WO2023155352A1 - 脂蛋白胆固醇检测方法和试剂盒 - Google Patents

脂蛋白胆固醇检测方法和试剂盒 Download PDF

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WO2023155352A1
WO2023155352A1 PCT/CN2022/100428 CN2022100428W WO2023155352A1 WO 2023155352 A1 WO2023155352 A1 WO 2023155352A1 CN 2022100428 W CN2022100428 W CN 2022100428W WO 2023155352 A1 WO2023155352 A1 WO 2023155352A1
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composition
reagent
lipoprotein cholesterol
kit
concentration
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PCT/CN2022/100428
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English (en)
French (fr)
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李元丽
芮海涛
李强
马腾飞
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北京沃森赛瑟生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/32—3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate, i.e. MBTH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the present invention relates to a method and a reagent for measuring lipoprotein cholesterol, in particular to a method and a reagent for detecting lipoprotein cholesterol related to the diagnosis of arteriosclerosis.
  • LDL Low-density lipoprotein
  • Examples of conventional methods for measuring small, dense LDL include ultracentrifugation, electrophoresis, and methods using high performance liquid chromatography. Since these methods require expensive equipment and a lot of measurement time, they are not convenient.
  • An example of a method for measuring small, dense LDL using an automatic analyzer is the following method (see Japanese Patent Publication No. 2003-28882A), which involves suspending or dissolving small-particle LDL using a difference in ionic strength, and then using a difference in absorbance to Small particle LDL was measured.
  • the difference in absorbance is based on turbidimetry according to the method described above. Therefore, cholesterol in small and dense LDL cannot be determined, so the specificity and accuracy are insufficient.
  • a method is known (see International Patent Publication WO2004/053500) which involves measuring the concentration of ⁇ in small, dense LDL by using a combination of a separating agent comprising polyanions and divalent cations and a reagent suitable for an automatic analyzer.
  • a separating agent comprising polyanions and divalent cations
  • a reagent suitable for an automatic analyzer cholesterol or triglycerides.
  • This method can measure lipid components in small and dense LDL more conveniently than ultracentrifugation or electrophoresis. But this method requires pretreatment of the sample and a step of separating LDL into small dense LDL and LDL other than such LDL.
  • the invention relates to a method for the fractional determination of lipoprotein cholesterol suitable for an automatic analyzer and reagents for such determination. In the case of clinical test results, it meets the linearity requirements of the reagent, with high accuracy and good stability.
  • composition comprises:
  • the composition also comprises a preservative.
  • the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
  • the present invention also provides a composition, characterized in that the composition comprises:
  • the preservative is one or more of sodium azide, Florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
  • the aforementioned composition is in an acidic environment.
  • the pH value is 0.5-4.5, more preferably, the pH value is 1-4.05.
  • the above composition further comprises one or more selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
  • the composition described above also comprises a surfactant.
  • the surfactant is selected from polyoxyethylene derivatives, preferably polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether (EMULGEN707, EMULGEN709), polyoxyethylene alkylphenyl ether, more preferably, Polyethylene glycol octylphenyl ether (Triton X-100), polyoxyethylene nonylphenyl ether (EMULGEN909).
  • the concentration of the 3-methylbenzothiazolone hydrazone or its salt is 0.1-1.0 g/L. More preferably, the concentration of the 3-methylbenzothiazolone hydrazone or its salt is 0.1-0.4 g/L.
  • the concentration of peroxidase is 2000-4000U/L.
  • the concentration of the surfactant is 20-35g/L.
  • the present invention also provides a kit, which is characterized in that the kit includes at least the reagent composition (ii), and the reagent composition (ii) includes the above-mentioned composition.
  • the kit further comprises a reagent composition (i), and the reagent composition (i) is used for effectively shielding interferences other than the lipoprotein to be tested.
  • the reagent composition (i) comprises phospholipase, preferably, at least one of phospholipase A2, phospholipase C, phospholipase D, lysophospholipase (LYPL) and sphingomyelinase; the concentration range of the preferred phospholipase 0.1U/mL-100U/mL.
  • effective shielding in the present invention is to act on the interfering components through physical or chemical means, so that the interfering components will not have a substantial impact on the subsequent detection steps, and effective shielding does not require that the interfering components be completely removed or excluded from the system outside.
  • the reagent composition (i) also includes a nonionic surfactant, preferably a nonionic surfactant of HLB 13-15, more preferably, a polyoxyethylene derivative or polyoxyethylene distyrylphenyl Ether, for example, EMULGEN B-66, EMULGEN A-90;
  • a nonionic surfactant preferably a nonionic surfactant of HLB 13-15, more preferably, a polyoxyethylene derivative or polyoxyethylene distyrylphenyl Ether, for example, EMULGEN B-66, EMULGEN A-90;
  • concentration of said nonionic surfactant is 1.5-2.0g/L.
  • the reagent composition (i) further comprises cholesterol esterase, cholesterol oxidase and hydrogen donor.
  • the reagent composition (i) also includes inorganic salt ions, selected from one or more of magnesium chloride, EDTA 2Na, EDTA 2K, sodium glutamate, sodium chloride or potassium chloride .
  • the reagent composition (i) further includes a stabilizer selected from one or more of BSA, sucrose or trehalose.
  • the hydrogen donor is an aniline derivative, preferably TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline).
  • the present invention also provides a preparation method of the above composition and/or kit.
  • the present invention also provides the application of the above composition and/or kit.
  • composition and/or kit are used in the preparation of products for detecting lipoprotein cholesterol.
  • the product includes a reagent or a test kit.
  • the detection of lipoprotein cholesterol includes detection of low-density lipoprotein cholesterol, small dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, remnant-like lipoprotein cholesterol or total lipoprotein cholesterol.
  • compositions and/or kit are also used in the preparation of products for diagnosing cardiovascular related diseases.
  • the product includes a reagent or a test kit.
  • cardiovascular-related diseases such as dyslipidemia, especially hyperlipidemia, hypercholesterolemia, atherosclerosis and other diseases, including low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein Detection of protein cholesterol, remnant lipoprotein cholesterol, or total lipoprotein cholesterol.
  • the present invention also provides a method for improving the linearity or accuracy of lipoprotein cholesterol detection, which is characterized by using the above composition and/or kit.
  • the present invention also provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or its salt, which is characterized by containing a preservative.
  • the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
  • the 3-methylbenzothiazolone hydrazone or its salt is preferably in an acidic environment.
  • the pH is 1.00-4.05. More preferably, it contains one or more acids or buffer systems selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
  • the present invention also provides a method for quantitatively determining lipoprotein cholesterol in a sample, which is characterized by using the above composition and/or the reagent composition (ii) in the kit.
  • the method includes the following steps: adding the reagent composition (ii) in the above composition and/or kit to detect target lipoprotein cholesterol. Further, the method includes the following steps:
  • it also includes adding cholesterol esterase, cholesterol oxidase and chromogen (hydrogen donor) in step (1) or (2).
  • the reagent composition (i) comprising adding the above kit in step (1).
  • the kit is used for the detection of low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, residual particle-like lipoprotein cholesterol or total lipoprotein cholesterol.
  • the step (1) can be further subdivided into multiple steps to effectively shield different interference components.
  • the present invention provides a method for measuring lipoprotein cholesterol suitable for an automatic analyzer and a reagent for such determination.
  • the method and the reagent can be carried out without pretreatment of the sample, and have good specificity, rapidity and It is convenient for analysis, and at the same time, it also ensures that the determination reagent can meet the linearity requirements of the reagent without affecting the clinical test results, and has the characteristics of high accuracy and good stability.
  • the present invention also provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or a salt thereof, a related composition and a kit.
  • Figure 1 shows the changes in the reactivity of 3-methylbenzothiazolone hydrazone after acceleration with different buffer types.
  • Fig. 2 is a random sample correlation analysis between the reagent of Example 10 and the commercially available reagent (Jiuqiang 20-0630).
  • Fig. 3 is a random sample correlation analysis between the reagent of Example 11 and the commercially available reagent (Jiuqiang 20-0630).
  • Fig. 4 is a random sample correlation analysis between the reagent of Example 12 and the commercially available reagent (Jiuqiang 20-0630).
  • the kit is divided into two reagent compositions, R1 and R2, and the specific formula is as follows:
  • Embodiment 2 Measuring method
  • the volume ratio of the reagent R1 to the reagent R2 is 3:150:50.
  • the correlation coefficient r should be ⁇ 0.990.
  • the absolute deviation of linearity should not exceed ⁇ 20 mg/L;
  • the linear relative deviation should not exceed ⁇ 10%.
  • Reagent blank Use purified water or a designated blank solution to add reagents as a sample test, which is an effective indicator for judging the quality of reagents.
  • Analytical Sensitivity The slope of the calibration curve.
  • Linearity The ability of a measurement result to be directly proportional to the value measured in the sample.
  • Linear range The test system can accept the linear range, that is, the nonlinear error is less than the specified error within this range.
  • Interference refers to the clinically significant deviation of the concentration of the analyte due to the influence of sample characteristics or other components in clinical chemistry.
  • Interference standard the maximum permissible interference deviation between the concentration of the measured substance and the true value, which may affect the doctor's
  • Anti-interference Determine the interfering properties of interfering substances.
  • the relative deviation of linearity should not exceed ⁇ 10%.
  • the low-end deviation is large, the linearity is poor, and the external quality assessment deviation is large, which cannot meet the requirements of accurate detection.
  • Embodiment 3 color source substance concentration screening
  • reagents containing different color source substances wherein TOOS is used as the color source material, and the concentrations are: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2mM.
  • concentrations are: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2mM.
  • the correlation coefficient r of the assay kit meets the requirements, but from Table 2, it can be seen that the low-end deviation of the reagent kit in Table 1 is relatively large, and the linearity is poor.
  • the present invention adjusts the concentration of the color source substance , when the concentration is 0.4mM-3.2mM, it does not conform to the range of 40-200mg/L, and the absolute deviation of linearity should not exceed ⁇ 20mg/L; Within the interval, the linear relative deviation should not exceed ⁇ 10%. It can be seen that the linearity of the kit reagents cannot be optimized by adjusting the concentration of the color source substance.
  • the linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents.
  • the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of the color source substance.
  • Embodiment 4 Surfactant concentration screening
  • kits components and contents in Table 1 prepare reagents containing different surfactants, wherein polyoxyethylene distyrenated phenyl ether (A90) is used as the surfactant, and the concentrations are: 0.35g/L, 0.7 g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8g/L.
  • concentrations are: 0.35g/L, 0.7 g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8g/L.
  • the linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of the surfactant.
  • the linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of 4-aminoantipyrine.
  • Triton-100 Prepare reagents containing different concentrations of Triton-100, the concentrations of Triton-100 are: 5g/L, 15g/L, 25g/L, 35g/L, 50g/L. Reagents with different concentrations of Triton-100 were used for linearity verification, and the linearity verification method was the same as above.
  • kit reagents in the prior art have poor linearity and do not meet the linearity standard of the reagents, and the linearity of the kit reagents cannot be optimized by adjusting the concentration of Triton-100.
  • Embodiment 7 replaces the reagent of 4-aminoantipyrine in embodiment 1 with 3-methylbenzothiazolone hydrazone
  • Accelerated calibration deviation calculation formula (37°C calibration response -4°C calibration response)/4°C calibration response.
  • the present inventors unexpectedly found that the linearity of the reagent is optimized after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone substrate, which meets the requirements of the linearity standard for the reagent.
  • reagents containing 3-methylbenzothiazolone hydrazone and PC-300 in R2 but with different buffer types, in which the concentration of 3-methylbenzothiazolone hydrazone is 0.4mol/L, and different types of buffers include: citric acid Buffer, Succinic Acid, Boric Acid, Tartaric Acid, Glycine, Hydrochloric Acid, Sulfuric Acid, Acetic Acid.
  • the correlation coefficient r of the assay reagent is in compliance with the regulations, as can be seen from Table 8 and Figure 1: in the range of pH 1.00-4.05, citric acid, succinic acid, boric acid, tartaric acid, glycine,
  • the acidic environment such as hydrochloric acid, acetic acid and its buffer system has a synergistic effect with the preservative PC-300, the stability of the reagent is significantly improved, and unexpected technical effects have been achieved.
  • the reagents using 4-aminoantipyrine in the prior art all do not meet the linearity requirements of the reagents, even by adjusting the concentration of the color source substance, the concentration of the surfactant, the concentration of 4-aminoantipyrine, and the concentration of Triton-100 After that, the linearity cannot be optimized, and the linearity cannot meet the requirements of reagent accuracy.
  • the linearity of the reagent is improved after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone, which meets the requirement of reagent accuracy, but the inventor finds that the stability of the system is not high.
  • PC-300 was selected as a preservative, which has the function of stabilizing the reagent system and has met the requirements of performance optimization. Further, by adjusting the pH value to the range of 1.00-4.05, it can produce a synergistic effect with PC-300, greatly improve the stability of the system, and achieve unexpected technical effects.
  • the correlation coefficient r using the 3-methylbenzothiazolone assay reagent of the present invention is in compliance with the regulations, and the absolute deviation and relative deviation in the low value area are very small, fully meeting the linearity requirement.
  • Reagent R1 Reagent R2: Buffer (PIPES) 100mmol/L PBS-citric acid 100mmol/L pH 2.74 Peroxidase 500KU/L Peroxidase 10000KU/L Cholesterol lipase 600KU/L 3-Methylbenzothiazolone hydrazone 0.8mol/L Cholesterol oxidase 500KU/L Emulgen 909 25g/L
  • PBS-citric acid 100mmol/L pH 2.74
  • the solvent is purified water Sodium azide 0.9g/L the
  • the solvent is purified water
  • Embodiment 13 Reagent of the present application and commercially available product correlation verification

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Abstract

一种适用于自动分析仪的分级测定脂蛋白胆固醇的试剂盒及检测方法以及用于此类测定的试剂，所述方法和试剂能够在无需预处理试样下进行，具有良好特异性的、快速且方便的分析，同时还保证测定试剂能在不影响临床检测结果的情况下，满足试剂线性标准，准确度高并且稳定性好的特点。

Description

脂蛋白胆固醇检测方法和试剂盒 技术领域
本发明涉及用于测定脂蛋白中胆固醇的方法和试剂，尤其涉及动脉硬化的诊断相关的脂蛋白胆固醇检测的方法和试剂。
背景技术
胆固醇是细胞的一种重要组分，也是临床上重要的组分，因为过高水平的胆固醇导致在内皮下间隙中的巨噬细胞摄取胆固醇后使巨噬细胞转化为泡沫细胞(foam cell)并继而导致动脉硬化的原发病灶的发展。低密度脂蛋白(LDL)在血液的胆固醇运输中起主要作用并且是动脉硬化的危险因素。已知小而密LDL，其在LDL中粒度特别小并且与标准LDL相比密度更高，具有多于正常LDL数倍水平的致动脉粥样化的能力。小而密LDL的增加是动脉硬化的主要危险因素之一。因此在临床上进行此类小而密LDL的分级测定(fractional measurement)是非常重要的。
用于测定小而密LDL的常规方法的实例包括超速离心法、电泳法以及使用高效液相色谱的方法。由于这些方法需要昂贵的设备和大量测量时间，因此并不方便。
一种利用自动分析仪测量小而密LDL的方法的实例是以下方法(参见日本专利公布第2003-28882A号)，该方法涉及利用离子强度差异将小颗粒LDL悬浮或溶解，然后利用吸光度差异对小颗粒LDL进行测定。然而，吸光度差异依照上述方法是基于浊度测定的。因此不能测定小而密LDL中胆固醇，因而特异性和准确度不足。
此外，已知一种方法(参见国际专利公布WO2004/053500)，该方法涉及通过使用包含聚阴离子和二价阳离子的分离剂与适合于自动分析仪的试剂的组合来测量小而密LDL中的胆固醇或三酰甘油。该方法能够比超速离心法或电泳法更方便地测量小而密LDL中的脂质组分。但是该方法需要预处理试样并需要将LDL分离成小而密LDL和除这种LDL之外的LDL的步骤。
现有技术中公开了一种方法(参见国际专利公布WO2007026829A1)，该方法涉及通过特定表面活性剂清除除小而密LDL之外的脂蛋白中的胆固醇，然后测定剩余的脂蛋白中的胆固醇的量。该方法能够在无需预处理试样下进行具有快速且方便的分析。然而，经实验发现，该方法测定试剂低端偏差较大，线性较差，室间质评偏差大，无法达到准确检测的要求。
发明内容
本发明涉及一种适用于自动分析仪的分级测定脂蛋白胆固醇的方法以及用于此类测定的试剂，所述方法和试剂能够在无需预处理试样下进行，同时保证测定试剂能在不影响临床检测结果的情况下，满足试剂线性要求，准确度高并且稳定性好。
本发明通过如下技术方案来实现：
本发明提供了一种组合物，其特征在于，所述组合物包含：
3-甲基苯并噻唑酮腙或其盐；和
过氧化物酶。
根据本发明，所述组合物还包含防腐剂。优选地，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。
本发明还提供了一种组合物，其特征在于，所述组合物包含：
3-甲基苯并噻唑酮腙或其盐；和
防腐剂。
根据本发明，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。
根据本发明，上述组合物在酸性环境中。
根据本发明，优选地，pH值为0.5-4.5，更优选地，pH值为1-4.05。
根据本发明，更优选地，上述组合物还包含选自柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系中的一种或多种。
根据本发明，上述组合物还包含表面活性剂。其中，所述表面活性剂选自聚氧乙烯衍生物，优选聚氧乙烯-聚氧丙烯共聚物、聚氧乙烯烷基醚(EMULGEN707，EMULGEN709)、聚氧乙烯烷基苯基醚，更优选，聚乙二醇辛基苯基醚(Triton X-100)，聚氧乙烯壬基苯基醚(EMULGEN909)。
根据本发明，所述3-甲基苯并噻唑酮腙或其盐的浓度为0.1-1.0g/L。更优选，所述3-甲基苯并噻唑酮腙或其盐的浓度为0.1-0.4g/L。过氧化物酶的浓度为2000-4000U/L。所述表面活性剂的浓度为20-35g/L。
本发明还提供了一种试剂盒，其特征在于，所述试剂盒至少包含试剂组合物(ii)，所述试剂组合物(ii)包含上述组合物。
根据本发明，所述试剂盒还包含试剂组合物(i)，所述试剂组合物(i)用于有效屏蔽除待测脂蛋白之外的干扰。优选地，所述试剂组合物(i)包含磷脂酶，优选，磷脂酶A2、磷脂酶C、磷脂酶D、溶血磷脂酶(LYPL)和鞘磷脂酶的至少一种；优选磷脂酶的浓度范围为0.1U/mL-100U/mL。
本发明所述有效屏蔽的定义为，通过物理或化学的手段作用于干扰成分，使干扰成分对后续的检测步骤不会产生实质影响，有效屏蔽并不要求将干扰成分完全清除或排除于体系之外。
根据本发明，所述试剂组合物(i)还包括非离子型表面活性剂，优选HLB 13-15的非离子表面活性剂，更优选，聚氧乙烯衍生物或聚氧乙烯二苯乙烯苯基醚，例如，EMULGEN B-66,EMULGEN A-90；优选所述非离子表面活性剂的浓度为1.5-2.0g/L。
根据本发明，所述试剂组合物(i)还包括胆固醇酯酶、胆固醇氧化酶和氢供体。
根据本发明，所述试剂组合物(i)还包括无机盐离子，选自：氯化镁、EDTA·2Na、EDTA·2K、谷氨酸钠、氯化钠或氯化钾中的一种或多种。
根据本发明，所述试剂组合物(i)还包括稳定剂，选自：BSA、蔗糖或海藻糖中的一种或多种。
根据本发明，所述氢供体为苯胺衍生物，优选TOOS(N-乙基-N-(2-羟基-3-磺基丙基)-3-甲基苯胺)。
本发明还提供了上述组合物和/或试剂盒的制备方法。
本发明还提供了上述组合物和/或试剂盒的应用。
根据本发明，所述组合物和/或试剂盒用于制备检测脂蛋白胆固醇的产品中的应用。
其中，所述产品包括试剂或试剂盒。所述检测脂蛋白胆固醇包括低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。
根据本发明，上述组合物和/或试剂盒还用于制备诊断心血管相关疾病的产品中的应用。
其中，所述产品包括试剂或试剂盒。所述诊断心血管相关疾病，例如血脂异常，尤其是高脂血症、高胆固醇血症、动脉粥样硬化等疾病，包括低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。
本发明还提供了一种提高脂蛋白胆固醇检测线性或准确性的方法，其特征在于，使用上述组合物和/或试剂盒。
本发明还提供了一种提高3-甲基苯并噻唑酮腙或其盐稳定性的方法，其特征在于，含有防腐剂。其中，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。
根据本发明，所述3-甲基苯并噻唑酮腙或其盐优选在酸性环境中。优选地，pH值为1.00-4.05。更优选地，包含选自柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系中的一种或多种酸或缓冲体系。
本发明还提供了一种用于定量测定样品中脂蛋白胆固醇的方法，其特征在于使用上述组合物和/或试剂盒中的试剂组合物(ii)。
根据本发明，该方法包括以下步骤：加入上述组合物和/或试剂盒中的试剂组合物(ii)对目标脂蛋白胆固醇进行检测。进一步，该方法包括以下步骤：
(1)有效屏蔽除待测脂蛋白之外的干扰成分；
(2)加入上述组合物和/或试剂盒中的试剂组合物(ii)对目标脂蛋白胆固醇进行检测。
根据本发明，还包括在步骤(1)或(2)中加入胆固醇酯酶、胆固醇氧化酶和色原(氢供体)。
根据本发明，包括在步骤(1)中加入上述试剂盒的试剂组合物(i)。
根据本发明，所述试剂盒用于低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。
根据本发明，所述步骤(1)可以进一步细分为多个步骤对不同的干扰成分进行有效屏蔽。
本发明的效果
本发明提供了一种适用于自动分析仪的测定脂蛋白胆固醇的方法以及用于此类测定的试剂，所述方法和试剂能够在无需预处理试样下进行，具有良好特异性的、快速且方便的分析，同时还保证测定试剂能在不影响临床检测结果的情况下，满足试剂线性要求，准确度高并且稳定性好的特点。此外，本发明还提供了一种提高3-甲基苯并噻唑酮腙或其盐稳定性的方法以及相关的组合物和试剂盒。
附图说明
图1为不同缓冲类型加速后3-甲基苯并噻唑酮腙反应度变化。
图2为实施例10的试剂与市售试剂(九强20-0630)随机样本相关性分析。
图3为实施例11的试剂与市售试剂(九强20-0630)随机样本相关性分析。
图4为实施例12的试剂与市售试剂(九强20-0630)随机样本相关性分析。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1试剂盒的制备
以现有技术中小而密低密度脂蛋白胆固醇检测试剂盒为例，试剂盒分为R1和R2两种试剂组合物，具体配方如下：
表1试剂盒的组分
Figure PCTCN2022100428-appb-000001
实施例2测定方法
(1)将待测的血清样本与试剂R1混匀，37℃恒温5min，测定吸光度A 1，再加入试剂R2，混匀，37℃恒温5min，测定吸光度A 2，计算ΔA＝A 2-A 1
(2)用全自动生化分析仪(日立7180)测定反应后的吸光度差值(主波长546nm，副 波长660nm，仪器读点16-34)；
(3)根据吸光度变化值计算出样本中sdLDL-C的浓度。
作为优选，步骤(1)中所述的血清样本，试剂R1与试剂R2的体积比为3:150:50。
(4)线性实验方法：
用接近线性范围上限的高浓度样品和接近线性范围下限的低浓度样品，混合成7个稀释浓度(稀释倍数见下表)，每个浓度测试3次取平均值，取其测定浓度值(即均值y i)与对应的理论浓度值或稀释倍数(xi)进行回归分析，求出线性回归方程(y＝Ax+B)，其中A是斜率，B是截距，计算线性回归相关系数r，结果应符合要求。
将自变量(xi，0值除外)代入线性回归方程，计算因变量(y i)的估计值(y′)，以及y i与y′的绝对偏差和相对偏差，结果应符合要求。
Figure PCTCN2022100428-appb-000002
绝对偏差(mg/L)＝|y i-y'|………………………(6)
线性稀释倍数：
序号 1 2 3 4 5 6 7
稀释比例(H：L) H 3:1 1:1 1:3 1:7 1:15 L
(1)、线性实验要求：
在[40，900]mg/L区间内，相关系数r应≥0.990。
在[40，200)mg/L区间内，线性绝对偏差应不超过±20mg/L；
在[200，900]mg/L区间内，线性相对偏差应不超过±10％。
样本来源：合作医院(成都中医药大学附属医院)
(2)、术语、定义、缩略词：
试剂空白(R)：使用纯化水或指定空白液加入试剂作为样品测试，是判定试剂质量的一个有效指标。
定标反应度(S)：校准品标识值在反应体系中所测吸光度变化(ΔA)，是建立浓度与吸光度转化关系的重要指标。
分析灵敏度：校准曲线的斜率。
线性:测量结果与样品中被测量的值直接成比例的能力。
线性范围：测试系统可接收线性的范围，即在此范围内非线性误差小于规定误差。
精密度：在规定条件下，对同一被测物进行连续多次测量所得结果之间的一致性。
干扰:指在临床化学中，被测物浓度因样品特性或其他成分的影响而出现的临床显著性偏差。
干扰标准：被测物浓度与真值间可产生的最大允许干扰偏差，此偏差可能影响医生的
医疗决定。
抗干扰：确定干扰物质的干扰性。
表1的试剂盒检测结果如下：
Figure PCTCN2022100428-appb-000003
经实验发现，该测定试剂盒的相关系数r是符合规定的，但是不符合在40-200mg/L区间
内，线性绝对偏差应不超过±20mg/L；也不符合在200-900mg/L区间内，线性相对偏差应不超过±10％。低端偏差较大，线性较差，室间质评偏差大，无法达到准确检测的要求。
实施例3色源物质浓度筛选
1.实验方法：
根据表1的试剂盒组分和含量，配制含有不同色源物质的试剂，其中色源物质使用TOOS，浓度分别为：0.4mM、0.8mM、1.6mM、2.0mM、3.2mM。用不同TOOS浓度的试剂进行线性验证，线性做法及要求同上。
2.实验结果：
表2色源物质浓度筛选
Figure PCTCN2022100428-appb-000004
Figure PCTCN2022100428-appb-000005
Figure PCTCN2022100428-appb-000006
Figure PCTCN2022100428-appb-000007
Figure PCTCN2022100428-appb-000008
经实验发现，该测定试剂盒的相关系数r是符合规定的，但是从表2中，可以看出，表1试剂盒的试剂低端偏差较大，线性较差，本发明调整色源物质浓度，在浓度为0.4mM-3.2mM，均不符合在40-200mg/L区间内，线性绝对偏差应不超过±20mg/L；在浓度为0.4mM-3.2mM，不符合在200-900mg/L区间内，线性相对偏差应不超过±10％。由此可见，通过调整色源物质浓度仍不可对试剂盒试剂的线性进行优化。
3.结论：
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整色源物质浓度仍不可对试剂盒试剂的线性进行优化。
实施例4表面活性剂浓度筛选
1.实验目的：优化试剂线性
2.实验方法：
根据表1的试剂盒组分和含量，配制含有不同表面活性剂的试剂，其中表面活性剂使用聚氧乙烯基联苯乙烯化苯基醚(A90)，浓度分别为：0.35g/L、0.7g/L、1.4g/L、1.75g/L、2.1g/L、2.8g/L。用不同A90浓度的试剂进行线性验证,线性验证方法同上。
3.实验结果
表3表面活性剂A90浓度筛选验证
Figure PCTCN2022100428-appb-000009
Figure PCTCN2022100428-appb-000010
Figure PCTCN2022100428-appb-000011
Figure PCTCN2022100428-appb-000012
Figure PCTCN2022100428-appb-000013
Figure PCTCN2022100428-appb-000014
Figure PCTCN2022100428-appb-000015
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表3中，可以看出，调整表面活浓度，仍不能使试剂线性满足要求。
4.结论：
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整表面活性剂浓度仍不可对试剂盒试剂的线性进行优化。
实施例5底物4-氨基安替比林浓度的筛选
配制含有不同4-氨基安替比林浓度的试剂，4-氨基安替比林浓度为：2mM、4mM、8mM、12mM，用不同4-氨基安替比林浓度的试剂进行线性验证,线性验证方法同上。
实验结果：
4-氨基安替比林浓度为2mM时试剂无反应度。其他4-氨基安替比林浓度的试剂验证结果如下：
表4 4-氨基安替比林浓度筛选验证
Figure PCTCN2022100428-appb-000016
Figure PCTCN2022100428-appb-000017
Figure PCTCN2022100428-appb-000018
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表4中，可以看出，4-氨基安替比林浓度调高浓度后也并不能使试剂满足线性要求。
4.结论：
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整4-氨基安替比林浓度仍不可对试剂盒试剂的线性进行优化。
实施例6曲拉通-100浓度筛选
配制含有不同曲拉通-100浓度的试剂，曲拉通-100的浓度为：5g/L、15g/L、25g/L、35g/L、50g/L。用不同曲拉通-100浓度的试剂进行线性验证,线性验证方法同上。
表5曲拉通-100浓度筛选验证
Figure PCTCN2022100428-appb-000019
Figure PCTCN2022100428-appb-000020
Figure PCTCN2022100428-appb-000021
Figure PCTCN2022100428-appb-000022
Figure PCTCN2022100428-appb-000023
Figure PCTCN2022100428-appb-000024
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表5中，可以看出，调整曲拉通-100的量也不能使试剂线性满足要求
4.结论：
现有技术中的试剂盒试剂线性较差，不符合试剂的线性标准，通过调整曲拉通-100浓度仍不可对试剂盒试剂的线性进行优化。
实施例7用3-甲基苯并噻唑酮腙替换实施例1中的4-氨基安替比林的试剂
配制R2中含有3-甲基苯并噻唑酮腙，不含有4-氨基安替比林的试剂，3-甲基苯并噻唑酮腙浓度为0.4mol/L，其余组分与实施例1相同.
实验说明：
加速定标偏差计算公式式：(37℃定标反应度-4℃定标反应度)/4℃定标反应度。
实验结果
表6 4-氨基安替比林换为3-甲基苯并噻唑酮腙
Figure PCTCN2022100428-appb-000025
Figure PCTCN2022100428-appb-000026
经实验发现，该测定试剂盒的相关系数r是符合规定的，从表6中可以看出，配完立刻测定效果很好，线性满足要求，但试剂稳定性不满足要求。
4.结论
本发明人意外的发现，将4-氨基安替比林替换为3-甲基苯并噻唑酮腙底物后试剂线性得到了优化，满足试剂线性标准的要求。
实施例8试剂2防腐剂不同的试剂
配制R2中含有3-甲基苯并噻唑酮腙，试用原缓冲类型(PIPES)，不同的防腐剂试剂，其中3-甲基苯并噻唑酮腙浓度为0.4mol/L，试剂分别为无防腐剂、叠氮钠1.0g/L、氟苯尼考1.0g/L、PC-300 1.0g/L。
表7不同类型防腐剂验证
Figure PCTCN2022100428-appb-000027
Figure PCTCN2022100428-appb-000028
经实验发现，该测定试剂盒的相关系数r是符合规定的，从表7中可以看出：不同防腐剂的添加对R2稳定性有不同影响，不加防腐试剂稳定性最差，其次是叠氮钠和氟苯尼考。本发明人意外的发现，PC-300对于试剂稳定性验证中表现异常的好，说明PC-300不仅有防腐剂的作用，还具有明显提高R2试剂稳定性的作用。
实施例9试剂2缓冲类型不同的试剂
配制R2中含有3-甲基苯并噻唑酮腙、PC-300，但缓冲类型不同的试剂，其中3-甲基苯 并噻唑酮腙浓度为0.4mol/L，不同类型的缓冲有：柠檬酸缓冲、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、硫酸、醋酸。
表8不同缓冲类型验证
Figure PCTCN2022100428-appb-000029
Figure PCTCN2022100428-appb-000030
Figure PCTCN2022100428-appb-000031
Figure PCTCN2022100428-appb-000032
Figure PCTCN2022100428-appb-000033
Figure PCTCN2022100428-appb-000034
经实验发现，该测定试剂的相关系数r是符合规定的，从表8和图1中可以看出：在pH值1.00-4.05的范围内，柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系等酸性环境，与防腐剂PC-300产生协同作用，试剂稳定性显著得到改善，取得了预料不到的技术效果。
总结：
采用现有技术中4-氨基安替比林的试剂均不符合试剂的线性要求，即使通过调整色源物质浓度、表面活性剂浓度、4-氨基安替比林浓度、曲拉通-100浓度后均不可对线性进行优化，线性均无法满足试剂准确性的要求。本发明将4-氨基安替比林替换为3-甲基苯并噻唑酮腙后试剂的线性得到了改善，满足试剂准确性的要求，但是发明人发现体系的稳定性不高。通过实验意外发现，选择PC-300作为防腐剂，兼具了稳定试剂体系的作用，已经满足了性能优化的要求。进一步，通过将pH值调整至1.00-4.05范围，能与PC-300产生协同作用，极大提高了体系的稳定性，取得了预料不到的技术效果。
实施例10试剂线性验证
按照表9组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表9试剂在全自动生化分析仪中进行测定。测定结果如表10所示。
表9试剂组分
试剂R1： 试剂R2：
缓冲液(PIPES)100mmol/L 硼酸缓冲(50mM pH＝4.05)
过氧化物酶500KU/L 过氧化物酶10000KU/L
胆固醇脂酶600KU/L 3-甲基苯并噻唑酮腙0.2mol/L
胆固醇氧化酶500KU/L Triton X-100(曲拉通)25g/L
TOOS 1.6mmol/L 氟苯尼考1.0g/L
Emulgen A-90 1.4g/L 其溶剂为纯化水
叠氮钠0.9g/L  
其溶剂为纯化水  
表10线性验证
Figure PCTCN2022100428-appb-000035
从表10可以看出，使用本发明的3-甲基苯并噻唑酮测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差均非常小，完全满足线性要求。
实施例11试剂线性验证
按照表11组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表11试剂在全自动生化分析仪中进行测定。测定结果如表12所示。
表11试剂组分
试剂R1： 试剂R2：
缓冲液(PIPES)100mmol/L PBS-柠檬酸100mmol/L pH 2.74
过氧化物酶500KU/L 过氧化物酶10000KU/L
胆固醇脂酶600KU/L 3-甲基苯并噻唑酮腙0.8mol/L
胆固醇氧化酶500KU/L Emulgen 909 25g/L
TOOS 1.6mmol/L PC-300 1.0g/L
Emulgen B-66 1.4g/L 其溶剂为纯化水
叠氮钠0.9g/L  
其溶剂为纯化水  
表12线性验证
Figure PCTCN2022100428-appb-000036
从表12可以看出，该测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差也非常小，完全满足线性要求。
实施例12试剂线性验证
按照表13组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表11试剂在全自动生化分析仪中进行测定。测定结果如表14所示。
表13试剂组分
试剂R1： 试剂R2：
缓冲液(PIPES)100mmol/L 盐酸95mM pH＝1.00
过氧化物酶500KU/L 过氧化物酶10000KU/L
胆固醇脂酶600KU/L 3-甲基苯并噻唑酮腙0.8mol/L
胆固醇氧化酶500KU/L Emulgen 709 25g/L
TOOS 1.6mmol/L PC-300 1.0g/L
Emulgen A-90 1.4g/L 其溶剂为纯化水
叠氮钠0.9g/L  
其溶剂为纯化水  
表14线性验证
Figure PCTCN2022100428-appb-000037
从表14可以看出，该测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差也非常小，完全满足线性要求。
实施例13本申请的试剂与市售产品相关性验证
分别采用本发明实施例10、11和12的试剂与市售试剂(九强20-0630)，使用实施例2的方法，采用日立7180全自动生化分析仪，对100份样本，按各自参数进行测定，并对测定值进行相关分析(横纵坐标单位mg/L)。
结果如图2-4所示，本发明的试剂与市售试剂相比，一致性程度高。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (17)

  1. 一种组合物，其特征在于，所述组合物包含：
    3-甲基苯并噻唑酮腙或其盐；
    过氧化物酶；
    和防腐剂；
    所述组合物的pH值为1.0-5.0。
  2. 根据权利要求1所述的组合物，其特征在于，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。
  3. 根据权利要求1或2所述的组合物，其特征在于，所述组合物还包含选自柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系中的一种或多种。
  4. 根据权利要求1或2所述的组合物，其特征在于，所述组合物还包含表面活性剂，所述表面活性剂选自聚氧乙烯衍生物，优选聚氧乙烯-聚氧丙烯共聚物、聚氧乙烯烷基醚、聚氧乙烯烷基苯基醚。
  5. 根据权利要求1或2所述的组合物，其特征在于，所述3-甲基苯并噻唑酮腙或其盐的浓度为0.1-1.0g/L。
  6. 根据权利要求1或2所述的组合物，其特征在于，过氧化物酶的浓度为2000-4000U/L。
  7. 根据权利要求4所述的组合物，其特征在于，所述表面活性剂的浓度为20-35g/L。
  8. 一种试剂盒，其特征在于，所述试剂盒至少包含试剂组合物(ii)，所述试剂组合物(ii)包含权利要求1-7中任一所述组合物；所述试剂盒还包含试剂组合物(i)，所述试剂组合物(i)包含磷脂酶，所述磷脂酶选自甘油磷脂酶或鞘磷脂酶的至少一种。
  9. 根据权利要求8所述试剂盒，其特征在于，所述磷脂酶的浓度范围为0.1U/mL-100U/mL。
  10. 根据权利要求8或9所述试剂盒，其特征在于，所述试剂组合物(i)还包括非离子型表面活性剂，所述非离子表面活性剂的HLB为13-15。
  11. 根据权利要求10所述试剂盒，其特征在于，所述试剂组合物(i)中的非离子表面活性剂选自聚氧乙烯衍生物或聚氧乙烯二苯乙烯苯基醚，所述非离子表面活性剂的浓度为1.5-2.0g/L。
  12. 根据权利要求8或9所述试剂盒，其特征在于，所述试剂组合物(i)还包括胆固醇酯酶、胆固醇氧化酶和氢供体。
  13. 根据权利要求1-7中任一所述组合物用于制备检测脂蛋白胆固醇的产品中的应用，所述检测脂蛋白胆固醇包括低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇。
  14. 根据权利要求1-7中任一所述组合物用于制备诊断心血管相关的产品中的应用；所述心血管相关疾病包括血脂异常、高胆固醇血症或动脉粥样硬化。
  15. 一种提高脂蛋白胆固醇检测线性或准确性的方法，其特征在于，使用权利要求1-7中任一所述的组合物。
  16. 一种用于定量测定样品中脂蛋白胆固醇的方法，其特征在于，使用权利要求1-7中任一所述的组合物。
  17. 根据权利要求16所述方法，其特征在于，包括以下步骤：
    (1)有效屏蔽除待测脂蛋白之外的干扰成分；
    (2)加入上述组合物和/或试剂盒中的试剂组合物(ii)对目标脂蛋白胆固醇进行检测。
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