阅读说明：本技术 定量测定小而密低密度脂蛋白胆固醇的方法和试剂盒 (Method and kit for quantitative determination of small dense low density lipoprotein cholesterol ) 是由 崔贤艳 许国和 范翠翠 于 2019-12-03 设计创作，主要内容包括：本发明公开了一种定量测定小而密低密度脂蛋白胆固醇的方法,首先在优先保护LDL-C的情况下,消除其它脂蛋白胆固醇,然后加入表面活性剂选择性的测定sd LDL-C。在该方法中,使用的试剂盒包括R1和R2两种独立液体试剂。试剂R1中含有缓冲液、表面活性剂、保护剂、胆固醇酯酶、胆固醇氧化酶、过氧化氢酶、显色底物、防腐剂、抗干扰成分；试剂R2中含有缓冲液、表面活性剂、过氧化物酶、显色剂、防腐剂。优点是：性能更为优良,准确性和特异性高、能有效抗干扰、线性范围广、性价比更高,为临床需要提供了一个很好的选择。(A method for quantitatively measuring small, dense and low-density lipoprotein cholesterol is disclosed, which comprises eliminating other lipoprotein cholesterol under the condition of preferential protection of LDL-C, and then adding surfactant to selectively measure sd LDL-C. In this method, a kit is used comprising two separate liquid reagents R1 and R2. The reagent R1 contains buffer solution, surfactant, protectant, cholesterol esterase, cholesterol oxidase, catalase, chromogenic substrate, preservative and anti-interference components; the reagent R2 contains buffer solution, surfactant, peroxidase, color developing agent and preservative. The advantages are that: the method has the advantages of more excellent performance, high accuracy and specificity, effective anti-interference, wide linear range and higher cost performance, and provides a good choice for clinical needs.)

1. A method for quantitatively determining small, dense low-density lipoprotein cholesterol, characterized by:

the method first eliminates other lipoprotein cholesterol in the case of preferential protection of LDL-C, and then adds a surfactant to selectively quantify sd LDL-C.

2. A kit for quantitatively determining small and dense low-density lipoprotein cholesterol, characterized in that:

the kit comprises two independent liquid reagents of R1 and R2, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1.

3. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 2, characterized in that:

the reagent R1 contains buffer solution, surfactant, protective agent, cholesterol esterase, cholesterol oxidase, catalase, chromogenic substrate, preservative and anti-interference components;

the reagent R2 contains buffer solution, surfactant, peroxidase, color developing agent and preservative.

4. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 3, characterized in that:

in the reagent R1: the surfactant is polyethylene glycol nonionic surfactant, the protective agent is polyethylene glycol nonionic surfactant, the preservative is one or more of potassium sorbate, gentamicin and Proclin series, the buffer solution is one or more of GOOD' S buffer and phosphate buffer, the chromogenic substrate is TOOS or DAOS, and the anti-interference component is ascorbic acid oxidase.

In the reagent R2: the surfactant is alkylbenzene sulfonate surfactant and polyacrylamide surfactant, the preservative is sodium azide, the buffer solution is one or more of GOOD' S buffer and phosphate buffer, and the color developing agent is 4-aminoantipyrine.

5. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 4, characterized in that: in the reagent R1, the concentration of the buffer solution in the solution is 10-200mmol/L, the pH range of the buffer solution is 6.0-8.0, the concentration of the surfactant in the solution is 0.01-2%, the concentration of the protective agent in the solution is 0.01-1%, the concentration of the cholesterol esterase in the reagent is 0.5-5KU/L, the concentration of the cholesterol oxidase in the reagent is 0.2-1KU/L, the concentration of the catalase in the reagent is 300-1500KU/L, the concentration of the chromogenic substrate in the solution is 0.5-6mmol/L, the concentration of the preservative in the solution is 0.05-0.2%, and the concentration of the anti-interference component in the solution is 0.5-8 KU/L.

6. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 5, characterized in that: in the reagent R1, the concentration of the buffer solution in the solution is 20-100mmol/L, the pH range of the buffer solution is 6.0-7.0, the concentration of the surfactant in the solution is 0.03-0.5%, the concentration of the cholesterol esterase in the reagent is 2-5KU/L, the concentration of the cholesterol oxidase in the reagent is 0.3-0.6KU/L, the concentration of the catalase in the reagent is 500-1200KU/L, the concentration of the chromogenic substrate in the solution is 1-4mmol/L, and the concentration of the anti-interference component in the solution is 2-5 KU/L.

7. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 4, characterized in that: in the reagent R2, the concentration of a buffer solution in the reagent is 10-200mmol/L, the pH range of the buffer solution is 6.0-8.0, the concentration of a surfactant in the solution is 0.01-2%, the concentration of peroxidase in the reagent is 1-20KU/L, the concentration of a color developing agent in the solution is 1-8mmol/L, and the concentration of a preservative in the solution is 0.05-0.2%.

8. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 7, characterized in that: in the reagent R2, the concentration of a buffer solution in the reagent is 20-50mmol/L, the pH range of the buffer solution is 6.0-7.0, the concentration of a surfactant in a solution is 0.05% -1%, the concentration of peroxidase in the reagent is 4-15KU/L, and the concentration of a color developing agent in the solution is 2-5 mmol/L.

9. The kit for quantitatively determining small, dense low-density lipoprotein cholesterol according to claim 4, characterized in that: the HLB value of the surfactant in the reagent R1 is 11-14, the HLB value of the protective agent is 12-13.5, and the HLB value of the surfactant in the reagent R2 is 11.5-13.5.

Technical Field

The invention relates to the field of biotechnology, in particular to a method for quantitatively measuring small and dense low-density lipoprotein cholesterol. The invention also relates to a kit for quantitatively determining the small and dense low-density lipoprotein cholesterol.

Background

Lipoproteins are roughly classified into Chylomicrons (CM), Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL) according to specific gravity. LDL has been widely used in clinical settings to assess the risk of atherosclerosis. However, recent studies have revealed that LDL has heterogeneity, and these are classified into: (1) low density, large particle low density lipoprotein (large and buoyant, lb LDL); (2) low-density lipoprotein (sd LDL) with high density and small particles is called small dense low-density lipoprotein for short. LDL is known to be one of the accepted risk factors of arteriosclerosis, however, sd LDL has more atherosclerosis-activating capability than lb LDL, so that the clinical detection of sd LDL is very important.

Methods for measuring small, dense low-density lipoproteins include ultracentrifugation, gradient gel electrophoresis, chemical precipitation, high performance liquid chromatography, and the like. These methods are generally complex to operate, have high requirements on laboratory conditions and operators, and are not strong in clinical applicability. Although the chemical precipitation method is relatively simple and convenient, the precipitation is not easy to be complete, so that the measurement result is not accurate enough, and the method is not suitable for wide clinical popularization.

Further, PCT patent WO2004053500A1 discloses a method for quantitatively determining cholesterol or triacylglycerol in sd LDL-C by using a separating agent (containing a divalent cation and a polyanion) in combination with a reagent suitable for a fully automated biochemical analyzer. This method, although capable of more convenient determination of sd LDL-C than electrophoresis or ultracentrifugation, does not avoid the need to pre-treat the sample and the need for separation of LDL into sd LDL and LDL other than sd LDL.

Recently, inventions granted to China and having publication Nos. CN101512012B (hereinafter referred to as document 1) and CN 101896620B (hereinafter referred to as document 2) disclose the development of a method for directly and quantitatively detecting sd LDL-C by utilizing a specific reaction between a surfactant and lipoprotein, respectively. The advent of such methods opened up new avenues for the direct determination of sd LDL-C in samples. In this method, non-sd LDL-C lipoprotein cholesterol is selectively dissociated using a specific surfactant, and the cholesterol ester released by the dissociation is decomposed so as not to participate in a color development reaction; the remaining sd LDL-C was then cleared and quantified. This method of directly measuring sd LDL-C can be called a homogeneous method, and is suitable for rapid measurement of a large clinical sample by a fully automated biochemical analyzer. At present, the reagent sold on the market based on the method is popularized and used in clinical laboratories of all levels of hospitals, and some domestic related commercial kits are also supplied.

Disclosure of Invention

The invention aims to provide a method for quantitatively measuring small and dense low-density lipoprotein cholesterol, which has the characteristics of effective interference resistance and nonspecific elimination. The invention also discloses a kit for quantitatively determining the small and dense low-density lipoprotein cholesterol.

The first technical scheme adopted by the invention is as follows:

a method for quantitatively determining small, dense low-density lipoprotein cholesterol,

the method first eliminates other lipoprotein cholesterol in the case of preferential protection of LDL-C, and then adds a surfactant to selectively quantify sd LDL-C.

The method has the innovation points that:

1. in the first step of the assay, rather than eliminating lipoprotein cholesterol except for small, dense, other lipoprotein cholesterol is eliminated in the case of preferential protection of LDL-C; in the second step of the assay, instead of adding a strong surfactant to eliminate all lipoprotein cholesterol, sd LDL-C was selectively measured. That is, long-term studies have found that the dissociation of lipoprotein cholesterol by a surfactant is not absolute, and under the guidance of the principle of the present invention, selective measurement of sd LDL-C can ensure that lipoprotein cholesterol other than sd LDL-C is not reacted as much as possible in the process of quantifying sd LDL-C, thereby improving the specificity and accuracy of sd LDL-C detection.

2. In the reagent R1, the selection of the surfactant and the protecting agent is focused, and the selection of the kind, concentration, and ratio of the preferred surfactant and protecting agent is focused. That is, it was found that selection of a surfactant and a protecting agent, and their concentrations and ratios play an important role in protecting LDL-C and eliminating lipoprotein cholesterol other than LDL-C, and that this is a technical key for improving the effect of the method for measuring sd LDL-C. This option has not previously been of high interest. That is, the selection of the surfactant and the protecting agent is highly compatible with the principle of measurement, and LDL-C is protected first, lipoprotein cholesterol other than LDL-C is eliminated, and then sd LDL-C is selectively measured. Therefore, the effects of high specificity and accuracy and avoiding the occurrence of false positive in clinical data are achieved.

3. The selection of the source of the enzyme for measuring cholesterol in the reagent, and the concentration and ratio in the reagent is of great importance. That is, the correlation between the enzymes for measuring cholesterol in the reagent is a key factor for accurately and quantitatively measuring sd LDL-C in a human serum sample by the method of the present invention. It was found that there are great differences in stability, potency, action effect, etc. of enzymes for cholesterol measurement from different sources.

4. The preservative added to the reagent R1 was Proclin series. The preservative is a novel biological preservative, and is found to have good compatibility with various enzymes, good low toxicity and good stability, and the stability of the reagent is greatly maintained.

5. The reagent R1 has a main effect of protecting LDL-C and eliminating lipoprotein cholesterol other than LDL-C from hydrolysis by enzymes in the reaction system. That is, the surfactant and the protecting agent in the reagent R1 can effectively protect LDL-C and eliminate VLDL-C, HDL-C, CM and the like at an appropriate ratio and concentration. At the same time, the reagent R2 acts to selectively dissociate sd LDL-C while ensuring that non-sd LDL-C species do not participate or participate to a lesser extent in the reaction. In the first stage, in order to study the selective dissociation of lipoproteins by several tens of surfactants such as cations, anions and nonionic surfactants, the hydrophilic-lipophilic value and critical micelle concentration are mainly considered, and in the first step, a surfactant having a low reactivity with the LDL fraction and a high reactivity with the CM and VLDL fractions is preferably selected as the surfactant in the reagent R1, and one or two of them may be used in combination. In the second step, a surfactant having a high reactivity with sd LDL and a low reactivity with lb LDL and other components is preferably selected. Namely, the effects of high specificity and accuracy are achieved. In addition, the method and the technology can well improve the linear range of the detection of the kit, and compared with similar products in the market, the existing kit has a higher linear detection range.

The second technical scheme adopted by the invention is as follows:

a kit for quantitatively determining small and dense low-density lipoprotein cholesterol,

the kit comprises two independent liquid reagents of R1 and R2, wherein the liquid reagents consist of a reagent R1 and a reagent R2 according to the volume ratio of 3: 1.

The reagent R1 contains buffer solution, surfactant, protective agent, cholesterol esterase, cholesterol oxidase, catalase, chromogenic substrate, preservative and anti-interference components;

the reagent R2 contains buffer solution, surfactant, peroxidase, color developing agent and preservative.

In the reagent R1: the surfactant is polyethylene glycol nonionic surfactant, the protective agent is polyethylene glycol nonionic surfactant, the preservative is one or more of potassium sorbate, gentamicin and Proclin series, the buffer solution is one or more of GOOD' S buffer and phosphate buffer, the chromogenic substrate is TOOS or DAOS, and the anti-interference component is ascorbic acid oxidase.

In the reagent R2: the surfactant is alkylbenzene sulfonate surfactant and polyacrylamide surfactant, the preservative is sodium azide, the buffer solution is one or more of GOOD' S buffer and phosphate buffer, and the color developing agent is 4-aminoantipyrine.

The surfactant in the reagent R1 is a polyethylene glycol nonionic surfactant, preferred specific examples of HLB include, but are not limited to, compounds having HLB of 11-14, such as alkylphenol polyoxyethylene ether, nonylphenol polyoxyethylene ether, and the like, and specific examples of the above surfactants include polyethylene glycol alkylphenyl ether JK-14, nonylphenol polyoxyethylene ether NP-7.

The surfactant in the reagent R1 is a polyethylene glycol nonionic surfactant, the protective agent in the reagent R1 is a polyethylene glycol nonionic surfactant, and preferred specific examples of HLB include, but are not limited to, compounds having HLB of 12-13.5, such as polyoxyethylene alkyl ether EM-701, and nonylphenol polyoxyethylene ether NP-10.

Preferred specific examples of the HLB, which is a surfactant in the agent R2, include, but are not limited to, compounds having an HLB of 11.5 to 13.5, including at least one of alkylbenzenesulfonates and polyacrylamides, such as HNL006 and HEAL-06.

In the reagent R1, the concentration of the buffer solution in the solution is 10-200mmol/L, preferably 20-100 mmol/L; the pH of the buffer is in the range of 6.0 to 8.0, preferably 6.0 to 7.0; the concentration of the surfactant in the solution is 0.01% -2%, preferably 0.03% -0.5%; the concentration of the protective agent in the solution is 0.01-1%; the concentration of the cholesterol esterase in the reagent is 0.5-5KU/L, preferably 2-5 KU/L; the concentration of cholesterol oxidase in the reagent is 0.2-1KU/L, preferably 0.3-0.6 KU/L; the concentration of the catalase in the reagent is 300-1500KU/L, preferably 500-1200 KU/L; the concentration of the chromogenic substrate in the solution is 0.5-6mmol/L, preferably 1-4 mmol/L; the concentration of the preservative in the solution is 0.05-0.2%; the concentration of the anti-interference component in the solution is 0.5-8KU/L, preferably 2-5 KU/L.

In the reagent R2, the concentration of the buffer solution in the reagent is 10-200mmol/L, preferably 20-50 mmol/L; the pH of the buffer is in the range of 6.0 to 8.0, preferably 6.0 to 7.0; the concentration of the surfactant in the solution is 0.01% -2%, preferably 0.05% -1%; the concentration of the peroxidase in the reagent is 1-20KU/L, preferably 4-15 KU/L; the concentration of the color developing agent in the solution is 1-8mmol/L, preferably 2-5 mmol/L; the concentration of the preservative in the solution is 0.05% -0.2%.

In conclusion, the method and the kit for quantitatively determining the small, dense and low-density lipoprotein cholesterol have relatively unique principles and characteristics, are more excellent in reagent performance, high in accuracy, capable of effectively resisting interference, eliminating non-specificity and higher in cost performance, and provide a good choice for clinical needs. Meanwhile, the reagent formula components are easy to obtain, the cost is low, large-scale industrial preparation can be met, and the requirements of clinical laboratories on sd LDL-C content determination can be met conveniently, reliably and efficiently.

Drawings

The invention is further illustrated with reference to the following figures and examples:

FIG. 1 is a diagram of the measurement of 11 serum samples mixed in gradient concentration by using Hitachi 7180 full-automatic biochemical analyzer,and the measured values are subjected to correlation analysis to form a graph. Wherein the X-axis represents the dilution concentration and the Y-axis represents the mean of the measured values. Coefficient of correlation R²0.999; the linear equation: 1.0173 x-0.0693.

FIG. 2 shows that 104 fresh serum samples (covering linear range, including normal and abnormal samples) were measured according to their respective parameters and correlation analysis was performed on the measured values using reagents A commercially available from the kit of the present invention and Hitachi 7180 full-automatic biochemical analyzer, respectively. Wherein the X-axis represents the measured value of the reagent of the present invention, and the Y-axis represents the measured value of the commercially available reagent A, in the fifth embodiment, the correlation coefficient: r²0.9647, linear equation: y 0.8809x + 0.0969.

Fig. 3 is a schematic diagram of a specific detection procedure of an embodiment of the present invention.

Detailed Description