CN113913492A - Small and dense low-density lipoprotein cholesterol detection kit and preparation method thereof - Google Patents
Small and dense low-density lipoprotein cholesterol detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a small and dense low-density lipoprotein cholesterol detection kit and a preparation method thereof. The small and dense low-density lipoprotein cholesterol detection kit can selectively react with specific small and dense low-density lipoprotein cholesterol (sdLDL-C) based on the action of a special protective agent and a surfactant in a reagent, so that the concentration of the sdLDL-C can be accurately detected.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnostic reagents, in particular to a small and dense low-density lipoprotein cholesterol detection kit and a preparation method thereof.
Background
Low Density Lipoproteins (LDL) are heterogeneous and consist of a range of particles that vary in size, density and chemical composition. LDL with smaller particles and higher Density in the LDL subcomponent is generally called small dense LDL (small dense lipid protein, sd LDL); LDL with larger particles and lower density is called large and light LDL; the subcomponent between the two is medium density LDL. Elevated plasma triglyceride levels promote the conversion of LDL from large, loose LDL to sd LDL, and it has been found that sd LDL has a stronger atherogenic effect than ordinary LDL in a subset of small, dense, low-density lipoproteins (sd LDL). Elevated LDL levels of low density lipoprotein cholesterol are independent atherosclerosis-actuating risk factors, and are based on the manifestation of a stronger effect of small, dense low density lipoprotein cholesterol sd LDL, high invasiveness of sd LDL into the vessel wall, low affinity with LDL receptors, longer plasma half-life and low tolerance to oxidative stress, resulting in sd LDL being more prone to atherosclerosis. Therefore, small, dense low-density lipoprotein cholesterol sd LDL is an important cardiovascular marker, and can predict the risk of coronary heart disease.
The current detection methods mainly comprise: sd LDL gradient gel electrophoresis (CGE method), sd LDL density gradient ultracentrifugation (DGUC method), sd LDL high performance liquid chromatography (HPLC method), etc. The methods all need special instruments and equipment, part of the instruments and equipment are very expensive, and the requirements of clinical batch detection cannot be met when the operation is complicated and waste, so the methods are not well popularized and popularized in clinical examination. The detection method popularized in clinic is a small and dense low-density lipoprotein cholesterol detection kit of an enzyme method, can be suitable for various full-automatic biochemical analyzers, has the characteristics of simple methodology principle, convenience in operation, short analysis time, high result precision, high stability and the like, and is particularly suitable for clinical batch detection.
The method for detecting the small and dense low-density lipoprotein cholesterol by adopting the enzyme method is characterized in that the method is based on the reactivity of a protective agent for inhibiting and protecting the small and dense low-density lipoprotein cholesterol and cholesterol esterase and cholesterol oxidase in reagent components, and has high inhibition effect and bottom, so that the method has great influence on the accuracy of a detection result. However, in the small and dense low-density lipoprotein cholesterol detection kit in the prior art, due to the fact that the reagent proportion is complex, and the inhibition protection effects of the protective agent on small and dense low-density lipoprotein cholesterol and cholesterol esterase and cholesterol oxidase in the components of the reagent are different, the detection result may have certain deviation, and therefore, a small and dense low-density lipoprotein cholesterol detection kit with more reasonable components and more accurate detection result is lacking in the market.
Disclosure of Invention
In order to solve the above problems, an object of the present invention is to provide a small and dense low-density lipoprotein cholesterol assay kit and a method for preparing the same, wherein the small and dense low-density lipoprotein cholesterol assay kit comprises a reagent R1 and a reagent R2, and the small and dense low-density lipoprotein cholesterol assay kit can selectively react with a specific small and dense low-density lipoprotein cholesterol (sd LDL-C) based on the action of a specific protective agent and a surfactant in the reagents, thereby accurately detecting the concentration of the sd LDL-C.
In order to achieve the technical effect, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a small, dense low density lipoprotein cholesterol assay kit comprising a reagent R1 and a reagent R2, wherein:
the R1 reagent includes: buffer solution, protective agent, active enzyme I, chromogen agent and preservative.
The R2 reagent includes: buffer solution, surfactant, active enzyme II, color developing agent and preservative.
Further, the buffer solution in the R1 reagent and the R2 reagent is one or more of Tris buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution or MOPS buffer solution in Good's buffer solution.
Further, the protective agent in the R1 reagent is one or more of polyoxyethylene fatty acid ester and Tween 80.
Preferably, the protective agent is a mixture of polyoxyethylene fatty acid ester and tween 80.
Further, the R1 reagent active enzyme I is cholesterol esterase, cholesterol oxidase, phospholipase and catalase; the chromogen agent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS).
Further, the chromogen agent N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS) in the R1 reagent.
Further, the surfactant in the R2 reagent is one or more of triton X-100, triton X-200 or triton X-405.
Further, the active enzyme II in the R2 reagent is Peroxidase (POD).
Further, the color developing agent in the R2 is 4-aminoantipyrine (4-AAP).
Further, the R1 reagent and the R2 reagent specifically comprise the following components:
r1 reagent:
r2 reagent:
in a second aspect, the present invention also provides a method for preparing a small, dense low-density lipoprotein cholesterol assay kit, comprising the steps of:
s1: preparation of R1 reagent:
adding the buffer solution into a container, adding the protective agent, the active enzyme I, the chromogen agent and the preservative according to the proportion, continuously stirring until the buffer solution is completely dissolved, and fixing the volume by using water;
s2: preparation of R2 reagent:
adding the buffer solution into a container, sequentially adding the surfactant and the color developing agent, stirring uniformly, sequentially adding the active enzyme II and the preservative, adding water to a constant volume, and stirring until the system is completely and uniformly mixed.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a small and dense low-density lipoprotein cholesterol detection kit, which is based on the action of a special protective agent and a surfactant, wherein the protective agent polyoxyethylene fatty acid ester and polyoxyethylene sorbitol fatty acid ester Tween (Tween)80 in an R1 reagent can protect and inhibit the reactivity of sd LDL with cholesterol esterase, cholesterol oxidase and phospholipase, and the cholesterol contained in non-sd LDL components, such as chylomicron CM, very low-density lipoprotein VLDL and intermediate-density lipoprotein IDL, and high-density lipoprotein HDL, is cleared firstly. After the reagent R2 is added, the surfactant can fully release the protected sd LDL, hydrogen peroxide is produced under the action of cholesterol esterase and cholesterol oxidase, the hydrogen peroxide is coupled with chromogens TOOS and 4-AAP under the action of peroxidase to generate purple-red quinone substances, the change of the absorbance can be detected by spectrophotometry, and finally the content of the sd LDL can be detected. The small and dense low-density lipoprotein cholesterol detection kit can selectively react with specific small and dense low-density lipoprotein cholesterol (sd LDL-C) based on the action of a special protective agent and a surfactant in a reagent, so that the concentration of the sd LDL-C can be accurately detected.
Drawings
FIG. 1 shows the result of the accuracy verification of the small and dense low density lipoprotein cholesterol assay kit provided in example 1 of the present invention;
FIG. 2 shows the result of the accuracy verification of the small and dense low density lipoprotein cholesterol assay kit provided in example 2 of the present invention;
FIG. 3 shows the result of the accuracy verification of the small and dense low density lipoprotein cholesterol assay kit provided in example 3 of the present invention;
FIG. 4 shows the result of the accuracy verification of the small and dense low density lipoprotein cholesterol assay kit provided in example 4 of the present invention;
FIG. 5 shows the results of stability verification of the small, dense low-density lipoprotein cholesterol assay kit provided in example 1 of the present invention;
FIG. 6 shows the results of stability verification of the small, dense low-density lipoprotein cholesterol assay kit provided in example 2 of the present invention;
FIG. 7 shows the results of stability verification of the small, dense low-density lipoprotein cholesterol assay kit provided in example 3 of the present invention;
FIG. 8 is a graph showing the results of stability verification of the small, dense low-density lipoprotein cholesterol assay kit provided in example 4 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. The raw materials used in the examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art, unless otherwise specified.
Example 1
The detection kit for the small and dense low-density lipoprotein cholesterol provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 specifically comprise the following components:
r1 reagent:
the R2 reagent:
the preparation method of the small and dense low-density lipoprotein cholesterol detection kit comprises the following steps:
s1: preparation of R1 reagent:
adding the buffer solution into a container, adding the protective agent, the active enzyme I, the chromogen agent and the preservative according to the proportion, continuously stirring until the buffer solution is completely dissolved, and fixing the volume by using water;
wherein the active enzyme I comprises 1600U/L cholesterol esterase, 2700U/L cholesterol oxidase 600U/L phospholipase and 1200KU/L catalase;
s2: preparation of R2 reagent:
adding the buffer solution into a container, sequentially adding the surfactant and the color developing agent, stirring uniformly, sequentially adding the active enzyme II and the preservative, adding water to a constant volume, and stirring until the system is completely and uniformly mixed.
Example 2
The detection kit for the small and dense low-density lipoprotein cholesterol provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 specifically comprise the following components:
r1 reagent:
r2 reagent:
the procedure for preparing the small, dense low-density lipoprotein cholesterol assay kit was the same as in example 1.
Example 3
The detection kit for the small and dense low-density lipoprotein cholesterol provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 specifically comprise the following components:
r1 reagent:
r2 reagent:
the procedure for preparing the small, dense low-density lipoprotein cholesterol assay kit was the same as in example 1.
Example 4
The detection kit for the small and dense low-density lipoprotein cholesterol provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 specifically comprise the following components:
r1 reagent:
r2 reagent:
the procedure for preparing the small, dense low-density lipoprotein cholesterol assay kit was the same as in example 1.
Comparative example 1
This comparative example 1 adopts the small and dense low-density lipoprotein cholesterol sd LDL assay kit approved by the State drug administration of Biotech, Inc. of Beijing Jiuqiang Biotech
Example 5
This example also provides a method for testing a small, dense low-density lipoprotein cholesterol (sd LDL-C) test kit:
in the detection process, taking a full-automatic beckman AU480 biochemical analyzer as an example, the main wavelength is set as follows: 600nm, and a secondary wavelength of 700 nm; the optical path of the cuvette is 1.0 cm; end point method (ENDPOINT); the reaction is raised.
The specific operation steps are shown in table 1:
TABLE 1 test procedure for the determination of small dense low density lipoprotein cholesterol
Before the sample is measured, calibration and quality control procedures are required, a matched calibrator is used as a calibration curve, and the calibration curve of the invention is calibrated by a 2-point linear method. The calibration curve is reproduced every 7 days, and when the reagent lot number is replaced, the instrument is replaced or maintained, and the quality control product or sample is abnormal.
And (3) quality control program: adopting a matched quality control product to perform a quality control experiment every day; and (3) repeatedly measuring each concentration quality control product for 10 times, calculating precision CV, and controlling the precision CV within the deviation range of the quality control product.
Calculating the small and dense low density lipoprotein cholesterol content of the sample according to the calibration curve by the following method:
the accuracy and stability performance evaluation was performed by using the detection kit preparation methods of examples 1 to 4 and comparative example 1:
the accuracy verification method comprises the following steps:
comparison test: referring to the method of EP9a2, a total of 40 fresh clinical samples were collected, with the selected sd LDL concentration in the linear range of reagents and evenly distributed. A comparative experiment was conducted using examples 1 to 3 and comparative example 1, and the correlation coefficient (r) and the linear regression equation of the comparative results were calculated.
TABLE 2 three examples the kit detects sd LDL content (mmol/L) in 40 samples
The accuracy of the statistical data is verified, and the analysis results are shown in the attached figures 1-4:
and (4) conclusion: as shown in the statistical data of fig. 1-4, according to the linear regression equation: y ═ ax + b, it can be seen from R2 of the three sets of comparison results that the correlation between the comparison results of example 3 and comparative example 1 is significantly better than that of examples 1 and 2, and example 4 illustrates that the accuracy of the small dense low density lipoprotein cholesterol sd LDL liquid double reagent prepared by example 3 using the method of the present invention is higher.
The stability test method is as follows:
the detection reagents prepared by the methods of examples 1-4 of the present invention were subjected to stability verification experiments. Each of the above 4 reagents was divided into two parts on average, one part was stored at 2-8 ℃ and the other part was stored in 37 ℃ water bath, and the measurement was performed every other day for one week with continuous monitoring, and the measured value of the serum sample (serum fixed value of 1.50mmol/L, allowable error range. + -. 10%) was observed, and the stability of the reagents was compared by continuous measurement.
TABLE 3 stability data units (mmol/L) of reagents prepared by the method of example 1
TABLE 4 stability data units (mmol/L) of reagents prepared by the method of example 2
TABLE 5 stability data units (mmol/L) of reagents prepared by the method of example 3
TABLE 6 stability data units (mmol/L) of reagents prepared by the method of example 4
The above statistical data are subjected to accuracy verification, and the analysis results are shown in the accompanying fig. 5-8:
and (4) conclusion: as can be seen from FIGS. 5, 6, 7 and 8, the reagents prepared by the methods of examples 1 to 4 had relatively good storage stability at 2 to 8 ℃. The reagent prepared by the method of example 3 was subjected to 37 ℃ water bath for one week, and although the sd LDL content was measured to be somewhat decreased, the decrease was not so large as to be within the allowable range, and was significantly superior to the reagents prepared by the methods of example 1, example 2, and example 4. The proportion and the dosage of the protective agent polyoxyethylene sorbitol fatty acid ester in R1 and the surfactant triton X-200 in R2 in the whole reaction process are proved to have important effect on the accuracy of detecting the content of sd LDL.
As can be seen from the performance evaluation experiments, the small and dense low-density lipoprotein cholesterol (sd LDL-C) liquid double reagent prepared by the method in the embodiment 3 of the invention has the advantages of high accuracy and good stability, and can meet the clinical requirements. Therefore, the detection of the content of small and dense low-density lipoprotein cholesterol sd LDL can predict the risk of coronary heart disease, and provide an important marker for cardiovascular diseases for clinicians so as to take preventive and therapeutic measures in time.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Claims (10)
1. A small, dense low density lipoprotein cholesterol assay kit comprising an R1 reagent and an R2 reagent, wherein:
the R1 reagent includes: buffer solution, protective agent, active enzyme I, chromogen agent and preservative;
the R2 reagent includes: buffer solution, surfactant, active enzyme II, color developing agent and preservative.
2. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the buffer solution in the R1 reagent and the R2 reagent is one or more of Tris buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution or MOPS buffer solution in Good's buffer solution.
3. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the protective agent in the R1 reagent is one or more of polyoxyethylene fatty acid ester and Tween 80.
4. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the R1 reagent active enzyme I is cholesterol esterase, cholesterol oxidase, phospholipase and catalase; the chromogen agent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS).
5. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the chromogen agent N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS) in the R1 reagent.
6. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the surfactant in the R2 reagent is one or more of triton X-100, triton X-200 or triton X-405.
7. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the active enzyme II in the R2 reagent is Peroxidase (POD).
8. The small, dense low-density lipoprotein cholesterol assay kit of claim 1 in which: the color developing agent in the R2 is 4-aminoantipyrine (4-AAP).
9. The small, dense low-density lipoprotein cholesterol assay kit of claim 1, wherein the R1 reagent and the R2 reagent specifically comprise the following components:
r1 reagent:
r2 reagent:
10. the method of claim 1, wherein the method comprises the steps of:
s1: preparation of R1 reagent:
adding the buffer solution into a container, adding the protective agent, the active enzyme I, the chromogen agent and the preservative according to the proportion, continuously stirring until the buffer solution is completely dissolved, and fixing the volume by using water;
s2: preparation of R2 reagent:
adding the buffer solution into a container, sequentially adding the surfactant and the color developing agent, stirring uniformly, sequentially adding the active enzyme II and the preservative, adding water to a constant volume, and stirring until the system is completely and uniformly mixed.
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