JP2017060522A - Quantitative method of cholesterol in high density lipoprotein (hdl) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separately or simultaneously quantifying cholesterol in all HDL-C and HDL subfractions of ApoE-containing HDL-C and ApoE-deficient HDL-C.SOLUTION: In the method for quantifying cholesterol in all HDL including ApoE containing HDL, cholesterol oxidase having a final concentration of 0.9 U/ml or more is added to a test sample in the presence of cholesterol esterase and peroxidase.SELECTED DRAWING: None

Description

  The present invention relates to a method for quantifying cholesterol (-C) in high density lipoprotein (HDL).

  Since HDL receives cholesterol from tissues including the arteriosclerotic wall, it is related to the removal action of cholesterol accumulated in cells and is a risk prevention factor for various arteriosclerosis including coronary arteriosclerosis. Levels are known to be useful guidelines for predicting the onset of arteriosclerotic diseases.

  HDL is a complex of a protein called apoprotein and a lipid component such as phospholipid, cholesterol, and neutral fat. ApoE containing HDL is due to the difference in the content ratio of one of its components, apolipoprotein E (ApoE). And ApoE deficient HDL subfractions. ApoE containing HDL has a strong ability to withdraw cholesterol and has an antiplatelet action, and has attracted attention as a super-good lipoprotein among HDL. Recently, as a therapeutic drug for dyslipidemia after statin, there is an expectation for a cholesterol ester transfer protein (CETP) inhibitor that increases HDL-C. CETP inhibitors are known to increase mainly HDL, particularly ApoE containing HDL.

  Methods for measuring cholesterol in HDL include, for example, separating HDL from other lipoproteins by ultracentrifugation and then subjecting it to cholesterol measurement, or measuring the color intensity by staining lipids after separation by electrophoresis The method of doing is known. However, these methods are troublesome in operation and have problems such as being unable to process a large number of specimens, and are rarely used on a daily basis.

  As a method for measuring cholesterol in HDL, there is a method in which a precipitant is added to a sample to aggregate lipoproteins other than HDL, which is removed by centrifugation, and cholesterol in the supernatant containing only the separated HDL is measured. . In this method, it is known that the reactivity with respect to HDL subfractions varies depending on the type of precipitant used. In the method using phosphotungstic acid-magnesium, dextran sulfate-magnesium, and heparin-calcium as the precipitant, ApoE Since containing HDL aggregates with lipoproteins such as VLDL and LDL and is removed as a precipitate fraction by centrifugation, it cannot be measured as an HDL fraction. On the other hand, in the method using heparin-manganese or polyethylene glycol (PEG), ApoE containing HDL does not aggregate and is measured as HDL. In any case, the method for separating and quantifying HDL using this precipitating agent is simpler than ultracentrifugation and electrophoresis, but it involves the operation of adding a precipitating agent to separate it. It is not satisfactory and requires a relatively large amount of specimen.

  In recent years, as a simple method for quantifying HDL-C, there is known a method for quantifying HDL-C with an automatic analyzer without pretreatment using a precipitant, and chemically modifying cholesterol esterase or cholesterol oxidase enzyme. A method of specifically capturing cholesterol in HDL in the presence of an inclusion compound such as cyclodextrin (see Patent Document 1), or forming an aggregate or complex with lipoproteins other than HDL, and then cholesterol in HDL There are known a method for catching selenium by enzymatic reaction (see Patent Documents 2 and 3), a method using a surfactant having 13 to 14 HLB specifically acting on HDL (see Patent Document 4), and the like.

Japanese Unexamined Patent Publication No. 7-301636 JP-A-8-131197 JP-A-8-201393 WO98 / 26090

  Although the method of quantifying HDL-C with the current automatic analyzer can easily measure HDL-C, the particle size is large and the reactivity with respect to ApoE containing HDL subfractions is not sufficient. Cannot be measured accurately. Especially in CETP deficiency, HDL may be high, but in these cases the particle size is large and the proportion of ApoE containing HDL is increasing. Negative error. In addition, when determining the therapeutic effect of drugs that increase HDL-C, such as CETP inhibitors, the results may be misleading. Therefore, a total HDL-C measurement technique for accurately measuring these HDL subfractions is important.

  An object of the present invention is to provide a method compatible with a general-purpose automatic analyzer for easily quantifying all HDL-C including HDL sub-fraction, particularly, ApoE containing HDL having a large particle size.

  As a result of intensive studies, the inventors of the present application have been able to increase the reactivity of ApoE containing HDL, particularly by increasing the particle size, by defining the concentration of cholesterol oxidase in the presence of a specific surfactant. It was found that C can be accurately quantified.

  As described above, the present invention provides a method for quantifying total HDL-C including ApoE containing HDL having a large particle size in a test sample.

That is, the present invention is as follows.
[1] A method for quantifying cholesterol in total HDL including ApoE containing HDL, characterized in that cholesterol oxidase having a final concentration of 0.9 U / ml or more is added to a test sample in the presence of cholesterol esterase and peroxidase. A method for quantifying cholesterol in total HDL including ApoE containing HDL.
[2] The method for quantifying cholesterol in total HDL including ApoE containing HDL according to [1], wherein the cholesterol oxidase is present in either the first reagent and / or the second reagent.

The present invention is as follows.
(1) A method for quantifying total cholesterol in the HDL comprising a step of mixing HDL containing ApoE-containing HDL with cholesterol esterase, peroxidase and cholesterol oxidase in the reaction solution, the cholesterol oxidase in the reaction solution The said method whose content of is 0.9 U / mL or more.
(2) The step described above includes at least a first step of removing subfractionated lipoproteins other than HDL and a second step of specifically quantifying total cholesterol in HDL including ApoE-containing HDL. the method of.
(3) The method according to (2), wherein the content of cholesterol oxidase in the second step of specifically quantifying total cholesterol in HDL including ApoE-containing HDL is 0.9 U / mL or more.
(4) The method according to any one of (1) to (3), wherein HDL containing ApoE-containing HDL is HDL contained in a collected blood sample.

  The conventional method for measuring cholesterol in HDL cannot accurately quantify cholesterol in ApoE-containing HDL, and as a result, it has been difficult to accurately quantify cholesterol in total HDL. In the method of the present invention, in the method for measuring cholesterol in HDL containing ApoE containing HDL using an enzyme, the reactivity of ApoE containing HDL to the enzyme is increased by using cholesterol oxidase at a specific concentration, and ApoE containing Cholesterol in HDL containing a large amount of HDL can be accurately quantified.

It is a figure which shows% bias with respect to 13% PEG precipitation method when it measures with the method of Example 1 of this invention with respect to HDL sample. It is a figure which shows% bias with respect to 13% PEG precipitation method when it measures with the method of Example 2 of this invention with respect to HDL sample.

Hereinafter, the present invention will be described in detail.
Cholesterol contained in lipoprotein includes ester-type cholesterol (cholesterol ester) and free-type cholesterol. In the present specification, the term “cholesterol” includes both of them.

  Lipoprotein is roughly divided into VLDL, LDL and HDL fractions. Among these, HDL can be classified into ApoE containing HDL and ApoE deficient HDL subfractions based on the difference in the content ratio of ApoE. Usually, ApoE containing HDL indicates that HDL contains ApoE, and ApoE deficient HDL indicates that it does not contain ApoE. HDL is divided into ApoE containing HDL and ApoE deficient HDL because these lipoproteins have different mechanisms of action against arteriosclerosis, and therefore it was necessary to measure them separately. In other words, ApoE-containing HDL has a strong ability to extract cholesterol, has an antiplatelet action, and acts as a good HDL. In addition, CETP inhibitors that increase HDL-C as a dyslipidemic drug are known to increase ApoE-containing HDL mainly. In addition, HDL in which ApoE is present or contained in a large amount is sometimes referred to as ApoE-rich HDL, which is also included in ApoE-containing HDL. Since the distribution of ApoE content present in HDL is continuous, it is not possible to clearly distinguish between ApoE containing HDL and ApoE deficient HDL depending on the ApoE content ratio in the lipoprotein. When HDL is fractionated by chromatography or the like using the amount as an index, a specific ApoE content may be determined, and ApoE-containing HDL and ApoE-deficient HDL may be distinguished based on the value.

  Moreover, HDL can be classified into HDL2 and HDL3 according to the difference in density, and there are ApoE containing HDL2 and ApoE containing HDL3, each having a different ApoE content. In addition, large HDL having a large particle size is deficient in apolipoprotein A1 (ApoA1) which is usually present and has a large number of ApoE, which is also included in ApoE containing HDL. The diameter of the lipoprotein particle size varies depending on the reporter, but VLDL is 30 nm to 80 nm (30 nm to 75 nm), LDL is 22 nm to 28 nm (19 nm to 30 nm), and HDL is 7 to 10 nm in diameter. Specific gravity is 1.006 or less for VLDL, 1.019 to 1.063 for LDL, and 1.063 to 1.21 for HDL.

  An object of the method of the present invention is to measure cholesterol in total HDL including ApoE containing HDL, that is, cholesterol in total HDL including both ApoE-containing HDL and ApoE-deficient HDL.

  The test sample to be subjected to measurement in the method of the present invention may be any sample as long as it may contain lipoproteins such as HDL, LDL, VLDL and CM, for example, serum, plasma, etc. A body fluid and its dilution can be mentioned, However, It is not limited to these.

  The method of the present invention is usually performed using an automatic analyzer. Although the number of steps of the method of the present invention is not limited, it is preferably performed by two steps, a first step and a second step. In the first step, lipoproteins other than HDL such as VLDL and LDL can be introduced outside the reaction system, and in the second step, cholesterol in all HDL including ApoE containing HDL can be measured. In the first step, In the second step, cholesterol in all HDL including ApoE containing HDL can be measured without introducing lipoproteins other than HDL such as VLDL and LDL out of the reaction system.

  For example, in the first step, lipoproteins other than HDL such as CM, VLDL, and LDL are introduced out of the reaction system. As a result, subfraction HDL of ApoE-containing HDL and ApoE-deficient HDL remains and reacts in the second step. That is, cholesterol esterase is reacted with a specimen sample, and the resulting cholesterol is reacted in the presence of a cholesterol-reactive enzyme such as cholesterol oxidase or cholesterol dehydrogenase to lead out of the reaction system. At this time, the reaction is caused to occur in the presence of a specific surfactant reactive to lipoproteins other than HDL to be introduced outside the reaction system. In this case, a surfactant that acts on HDL is not added to the first step. Here, guiding cholesterol of lipoproteins out of the reaction system includes subfractionated HDL of ApoE-containing HDL and ApoE-deficient HDL in which cholesterol contained in lipoproteins such as CM, VLDL and LDL is the measurement target This refers to eliminating or aggregating cholesterol contained in lipoproteins other than HDL, or inhibiting them from reacting in a later step so as not to affect the quantification of HDL-C.

  Erasing means decomposing lipoprotein cholesterol in the specimen sample so that the degradation product is not detected in the next step. Examples of a method for eliminating lipoprotein cholesterol include a method in which hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase is decomposed into water and oxygen using catalase. In addition, the hydrogen donor and the generated hydrogen peroxide may be reacted with peroxidase to convert to colorless quinone, but is not limited thereto.

  In the first step, lipoproteins other than HDL such as CM, VLDL, and LDL are not led out of the reaction system, and in the second step, all lipoproteins containing ApoE containing HDL are included under the condition that the lipoproteins other than HDL do not react. Only HDL may be reacted, and sub-fraction HDL of ApoE-containing HDL and ApoE-deficient HDL may be reacted in the second step.

  The present inventors have found that there are conditions that increase the reactivity to ApoE containing HDL in the presence of a surfactant. For example, as shown in Example 1, reactivity to a specimen containing ApoE containing HDL with a high HDL-C value was examined. As a result, it was confirmed that the reactivity of ApoE containing HDL increases when cholesterol oxidase is used at a high concentration. As a result, in the second step, all HDL containing ApoE containing HDL reacts and cholesterol in all HDL containing ApoE containing HDL can be quantified.

  Cholesterol oxidase may be added alone to either the first reagent (added in the first step) or the second reagent (added in the second step), or may be present in both. However, in the first step, lipoproteins other than HDL are reacted with a sample in the presence of cholesterol esterase in the presence of a surfactant reactive with lipoproteins other than HDL, and the resulting cholesterol is converted to cholesterol oxidase. In the case where the reaction is conducted in the presence of the acid and led out of the reaction system, it must be present in the first reagent.

  When the cholesterol oxidase is added and used only in the first step, the cholesterol oxidase concentration in the first reagent is 1.2 to 3.2 U / ml (in the mixed solution when the first reagent and the second reagent are added). The final concentration is preferably 0.9 to 2.4 U / ml), and 1.6 to 2.0 U / ml (final concentration when the first and second reagents are added is 1.2 to 1.5 U / ml) Is particularly preferred. When cholesterol oxidase is added and used only in the second step, the cholesterol oxidase concentration in the second reagent is 1.2 to 7.2 U / ml (the final concentration in the mixed solution when the first reagent and the second reagent are added). The concentration is preferably 0.3 to 1.8 U / ml) and preferably 1.6 to 3.6 U / ml (the final concentration when the first and second reagents are added is 0.4 to 0.9 U / ml). Particularly preferred. When present in both the first reagent and the second reagent, the final concentration in the mixed solution after the addition of the second reagent is preferably 0.9 to 2.4 U / ml, particularly preferably 1.2 to 1.5 U / ml. .

  In the reaction of the present invention, in addition to the above-mentioned cholesterol oxidase, it is necessary to use a surfactant that reacts with HDL, that is, a surfactant that reacts with both ApoE containing HDL and ApoE deficient HDL. Surfactants that react with HDL have an ApoE containing HDL reaction rate / ApoE deficient HDL reaction rate ratio of 0.7 or more, preferably less than 1.3, preferably an ApoE containing HDL reaction rate / ApoE deficient HDL reaction rate ratio of 0.8 or more. , Surfactants of less than 1.2.

  Here, the reactivity of a surfactant to lipoprotein can be evaluated by using the degree of cholesterol reaction as an index when cholesterol esterase and cholesterol oxidase are allowed to act on lipoprotein in the presence of the surfactant. it can. Specifically, for example, the reactivity of the surfactant with respect to lipoprotein can be measured by adding various surfactants to the second reagent and performing the measurement by the method shown in Example 1 below. . Measure the reaction amount of the surfactant to ApoE-containing HDL and the reaction amount of the surfactant to ApoE-deficient HDL, and the ratio to the total cholesterol amount in each sample (“ApoE-containing HDL reaction rate / ApoE-deficient HDL response rate ratio ”). Accordingly, the ratio of ApoE-containing HDL reaction rate / ApoE-deficient HDL reaction rate can be calculated.

  Specifically, sodium alkylbenzene sulfonate is used as an anionic surfactant, and polyoxyethylene monolaurate, lauryl alcohol alkoxylate, polyoxyethylene laurylamine, polyoxyethylene benzylphenyl ether derivatives are used as nonionic surfactants. Polyoxyethylene polyoxypropylene block polymer having a molecular weight of less than 1700, polyoxyethylene nonylphenyl ether having an HLB of 13.0 or more and less than 14.5, and as an amphoteric surfactant, alkyldimethylaminoacetic acid betaine and alkylcarboxymethylhydroxyethylimidazolium betaine Can be mentioned.

  As an example of a specific surfactant, as an anionic surfactant, New Rex Soft 60-N, New Rex Powder F, New Rex Paste H (made by Nippon Oil & Fats Co., Ltd.), Neo-Perex No. 1-F, Neoperex G-65, Emar NC-35 (above, manufactured by Kao Corporation), Examples of nonionic surfactants include Adecatol LB70, Adecatol LB-103, Adecatol LB-93 (Asahi Denka Co., Ltd.), Dispanol K -3, Nonion L-4, Nonion MN-811, Nonion NS-210, Nonion NS-212, Naimine L-202, Pronon 102, Pronon 204 (above made by NOF Corporation), Nonipole 85, Nonipol 95, Nonipol 100, Nonipol 120 (manufactured by Sanyo Kasei Co., Ltd.), Emulgen B66 (Kao), amphoteric surfactants as examples of Anhitoal 24B (Kao Corporation), Nissan Anon BF, Nissan Anon GLM-R-LV, Nissan Anon LG Manufactured by NOF Corporation), and the like.

  In the method of the present invention, a surfactant having reactivity with both ApoE-containing HDL and ApoE-deficient HDL can be added and used in the second step. When cholesterol oxidase is not added in the first step, a surfactant having reactivity with both ApoE-containing HDL and ApoE-deficient HDL may be added in the first step. In this case, the concentration in the first step is preferably 0.05 g / L to 2.0 g / L, more preferably 0.1 g / L to 1.0 g / L. The concentration in the second step is preferably 0.15 g / L to 6.0 g / L, and more preferably 0.3 g / L to 3.0 g / L.

  Furthermore, when the above surfactant is used in the second step, lipoproteins such as CM, VLDL and LDL other than HDL can be eliminated in the first step, and the specificity for HDL-C can be increased.

  Examples of methods for eliminating lipoproteins other than HDL include a method using catalase and a method for forming colorless quinone. Cholesterol esterase and cholesterol oxidase are allowed to act on the test sample in the absence of a surfactant that acts on HDL, and the resulting hydrogen peroxide derived from lipoproteins other than HDL is removed. By the action of cholesterol esterase, ester-type cholesterol in lipoprotein is hydrolyzed to produce free cholesterol and fatty acid. Next, the resulting free cholesterol and free cholesterol originally present in the lipoprotein are oxidized by the action of cholesterol oxidase to produce cholestenone and hydrogen peroxide. Lipoprotein can be eliminated by removing the generated hydrogen peroxide. As a method for removing hydrogen peroxide, for example, a method in which catalase is allowed to act to decompose into water and oxygen, and peroxidase is used, for example, DAOS (N-ethyl-N- (2-hydroxysulfopropyl) -3,5- Examples thereof include a method of converting hydrogen peroxide into colorless quinone by reacting with a phenolic or aniline-based hydrogen donor compound that reacts with hydrogen peroxide to produce colorless quinone, such as dimethyoxyaniline). It is not limited to.

  By the above method, cholesterol in lipoproteins other than HDL is largely eliminated in the first step, and in the HDL subfraction of HDL or ApoE-containing HDL and / or ApoE-deficient HDL by the reaction in the second step. Only more cholesterol is quantified more specifically.

  The first step is performed in the absence of a surfactant that acts on HDL. By doing so, cholesterol in HDL hardly reacts, and cholesterol in other lipoproteins such as LDL, VLDL, CM reacts and is erased. As a result, cholesterol in HDL is selectively quantified in the next second step.

  The concentration of cholesterol esterase in the reaction solution in the first step is preferably about 0.2 to 1.0 U / ml. Further, the concentration of catalase is preferably about 40 to 100 U / ml, and the concentration of peroxidase is preferably about 0.4 to 1.0 U / ml. The concentration of the compound that reacts with hydrogen peroxide to produce colorless quinone is preferably about 0.4 to 0.8 mmol / l.

  The reaction in the first step is preferably carried out in a buffer solution having a pH of 5 to 8, and phosphate, glycine, Tris and Good buffers are preferred as the buffer solution. In particular, Bis-Tris, PIPES, MOPSO, BES, HEPES, and POPSO, which are good buffers, are preferable, and the concentration of the buffer is preferably about 10 to 500 mM.

  In the first step, a divalent metal ion may be included in the reaction solution in order to increase the elimination efficiency of lipoproteins other than HDL. As the divalent metal ion, copper ion, iron ion and magnesium ion can be preferably used, but magnesium ion is particularly preferable. The concentration of the divalent metal ion is preferably about 5 to 200 mM.

  Furthermore, lipoprotein hydrolase can be optionally added to the reaction solution in the first step. It is preferable to add this enzyme because cholesterol in VLDL is particularly easily reacted. The concentration of the enzyme in the reaction solution is preferably about 5.0 to 10.0 U / ml.

  The reaction temperature in the first step is suitably about 25 ° C to 40 ° C, and most preferably 37 ° C. The reaction time may be about 2 to 10 minutes.

  The enzymatic determination method of cholesterol in the present invention itself is well known in this field. For example, hydrogen peroxide is generated from cholesterol ester and free cholesterol by the action of cholesterol esterase and cholesterol oxidase as in the first step. This can be done by quantifying hydrogen peroxide. The quantification of hydrogen peroxide can be carried out, for example, by reacting with a compound that forms quinone dye by reacting with hydrogen peroxide in the presence of peroxidase, and measuring the amount of quinone dye produced by absorbance measurement or the like. it can. The quinone dye can be formed by using, for example, hydrogen peroxide, 4-aminoantipyrine, and a phenol or aniline hydrogen donor.

  Among the hydrogen donor compounds, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline are used as aniline-based hydrogen donor compounds. (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS) N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl)- 3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2- Roxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) and N-sulfopropylaniline (HALPS) ) And the like.

  The concentration of the compound that forms the quinone dye is not particularly limited. For example, 4-aminoantipyrine is preferably 0.1 to 2.0 mM, more preferably 0.5 to 1.5 mM, based on the entire reaction mixture. For phenolic or aniline hydrogen donor compounds, 0.5 to 2.0 mmol / L is preferred. The concentration of peroxidase is not particularly limited, but is preferably 0.4 to 5 U / ml with respect to the entire reaction mixture. The preferred reaction conditions (reaction temperature, reaction time, buffer solution, pH) in the second step are the same as the preferred reaction conditions in the first step.

  In the first step, when the generated hydrogen peroxide is decomposed with catalase, it is necessary to inhibit this catalase in the second step, so that a catalase inhibitor such as sodium azide is added in the second step. Used to inhibit catalase.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example. In this example, “%” means “% by weight” unless otherwise specified.

Example 1
As an example of confirming the action effect of cholesterol oxidase in HDL-C measurement, a reagent having the following composition was prepared.
First reagent
BES buffer (pH 7.0) 100 mmol / l
HDAOS 0.7 mmol / l
Cholesterol esterase 1.4 U / ml
Cholesterol oxidase 0.8 U / ml
Catalase 600U / ml
Magnesium chloride 10 mmol / l
Second reagent
BES buffer (pH 7.0) 100 mmol / l
4-Aminoantipyrine 4.0 mmol / l
Peroxidase 2.4 U / ml
Cholesterol oxidase 0 U / ml, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 2.4, 4.8, 9.6 U / ml
Sodium azide 0.1%
Surfactant (polyoxyethylene benzylphenyl derivative) 1.0%

  A high HDL-C sample containing 54.0 mg / ml of ApoE containing HDL-C using a reagent in which the cholesterol oxidase concentration in the second reagent was varied in the range of 0 U / ml to 9.6 U / ml (Total HDL-C 195.8 mg / ml) was measured. After mixing 180 μl of the first reagent with 2.4 μl of the sample and reacting at 37 ° C. for 5 minutes, reacting 60 μl of the second reagent at 37 ° C. for 5 minutes and measuring the absorbance at the main wavelength / sub wavelength = 600 nm / 700 nm. Cholesterol concentration was determined from a calibration curve. Similar samples were measured using a 13% PEG precipitation method (J Lipid Research 38, 1204-16: 1997) which can measure total HDL-C including ApoE containing HDL as a comparative control.

  Since the volume of the sample to be used is very small relative to the volume of the reagent, when the volume of the sample is ignored, the volume ratio of the reagent first reagent to the second reagent is 3: 1. Thus, if the first reagent contains C1 U / ml cholesterol oxidase and the second reagent contains C2 U / ml cholesterol oxidase, the final concentration of cholesterol oxidase is C1 × 3/4 + C2 × 1/4 U / ml. In this example, since the first reagent concentration is 0.8 U / ml and the second reagent concentration is 0 U / ml to 9.6 U / ml, the final concentration is 0.6 U / ml to 3 U / ml.

  The results are shown in FIG. FIG. 1 shows the results for the final concentration of cholesterol oxidase in% bias. The% bias can be obtained by (Example measurement value−reference method measurement value) / reference method measurement value × 100.

  As shown in FIG. 1, when cholesterol oxidase is not added to the second reagent (final concentration 0.6 U / ml), the measured value of the high HDL-C sample is lower than that of the 13% PEG precipitation method. On the other hand, when the cholesterol oxidase was added to the second reagent, the decrease in the measured value was improved. When the concentration of the second reagent was 0.8 U / ml (final concentration 0.8 U / ml) or more, the reaction difference from the control method Showed good reactivity with less than 1%.

Example 2
Using a reagent in which cholesterol oxidase in Example 1 was added only in the first reagent, a high HDL-C sample (Total HDL-C 195.8 mg / ml) containing an ApoE containing HDL-C concentration of 54.0 mg / ml was obtained. It was measured. The concentration of cholesterol oxidase in the first reagent is 0.8 U / ml, 0.85, 0.9, 0.85, 1.0, 1.05, 1.1, 1.15, 1.2, 2.4, 4.8 and 9.6 U / ml, and the final concentration is 0.6 U / ml, 0.638, 0.675, 0.713, 0.75, 0.788, 0.825, 0.863, 0.9, 1.8, 3.6, and 7.2 U / ml. Other conditions were the same as in Example 1.

  As shown in FIG. 2, when the concentration of the first reagent is 1.05 U / ml or more (final concentration 0.0.863 U / ml) or more, the reaction difference from the control method is less than 1%, indicating good reactivity. became.

Claims (2)

  When quantifying cholesterol in total HDL, including ApoE containing HDL, add cholesterol oxidase at a final concentration of 0.9 U / ml or more to the test sample in the presence of cholesterol esterase, peroxidase, and polyoxyethylenebenzylphenyl derivative A method for increasing the reactivity of ApoE containing HDL to an enzyme so that cholesterol in all HDL including ApoE containing HDL can be accurately quantified.   The cholesterol oxidase is present in either the first reagent and / or the second reagent, so that the cholesterol in the total HDL containing ApoE containing HDL can be accurately quantified. A method of increasing reactivity to enzymes.

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