CN1632541A - Reagent for measuring cholesterol in high-density lipoprotein and its preparation method - Google Patents

Abstract

This invention relates to a test agent and its process method to test the cholesterin in the lipoprotein with high intensity in blood serum. This invention aims to provide an improvement of the agent and its process method by which we can get the agent able to automatic biochemical analysis device in each type. The technique project and the contents are the flowing: a, high combining enzyme compound; b, surface activity agent; c, some antiseptic agent, stabilization agent and color source.

Description

Translated fromChinese

高密度脂蛋白中胆固醇的测定试剂及制备方法Reagent for measuring cholesterol in high-density lipoprotein and its preparation method

技术领域technical field

本发明涉及一种测定血清成分试剂的制备方法及试剂，特别是测定血清中的高密度脂蛋白中胆固醇试剂的制备方法与试剂，可广泛应用在医学及生物化学技术领域。The invention relates to a preparation method and a reagent for measuring serum components, in particular to a preparation method and a reagent for measuring high-density lipoprotein cholesterol in serum, which can be widely used in the technical fields of medicine and biochemistry.

背景技术Background technique

心脑血管疾病的发生率与血清高密度脂蛋白胆固醇(HDL-C)呈负相关已经流行病学和相关临床证实，检测高密度脂蛋白胆固醇还可广泛应用于相关的临床诊治，并作为脂类代谢异常的危险性指标之一。因此高密度脂蛋白胆固醇的准确测定至关重要，特别是在高密度脂蛋白胆固醇下限，极小的误差可能造成相对较大的影响，而且高密度脂蛋白胆固醇的结果直接影响低密度脂蛋白胆固醇(LDL-C)的计算值。目前，对高密度脂蛋白胆固醇的测定方法有已知的超速离心法和电泳法以及中华人民共和国卫生部医政司编制的《全国临床检验操作规程》第二版279页所推荐的沉淀法。超速离心法是根据脂蛋白的比重不同，用超速离心器把脂蛋白进行分类，再对胆固醇进行测定。该方法过程分超速离心和胆固醇检测两步，费用高、耗时大。电泳法需要借助一些如琼脂糖凝胶等做为载体分离，操作极其繁杂。沉淀法是使用多聚阴离子和二价金属离子试剂沉淀大颗粒的脂蛋白，然后离心，用其上清液测定其含量。该方法虽简便，但不适用于全自动、大批量样本检测，而且对高甘油三酯血症患者，会使高密度脂蛋白胆固醇结果偏高，当高甘油三酯大于5mmol/L时会使计算LDL-C的Friedwald公式受其限制。上述这些已知的方法由于都不能在全自动上直接检测，近几年，直接在全自动上进行检测HDL-C试剂也相继出现，如中国专利(申请号99116565.9)他们利用聚阴离子复合物与血清中LDL(低密度脂蛋白)、VLDL(极低密度脂蛋白)、CM(乳糜微粒)反应，生成不溶于水的复合物，而HDL-C仍以溶液状态存在，在胆固醇试剂的存在下进行其测定，本方法中添加了胆酸钠，可能是为了提高专一性。在本发明中不仅仅HDL起反应，LDL、VLDL等也缓慢地起反应，所以要想得到完全的HDL的反应终点是不可能的。并且特异性也不一定好。The incidence of cardiovascular and cerebrovascular diseases is negatively correlated with serum high-density lipoprotein cholesterol (HDL-C), which has been confirmed by epidemiology and related clinics. The detection of high-density lipoprotein cholesterol can also be widely used in relevant clinical diagnosis and treatment, and as a lipid One of the risk indicators of metabolic abnormalities. Therefore, the accurate determination of high-density lipoprotein cholesterol is very important, especially at the lower limit of high-density lipoprotein cholesterol, a very small error may cause a relatively large impact, and the result of high-density lipoprotein cholesterol directly affects low-density lipoprotein cholesterol Calculated value of (LDL-C). At present, there are known ultracentrifugation and electrophoresis methods for the determination of high-density lipoprotein cholesterol, as well as the precipitation method recommended on page 279 of the second edition of the "National Clinical Laboratory Operation Procedures" compiled by the Medical Administration Department of the Ministry of Health of the People's Republic of China. The ultracentrifugation method is to classify the lipoproteins with an ultracentrifuge according to the specific gravity of the lipoproteins, and then measure the cholesterol. The process of this method is divided into two steps of ultracentrifugation and cholesterol detection, which is costly and time-consuming. Electrophoresis requires the use of some such as agarose gel as a carrier for separation, and the operation is extremely complicated. The precipitation method is to use polyanions and divalent metal ion reagents to precipitate large-particle lipoproteins, then centrifuge, and use its supernatant to determine its content. Although this method is simple, it is not suitable for automatic and large-scale sample detection, and for patients with hypertriglyceridemia, the result of high-density lipoprotein cholesterol will be high, and when the high triglyceride is greater than 5mmol/L, it will cause The Friedwald formula for calculating LDL-C is limited by it. Above-mentioned these known methods all can not directly detect on automatic, in recent years, directly detect HDL-C reagent also to appear on automatic, as Chinese patent (application number 99116565.9) they utilize polyanion complex and LDL (low-density lipoprotein), VLDL (very low-density lipoprotein), and CM (chylomicrons) in serum react to form water-insoluble complexes, while HDL-C still exists in a solution state, and in the presence of cholesterol reagents For its determination, sodium cholate was added to this method, possibly to improve specificity. In the present invention, not only HDL reacts, but also LDL, VLDL, etc. react slowly, so it is impossible to obtain a complete HDL reaction end point. And the specificity is not necessarily good.

发明内容Contents of the invention

本发明所要解决的技术问题是克服上述现有技术的不足，提供一种测定高密度脂蛋白中胆固醇的试剂的制备方法及试剂的改进，用该方法所制得的试剂应特异性好、检测简便、测试准确率高，并能够在各种型号的全自动生化分析仪上对高密度脂蛋白中胆固醇进行检测。The technical problem to be solved by this invention is to overcome the above-mentioned deficiencies in the prior art, provide a kind of preparation method and the improvement of the reagent of the cholesterol in the mensuration high-density lipoprotein, the reagent prepared by this method should have good specificity, detect It is simple and has high test accuracy, and can detect cholesterol in high-density lipoprotein on various types of automatic biochemical analyzers.

本发明采用了以下技术方案：The present invention adopts following technical scheme:

高密度脂蛋白中胆固醇的测定试剂，包括适量防腐剂、稳定剂和色原，该试剂还包括以下成分：Reagent for the determination of cholesterol in high-density lipoprotein, including appropriate amount of preservatives, stabilizers and chromogens, the reagent also includes the following components:

A、高亲合性酶化合物，该酶化合物，由酶和化合物反应而得，其中：A, high-affinity enzyme compound, the enzyme compound is obtained by the reaction of enzyme and compound, wherein:

化合物是甾类糖苷、三萜烯糖苷、疏水蛋白质、蛋白质毒素和多烯抗菌素等化合物中的一种或任意比例混合的多种与活化剂混合、活化后的活化物，化合物的浓度为1-50毫克/毫升；The compound is one of steroidal glycosides, triterpene glycosides, hydrophobic proteins, protein toxins and polyene antibiotics, etc., or a variety of activators mixed with activators and activated in any proportion. The concentration of the compound is 1- 50 mg/ml;

酶是1、胆固醇酯酶和胆固醇氧化酶，二者比例10∶1-1∶10、The enzyme is 1, cholesterol esterase and cholesterol oxidase, the ratio of the two is 10:1-1:10,

    2、脂蛋白脂酶和胆固醇氧化酶，二者比例10∶1-1∶10中的任一组与缓冲液混合、溶解后的溶解物，缓冲液的PH值为5-9，缓冲液的摩尔配比是50-200mmol/L，所述的缓冲液是GOOD’S缓冲液或磷酸盐缓冲液或三羟甲基氨基甲烷缓冲液或柠檬酸缓冲液或硼酸盐缓冲液；2. Lipoprotein lipase and cholesterol oxidase, any group in the ratio of 10:1-1:10 is mixed with the buffer, the lysate after dissolution, the pH value of the buffer is 5-9, and the pH of the buffer is 5-9. The molar ratio is 50-200mmol/L, and the buffer is GOOD'S buffer or phosphate buffer or tris buffer or citric acid buffer or borate buffer;

上述化合物与酶二种成分反应时的摩尔配合比为1∶1-20∶1；The molar mixing ratio of the above compound and the two components of the enzyme when reacting is 1:1-20:1;

B、表面活性剂，其混合比例与A成分的重量百分比是0.003-30，所述的表面活性剂是阳离子表面活性剂、阴离子表面活性剂、两性离子表面活性剂和非离子表面活性剂中的一种或任意比例混合的多种。B, surfactant, its mixing ratio and the weight percent of A component are 0.003-30, and described surfactant is cationic surfactant, anionic surfactant, zwitterionic surfactant and nonionic surfactant One or multiple mixed in any proportion.

所述的酶优先选择胆固醇酯酶和胆固醇氧化酶。The enzymes are preferably cholesterol esterase and cholesterol oxidase.

该试剂还包括适量催化剂。The reagent also includes a suitable amount of catalyst.

所述的表面活性剂的混合比例为0.01-10重量百分比。The mixing ratio of the surfactant is 0.01-10% by weight.

该试剂中还有适量抗干扰物质。There is also an appropriate amount of anti-interference substances in the reagent.

所述的高密度脂蛋白中胆固醇的测定试剂的制备方法包括以下步骤：The preparation method of the assay reagent of cholesterol in described high-density lipoprotein comprises the following steps:

一、化合物活化1. Compound activation

选择所述的甾类糖苷、三萜烯糖苷、疏水蛋白质、蛋白质毒素和多烯抗菌素等化合物中的一种或任意比例混合的多种，在水介质中用公知的叠氮法或碳化二亚胺法或丁二酰亚胺法或高碘酸盐氧化法进行活化，活化条件为：Select one of the compounds such as steroidal glycosides, triterpene glycosides, hydrophobic proteins, protein toxins and polyene antibiotics or a mixture of them in any proportion, and use the known azide method or carbodioxide in the aqueous medium Amine method or succinimide method or periodate oxidation method for activation, the activation conditions are:

化合物与活化剂的摩尔比为1∶1-1∶10，温度0-25℃，PH在4-11范围内，时间0.1-24小时。The molar ratio of the compound to the activator is 1:1-1:10, the temperature is 0-25° C., the pH is in the range of 4-11, and the time is 0.1-24 hours.

二、酶溶解2. Enzyme dissolution

选择所述的胆固醇酯酶和胆固醇氧化酶，或脂蛋白脂酶和胆固醇氧化酶中的任一组，溶解在缓冲液中；Selecting the cholesterol esterase and cholesterol oxidase, or any group of lipoprotein lipase and cholesterol oxidase, dissolved in the buffer;

三、混合反应3. Mixed reaction

活化后的化合物与溶解后的酶混合，该溶液在0-25℃的温度下反应18-24小时；The activated compound is mixed with the dissolved enzyme, and the solution is reacted at a temperature of 0-25°C for 18-24 hours;

四、配置：4. Configuration:

反应后的酶化合物与表面活性剂混合，并加入防腐剂、稳定剂、用于显色测定的色原和催化剂。The reacted enzyme compound is mixed with a surfactant, and preservatives, stabilizers, chromogens and catalysts for chromogenic assays are added.

所述的甾类糖苷、三萜烯糖苷或多烯抗生素预先置于以下有机溶剂混合物中溶解：二甲亚砜或甲酰替二甲胺或六甲醇与甲苯按2∶1混合的混合物，或者，甲酰替二甲胺与乙醇按2∶1混合的混合物；化合物与有机溶剂混合物的重量比例为1∶30-1∶0.01。The steroidal glycoside, triterpene glycoside or polyene antibiotic is predissolved in the following organic solvent mixture: dimethyl sulfoxide or formyl dimethylamine or a mixture of hexamethanol and toluene in a ratio of 2:1, or , A mixture of formyl dimethylamine and ethanol at a ratio of 2:1; the weight ratio of the compound to the organic solvent mixture is 1:30-1:0.01.

该试剂中还加入抗干扰剂物质。Anti-interfering substances are also added to this reagent.

本发明所提供的高密度脂蛋白中胆固醇测定试剂的制备方法及试剂，能够直接在各种型号的全自动生化分析仪上对高密度脂蛋白中胆固醇进行检测，省却了样本离心、沉淀等繁杂步骤，大幅度减轻了检测工作量、减少了检测时间、降低了检测成本；而且试剂的特异性好，检测的准确率较高，与国家推荐的沉淀法的检测效果比较，相关性达到0.98以上。The preparation method and reagents for the determination of cholesterol in high-density lipoprotein provided by the present invention can directly detect cholesterol in high-density lipoprotein on various types of automatic biochemical analyzers, eliminating the need for sample centrifugation, precipitation, etc. steps, greatly reducing the detection workload, reducing the detection time, and reducing the detection cost; and the specificity of the reagent is good, and the detection accuracy is high. Compared with the detection effect of the precipitation method recommended by the country, the correlation reaches more than 0.98 .

附图说明Description of drawings

图1至图3分别是本发明的实施例1至实施例3的测得值与沉淀法(《全国临床检验操作规程》第二版279页所推荐)的测得值的相关性示意图。Fig. 1 to Fig. 3 are respectively the correlation schematic diagram of the measured value of theembodiment 1 to embodiment 3 of the present invention and the measured value of the precipitation method (recommended on page 279 of the second edition of "National Clinical Laboratory Operation Regulations").

具体实施方式Detailed ways

经研究发现：用经制备的高亲合性胆固醇酯酶化合物和高亲合性胆固醇氧化酶化合物及对高密度脂蛋白中胆固醇具有较强作用的表面活性剂配制的试剂用来测定经超速离心分离出的HDL、LDL、VLDL及CM各组份脂蛋白时，上述各组份脂蛋白胆固醇的反应性显示出不同，且不同组份的选择性和反应性为HDL＞LDL＞VLDL＞CM。当表面活性剂浓度达一定时，LDL、VLDL、CM在很长时间内不参加测定HDL的反应。After research, it was found that the reagent prepared with the prepared high-affinity cholesterol esterase compound and high-affinity cholesterol oxidase compound and a surfactant that has a strong effect on cholesterol in high-density lipoprotein was used to determine the ultracentrifugation When separating the lipoproteins of HDL, LDL, VLDL and CM, the reactivity of lipoprotein cholesterol of the above-mentioned components is different, and the selectivity and reactivity of different components are HDL>LDL>VLDL>CM. When the concentration of surfactant reaches a certain level, LDL, VLDL and CM will not participate in the reaction of measuring HDL for a long time.

由此，本发明提供的试剂及其制备方法是：Thus, reagent provided by the invention and preparation method thereof are:

一、选用高亲合性酶化合物：1. Select high-affinity enzyme compounds:

该酶化合物，由酶和化合物制备而得，其中：The enzyme compound is prepared from enzymes and compounds, wherein:

1、化合物选自甾类糖苷，该甾类糖苷在其结构中具有作为配基的呋甾烷醇或螺甾烷醇的甾环酶，以及具有含3-10个线状或分支结构单糖的低聚糖酶。或者选自三萜烯糖苷，该三萜烯糖苷在其结构中具有α-或β-胡椒烷、羊毛脂甾烷等的配基，以及具有由2-8个分支或线状结构基构成的低聚糖，或者选自能够有选择地与胆固醇形成复合物的疏水蛋白质，或者选自能够有选择地与胆固醇形成复合物的蛋白质毒素，或者选自能够有选择地与胆固醇形成复合物的多烯抗菌素。上述化合物可以单独使用也可以两种或几种混合使用，没有特别的限制，只要制得的酶化合物及对高密度脂蛋白中胆固醇具有较强作用的表面活性剂配制的试剂对高密度脂蛋白中胆固醇的反应比其它脂蛋白胆固醇的反应性强。1. The compound is selected from the group consisting of steroidal glycosides, which have a steroidal cyclase with furostanol or spirostanol as a ligand in their structure, and a monosaccharide containing 3-10 linear or branched structures of oligosaccharides. Or selected from triterpene glycosides, the triterpene glycosides have ligands such as α- or β-piperane, lanolin sterane, etc. in their structure, and have 2-8 branched or linear structural groups. Oligosaccharides, or selected from hydrophobic proteins capable of selectively forming complexes with cholesterol, or selected from protein toxins capable of selectively forming complexes with cholesterol, or selected from polysaccharides capable of selectively forming complexes with cholesterol Antibiotics. The above compounds can be used alone or in combination of two or more. There is no special limitation, as long as the prepared enzyme compound and the reagent prepared by a surfactant having a strong effect on cholesterol in HDL have a strong effect on HDL Cholesterol is more reactive than other lipoprotein cholesterols.

本发明中使用的甾类糖苷实例有：脱糖毛地黄皂苷、天门冬苷、龙舌苷、羊毛蜡苷等等。Examples of steroidal glycosides used in the present invention are: desugared digitonin, aspartoside, agavein, lanoside and the like.

三萜烯糖苷的实例有：七叶素、茶皂苷等。Examples of triterpene glycosides include aescin, tea saponin and the like.

疏水蛋白质实例有：脂蛋白的脱水蛋白质、溶酶体的蛋白质等等。Examples of hydrophobic proteins are: lipoprotein dehydrated proteins, lysosomal proteins, and the like.

蛋白质毒素可由细菌、海洋微生物等获得，作为实例有：链球菌溶血素O、脑酮溶细胞素等。Protein toxins can be obtained from bacteria, marine microorganisms, etc., as examples: streptolysin O, cerebroketolysin, and the like.

多烯抗菌素实例有：菲律宾菌等。Examples of polyene antibiotics include Philippine bacteria and the like.

2、酶选用胆固醇酯酶和胆固醇氧化酶，或脂蛋白脂酶和胆固醇氧化酶中任意一组。当然也可以选用其它的酶，只要制得的高亲合性酶化合物及对高密度脂蛋白中胆固醇具有较强作用的表面活性剂配成的试剂对高密度脂蛋白中胆固醇的反应比其它脂蛋白胆固醇的反应性强。为了提高检测的专一性，本发明中优先使用胆固醇酯酶和胆固醇氧化酶，胆固醇酯酶和胆固醇氧化酶的来源不作具体限制。2. The enzyme is selected from any group of cholesterol esterase and cholesterol oxidase, or lipoprotein lipase and cholesterol oxidase. Of course, other enzymes can also be used, as long as the prepared high-affinity enzyme compound and the surfactant with a strong effect on the cholesterol in the HDL are more reactive to the cholesterol in the HDL than other lipids. Protein cholesterol is highly reactive. In order to improve the specificity of detection, cholesterol esterase and cholesterol oxidase are preferably used in the present invention, and the sources of cholesterol esterase and cholesterol oxidase are not specifically limited.

二、高亲合胆固醇酶化合物的制备：Two, the preparation of high-affinity cholesterolase compound:

1、将上述化合物中的一种或几种混合物在水介质中进行活化1. Activate one or more mixtures of the above compounds in aqueous medium

将上述化合物中的一种或几种混合物在水介质中用公知的叠氮法、碳化二亚胺法、丁二酰亚胺法或高碘酸盐氧化法进行活化，选用的方法最好能够最大地保持制备的酶化合物的活性。活化条件为：化合物与活化剂的摩尔比为1∶1-1∶10，化合物的浓度为1-50毫克/毫升，温度0-25℃，PH在4-11范围内，时间0.1-24小时。众所周知，其中的活化剂，在叠氮法、碳化二亚胺法、丁二酰亚胺法或高碘酸盐氧化法中分别是叠氮化物、对环己基-2-(4-吗啉)乙基-碳化二亚胺-甲基-对甲苯磺酸盐、N-羟基琥珀酰亚胺以及高碘酸钠。上面所描述的某些化合物，如甾类糖苷、三萜烯糖苷或多烯抗生素较难溶于水，为使它们完全溶解可预先置于以下不给出质子的极性有机溶剂混合物中溶解：二甲亚砜或甲酰替二甲胺或六甲醇与甲苯按2∶1混合的混合物、甲酰替二甲胺与乙醇按2∶1混合的混合物。化合物与有机溶剂混合物的重量比例为1∶30-1∶0.01。One or more mixtures of the above-mentioned compounds are activated in an aqueous medium by the known azide method, carbodiimide method, succinimide method or periodate oxidation method, and the selected method is preferably able to The activity of the prepared enzyme compound is maintained maximally. The activation conditions are: the molar ratio of the compound to the activator is 1:1-1:10, the concentration of the compound is 1-50 mg/ml, the temperature is 0-25°C, the pH is in the range of 4-11, and the time is 0.1-24 hours . As we all know, the activators are azide, p-cyclohexyl-2-(4-morpholine) in the azide method, carbodiimide method, succinimide method or periodate oxidation method, respectively. Ethyl-carbodiimide-methyl-p-toluenesulfonate, N-hydroxysuccinimide, and sodium periodate. Some of the compounds described above, such as steroidal glycosides, triterpene glycosides or polyene antibiotics, are poorly soluble in water. To fully dissolve them, they can be pre-dissolved in the following non-proton-donating polar organic solvent mixture: Dimethyl sulfoxide or formyl dimethylamine or a mixture of hexamethanol and toluene at a ratio of 2:1, and a mixture of formyl dimethylamine and ethanol at a ratio of 2:1. The weight ratio of the compound to the organic solvent mixture is 1:30-1:0.01.

2、所述的胆固醇酯酶或胆固醇氧化酶溶解在PH为5-9范围的缓冲液中2. The cholesterol esterase or cholesterol oxidase is dissolved in a buffer solution with a pH of 5-9

胆固醇酯酶或胆固醇氧化酶溶解在PH为5-9范围的缓冲液中。为了提高胆固醇酶化合物的亲合性，还可向溶液中加入能改变胆固醇酯酶或胆固醇氧化酶化合物中的氨基的高分子聚合物。检测其活性后直接使用。Cholesterol esterase or cholesterol oxidase is dissolved in a buffer with a pH in the range of 5-9. In order to improve the affinity of the cholesterolase compound, a high molecular polymer capable of changing the amino group in the cholesterol esterase or cholesterol oxidase compound can also be added to the solution. Use directly after testing its activity.

3、混合反应3. Mixed reaction

活化后的化合物与溶解后的酶按1∶1-20∶1(摩尔比)的比例混合，该溶液在0-25℃的温度下反应18-24小时；The activated compound is mixed with the dissolved enzyme in a ratio of 1:1-20:1 (molar ratio), and the solution is reacted at a temperature of 0-25°C for 18-24 hours;

三、配置：3. Configuration:

在制得的酶化合物中加入表面活性剂，表面活性剂的混合比例为前述成分重量百分比的0.003-30，优先混合比例为0.01-10；还可加入适量防腐剂、稳定剂和用于显色测定的色原，还可考虑加入适量催化剂和抗干扰物质。A surfactant is added to the prepared enzyme compound, the mixing ratio of the surfactant is 0.003-30 of the weight percentage of the aforementioned ingredients, and the preferred mixing ratio is 0.01-10; an appropriate amount of preservatives, stabilizers and color-developing agents can also be added For the measured chromogen, it is also possible to consider adding an appropriate amount of catalyst and anti-interference substances.

本发明中使用的对高密度脂蛋白胆固醇具有较强作用的表面活性剂可以是阳离子表面活性剂、阴离子表面活性剂、两性离子表面活性剂或非离子表面性性剂，只要对高密度脂蛋白胆固醇具有较强作用，作为阳离子表面活性剂的实例有：月桂酰胺基咪唑啉、棕榈酰胺基咪唑啉、聚环氧乙烷十二烷胺、聚氧乙烯十六烷胺；作为阴离子表面活性剂的实例有：十二(烷)醇聚氧乙烯醚磷酸混合脂、PAM共聚物、十二烷基二苯醚二磺酸钠、棕榈酰甲基硫磺酸钠；作为非离子表面活性剂的实例有：磺基咪唑啉甜菜碱、烷基聚氧乙烯甜菜碱；作为非离子表面活性剂的实例子有：聚氧乙烯烷基醚、聚氧乙烯苯基醚、聚氧乙烯烷基苯基醚、二苄基联苯基聚氧乙烯醚、失水山梨醇聚氧乙烯、聚氧乙烯-聚氧丙烯聚醚、三苄基酚聚氧乙烯醚等。这里优选非离子表面活性剂(如：Tween-80、Triton X-100、GenapolX-081、Thesit等)。表面活性剂可以单独使用也可以混合使用，其用量如前所述。The surfactant used in the present invention that has a stronger effect on high-density lipoprotein cholesterol can be cationic surfactant, anionic surfactant, zwitterionic surfactant or nonionic surfactant, as long as it is effective on high-density lipoprotein Cholesterol has a strong effect. Examples of cationic surfactants are: lauryl amido imidazoline, palmito amido imidazoline, polyethylene oxide dodecylamine, polyoxyethylene cetylamine; as anionic surfactant Examples are: dodecyl alcohol polyoxyethylene ether phosphoric acid mixed fat, PAM copolymer, sodium dodecyl diphenyl ether disulfonate, sodium palmitoyl methylsulfonate; as examples of nonionic surfactants There are: sulfoimidazoline betaine, alkyl polyoxyethylene betaine; examples of nonionic surfactants are: polyoxyethylene alkyl ether, polyoxyethylene phenyl ether, polyoxyethylene alkylphenyl ether , Dibenzyl biphenyl polyoxyethylene ether, sorbitan polyoxyethylene, polyoxyethylene-polyoxypropylene polyether, tribenzylphenol polyoxyethylene ether, etc. Preferred nonionic surfactants (such as: Tween-80, Triton X-100, GenapolX-081, Thesit etc.) here. Surfactants can be used alone or in combination, and their consumption is as described above.

在本发明中为了保持酶化合物和试剂的稳定性，可以加入稳定剂、防腐剂或防霉剂或其它的醇类，如乙二胺四乙酸钠(EDTA.2Na)、牛血清白蛋白(BSA)、聚乙二醇(PEG)、二价金属离子。防腐剂或防霉剂的种类和用量可多可少不作限定，一般使用范围0.1-20mmol/L。In the present invention, in order to keep the stability of enzyme compound and reagent, can add stabilizer, antiseptic or antifungal agent or other alcohols, as sodium ethylenediamine tetraacetate (EDTA.2Na), bovine serum albumin (BSA ), polyethylene glycol (PEG), divalent metal ions. The type and amount of preservative or antifungal agent can be more or less, and the general use range is 0.1-20mmol/L.

在本发明中用于显色测定的色原有：2.4.6-三溴-3-羟基苯甲酸(TBHBA)、3.5-二氯-2-羟基苯甲酸(DHBS)、N-乙基-N-(3-磺丙基)-3-甲基苯胺钠盐(TOPS)、N-(2-羟基-3-磺丙基)-3-5-2甲氧基苯胺(HDAOS)、TODP、TOOS等。色原的种类和用量也可多可少不作限定，一般使用范围0.01-10mmol/L。The chromogens used for colorimetric determination in the present invention: 2.4.6-tribromo-3-hydroxybenzoic acid (TBHBA), 3.5-dichloro-2-hydroxybenzoic acid (DHBS), N-ethyl-N -(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-(2-hydroxy-3-sulfopropyl)-3-5-2methoxyaniline (HDAOS), TODP, TOOS wait. The type and amount of chromogen can be more or less without limitation, and the general use range is 0.01-10mmol/L.

本发明所使用的缓冲液，可以是GOOD’S缓冲液、磷酸盐缓冲液、三羟甲基氨基甲烷缓冲液、柠檬酸缓冲液、硼酸盐缓冲液等，缓冲液的种类和浓度因具体成份而定，这里不作限制，只要在PH为5-9的范围内保证缓冲作用且不抑制酶活性的一种或其混合物。优先选用50-200mmol/L的GOOD’S缓冲液。The buffer used in the present invention can be GOOD'S buffer, phosphate buffer, tris buffer, citric acid buffer, borate buffer, etc. The type and concentration of the buffer vary due to the specific components. It is determined, and there is no limitation here, as long as the buffering effect is guaranteed within the pH range of 5-9 and one or a mixture thereof does not inhibit enzyme activity. 50-200mmol/L GOOD'S buffer is preferred.

催化剂的种类和用量也可多可少不作限定，一般使用范围也为10U/L-10000U/L。The type and amount of the catalyst can be more or less without limitation, and the general use range is also 10U/L-10000U/L.

本发明中为了防止其它非测量物质的干扰，可加入抗坏血酸氧化酶等抗干扰物质。该物质的种类和用量也的种类和用量可多可少不作限定，一般使用范围10U/L-10000U/L。In the present invention, in order to prevent the interference of other non-measurement substances, anti-interference substances such as ascorbate oxidase can be added. The type and amount of the substance can be more or less without limitation, and the general use range is 10U/L-10000U/L.

本发明所采用的所有生化用品均可外购获得。All the biochemical products used in the present invention can be obtained by outsourcing.

本发明所提供的高密度中胆固醇的试剂，在测试时全自动生化分析仪上的主要参数如下：The reagent of the high-density medium cholesterol provided by the present invention, the main parameter on the full-automatic biochemical analyzer during test is as follows:

    Test Test     HDL-C HDL-C     Assay code Assay code     2point 2 points     Assay point Assay point     15-34 15-34     Wave(sub/main) Wave(sub/main)     700nm/546nm 700nm/546nm     Sample volume Sample volume     3μl 3μl     R1 volume R1 volume     225μl 225μl     R2 volume R2 volume     75μl 75μl     Calib.type Calib.type     Linear Linear     Calib/span.point Calib/span.point     2/2 2/2

实施例1：本实施例中的高亲合酶胆固醇酯酶化合物与高亲合酶胆固醇氧化酶化合物由甾类糖苷化合物中的龙舌苷甾类糖苷与酶制备；配置方式如前所述，其中：Example 1: The high-affinity enzyme cholesterol esterase compound and the high-affinity enzyme cholesterol oxidase compound in this example are prepared from the agave steroid glycoside and enzyme in the steroid glycoside compound; the configuration method is as described above, in:

化合物活化的活化条件为：The activation conditions for compound activation are:

化合物与活化剂的摩尔比为1∶1，温度15℃，PH值为8，时间10小时。The molar ratio of the compound to the activator is 1:1, the temperature is 15° C., the pH value is 8, and the time is 10 hours.

缓冲液的摩尔配比是100mmol/L，缓冲液的PH值为7。The molar ratio of the buffer solution is 100mmol/L, and the pH value of the buffer solution is 7.

活化后的化合物与溶解后的酶混合，该溶液在15℃的温度下反应20小时。The activated compound was mixed with the dissolved enzyme, and the solution was reacted at a temperature of 15° C. for 20 hours.

所述的龙舌苷预先置于以下有机溶剂混合物中溶解：甲酰替二甲胺与甲苯按2∶1混合的混合物，化合物与有机溶剂混合物的重量比例为1∶0.01。The agave glycoside is pre-dissolved in the following organic solvent mixture: a mixture of formyl dimethylamine and toluene at a ratio of 2:1, and the weight ratio of the compound to the organic solvent mixture is 1:0.01.

试剂IReagent I

高亲合酶胆固醇酯酶化合物             500U/LHigh affinity enzyme cholesterol esterase compound 500U/L

高亲合酶胆固醇氧化酶化合物           5000U/LHigh affinity enzyme cholesterol oxidase compound 5000U/L

缓冲液GOOD’S(PH：6.7)               100mmol/LBuffer GOOD’S (PH: 6.7) 100mmol/L

稳定剂BSA                            1g/LStabilizer BSA 1g/L

稳定剂聚乙二醇                       10g/LStabilizer polyethylene glycol 10g/L

稳定剂EDTA.2Na                       2mmol/LStabilizer EDTA.2Na 2mmol/L

色原TOPS                             2mmol/LChromogen TOPS 2mmol/L

防腐剂NaN3                           7.5mmol/LPreservative NaN3 7.5mmol/L

催化剂辣根过氧化物酶                 950U/LCatalyst horseradish peroxidase 950U/L

抗干扰物质抗坏血酸氧化酶             1000U/LAnti-interference substance ascorbate oxidase 1000U/L

试剂IIReagent II

表面活性剂聚氧乙烯烷基苯基醚         500g/LSurfactant polyoxyethylene alkylphenyl ether 500g/L

缓冲液GOOD’S(PH：6.7)               100mmol/LBuffer GOOD’S (PH: 6.7) 100mmol/L

防腐剂NaN3                           7.5mmol/LPreservative NaN3 7.5mmol/L

色原4-AAP                            0.25mmol/LChromogen 4-AAP 0.25mmol/L

稳定剂BSA                            1g/LStabilizer BSA 1g/L

稳定剂EDTA.2Na                       2mmol/LStabilizer EDTA.2Na 2mmol/L

上机测试时试剂I、试剂II同时混合加入测试。When testing on the machine, reagent I and reagent II are mixed and added to the test at the same time.

表1为本实施例1的上机测试值与沉淀法的测定值对照表。Table 1 is a comparison table between the test values on the machine and the measured values of the precipitation method in thepresent embodiment 1.

图1根据表1数据制成，本实施例1测得值与沉淀法(《全国临床检验操作规程》第二版279页所推荐)的测得值的相关性r为0.992，说明相关性良好。Fig. 1 is made according to table 1 data, and the correlation r of the measured value ofpresent embodiment 1 measured value and precipitation method (recommended on page 279 of the second edition of "National Clinical Testing Operation Regulations") is 0.992, illustrating that the correlation is good .

实施例2Example 2

本实施例中使用胆固醇酯酶化合物、胆固醇氧化酶化合物由三萜烯糖苷类化合物的中的茶皂苷与酶制备；配置方式如前所述，其中：In this embodiment, cholesterol esterase compound and cholesterol oxidase compound are used to prepare from tea saponin and enzyme in triterpene glycoside compounds; the configuration method is as described above, wherein:

化合物活化的活化条件为：The activation conditions for compound activation are:

化合物与活化剂的摩尔比为1∶5，温度0℃，PH值为4，时间24小时。The molar ratio of the compound to the activator is 1:5, the temperature is 0° C., the pH value is 4, and the time is 24 hours.

缓冲液的摩尔配比是50mmol/L，缓冲液的PH值为9。The molar ratio of the buffer solution is 50mmol/L, and the pH value of the buffer solution is 9.

活化后的化合物与溶解后的酶混合，该溶液在0℃的温度下反应24小时；The activated compound is mixed with the dissolved enzyme, and the solution is reacted at 0°C for 24 hours;

所述的茶皂苷预先置于以下有机溶剂混合物中溶解：甲酰替二甲胺与乙醇按2∶1混合的混合物；化合物与有机溶剂混合物的重量比例为1∶15。The tea saponin is predissolved in the following organic solvent mixture: a mixture of formyl dimethylamine and ethanol at a ratio of 2:1; the weight ratio of the compound to the organic solvent mixture is 1:15.

试剂IReagent I

胆固醇酯酶化合物用量为              5000U/LThe dosage of cholesterol esterase compound is 5000U/L

胆固醇氧化酶化合物用量为            4000U/LThe dosage of cholesterol oxidase compound is 4000U/L

缓冲液GOOD’S(PH：5)                50mmol/LBuffer GOOD’S (PH: 5) 50mmol/L

稳定剂BSA                           1g/LStabilizer BSA 1g/L

稳定剂Mgcl₂                        5mmol/LStabilizer Mgcl₂ 5mmol/L

稳定剂EDTA.2Na                      2mmol/LStabilizer EDTA.2Na 2mmol/L

色原TOOS                            2.2mmol/LChromogen TOOS 2.2mmol/L

防腐剂NaN3                          7.5mmol/LPreservative NaN3 7.5mmol/L

催化剂辣根过氧化物酶                950U/LCatalyst horseradish peroxidase 950U/L

抗干扰物质抗坏血酸氧化酶            1000U/LAnti-interference substance ascorbate oxidase 1000U/L

试剂IIReagent II

表面活性剂聚氧乙烯-聚氧丙烯聚醚     600g/LSurfactant polyoxyethylene-polyoxypropylene polyether 600g/L

缓冲液GOOD’S(PH：6.7)              50mmol/LBuffer GOOD’S (PH: 6.7) 50mmol/L

防腐剂NaN3                          7.5mmol/LPreservative NaN3 7.5mmol/L

色原4-AAP                           0.20mmol/LChromogen 4-AAP 0.20mmol/L

稳定剂BSA                           1g/LStabilizer BSA 1g/L

稳定剂EDTA.2Na                      2mmol/LStabilizer EDTA.2Na 2mmol/L

上机测试时试剂I、试剂II同时混合加入测试。When testing on the machine, reagent I and reagent II are mixed and added to the test at the same time.

表2为本实施例2的上机测试值与沉淀法的测定值对照表。Table 2 is a comparison table between the test values on the computer and the measured values of the precipitation method in Example 2.

图2根据表2数据制成，本实施例2测得值与沉淀法(《全国临床检验操作规程》第二版279页所推荐)的测得值的相关性r为0.994，说明相关性良好。Fig. 2 is made according to table 2 data, and the correlation r of the measured value of the measured value ofpresent embodiment 2 and precipitation method (recommended on page 279 of the second edition of "National Clinical Testing Operation Regulations") is 0.994, illustrating that the correlation is good .

实施例3Example 3

本实施例中使用胆固醇酯酶化合物、胆固醇氧化酶化合物由由三萜烯糖苷类化合物的中的茶皂苷与酶制备；配置方式如前所述。In this example, the cholesterol esterase compound and the cholesterol oxidase compound are prepared from tea saponin and enzymes in triterpene glycoside compounds; the configuration method is as described above.

化合物活化的活化条件为：The activation conditions for compound activation are:

化合物与活化剂的摩尔比为1∶10，温度25℃，PH值11，时间0.1小时。The molar ratio of the compound to the activator is 1:10, the temperature is 25° C., the pH value is 11, and the time is 0.1 hour.

缓冲液的摩尔配比是200mmol/L，缓冲液的PH值为5。The molar ratio of the buffer solution is 200mmol/L, and the pH value of the buffer solution is 5.

活化后的化合物与溶解后的酶混合，该溶液在25℃的温度下反应18小时；The activated compound is mixed with the dissolved enzyme, and the solution is reacted at a temperature of 25° C. for 18 hours;

所述的茶皂苷预先置于以下有机溶剂混合物中溶解：六甲醇与甲苯按2∶1混合的混合物，化合物与有机溶剂混合物的重量比例为1∶30。The tea saponin is pre-dissolved in the following organic solvent mixture: a mixture of hexamethanol and toluene at a ratio of 2:1, and the weight ratio of the compound to the organic solvent mixture is 1:30.

试剂IReagent I

胆固醇酯酶化合物用量为            6000U/LThe dosage of cholesterol esterase compound is 6000U/L

胆固醇氧化酶化合物用量为          600U/LThe dosage of cholesterol oxidase compound is 600U/L

缓冲液GOOD’S(PH：9)              200mmol/LBuffer GOOD’S (PH: 9) 200mmol/L

稳定剂BSA                         1g/LStabilizer BSA 1g/L

稳定剂Mgcl₂                      5mmol/LStabilizer Mgcl₂ 5mmol/L

稳定剂EDTA.2Na                    2mmol/LStabilizer EDTA.2Na 2mmol/L

色原HDAOS                         3mmol/LChromogen HDAOS 3mmol/L

防腐剂NaN3                        7.5mmol/LPreservative NaN3 7.5mmol/L

催化剂辣根过氧化物酶              950U/LCatalyst horseradish peroxidase 950U/L

抗干扰物质抗坏血酸氧化酶          1000U/LAnti-interference substance ascorbate oxidase 1000U/L

试剂IIReagent II

表面活性剂三苄基酚聚氧乙烯醚      12g/LSurfactant tribenzylphenol polyoxyethylene ether 12g/L

缓冲液GOOD’S(PH：6.7)            200mmol/LBuffer GOOD’S (PH: 6.7) 200mmol/L

防腐剂NaN3                        7.5mmol/LPreservative NaN3 7.5mmol/L

色原4-AAP                       0.15mmol/LChromogen 4-AAP 0.15mmol/L

稳定剂BSA                       1g/LStabilizer BSA 1g/L

稳定剂EDTA.2Na                  2mmol/LStabilizer EDTA.2Na 2mmol/L

上机测试时试剂I、试剂II同时混合加入测试。When testing on the machine, reagent I and reagent II are mixed and added to the test at the same time.

表3为本实施例3的上机测试值与沉淀法的测定值对照表。Table 3 is a comparison table between the test values on the machine of the present embodiment 3 and the measured values of the precipitation method.

图3根据表3数据制成，本实施例3测得值与沉淀法(《全国临床检验操作规程》第二版279页所推荐)的测得值的相关性r为0.989，说明相关性良好。Fig. 3 is made according to table 3 data, and the correlation r of the measured value of the measured value of present embodiment 3 and precipitation method (" national clinical examination operating procedure " second edition 279 pages is recommended) is 0.989, illustrates that correlation is good .

表1     沉淀法测定值(mmol/L) 本发明方法(实施例1)测定值(mmol/L)     1.2 1.2     1.61 1.56     1.1 1.12     1.62 1.63     0.62 0.61     1.45 1.5     1.55 1.54     1.77 1.84     1.04 1.01     1.57 1.54     1.12 1.04     1.06 0.95     1.68 1.63     1.21 1.22     1.46 1.46     1.11 1.04     1.72 1.7     1.38 1.35     1.27 1.25     0.85 0.77     0.91 0.96     1.49 1.5     0.87 0.91     1.0 0.98     1.79 1.75     1.06 1.1     0.83 0.8     0.99 0.98     0.96 0.95     1.31 1.3     1.43     1.4     1.25     1.23     0.87     0.86Table 1 Determination value of precipitation method (mmol/L) Method of the present invention (embodiment 1) measured value (mmol/L) 1.2 1.2 1.61 1.56 1.1 1.12 1.62 1.63 0.62 0.61 1.45 1.5 1.55 1.54 1.77 1.84 1.04 1.01 1.57 1.54 1.12 1.04 1.06 0.95 1.68 1.63 1.21 1.22 1.46 1.46 1.11 1.04 1.72 1.7 1.38 1.35 1.27 1.25 0.85 0.77 0.91 0.96 1.49 1.5 0.87 0.91 1.0 0.98 1.79 1.75 1.06 1.1 0.83 0.8 0.99 0.98 0.96 0.95 1.31 1.3 1.43 1.4 1.25 1.23 0.87 0.86

表2     沉淀法测定值(mmol/L)  本发明方法(实施例2)测定值(mmol/L)     0.94  0.92     1.08  1.04     0.97  1.01     0.86  0.89     0.94  0.96     1.41  1.28     1.17  1.21     0.89  0.85     1.68  1.64     0.89  0.94     0.83  0.82     1.13  1.09     1.49  1.5     1.4  1.4     0.84  0.9     0.79  0.78     1.12  1.1     1.12  1.2     0.91  0.9     1.03  1.0     1.02  1.0     1.29  1.27     0.88  0.88     1.59  1.54     1.06  1.09     1.23  1.2     1.5  1.48     0.38  0.36     1.22  1.18     2.13  2.1     0.43  0.41     1.52  1.5     1.36  1.36Table 2 Determination value of precipitation method (mmol/L) Method of the present invention (embodiment 2) measured value (mmol/L) 0.94 0.92 1.08 1.04 0.97 1.01 0.86 0.89 0.94 0.96 1.41 1.28 1.17 1.21 0.89 0.85 1.68 1.64 0.89 0.94 0.83 0.82 1.13 1.09 1.49 1.5 1.4 1.4 0.84 0.9 0.79 0.78 1.12 1.1 1.12 1.2 0.91 0.9 1.03 1.0 1.02 1.0 1.29 1.27 0.88 0.88 1.59 1.54 1.06 1.09 1.23 1.2 1.5 1.48 0.38 0.36 1.22 1.18 2.13 2.1 0.43 0.41 1.52 1.5 1.36 1.36

表3     沉淀法测定值(mmol/L)  本发明方法(实施例3)测定值(mmol/L)     1.5  1.43     1.48  1.31     1.08  0.96     1.12  0.99     0.88  0.83     1.27  1.06     2.0  1.79     1.11  1.0     1.04  0.87     1.72  1.49     1.1  0.91     0.88  0.85     1.42  1.27     1.54  1.38     1.94  1.72     1.18  1.11     1.66  1.46     1.39  1.21     1.86  1.68     1.08  1.06     1.19  1.12     1.75  1.57     1.15  1.04     2.1  1.77     1.75  1.55     1.72  1.45     1.86  1.62     1.28  1.1     1.78  1.61     1.37  1.2table 3 Determination value of precipitation method (mmol/L) Method of the present invention (embodiment 3) measured value (mmol/L) 1.5 1.43 1.48 1.31 1.08 0.96 1.12 0.99 0.88 0.83 1.27 1.06 2.0 1.79 1.11 1.0 1.04 0.87 1.72 1.49 1.1 0.91 0.88 0.85 1.42 1.27 1.54 1.38 1.94 1.72 1.18 1.11 1.66 1.46 1.39 1.21 1.86 1.68 1.08 1.06 1.19 1.12 1.75 1.57 1.15 1.04 2.1 1.77 1.75 1.55 1.72 1.45 1.86 1.62 1.28 1.1 1.78 1.61 1.37 1.2

Claims (9)

1, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) comprises an amount of antiseptic, stabilizing agent and chromogen, it is characterized in that this reagent also comprises following composition:

A, high affinity enzyme compound, this enzyme compound, by the reaction of enzyme and compound and get, wherein,

Compound is the multiple and activator mix of a kind of or arbitrary proportion mixing in the compounds such as steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotics, the activator after the activation, and compound concentrations is the 1-50 mg/ml;

Enzyme is 1, cholesterol esterase and cholesterol oxidase, the two ratio 10: 1-1: 10,

2, lipoprotein lipase and cholesterol oxidase, the two ratio 10: 1-1: 10

In the arbitrary group of dissolved matter after mixing, dissolve with damping fluid, the pH value of damping fluid is 5-9, the mole proportioning of damping fluid is 50-200mmol/L, and described damping fluid is GOOD ' S damping fluid or phosphate buffer or TRIS buffer or citrate buffer solution or borate buffer solution;

Mole match ratio during two kinds of composition reactions of above-claimed cpd and enzyme is 1: 1-20: 1;

B, surfactant, its blending ratio is the 0.003-30 of the percentage by weight of A composition, described surfactant be a kind of or arbitrary proportion in cationic surfactant, anionic surfactant, zwitterionic surfactant and the non-ionic surfactant mix multiple.

2, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1 is characterized in that described enzyme preferentially selects cholesterol esterase and cholesterol oxidase.

3, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1 is characterized in that this reagent also comprises proper catalyst.

4, according to the mensuration reagent of cholesterol in claim 1 or the 2 or 3 described high-density lipoprotein (HDL), the blending ratio that it is characterized in that described surfactant is the 0.01-10 percentage by weight.

5, the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 4 is characterized in that also having an amount of anti-interference material in this reagent.

6, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 1 is characterized in that this method may further comprise the steps:

One, compound activating

That selects that a kind of or arbitrary proportion in the compounds such as described steroid glycoside, triterpene glucoside, hydrophobic protein, archon and polyene antibiotics mixes is multiple, in aqueous medium, activate with known azide method or carbodiimide method or succimide method or periodate oxidation method, activation condition is: the mol ratio of compound and activator is 1: 1-1: 10, temperature 0-25 ℃, PH in the 4-11 scope, time 0.1-24 hour.

Two, enzyme dissolving

Select described cholesterol esterase and cholesterol oxidase, or in lipoprotein lipase and the cholesterol oxidase arbitrary group, be dissolved in the damping fluid;

Three, hybrid reaction

Compound after the activation with the dissolving after enzyme mix, this solution reacted 18-24 hour under 0-25 ℃ temperature;

Four, configuration:

Reacted enzyme compound mixes with surfactant, and adds antiseptic, stabilizing agent, is used for the chromogen of chromogenic assay.

7, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 6, it is characterized in that described steroid glycoside, triterpene glucoside or polyene antibiotic place following ORGANIC SOLVENT MIXTURES to dissolve in advance: dimethyl sulfoxide or formyl for dimethylamine or pregnancy alcohol and toluene by the potpourri that mixes at 2: 1, perhaps, by the potpourri that mixes at 2: 1, the part by weight of compound and ORGANIC SOLVENT MIXTURES is 1 to formyl: 30-1: 0.01 for dimethylamine and ethanol.

8, according to the preparation method of the mensuration reagent of cholesterol in claim 6 or the 7 described high-density lipoprotein (HDL), it is characterized in that also adding in this reagent catalyzer.

9, the preparation method of the mensuration reagent of cholesterol in the high-density lipoprotein (HDL) according to claim 8 is characterized in that also adding in this reagent anti-interference material.

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