KR19980086568A - Determination of LDL-Cholesterol - Google Patents

Abstract

Cholesterol in low density lipoproteins can be specifically and accurately measured using an autoanalyzer directly in the presence of one or more components and amphoteric surfactants selected from cyclodextrins and derivatives thereof.

Description

Measurement of LDL-cholesterol

The present invention relates to a method for measuring cholesterol in low density lipoprotein (hereinafter referred to as LDL) present in biological samples such as serum and plasma.

The major components of serum lipids are cholesterol, triglycerides, phospholipids, and the like. These serum lipids bind to apoproteins and form circulating lipoproteins in the blood. Lipoproteins can be classified into high density lipoproteins (HDL), low density lipoproteins (LDL), ultra low density lipoproteins (VLDL), and fine particles (chylomicron, CM) according to the difference in density. Among these lipoproteins, HDL has a role of transporting excess cholesterol precipitated on the tissue to the liver, thereby inhibiting atherosclerosis. In contrast, LDL is the main carrier of cholesterol from the liver to each tissue. The increase in LDL seems to be closely associated with the development of atherosclerosis.

Thus, cholesterol in LDL (hereinafter referred to as LDL-cholesterol) is considered a risk factor for atherosclerosis and ischemic heart disease (coronary atherosclerosis). Therefore, the content of LDL-cholesterol is an important indication of the diagnosis, treatment and prevention of such diseases.

As a measuring method of LDL-cholesterol, precipitation, ultracentrifugation, electrophoresis and Friedewald method are known. Among these methods, sedimentation, ultracentrifugation and electrophoresis are pretreatment steps such as the separation of unnecessary lipoproteins except LDL from LDL by sedimentation / supercentrifugation, ultracentrifugation or electrophoresis. This leads to complicated procedures. Therefore, these methods have the disadvantage that direct measurement using only the automatic analyzer which is widely used in the clinical trial field is impossible.

In contrast, the Friedwald method, known as the Friedwald equation, in which total cholesterol, HDL-cholesterol and triglyceride values are used for calculations, is an accurate amount of LDL-cholesterol when using a sample containing at least 500 mg / dl of triglycerides. There is a problem that cannot be measured.

In order to solve the above problem, various methods have been developed. For example, JP-A-280812 uses coagulants and / or antibodies to coagulate LDL and introduce it into another reaction system that does not belong to a quantitative reaction to remove (consume and consume) cholesterol contained in other lipoproteins other than LDL. ), A method in which the coagulated LDL is dissolved to the extent that a quantitative reaction using a surfactant and / or an inorganic salt can be carried out, and the absorbance of the solution is measured by carrying out a quantitative reaction of LDL-cholesterol.

However, since the method uses a 3-reagent system or a 4-reagent system in the measurement, it can be applied only to a few autoanalyzers that can use such a multi-reagent system. Many autoanalyzers commonly used in clinical trials cannot be used because they can only be used in two-reagent methods. In addition, the method has a problem that the reproducibility of the measured value is lowered because many reagents are used.

To measure LDL-cholesterol without complicated pretreatment, the method disclosed in JP-A-165800 can be used. According to this method, however, the preparation of the reagents is complicated because there are limitations on the usable concentrations of cholesterol esterases and surfactants in the reagents, for example. In addition, the measurement conditions, such as pH and measurement interval, must be set strictly. In addition, since cholesterol in HDL reacts to some extent, LDL-cholesterol can only be measured by kinetic measurements, ie rate assays. Therefore, this method can be called a measurement method.

On the other hand, WO 96/29599 discloses a method for measuring cholesterol in LDL or VLDL in the presence of sugar compounds and / or nonionic or anionic surfactants, but this method is less accurate.

[Summary of invention]

An object of the present invention is to provide a method for directly measuring LDL-cholesterol in a biological sample using an automatic analyzer or the like, without preliminary treatment of separating LDL from unnecessary lipoproteins except LDL, which is essential in the above known methods.

The present invention relates to a method for measuring cholesterol in a low density lipoprotein present in a live sample by optically measuring the reaction product of the live sample with a reagent.

The method is characterized in that the living sample is reacted with cholesterol oxidase or cholesterol dehydrogenase in the presence of at least one component and amphoteric surfactant selected from the group consisting of cyclodextrins and derivatives thereof.

The present invention also treats a live sample with a first reagent containing at least one component selected from the group consisting of cyclodextrins and derivatives thereof, measures the absorbance or permeability of the resulting solution, and the obtained solution is subjected to cholesterol oxidase. Treatment with a second reagent solution containing, measuring another absorbance or permeability of the final solution obtained, and defining cholesterol levels in the living sample based on the absorbance or permeability data measured above. A method for measuring cholesterol in low density lipoproteins, wherein a coupler, developer, peroxidase, amphoteric surfactant, and cholesterol esterase are contained in one or more of the first or second reagents.

The present invention also relates to a method of determining cholesterol levels in low density lipoproteins present in living samples using cholesterol dehydrogenase instead of cholesterol oxidase.

In another aspect, the present invention, the living sample is reacted with cholesterol oxidase or cholesterol dehydrogenase in the presence of at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, and the cholesterol level is optically measured. It relates to a method for measuring cholesterol in a living sample.

In addition, the present invention provides a reagent used in the above method.

[Description of Preferred Embodiment]

In order to determine the amount of cholesterol in low density lipoproteins in living samples such as serum and plasma without pretreatment to separate LDL-cholesterol from unnecessary lipoproteins, except for LDL, the present inventors have used amphoteric surfactants and cyclodextrins and / or their When measuring LDL-cholesterol in a live sample in the presence of a derivative or in the presence of at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, the amount of cholesterol in the LDL is known in It was found that an automatic analyzer was obtained directly and accurately without treatment, and the present invention was completed.

In accordance with the present invention, LDL-cholesterol in live samples can be measured accurately using, for example, an autoanalyzer in the presence of amphoteric surfactants and cyclodextrins and / or derivatives thereof.

One method of determining the LDL-cholesterol level in a live sample according to the present invention is to provide a live sample with cholesterol oxidase or cholesterol dehydrogena in the presence of at least one component and amphoteric surfactant selected from the group consisting of cyclodextrins and derivatives thereof. Reacting with the agent and optically measuring the amount of cholesterol, such as measuring the optical change in a conventional manner.

Another method of measuring the LDL-cholesterol value in a live sample according to the present invention is to treat the live sample with a first reagent containing at least one component selected from the group consisting of cyclodextrin and its derivatives, and to obtain optical Change, such as absorbance or transmittance, are treated with a second reagent containing cholesterol oxidase (hereinafter referred to as COD), and another optical change, such as absorbance or transmittance, of the final solution obtained, A method of defining cholesterol values in a living sample based on the optical change data measured above, wherein at least one of the first reagent or the second reagent is used as a coupler, developer, peroxidase, amphoteric surfactant, and Contains cholesterol esterases.

A third method for measuring the LDL-cholesterol value in a live sample according to the present invention is to treat a live sample with a first reagent containing at least one component selected from the group consisting of cyclodextrin and derivatives thereof, Optical changes such as absorbance, transmittance, etc. are measured and the resulting solution is treated with a second reagent containing cholesterol dehydrogenase (hereinafter referred to as CHD) and another optical change of the final solution obtained such as absorbance or A method comprising measuring the transmittance and defining cholesterol values in a living sample based on the data of the optical change measured above, wherein at least one of the first reagent or the second reagent is bound to nicotinamide adenine dinucleotide (phosphate). ), Amphoteric surfactants and cholesterol esterases There is oil.

The reagent composition for measuring LDL-cholesterol according to the present invention contains cholesterol oxidase or cholesterol dehydrogenase, amphoteric surfactants and cyclodextrins and / or derivatives thereof.

Another reagent composition for measuring LDL-cholesterol according to the present invention contains:

(a) a first reagent containing cyclodextrin and / or a derivative thereof,

(b) a second reagent containing COD and

(c) Couplers, developers, peroxidases (hereinafter referred to as PODs), amphoteric surfactants and cholesterol esterases (hereinafter referred to as CHEs) contained in one or more of the first or second reagents.

The third reagent composition for measuring LDL-cholesterol according to the present invention contains:

(a) a first reagent containing cyclodextrin and / or a derivative thereof, an amphoteric surfactant and CHE,

(b) a second reagent containing COD and CHE and

(c) Couplers, developers, and PODs contained in one or more of the first or second reagents.

The reagent according to the invention comprises a first container comprising a cyclodextrin and / or a derivative thereof, an amphoteric surfactant, CHE and a first reagent containing a coupler (or developer) and a COD, CHE, POD and developer (or coupler) It can be used in the form of a kit consisting of a second container containing a second reagent containing).

When using at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin as derivatives of the cyclodextrin, the use of amphoteric surfactants can be omitted.

In this case, the method of the present invention specifically reacts the living sample with cholesterol oxidase or cholesterol dehydrogenase in the presence of at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. Optically measuring cholesterol levels, such as measuring optical changes in a conventional manner.

Another method of measuring the LDL-cholesterol value in a live sample according to the present invention is to provide a live sample with a first reagent containing at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. Treatment, measuring optical changes, such as absorbance or transmittance of the obtained solution, treating the obtained solution with a second reagent containing cholesterol oxidase, and measuring another optical change, such as absorbance or transmittance of the final solution obtained. Wherein the cholesterol value in the living sample is defined on the basis of the optical change data measured above, wherein at least one of the first reagent or the second reagent contains a coupler, a developer, a peroxidase and a cholesterol esterase. It is.

A third method of measuring the LDL-cholesterol value in a live sample according to the present invention is a first reagent containing at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin in the live sample. , The optical change of the obtained solution, such as absorbance or permeability, the treated solution with a second reagent containing cholesterol dehydrogenase, and another optical change of the final solution obtained, such as absorbance Or measuring the transmittance and defining cholesterol values in the living sample based on the data of the optical change measured above, wherein at least one of the first or second reagent is nicotinamide adenine dinucleotide (phosphate) , And cholesterol esterases.

The reagents according to the invention contain cholesterol oxidase or cholesterol dehydrogenase and at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin.

Another reagent composition according to the present invention contains:

(a) a first reagent containing at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin,

(b) a second reagent containing COD and

(c) Couplers, developers, PODs and CHEs contained in one or more of the first or second reagents.

The third reagent composition according to the present invention contains:

(a) a first reagent containing CHE and at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin,

(b) a second reagent containing COD and

(c) Couplers, developers, and PODs contained in one or more of the first or second reagents.

The reagent according to the present invention comprises a first container comprising a first reagent containing at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, CHE and a coupler (or developer) And a second container comprising a second reagent containing COD, CHE, POD, and a coupler (or developer).

As the amphoteric surfactant, any can be used as long as it can prevent cholesterol contained in lipoproteins other than LDL in the presence of cyclodextrin and / or its derivatives from being involved in the cholesterol measurement reaction. Examples of such amphoteric surfactants include betaine derivatives such as alkylbetaines such as lauryl betaine, stearyl betaine, lauryldimethylammonium betaine, coconut betaine, coconut oil fatty acid amidopropyl betaine, And lauric acid amidopropyl betaine, imidazolinium betaine derivatives such as lauryl carboxymethyl hydroxyethyl imidazolinium betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazoli Nium betaine, 2-alkyl-N-carboxyethyl-N-hydroxyethyl imidazolinium betaine, and 2-undecyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, sulfobe Tine derivatives such as N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, N-dodecyl-N , N-dimethyl-3-ammonio-1-propanesulfonic acid, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, N-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, aminocarboxylic acid derivatives such as alkylglycine, alkyldi (aminoethyl) glycine, dioctylpolyaminoethyl glycine, N-alkylpoly Amino-ethyl glycine and β-alanine derivatives, imidazoline derivatives such as bis (2-undecyl-N-hydroxyethylimidazoline) chloroacetic acid complexes and alkylimidazoline derivatives, amine oxide derivatives such as lauryl Dimethylamine oxide.

The amphoteric surfactants can be used alone or in mixtures thereof. Such amphoteric surfactants may be commercially available or may be synthesized by conventional methods.

The concentration of amphoteric surfactants in the presence of cyclodextrins is not particularly limited so long as the amphoteric surfactants can prevent cholesterol contained in lipoproteins other than LDL from being involved in cholesterol measurement reactions. none. The preferred concentration of amphoteric surfactant in the final (reaction) solution when measuring cholesterol is 0.0001-10% (w / v), more preferably 0.001-1% (w / v).

When implementing the two-reagent method to measure LDL-cholesterol measurements, the concentration of amphoteric surfactant in each reagent varies with the amount of sample, first reagent, and second reagent. When the amphoteric surfactant is contained only in the first reagent, the concentration of the amphoteric surfactant is preferably selected in the range of 0.0002 to 20% (w / v), more preferably 0.002 to 2% (w / v). ) Is selected from the range. When the amphoteric surfactant is contained only in the second reagent, the concentration of the amphoteric surfactant is preferably selected in the range of 0.0002 to 20% (w / v), more preferably 0.002 to 2% (w / v). Is selected from the range of. In addition, when the amphoteric surfactant is contained in both the first reagent and the second reagent, the concentration of the amphoteric surfactant is preferably 0.0001 to 20% (w / v) in the first reagent, more preferably 0.001 To 2% (w / v), preferably 0.0001 to 20% (w / v) in the second reagent, and more preferably 0.001 to 20% (w / v).

As cyclodextrins and / or derivatives thereof, any may be used as long as they can prevent cholesterol contained in lipoproteins except LDL from being involved in the cholesterol measurement reaction in the presence of an amphoteric surfactant.

Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and the like.

Examples of cyclodextrin derivatives include alkylated cyclodextrins such as 2,6-di-O-methyl-α-cyclodextrin, 2,3,6-tri-O-methyl-α-cyclodextrin, 2,6-di-O -Ethyl-α-cyclodextrin, 2,3,6-tri-O-ethyl-α-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, 2,3,6-tri-O- Methyl-β-cyclodextrin, 2,6-di-O-ethyl-β-cyclodextrin, 2,3,6-tri-O-ethyl-β-cyclodextrin, 2,6-di-O-methyl-γ -Cyclodextrin, 2,3,6-tri-O-methyl-γ-cyclodextrin, 2,6-di-O-ethyl-γ-cyclodextrin and 2,3,6-tri-O-ethyl-γ- Cyclodextrins, hydroxyalkylated cyclodextrins such as 2-hydroxyethyl-α-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 3-hydroxypropyl-α-cyclodextrin, 2,3-dihydroxy Propyl-α-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxy Cypropyl-β-cyclodextrin, 2,3-dihydroxypropyl-β-cyclodextrin, 2-hydroxyethyl-γ-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, 3-hydroxypropyl- γ-cyclodextrin and 2,3-dihydroxypropyl-γ-cyclodextrin, acylated cyclodextrins such as 2,3,6-tri-O-acetyl-α-cyclodextrin, 2,3,6-tri- O-acetyl-β-cyclodextrin and 2,3,6-tri-O-acetyl-γ-cyclodextrin, sugar-modified cyclodextrins such as 6-O-α-D-glucosyl-α-cyclodextrin, 6 -O-α-maltosyl-α-cyclodextrin, 6-O-α-D-glucosyl-β-cyclodextrin, 6-O-α-maltosyl-β-cyclodextrin, 6-O-α-D -Glucosyl- [gamma] -cyclodextrin and 6-O- [alpha] -maltosyl- [gamma] -cyclodextrin, carboxyalkylated cyclodextrins such as O-carboxymethyl- [alpha] -cyclodextrin, O-carboxymethyl- [beta] -cyclodextrin and O -Carboxymethyl-γ-shi As dextrin, cyclodextrin polymers, such as poly -α- cyclodextrin, poly -β- cyclodextrin and poly have -γ- cyclodextrin. As said cyclodextrin and its derivative (s), a commercially available thing can be used. See also US Pat. Nos. 3,453,258, 3,453,259, Polymer Journal, 13, 8, pp. 777-781, 1981, JP-A 61-266401, JP-A 63-122701 and JP- Synthesis by the known method disclosed in A 62-243602 can also be used in the present invention.

The concentration of cyclodextrin and / or derivatives thereof described above is not particularly limited so long as the water can prevent cholesterol contained in lipoproteins other than LDL from being involved in the cholesterol measurement reaction. The preferred concentration of cyclodextrin and / or its derivatives in the final (reaction) solution when measuring cholesterol is 0.0001-10% (w / v), more preferably 0.001-1% (w / v).

In addition, when implementing the two-reagent method to measure LDL-cholesterol, the concentration of cyclodextrin and its derivatives in the first reagent varies with the amount of sample, first reagent, and second reagent. Usually the concentration is in the range of 0.0002 to 20% (w / v), preferably in the range of 0.002 to 2% (w / v).

Such cyclodextrins and derivatives thereof can be used alone or in mixtures thereof.

According to the measuring method of the present invention, in addition to the amphoteric surfactant and cyclodextrin and derivatives thereof, conventional reagents used for measuring cholesterol can also be used.

That is, LDL-cholesterol in live samples such as serum, plasma and the like can be measured specifically by conventional methods in the presence of cyclodextrins and / or derivatives thereof, such as using enzymatic reactions.

The conventional method described above (oxidative color producing process) decomposes cholesterol esters in a sample with CHE to obtain free cholesterol and fatty acids, while converting the product into COD together with the existing free cholesterol. Oxidation yields choles-4-en-3-one and hydrogen peroxide, conducts an oxidative color reaction of hydrogen peroxide and oxidizing colorant produced in the presence of POD, and colorimetric determination of the resulting oxidative dye. It consists of

Another conventional method (ultraviolet ray measuring method) is to decompose the cholesterol ester in the sample with CHE to obtain free cholesterol and fatty acid, and the product is nicotinamide adenine in the presence of free cholesterol and CHD which existed from the beginning. Reaction with dinucleotide or its phosphate (hereinafter referred to as NAD (P)) and the resulting NAD (P) H measured by ultraviolet light of 340 nm.

There is no particular limitation on the source of COD used in the present invention. Those conventionally used in the art can be used, for example, those derived from animal organs such as microorganisms such as Nocardia Genera , Pseudomonas Genera , and pancreas of cattle. The amount of COD in the final reaction solution for measuring cholesterol is preferably from 0.02 to 10 u / ml, more preferably from 0.1 to 2 u / ml.

There is no particular limitation on the source of CHE used in the present invention. Those commonly used in the art may be used, for example, those derived from animal organs such as microorganisms such as Candida Genera , Pseudomonas Genera , and pancreas of cattle. The amount of CHE used in the final reaction solution in measuring cholesterol is preferably from 0.02 to 10 u / ml, more preferably from 0.1 to 2 u / ml.

There is no particular limitation on the source of POD used in the present invention. Those conventionally used in the art can be used, for example, horseradish, plants such as radish, fungi, microorganisms such as yeast, and those derived from animal white blood cells and thyroid gland. The amount of POD used in the final reaction solution in measuring cholesterol is preferably 0.01 to 50 u / ml, more preferably 0.1 to 5 u / ml.

As the oxidizing color developer used in the present invention, those which can develop color when reacted with hydrogen peroxide in the presence of POD can be used. Examples thereof include a combination of a developer capable of producing a dye by oxidative condensation with a coupler and a coupler such as 4-aminoantipyrine (4-AA), such as a phenolic compound, a naphthol compound, or an aniline compound with 4-AA. Colorants which can be colored by themselves, such as combinations, combinations of aniline compounds and 3-methyl-2-benzothiazolinone hydrozone, such as 2,2'-azinobis (3-ethylbenzothiazoline -6-sulfonic acid), triphenylmethane leuco dyes, diphenylamine derivatives, benzidine derivatives, triallylimidazole derivatives, leucomethylene blue derivatives and o-phenylenediamine derivatives.

As a developing solution, phenolic compounds such as phenol, p-chlorophenol and 2,4-dichlorophenol, naphthol compounds such as 1-naphthol, 1-naphthol-2-sulfonic acid, and 1-naphthol-2-car Acids, aniline compounds such as N, N-diethylaniline, N-ethyl-N- (β-hydroxyethyl) -m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N- (2 -Hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) and N-ethyl- N- (3-methylphenyl) -N'-succinyl-ethylenediamine (EMSE).

The amount of coupler used when used in combination with a developer depends on the type of coupler and the type of developer to be combined. The concentration of the coupler in the final (reaction) solution when measuring cholesterol is preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM. When 4-AA is used as the coupler, the concentration of 4-AA in the final reaction solution when measuring cholesterol is preferably 0.01 to 50 mM, more preferably 0.1 to 5 mM.

The amount of developer used depends on the type of developer and the type of coupler to be combined. The concentration of the developer in the final reaction solution when measuring cholesterol is preferably 0.01 to 50 mM, more preferably 0.1 to 5 mM.

Examples of the triphenylmethane leuco dye include leuco malachite green, bis (p-diethylaminophenyl) -2-sulfophenylmethane, bis (p-diethylaminophenyl) -3,4-disulfopropoxyphenylmethane di Sodium salts and the like.

Examples of diphenylamine derivatives include bis [4-di (2-butoxyethyl) amino-2-methylphenyl] amine, N, N-bis (4-diethylamino-2-methylphenyl) -N'-p-toluene Sulfonyl urea and the like.

Examples of leucomethylene blue derivatives include 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium salt, and the like.

Examples of benzidine derivatives include benzidine, o-toluidine, o-diazinidine, 3,3'-diamino-benzidine, 3,3 ', 5,5'-tetraaminobenzidine and the like.

Examples of triallylimidazole derivatives include 2- (4-carboxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole, and the like.

The amount of such colorant to be used is appropriately selected from a range commonly used in the art.

There is no particular limitation on the source of CHD used in the present invention. Commonly used ones such as those derived from Nocardia Genera and the like can be used. The amount used in the final reaction solution for the determination of cholesterol is preferably 0.1 to 100 u / ml, more preferably 1 to 50 u / ml.

There is no particular limitation on the NAD (P) used in the present invention, and those commonly used in the art may be used. The amount of NAD (P) used in the final reaction solution for cholesterol measurement is preferably 0.02 to 50 mM, more preferably 0.1 to 10 mM.

The reagent for measuring LDL-cholesterol used in the present invention is used to measure LDL-cholesterol in a sample derived from a living body such as serum or plasma, except that an amphoteric surfactant and cyclodextrin and / or a derivative thereof are used. It is commonly used. Commonly used reagents include CHE, CHD, NAD (P), etc., which are used in violet light measurement methods, such as COD, CHE, POD, and oxidizing colorants used in the oxidative color development method. These reagents are used at concentrations suitable for measuring LDL-cholesterol.

The reagent for measuring LDL-cholesterol used in the present invention can be prepared by the 1-reagent method, the 2-reagent method, or the 3- or more reagent method. When the reagent is separated into two or more reagents, the following compositions are preferred in consideration of specificity, accuracy of measurement, and the like.

For the oxidative color development method, the first reagent contains cyclodextrin and / or its derivatives, and the second reagent contains COD. At least one of the first reagent or the second reagent may contain other reagents such as enzymes such as CHE, POD, amphoteric surfactants, couplers and developer.

In the case of the ultraviolet measurement method, the first reagent contains cyclodextrin and / or its derivatives, the second reagent contains CHD, and at least one of the first reagent or the second reagent contains another reagent such as NAD (P). It may be contained.

When the method of the present invention is carried out in a two-reagent method, the sample is treated using a first reagent containing cyclodextrin and / or a derivative thereof, such as by mixing the sample with the first reagent, and treating the treated sample. React with a second reagent containing COD or CHD in the presence of cyclodextrin and / or its derivatives and amphoteric surfactants. This step is preferred in view of specificity, measurement accuracy and the like.

For the oxidative color development method, the following reagent compositions are preferably used:

(Iii) a coupler, developer, POD contained in at least one of a first reagent containing cyclodextrin and / or a derivative thereof and an amphoteric surfactant, a second reagent containing COD and a first reagent or second reagent, and CHE,

(Ii) a coupler, a developer, a POD and an amphoteric interface contained in at least one of the cyclodextrin and / or its derivatives and the first reagent containing CHE, the second reagent containing COD and the first or second reagent. Activator,

(Iii) a cyclodextrin and / or a derivative thereof, a first reagent containing an amphoteric surfactant and CHE, a second reagent containing COD and a coupler, a developer contained in at least one of the first reagent or the second reagent, and POD.

For the ultraviolet measurement method, the following reagent composition is preferably used:

(Iii ′) NAD (P) contained in at least one of a first reagent containing cyclodextrin and / or a derivative thereof and an amphoteric surfactant, a second reagent containing CHD and a first reagent or second reagent, and CHE,

(Ii ') NAD (P) and an amphoteric interface contained in at least one of cyclodextrin and / or derivatives thereof and a first reagent containing CHE, a second reagent containing CHD and a first or second reagent. Activator,

(Iii ′) NAD contained in at least one of a cyclodextrin and / or a derivative thereof, a first reagent containing amphoteric surfactant and CHE, a second reagent containing CHD and a first reagent or second reagent (P ).

When the reagent for measuring LDL-cholesterol according to the present invention is prepared for the 2-reagent method, the amphoteric surfactant and / or CHE is cyclodextrin and / or to improve the specificity of LDL-cholesterol and the measurement accuracy of LDL-cholesterol. Or coexist in the first reagent with a derivative thereof.

Moreover, in the said reagent composition, when an amphoteric surfactant coexists with CHE, there exists a possibility that the stability of CHE activity by an amphoteric surfactant may fall. Therefore, in this case, it is preferable to include CHE in both the first reagent and the second reagent. For reagents for the oxidative color development method, the first reagent contains cyclodextrin and / or its derivatives, amphoteric surfactants and CHE, the second reagent contains COD and CHE, the first reagent or the second reagent At least one contains a coupler, a developer, and a POD. In the case of reagents for UV measurement methods, the first reagent contains cyclodextrin and / or its derivatives, amphoteric surfactants and CHE, the second reagent contains CHD and CHE, the first reagent or the second reagent At least one contains NAD (P). In addition, in the reagent combination for the oxidation-coloring method, it is preferable that one of the coupler and the developer is contained in the first reagent and the other is contained in the first reagent.

The reagent for measuring LDL-cholesterol according to the present invention may be contained in one or more buffers. The buffer used in the present invention may vary depending on the combination of various enzymes and oxidizing agents, but conventional buffers frequently used in the art may be used. Preference is given to using buffers having a buffering effect in the range of pH 5 to 11. The buffer may preferably be used at a concentration of 1 mM to 5 M, more preferably 5 mM to 1 M.

Given the specificity for LDL-cholesterol, examples of preferred buffers include aminoethanesulfonic acid derivatives such as N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxy Ethyl) -2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) , 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) and N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), amino Propanesulfonic acid derivatives such as N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 3- [N, N-bis (2-hydride) Hydroxyethyl) amino] -2-hydroxy-propanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), 2-hydroxy-3- [ 4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), 3-morpholi Nopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris ( Aliphatic having sulfonic acid groups or carboxyl groups such as hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) and 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), glycine derivatives Amines such as N- (2-acetamido) iminodiacetic acid (ADA), N, N-bis (2-hydroxyethyl) glycine (bisine), N- [tris (hydroxymethyl) methyl] glycine (tri God).

The reagent for measuring LDL-cholesterol according to the present invention may contain one or more ionic compounds such as polyanions such as dextran sulfate, heparin, heparin sulfate, tungsten phosphate, alone or as a mixture thereof. . In addition, divalent cations such as Mg ²⁺ , Mn ²⁺ and Ca ²⁺ (or one or more metal salts capable of producing the divalent cations) can be used in combination with the above ionic compounds. There is no particular limitation on the concentration of such additives. The concentration of the ionic compound in the final (reaction) solution is preferably 0.01 to 10% (w / v). The concentration of divalent cations in the final (reaction) solution is preferably 0.1 to 200 mM.

The reagent for measuring LDL-cholesterol according to the present invention may contain one or more polyclonal or monoclonal antibodies to prevent cholesterol in lipoproteins related to the cholesterol measurement response except LDL. have.

Examples of such antibodies include anti-apolipoprotein A antibodies, anti-apolipoprotein C antibodies, anti-apolipoprotein E antibodies, anti-a-lipoprotein antibodies, and the like.

If the antibody can prevent cholesterol contained in lipoproteins other than LDL from being involved in the cholesterol measurement response, the concentration is not particularly limited. The antibody is preferably added at a concentration of 0.001 to 10 mgAb / ml, more preferably 0.01 to 1 mgAb / ml in the final (reaction) solution.

The reagent for measuring LDL-cholesterol according to the present invention may also contain one or more surfactants other than amphoteric surfactants commonly used in the art, and examples thereof include polyoxyethylene isooctylphenyl ether and polyoxy. Nonionic surfactants such as ethylene alkylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyethyl glycol monolaurate and polyethylene lauryl ether, stearyl trimethylammonium chloride and alkyl Cationic surfactants such as benzyldimethylammonium chloride, anionic surfactants such as cholic acid, deoxycholic acid and polyoxyethylene alkylphenyl ether sodium sulfonate. The surfactants can be used alone or in mixtures thereof. There is no particular restriction on the concentration of such surfactants, but it is preferred to range from 0.0001 to 10% (w / v), more preferably 0.001 to 1% (w / v) in the final (reaction) solution.

When the LDL-cholesterol assay according to the present invention is a two-reagent method, it can be measured as follows:

1) Oxidation Coloration Method

Live samples such as serum, plasma, etc. may be prepared by, for example, a first reagent containing amphoteric surfactants, cyclodextrins and / or derivatives thereof, CHEs and couplers (or developer), and, if desired, one or more buffers, antibodies, ionic compounds. The sample is treated by mixing with a divalent cation, and then reacted at 2 to 40 ° C. for 1 to 30 minutes to measure absorbance (OD ₁ ). The reaction solution is then mixed with a second reagent containing, for example, COD, CHE, POD, and developer (or coupler), and, if necessary, mixed with one or more buffers and the like, and then treated the sample at 1 to 60 ° C. at 2 to 40 ° C. The reaction is carried out for a minute to measure the absorbance (OD ₂ ). The absorbance (OD ₃ ) is obtained by subtracting the value derived from OD ₁ (eg, OD ₁ multiplied by a correction constant) from OD ₂ . And applying the obtained OD _3, the calibration curve using standard solution or the like having a LDL- cholesterol and cholesterol content of known showing the relationship between the OD ₃ obtained in the same manner as described above.

2) UV measurement method

Live samples such as serum, plasma, etc. are mixed, for example, with a first reagent containing amphoteric surfactants, cyclodextrins and / or derivatives thereof, and CHE, and, if necessary, one or more buffers, antibodies, ionic compounds, divalent cations and the like. The sample is treated, and then reacted at 2 to 40 ° C. for 1 to 30 minutes to measure absorbance (OD ₁ ′). The reaction solution is then mixed with a second reagent containing, for example, CHD, CHE, and NAD (P), and, if necessary, mixed with one or more buffers and the like, and then the sample is reacted at 2 to 40 ° C. for 1 to 60 minutes. Absorbance (OD ₂ ′) is measured at 240 nm. The absorbance (OD ₃ ′) is obtained by subtracting the value derived from OD ₁ ′ (eg OD ₁ ′ multiplied by a correction constant) from OD ₂ ′. 'A, by using the standard solution or the like having a LDL- cholesterol and cholesterol content of the known ₃ OD obtained in the same manner as described above, the obtained OD ₃ is applied to a calibration curve showing the relationship between. In this way, LDL-cholesterol values in living samples can be obtained.

When the LDL-cholesterol assay according to the present invention is a 1-reagent method, it can be measured as follows:

1) Oxidation Coloration Method

Live samples such as serum, plasma, etc. may be prepared by, for example, CHE, COD, POD, oxidizing colorants, amphoteric surfactants and cyclodextrins and / or derivatives thereof and, if necessary, one or more buffers, antibodies, ionic compounds, divalent cations, The mixture is then reacted at 2 to 40 ° C. for 1 to 30 minutes to measure absorbance (OD _S ). A blank value (OD _B1 ) is obtained by using the same reagent as above and performing the same method as above except that physiological saline is used instead of the live sample. The absorbance (OD _R ) is obtained by subtracting OD _B1 from OD _S. Apply the resulting OD _R, a calibration curve using a standard solution, and the like having a LDL- cholesterol and cholesterol content of known showing the relationship between the OD _R obtained in the same manner as described above. In this way, the cholesterol value in a biological sample can be obtained.

2) UV measurement method

Live samples such as serum, plasma, etc. may be prepared by, for example, CHE, CHD, NAD (P), amphoteric surfactants and cyclodextrins and / or derivatives thereof, and, if desired, one or more buffers, antibodies, ionic compounds, divalent cations, etc. The mixture is then reacted at 2 to 40 ° C. for 1 to 30 minutes to measure absorbance (OD _S ′) at 340 nm. A blank value (OD _B1 ′) is obtained by using the same reagents as above and performing the same method as above except that physiological saline is used instead of the live sample. Absorbance (OD _R ') is obtained by subtracting OD _B1 ' from OD _S '. 'A, by using the standard solution or the like having a LDL- cholesterol levels and cholesterol content of known OD _R obtained in the same manner as described above, the resultant OD _R is applied to a calibration curve showing the relationship between. In this way, LDL-cholesterol values in living samples can be obtained.

In this case, other optical changes, such as transmittance, may be used instead of absorbance as the optical change.

The kit for measuring LDL-cholesterol according to the present invention is used for LDL-cholesterol in living samples such as serum and plasma, which has the following configuration:

1) Oxidation color development method

A first container comprising a cyclodextrin and a derivative thereof, an amphoteric surfactant, CHE, and a first reagent containing a coupler (or developer), and

A second container comprising a second reagent containing COD, CHE, POD and developer (or coupler).

2) UV measurement method

A second container comprising a first container comprising cyclodextrin and derivatives thereof, an amphoteric surfactant, and a first reagent containing CHE and a second reagent containing CHD, NAD (P), and CHE.

Examples of preferred embodiments and individual components are as described above. It goes without saying that the kit can be combined with LDL-cholesterol standard.

The method for measuring LDL-cholesterol according to the present invention is carried out in the presence of an amphoteric surfactant and cyclodextrin and / or derivatives thereof, or at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. In the presence of a lipoprotein excluding LDL, such as HDL, VLDL, CM, etc., cholesterol can react specifically and the LDL-cholesterol can react specifically, thus making endpoint analysis impossible, which is not possible with conventional methods. LDL-cholesterol can be measured by an end point assay. In addition, there is no need to use pure LDL-cholesterol for standard materials. Instead, a standard solution containing standard serum or cholesterol with the same viscosity and specific gravity as normal serum can be used.

The invention will be illustrated by the following examples, but is not limited thereto.

Example

Example 1

Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi), cholesterol in various lipoproteins fractionated by ultracentrifugation according to the method of the present invention for studying the reactivity of cholesterol is measured.

[sample]

As the sample, LDL fraction, HDL fraction, VLDL fraction and CM fraction obtained from serum by fractionation using a known ultracentrifugation method are used.

Cholesterol in each sample is measured in advance according to the standard method described in the kit guide using a commercially available total cholesterol measurement reagent kit (L-type Wako CHO.H, trade name, available from Wako Pure Chemical Industries).

[reagent]

(Reagent 1)

R-1 : 50 mM piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) -NaOH buffer (pH 7.0) containing:

(Iii) 2,6-di-O-methyl-α-cyclodextrin (manufactured by Wako Pure Chemical Industries, hereinafter DM-α-CD) 0.06% (w / v),

(Ii) N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline Na salt (HDAOS, manufactured by Dojindo Laboratories) 0.6 mM, and

Na ₂ SO ₄ 0.4 M

R-2 : 50 mM PIPES-NaOH buffer (pH 7.0) containing:

(Iii) COD (manufactured by Amano Pharmaceutical) 4 u / ml,

(Ii) CHE (manufactured by Asahi Kasei Kogyo K.K.) 4 u / ml,

(Iii) POD (manufactured by Toyobo) 6 u / ml, 4-AA 3 mM, and

(Iii) 0.01% (w / v) of the amphoteric surfactant shown in Table 1.

(Amphoteric Surfactant)

Use the following:

Lipomin LA (trademark, manufactured by Lion),

Aminocarboxylic acid derivatives

Sotazoline LPB-R (trade name, manufactured by Kawa-Ken Fine Chemical),

Lauric Acid Amidopropyl Betaine

Sotazoline CL (trade name, manufactured by Kawa-Ken Fine Chemical),

2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine

Anhitol 24B (trade name, manufactured by Kao Corporation),

Lauryl betaine

Enadicol L-30AN (trade name, product made in Lion),

Sodium N-lauroyl-N-methyl-β-alanine

Zwittergent 3-08 (trade name, manufactured by Wako Pure Chemical Industries),

N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid

(Reagent 2)

R-1 : same as reagent 1

R-2 : Same as Reagent 1 except for using the following surfactant instead of the amphoteric surfactant

(Surfactants)

Emalgen A-90 (trade name, manufactured by Kao Corporation),

Polyoxyethylene derivatives

Emalgen 709 (trade name, manufactured by Kao Corporation),

Polyoxyethylene higher alcohol ether

Emalgen X-100 (trade name, manufactured by Rhom Haas),

Polyoxyethylene alkylphenyl ether

(Reagent 3)

R-1 : 50 mM PIPES-NaOH buffer, pH 7.0, containing 0.6 mM HDAOS and 0.4 M Na ₂ SO ₄ .

R-2 : 50 mM PIPES-NaOH buffer (pH 7.0) containing:

(Iii) COD (manufactured by Amano Pharmaceutical) 4 u / ml,

(Ii) CHE (manufactured by Asahi Kasei Kogyo K.K.) 4 u / ml,

(Iii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) 4-AA 3 mM.

[Measuring conditions]

In order to measure cholesterol in each sample, measurement parameters are set as follows.

Measurement method 2 Point end [16]-[34]

Sample volume 3 μl

R-1 270 μl

R-2 90 μl

700/600 nm wavelength

Measuring temperature 37 ℃

Standard substance concentration 100 mg / dl

[result]

The conversion (%) of each lipoprotein can be obtained by substituting the cholesterol values obtained from each sample and the cholesterol values in each sample using commercially available kits:

% Conversion = (cholesterol value obtained from sample /

Cholesterol level obtained using a commercial kit) × 100

The results are shown in Table 1.

Surfactants Degree of conversion of lipoprotein (%) LDL HDL VLDL CM Reagent 1 Lipomin LA Amphoteric 94 4 6 4 Soptazoline LBP-R 〃 101 0 13 5 Soptazoline CL 〃 100 0 0 2 Anhitol 24B 〃 98 0 8 3 Enadicall L-30AN 〃 98 0 11 7 Zwitter 3-08 〃 100 One 9 6 Reagent 2 Emalgen A-90 Nonionic 65 54 51 20 Emalgen 709 〃 86 45 24 25 Triton X-100 〃 93 42 0 19 Reagent 3 No surfactant added DM-α-CD 103 86 99 98

As is clear from the results in Table 1, when using Reagent 1, cholesterol is measured in the presence of amphoteric surfactant and DM-α-CD, so lipoproteins except LDL are less likely to react, thus specificizing cholesterol in LDL. It is possible to measure by means of In contrast, when measured in the presence of a nonionic surfactant with DM-α-CD (Reagent 2) and in the absence of a surfactant with DM-α-CD (Reagent 3), lipoproteins except LDL also react, and thus LDL-cholesterol cannot be measured specifically.

As mentioned above, in order to specifically measure cholesterol in LDL, it is necessary to carry out the measurement in the presence of amphoteric surfactants and cyclodextrins and / or derivatives thereof.

Example 2

Another typical example of a kit for measuring LDL-cholesterol in a live sample is as follows.

(1) first reagent (pH 6-8)

Cyclodextrins and / or derivatives thereof, amphoteric surfactants, CHEs and couplers (or developer)

(2) second reagent (pH 6-8)

COD,

CHE,

POD and developer (or coupler)

Using the kit, the same results as in Example 1 were obtained.

Example 3

Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi), cholesterol in various lipoproteins fractionated by ultracentrifugation according to the method of the present invention for studying the reactivity of cholesterol is measured.

[sample]

As the sample, LDL fraction, HDL fraction, VLDL fraction and CM fraction obtained from serum by fractionation using a known ultracentrifugation method are used.

Cholesterol in each sample is measured in advance according to the standard method described in the kit guide using a commercially available total cholesterol measurement reagent kit (L-type Wako CHO.H, trade name, available from Wako Pure Chemical Industries).

[reagent]

R-1 : 100 mM PIPES-NaOH buffer (pH 7.0) containing:

(Iii) HDAOS 0.6 mM,

(Ii) Na ₂ SO ₄ 0.4 M, and

(Iii) Cyclodextrin derivatives of Table 2.

R-2 : 100 mM PIPES-NaOH buffer (pH 7.0) containing:

(Iii) COD (manufactured by Amano Pharmaceutical) 4 u / ml,

(Ii) CHE (manufactured by Asahi Kasei Kogyo K.K.) 4 u / ml,

(Iii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) 4-AA 3 mM.

[Cyclodextrin derivative]

DM-α-CD (manufactured by Wako Pure Chemical Industries)

HP-α-CD (hydroxypropyl-α-cyclodextrin) (manufactured by Wako Pure Chemical Industries)

Poly-β-CD (poly-β-cyclodextrin) (manufactured by Wako Pure Chemical Industries)

[Measuring conditions]

Measurement parameters for measuring cholesterol in each sample are set as follows.

Measurement method 2 Point end [16]-[34]

Sample volume 3 μl

R-1 270 μl

R-2 90 μl

700/600 nm wavelength

Measuring temperature 37 ℃

Standard substance concentration 100 mg / dl

[result]

The percent conversion of each lipoprotein is obtained in the same manner as described in Example 1.

The results are shown in Table 2.

Cyclodextrin Derivatives (Concentration in R-1) Degree of conversion of lipoprotein (%) LDL HDL VLDL CM No addition 100 58 100 100 DM-α-CD (0.06%) 102 33 3 2 HP-α-CD (0.1%) 96 40 15 21 DM-α-CD (0.1%) poly-β-CD (0.01%) 104 9 0 0

As is clear from the results in Table 2, when cholesterol is measured in the presence of a specific cyclodextrin derivative, the reaction of lipoproteins except LDL is remarkably suppressed. In particular, when DM-α-CD and poly-β-CD are used together, lipoproteins except LDL hardly react. In contrast, when measured in the absence of cyclodextrin derivatives, the response of VLDL and CM is not inhibited at all, thus making it impossible to specifically measure LDL-cholesterol.

As described above, when measuring cholesterol in lipoproteins in the presence of at least one compound selected from the group consisting of DM-α-CD, poly-β-CD, the cholesterol in the LDL can be specifically measured.

Example 4

Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi), cholesterol in various lipoproteins fractionated by ultracentrifugation according to the method of the present invention for studying the reactivity of cholesterol is measured.

[sample]

Same as Example 3.

[reagent]

R-1 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) 4-AA 1.2 mM,

(Ii) CHE (manufactured by Asahi Kasei Kogyo K.K.) 1 u / ml, and

(Iii) cyclodextrin derivatives of Table 3.

R-2 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) COD (manufactured by Amano Pharmaceutical) 4 u / ml,

(Ii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) HDAOS 1.2 mM.

[Cyclodextrin derivative]

Poly-β-CD (manufactured by Wako Pure Chemical Industries)

[Measuring conditions]

Same as Example 3.

[result]

The percent conversion of each lipoprotein is obtained in the same manner as described in Example 3.

The results are shown in Table 3.

Cyclodextrin Derivatives (Concentration in R-1) Degree of conversion of lipoprotein (%) LDL HDL VLDL CM No addition 75 97 75 38 Poly-β-CD (0.13%) 99 10 32 16

As apparent from the results in Table 3, when measuring cholesterol in the presence of a first reagent containing poly-β-CD and CHE, the reaction with LDL was not affected at all, and with lipoproteins except LDL. Is remarkably suppressed, enabling specific measurement of LDL-cholesterol. Conversely, when the first reagent contains only CHE, the reaction with lipoproteins except LDL, particularly HDL and VCDL, is hardly inhibited, and the reaction with LDL is significantly suppressed, so that LDL-cholesterol can be specifically measured. It becomes impossible.

Example 5

LDL-cholesterol in serum was measured using a Hitachi 7170 automatic analyzer (manufactured by Hitachi).

[sample]

Eight samples are taken from fresh human serum.

[reagent]

(Reagent 1)

R-1 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) 4-AA 1.2 mM,

(Ii) 0.13% poly-β-CD

R-2 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iv) COD (manufactured by Toyobo) 4 u / ml,

(Ii) CHE (manufactured by Amano Pharmaceutical) 4 u / ml,

(Iii) POD (manufactured by Toyobo) 6 u / ml,

(Iii) HDAOS 1.2 mM, and

Lipomin LA 0.4%

(Reagent 2)

R-1 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) 3-AA 1.2 mM,

(Ii) CHE (manufactured by Amano Pharmaceutical) 1 u / ml,

(Iii) 0.13% poly-β-CD, and

(Iii) lipomin LA (trade name of aminocarboxylic acid derivative, manufactured by Lion)

R-2 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iv) COD (manufactured by Toyobo) 4 u / ml,

(Ii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) HDAOS 1.2 mM

(Reagent 3)

R-1 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) 4-AA 1.2 mM,

(Ii) 0.13% poly-β-CD,

(Iii) CHE (manufactured by Amano Pharmaceutical) 1 u / ml, and

Lipomin LA 0.1%

R-2 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iv) COD (manufactured by Toyobo) 4 u / ml,

(Ii) CHE (manufactured by Amano Pharmaceutical) 4 u / ml,

(Iii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) HDAOS 1.2 mM

(Reagent 4)

R-1 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iii) 4-AA 1.2 mM,

(Ii) 1 u / ml of CHE (manufactured by Amano Pharmaceutical), and

Lipomin LA 0.1%

R-2 : 25 mM PIPES-NaOH buffer (pH 7.2) containing:

(Iv) COD (manufactured by Toyobo) 4 u / ml,

(Ii) 6 u / ml POD (manufactured by Toyobo), and

(Iii) HDAOS 1.2 mM

[Measuring conditions]

Same as Example 3.

[result]

The results are shown in Table 4.

Reference Example 1

For the serum samples used in Example 5, cholesterol in the LDL is calculated by Friedard's equation by conventional methods.

Sample number Example 5 Reference Example 1 (mg / dl) Reagent 1 (mg / dl) Reagent 2 (mg / dl) Reagent 3 (mg / dl) Reagent 4 (mg / dl) One 155 136 135 186 130 2 118 86 87 141 81 3 104 87 89 133 75 4 132 104 106 157 101 5 101 77 79 129 70 6 155 136 137 192 130 7 184 167 167 219 161 8 159 139 140 198 138 medium 138.5 116.5 117.4 169.4 110.8 Association Constant with Reference Example 1 0.994 0.996 0.996 0.998 - Slope of the regression line 1.13 1.04 1.05 0.995 - Y intercept of the regression line -46.4 -9.90 -13.41 -57.73 -

As is apparent from the results in Table 4, the LDL-cholesterol values obtained using the reagents according to the present invention containing poly-β-CD (i.e., reagents 1 to 3) are the reagents containing no poly-β-CD ( That is, when compared with the value obtained using Reagent 4), it is closer to the LDL-cholesterol value calculated by the conventional Friedwald equation shown in Reference Example 1. In other words, LDL-cholesterol can be measured specifically by the present invention.

In addition, when poly-β-CD is used with amphoteric surfactants and if necessary with CHE (Reagents 2 and 3), the LDL-cholesterol values obtained are compared to those using poly-β-CD alone. It is closer to the value obtained by calculating.

Example 6

LDL-cholesterol in serum was measured using a Hitachi 7170 automatic analyzer (manufactured by Hitachi).

[sample]

Fifteen samples are taken from fresh human serum.

[reagent]

R-1 : 25 mM 2-hydroxy-N-tris (hydroxymethyl) -methyl-3-aminopropanesulfonic acid (TAPSO) -NaOH buffer containing pH 7.0:

(Iii) HDAOS 0.6 mM,

(Ii) CHE (manufactured by Amano Pharmaceutical) 2 u / ml,

(Iii) 0.125% of poly-β-CD (poly-β-cyclodextrin) manufactured by Wako Pure Chemical, and

(Iii) 0.15% of surfactant in Table 5

R-2 : 25 mM TAPSO-NaOH buffer (pH 7.0) containing:

(Iv) COD (manufactured by Toyobo) 4 u / ml,

(Ii) 10 u / ml POD (manufactured by Toyobo), and

(Iii) 4-AA 6 mM

[Surfactants]

Lipomin LA (trade name, manufactured by Kao Corporation)

EMALEX 712 (trade name, manufactured by Nihon Emulsion),

Polyoxyethylene lauryl ether

EMALEX 1615 (trade name, manufactured by Nihon Emulsion),

Polyoxyethylene hexyldecyl ether

EMALEX NP-20 (trade name, product made by Nihon Emulsion),

Polyoxyethylene nonylphenyl ether

[Measuring conditions]

Same as Example 3.

[result]

The results are shown in Table 5.

Reference Example 1

For the serum samples used in Example 5, cholesterol in the LDL is calculated by the Friedwald equation by conventional methods.

Sample number Example 6 Cholesterol value (mg / dl) Surfactants Reference Example 2 No addition Lipomin LA EMALEX 712 EMALEX 1615 EMALEX NP-20 One 220.3 153.2 94.6 207.9 189.8 149.3 2 213.1 154.2 102.7 230.1 218.1 160.0 3 203.5 147.1 85.4 205.5 192.6 166.6 4 230.8 160.2 93.8 227.2 199.6 179.1 5 233.7 166.6 95.8 227.5 208.1 193.2 6 259.2 180.0 99.4 245.4 221.5 203.8 7 94.0 105.1 73.9 168.8 160.8 122.3 8 191.3 145.4 93.5 213.9 202.1 158.0 9 142.3 126.3 91.1 191.6 186.1 127.8 10 177.9 147.4 95.0 211.1 205.2 150.0 11 204.5 149.9 93.7 213.8 194.8 147.1 12 294.3 238.5 124.3 248.9 249.2 247.0 13 237.7 170.2 102.1 234.1 219.6 175.3 14 291.5 202.7 123.4 273.9 260.4 224.2 15 253.5 171.1 106.2 247.4 238.8 189.9 medium 216.5 161.2 98.3 223.1 209.8 172.9 Association Constant with Reference Example 1 0.914 0.959 0.848 0.881 0.878 - Slope of the regression line 0.598 1.063 2.270 1.174 1.171 - Y intercept of the regression line 43.437 1.571 -50.338 -89.035 -72.813 -

As is evident from the results in Table 5, LDL-cholesterol values obtained using the reagents according to the invention containing poly-β-CD and lipomin LA were prepared using reagents containing poly-β-CD and nonionic surfactants. When compared with the values obtained using, it is closer to the LDL-cholesterol value calculated by the conventional Friedwald equation shown in Reference Example 2. In other words, LDL-cholesterol can be measured specifically by the present invention.

Example 7

Typical examples of kits for measuring LDL-cholesterol in living samples such as serum and plasma are as follows.

(1) first reagent (pH 6-8)

At least one compound selected from the group consisting of DM-α-CD, HP-α-CD and poly-β-CD,

CHE and

Coupler (or developer)

(2) second reagent (pH 6-8)

COD,

CHE,

POD and

Developer (or Coupler)

Using the kit, the same results as in Example 3 were obtained.

With the present invention, LDL-cholesterol in live samples can be measured specifically and accurately by applying one or more components and amphoteric surfactants selected from specific reagents according to the invention, ie cyclodextrins and / or derivatives thereof. . In addition, by applying the present invention, LDL-cholesterol can be measured directly using a multipurpose automatic analyzer, which was not possible with the conventional method.

Claims (34)

The living product is reacted with a cholesterol oxidase or cholesterol dehydrogenase in the presence of an amphoteric surfactant and at least one component selected from the group consisting of cyclodextrins and derivatives thereof as a reagent. A method of measuring cholesterol in low density lipoproteins present in living samples by optical measurements. The method of claim 1, wherein the cyclodextrin derivative is at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. The method of claim 1, wherein said cyclodextrin derivative is poly-β-cyclodextrin. The method of claim 1, wherein the amphoteric surfactant is selected from the group consisting of alkyl betaine derivatives, imidazolinium betaine derivatives, sulfobetaine derivatives, aminocarboxylic acid derivatives, imidazoline derivatives and amine oxide derivatives. At least one compound. The method of claim 1, wherein the amphoteric surfactant is an aminocarboxylic acid derivative, lauric acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, lauryl At least one compound selected from the group consisting of betaine, sodium N-lauroyl-N-methyl-β-alanine and N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid How to. A method for measuring cholesterol in a low density lipoprotein present in a living sample, wherein the living sample is treated with a first reagent containing at least one component selected from the group consisting of cyclodextrin and derivatives thereof, and the absorbance or permeability of the solution obtained is determined. Measured, and the resulting solution was treated with a second reagent solution containing cholesterol oxidase, another absorbance or permeability of the final solution obtained was determined, and the cholesterol value in the live sample was based on the absorbance or permeability data measured above. A method, comprising: a coupler, a developer, a peroxidase, an amphoteric surfactant, and a cholesterol esterase in one or more of the first or second reagents. 7. The method of claim 6 wherein the concentration of cyclodextrin or derivative thereof is 0.0001 to 10% (w / v) in the final solution. 7. The method of claim 6, wherein the concentration of amphoteric surfactant is from 0.0001 to 10% (w / v) in the final solution. A method for measuring cholesterol in a low density lipoprotein present in a living sample, wherein the living sample is treated with a first reagent containing at least one component selected from the group consisting of cyclodextrin and derivatives thereof, and the absorbance or permeability of the solution obtained is determined. Measured, and the obtained solution was treated with a second reagent solution containing cholesterol dehydrogenase, another absorbance or transmittance of the final solution obtained was measured, and the live sample was based on the absorbance or transmittance data measured above. And defining cholesterol levels in the body, wherein at least one of the first or second reagent contains nicotinamide adenine dinucleotide (phosphate), an amphoteric surfactant and a cholesterol esterase. 10. The method of claim 9, wherein the concentration of cyclodextrin or derivative thereof is 0.0001 to 10% (w / v) in the final solution. 10. The method of claim 9, wherein the concentration of amphoteric surfactant is from 0.0001 to 10% (w / v) in the final solution. A reagent composition for measuring cholesterol in low density lipoproteins, characterized by containing at least one component selected from the group consisting of cholesterol oxidase or cholesterol dehydrogenase, amphoteric surfactant, and cyclodextrin and derivatives thereof. 13. The reagent composition of claim 12, wherein the cyclodextrin derivative is at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. 13. The reagent composition of claim 12, wherein said cyclodextrin derivative is poly-β-cyclodextrin. The method of claim 12, wherein the amphoteric surfactant is selected from the group consisting of alkyl betaine derivatives, imidazolinium betaine derivatives, sulfobetaine derivatives, aminocarboxylic acid derivatives, imidazoline derivatives and amine oxide derivatives. Reagent composition, characterized in that at least one compound. 13. The method of claim 12, wherein the amphoteric surfactant is an aminocarboxylic acid derivative, lauric acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, lauryl At least one compound selected from the group consisting of betaine, sodium N-lauroyl-N-methyl-β-alanine and N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid Reagent composition. 13. The reagent composition of claim 12, wherein the concentration of cyclodextrin or derivative thereof is 0.0002 to 20% (w / v). 13. The reagent composition of claim 12, wherein the concentration of the amphoteric surfactant is from 0.0001 to 20% (w / v). (a) a first reagent containing at least one component selected from the group consisting of cyclodextrins and derivatives thereof, (b) a second reagent containing cholesterol oxidase and (c) A reagent composition for measuring cholesterol in low density lipoprotein, comprising a coupler, a developer, a peroxidase, an amphoteric surfactant, and a cholesterol esterase contained in at least one of the first and second reagents. 20. The reagent composition of claim 19, wherein said cyclodextrin derivative is at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. 20. The reagent composition of claim 19, wherein said cyclodextrin derivative is poly-β-cyclodextrin. 20. The method of claim 19, wherein the amphoteric surfactant is selected from the group consisting of alkyl betaine derivatives, imidazolinium betaine derivatives, sulfobetaine derivatives, aminocarboxylic acid derivatives, imidazoline derivatives and amine oxide derivatives. Reagent composition, characterized in that at least one compound. 20. The method of claim 19, wherein the amphoteric surfactant is an aminocarboxylic acid derivative, lauric acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, lauryl At least one compound selected from the group consisting of betaine, sodium N-lauroyl-N-methyl-β-alanine and N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid Reagent composition. 20. The reagent composition of claim 19, wherein the concentration of cyclodextrin or derivative thereof is 0.0002 to 20% (w / v). 20. The reagent composition of claim 19, wherein the concentration of amphoteric surfactant is from 0.0001 to 20% (w / v). (a) a first container comprising an amphoteric surfactant, a cholesterol esterase, a coupler or a developer, and a first reagent containing at least one component selected from the group consisting of cyclodextrins and derivatives thereof (b) A kit for measuring cholesterol in low density lipoprotein, comprising a second container comprising a developer or coupler, a cholesterol oxidase, a cholesterol esterase, and a second reagent containing a peroxidase. The kit according to claim 26, wherein the cyclodextrin derivative is at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin. 27. The kit of claim 26, wherein said cyclodextrin derivative is poly-β-cyclodextrin. The living sample is reacted with cholesterol oxidase or cholesterol dehydrogenase in the presence of at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, and the cholesterol level is measured optically. A method for measuring cholesterol in low density lipoproteins present in a sample. A method for measuring cholesterol in low density lipoproteins present in a living sample, wherein the living sample is treated with a first reagent containing at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, Measure the absorbance or permeability of the solution obtained, treat the obtained solution with a second reagent solution containing cholesterol oxidase, measure the other absorbance or permeability of the final solution obtained, and the absorbance or permeability data measured above. And defining cholesterol levels in the living sample based on which at least one of the first or second reagent contains a coupler, a developer, a peroxidase, and a cholesterol esterase. A method for measuring cholesterol in low density lipoproteins present in living samples, wherein the living sample is treated with a first reagent containing at least one component selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin The absorbance or permeability of the solution obtained, treating the obtained solution with a second reagent solution containing cholesterol dehydrogenase, and measuring the other absorbance or permeability of the final solution obtained, measured above And defining cholesterol levels in the living sample based on one absorbance or permeability data, wherein at least one of the first or second reagent contains nicotinamide adenine dinucleotide (phosphate) and cholesterol esterase. A reagent composition for measuring cholesterol in low density lipoproteins, comprising at least one compound selected from the group consisting of cholesterol oxidase or cholesterol dehydrogenase, and dimethyl-α-cyclodextrin and poly-β-cyclodextrin. (a) a first reagent containing at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, (b) a second reagent containing cholesterol oxidase and (c) A reagent composition for measuring cholesterol in a low density lipoprotein, comprising a coupler, a developer, a peroxidase and a cholesterol esterase contained in at least one of the first and second reagents. (a) a first container comprising a cholesterol esterase, coupler or developer and a first reagent containing at least one compound selected from the group consisting of dimethyl-α-cyclodextrin and poly-β-cyclodextrin, and (b) A kit for measuring cholesterol in low density lipoprotein, comprising a second container comprising a developer or coupler, a cholesterol oxidase, a cholesterol esterase, and a second reagent containing a peroxidase.