and just can detect, and the acquisition high accuracy.
To achieve these goals, the invention provides following technical scheme:
A kind of method that detects LDL-C, be the mixed liquor of 6.1-6.2 with phosphotungstic acid and agaropectin composition pH value, testing sample mixes with mixed liquor and PEG 20000 under the effect of magnesium ion, obtain suspension, then detect absorbance under the 630nm wavelength, with through the standard solution of above-mentioned same treatment than turbid, calculate the concentration of low density lipoprotein cholesterol of testing sample.
Wherein, sample concentration of low density lipoprotein cholesterol (mmol/L)=concentration of standard solution * A
Sample/ A
Mark, the volume ratio of described testing sample, mixed liquor and PEG 20000 is 1: 48: 12.
In mixed liquor of the present invention, the concentration of agaropectin is 0.2-0.4g/L, and in described mixed liquor, the concentration of phosphotungstic acid is 4-5g/L, and described magnesium ion concentration is 2-4g/L, preferably adopts magnesium chloride solution, and described PEG 20000 concentration is 6-10g/L.
Existing precipitation agent method is to utilize low-density lipoprotein (LDL) and polyanion and bivalent cation to react the principle that generates the deposit seed suspension, detects the concentration of LDL-C by turbidimetry.But, because the precipitation agent that adopts in existing precipitation agent method is nonspecific, so in blood sample, globulin, high-density lipoprotein (HDL) etc. disturb albumen can generate suspension with its reaction equally, detection is caused interference, thereby need to remove in advance to disturb albumen, so just make and detect the very complicated that becomes.In addition, not thorough due to what disturb albumen to remove, make the testing result of existing precipitation agent method deviation also occur.The present invention is directed to this problem, select a kind of novel associating precipitation agent, i.e. phosphotungstic acid, magnesium chloride and PEG 20000.This co-precipitation agent only has the specificity with the LDL reaction, thereby can remove foreign protein, make detect simple, accurately.Wherein, phosphotungstic acid is polyanion, and the magnesium ion in magnesium chloride is bivalent cation, and PEG 20000 has the effect of co precipitation.Simultaneously, the present invention also adds agaropectin, agaropectin when detecting be colloidal sol long-chain macromolecule material, and the deposit seed that can impel reaction to generate is dispersed in solution, prevents the bulky grain precipitation, makes detection more accurate.
(LDL-C) is combined on LDL due to LDL-C, so the suspension absorbance that LDL reaction produces is proportional with the concentration of LDL-C, need only by with standard solution than the turbid concentration that can detect LDL-C.Standard solution of the present invention is that concentration of low density lipoprotein cholesterol is deactivation human serum or the pig serum of 2.6mmol/L.
The present invention also provides a kind of kit that detects LDL-C, comprising:
The phosphotungstic acid of the magnesium chloride of 2-4g/L, the PEG 20000 of 6-10g/L, 4-5g/L and the agaropectin of 0.2-0.4g/L.
As preferably, kit of the present invention also comprises: the NaOH of 7-9g/L, the Sodium azide of 0.5-1.0g/L and standard solution.Wherein, NaOH is used for regulating the pH value of reagent when detecting, and Sodium azide is stable as the antiseptic guarantee reagent, and described standard solution is that concentration of low density lipoprotein cholesterol is deactivation human serum or the pig serum of 2.6mmol/L.
Kit of the present invention can be mixed with double reagent, also can be mixed with single reagent.When being mixed with double reagent, form reagent 1 by the phosphotungstic acid of 4-5g/L, the NaOH of 7-9g/L, the agaropectin of 0.2-0.4g/L and the Sodium azide of 0.5-1.0g/L; Form reagent 2 by the magnesium chloride of 2-4g/L, the PEG 20000 of 6-10g/L and the Sodium azide of 0.5-1.0g/L; The deactivation human serum or the pig serum that are 2.6mmol/L by concentration of low density lipoprotein cholesterol form standard solution.
When being mixed with single reagent, form single agents by the phosphotungstic acid of 4-5g/L, the NaOH of 7-9g/L, the agaropectin of 0.2-0.4g/L, the magnesium chloride of 2-4g/L, the PEG 20000 of 6-10g/L and the Sodium azide of 0.5-1.0g/L; The deactivation human serum or the pig serum that are 2.6mmol/L by concentration of low density lipoprotein cholesterol form standard solution.
The method of the invention detects LDL-C concentration and has high accuracy and precision, and test findings shows, the result that the method for the invention detects and employing Japan and light imported L DL-C liquid double reagent kit (inhibition method) testing result no significant difference.In addition, the present invention carries out repeatability to same sample and detects, and the standard rate (coefficient of variation) between each result is 1.46%, less than 3% of standard.Above-mentioned test findings shows that the present invention has very high accuracy and precision.
By above technical scheme as can be known, the method for the invention can be removed foreign protein, and specific and LDL reaction makes the detection of LDL-C simply, fast, accurately, can be widely used in the detection of LDL-C.
Embodiment
The invention discloses a kind of method and kit that detects LDL-C, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.The method of the invention and kit are described by preferred embodiment, the related personnel obviously can change methods and applications as herein described within not breaking away from content of the present invention, spirit and scope or suitably change and combination, realizes and use the technology of the present invention.
Below be described further with regard to a kind of kit and method that detects LDL-C provided by the present invention.
Embodiment 1: the method for the invention
Phosphotungstic acid and agaropectin are formed mixed liquor, and (phosphotungstic acid concentration is 4.4g/L, agaropectin concentration is 0.25g/L, pH 6.1), be in the magnesium chloride solution of 3g/L in concentration, testing sample and mixed liquor and PEG 20000 (8g/L) are by volume, testing sample: mixed liquor: the ratio of PEG 20000=1: 48: 12 is mixed, and the suspension that obtains detects absorbance A under the 630nm wavelength
Sample, then with A through the standard solution (concentration of low density lipoprotein cholesterol is the deactivation human serum of 2.6mmol/L) of above-mentioned same treatment
MarkThan turbid, calculate the LDL-C concentration of testing sample, formula is sample LDL-C concentration (mmol/L)=concentration of standard solution (2.6mmol/L) * A
Sample/ A
Mark
Embodiment 2: the method for the invention
Phosphotungstic acid and agaropectin are formed mixed liquor, and (phosphotungstic acid concentration is 4g/L, agaropectin concentration is 0.3g/L, pH 6.2), be in the magnesium chloride solution of 2g/L in concentration, testing sample and mixed liquor and PEG 20000 (6g/L) are by volume, testing sample: mixed liquor: the ratio of PEG 20000=1: 48: 12 is mixed, and the suspension that obtains detects absorbance A under the 630nm wavelength
Sample, then with A through the standard solution (concentration of low density lipoprotein cholesterol is the deactivation human serum of 2.6mmol/L) of above-mentioned same treatment
MarkThan turbid, calculate the LDL-C concentration of testing sample, formula is sample LDL-C concentration (mmol/L)=concentration of standard solution (2.6mmol/L) * A
Sample/ A
Mark
Embodiment 3: the method for the invention
Phosphotungstic acid and agaropectin are formed mixed liquor, and (phosphotungstic acid concentration is 5g/L, agaropectin concentration is 0.4g/L, pH 6.1), be in the magnesium chloride solution of 4g/L in concentration, testing sample and mixed liquor and PEG 20000 (10g/L) are by volume, testing sample: mixed liquor: the ratio of PEG 20000=1: 48: 12 is mixed, and the suspension that obtains detects absorbance A under the 630nm wavelength
Sample, then with A through the standard solution (concentration of low density lipoprotein cholesterol is the deactivation human serum of 2.6mmol/L) of above-mentioned same treatment
MarkThan turbid, calculate the LDL-C concentration of testing sample, formula is sample LDL-C concentration (mmol/L)=concentration of standard solution (2.6mmol/L) * A
Sample/ A
Mark
Embodiment 4: the accuracy of the method for the invention (degree of conformity) is analyzed
Test apparatus: Olympus400 automatic clinical chemistry analyzer;
Test sample: 20 examinee's blood samples;
Control methods kit: Japan and light imported L DL-C liquid double reagent kit (inhibition method);
The setting of Olympus400 automatic clinical chemistry analyzer parameter sees Table 1.
Table 1 Olympus400 automatic clinical chemistry analyzer parameter
| Method | Wavelength | The Direction of Reaction | Temperature | Measuring Time | Concentration of standard solution | Sample size | Linear |
| End-point method | 630nm | + | 37℃ | 300s | 2.6mmol/L | 5μL | 1.3-5.2mmol/L |
The detection method of employing embodiment 1 and Japan and light imported L DL-C liquid double reagent kit detect respectively above-mentioned 20 examinee's blood samples.Concrete outcome sees Table 2.
Table 2 analysis result (mmol/L)
Result shows, the R value that calculates according to testing result is 0.993, the R value of embodiment 2,3 detection methods shows the method for the invention testing result and Japan and light kit testing result no significant difference equally greater than 0.99, has high accuracy (degree of conformity).
Embodiment 5: the precision analysis of the method for the invention
Test apparatus: Hitachi's 7080 automatic clinical chemistry analyzers;
Test sample: any blood serum sample;
Hitachi's 7080 automatic clinical chemistry analyzers are set: temperature is 37 ℃, and reaction method is end-point method, and the mensuration wavelength is 630nm, and the Direction of Reaction is positive reaction, hatches 180s.
To same testing sample duplicate detection 10 times, wherein, the testing result of embodiment 1 detection method sees Table 3 to the detection method that adopts embodiment 1 to embodiment 3 respectively.
Table 3 test sample LDL-C concentration (10 times) and standard rate (coefficient of variation)
The result demonstration, the standard rate is 1.46%, less than 3% of standard, the standard rate of the testing result of embodiment 2,3 detection methods shows that equally less than 3% the method for the invention has high precision.
Embodiment 6: the linear analysis of the method for the invention
Test apparatus: Hitachi's 7080 automatic clinical chemistry analyzers;
Test sample: high LDL-C blood serum sample (5.2mmol/L);
Hitachi's 7080 automatic clinical chemistry analyzers are set: temperature is 37 ℃, and reaction method is end-point method, and the mensuration wavelength is 630nm, and the Direction of Reaction is positive reaction, hatches 180s.
High LDL-C serum samples diluted is become 4 different concentration, be followed successively by 1.30mmol/L, 2.60mmol/L, 3.90mmol/L, 5.20mmol/L, adopt the detection method of embodiment 1 to embodiment 3 to carry out 2 detections to each concentration of above-mentioned sample, calculate the R value according to the formula in Excel, wherein, the testing result of embodiment 1 detection method sees Table 4.
Table 4 linear test result
Result shows, the R value that calculates according to testing result is 0.998, equally greater than 0.99, shows that the method for the invention has the good linearity close to the R value of 1, embodiment 2,3 detection methods in the scope of 1.30-5.20mmol/L.
Embodiment 7: kit of the present invention
The agaropectin of the phosphotungstic acid of reagent 1:4.4g/L, the NaOH of 8g/L, 0.25g/L and the Sodium azide of 0.75g/L;
The Sodium azide of the magnesium chloride of reagent 2:3g/L, the PEG 20000 of 8g/L and 0.75g/L;
Titer: concentration of low density lipoprotein cholesterol is the human serum of 2.6mmol/L.
Embodiment 8: kit of the present invention
The agaropectin of the phosphotungstic acid of single reagent: 4g/L, the NaOH of 7g/L, 0.3g/L, the Sodium azide of 0.5g/L, the magnesium chloride of 2g/L and the PEG 20000 of 6g/L;
Titer: concentration of low density lipoprotein cholesterol is the pig serum of 2.6mmol/L.
Embodiment 9: kit of the present invention
The agaropectin of the phosphotungstic acid of reagent 1:5g/L, the NaOH of 9g/L, 0.4g/L and the Sodium azide of 1.0g/L;
The Sodium azide of the magnesium chloride of reagent 2:4g/L, the PEG 20000 of 10g/L and 1.0g/L;
Titer: concentration of low density lipoprotein cholesterol is the human serum of 2.6mmol/L.
The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. method that detects LDL-C, it is characterized in that, be the mixed liquor of 6.1-6.2 with phosphotungstic acid and agaropectin composition pH value, in described mixed liquor, the concentration of agaropectin is 0.2-0.4g/L, the concentration of phosphotungstic acid is 4-5g/L, testing sample mixes with mixed liquor and PEG 20000 under the effect of magnesium ion, obtain suspension, then detect absorbance under the 630nm wavelength, with through the standard solution of above-mentioned same treatment than turbid, calculate the concentration of low density lipoprotein cholesterol of testing sample;
Wherein, described magnesium ion concentration is 2-4g/L, and described PEG 20000 concentration is 6-10g/L.
2. method according to claim 1, is characterized in that, the volume ratio of described testing sample, mixed liquor and PEG 20000 is 1:48:12.
3. method according to claim 1, is characterized in that, described standard solution is that concentration of low density lipoprotein cholesterol is deactivation human serum or the pig serum of 2.6mmol/L.
4. a kit that detects LDL-C, is characterized in that, comprising:
The phosphotungstic acid of the magnesium chloride of 2-4g/L, the PEG 20000 of 6-10g/L, 4-5g/L and the agaropectin of 0.2-0.4g/L.
5. kit according to claim 4, is characterized in that, also comprises:
The NaOH of 7-9g/L, the Sodium azide of 0.5-1.0g/L and standard solution.
6. kit according to claim 5, is characterized in that, described standard solution is that concentration of low density lipoprotein cholesterol is deactivation human serum or the pig serum of 2.6mmol/L.
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| CN102749459A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Lipoprotein (alpha) detection kit and preparing method thereof |
| CN106596209B (en) * | 2015-10-14 | 2020-05-12 | 杭州量康科技有限公司 | Dry blood sample pretreatment and detection method |
| CN108949902A (en) * | 2018-08-31 | 2018-12-07 | 山东博科生物产业有限公司 | A kind of serum small and dense low-density lipoprotein cholesterin detection reagent box |
| CN109580303A (en) * | 2018-12-06 | 2019-04-05 | 潍坊泽成生物技术有限公司 | The preparation method of low-density lipoprotein |
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