JP2010063468A - Method for measuring cholesterol in low-density lipoprotein and reagent therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring cholesterol in a low-density lipoprotein (LDL) in biological materials such as blood serum and plasma, and a reagent therefor. <P>SOLUTION: The method for measuring cholesterol in a low-density lipoprotein by enzymatic reaction includes a first reaction for preferentially eliminating cholesterol in a high-density lipoprotein, ultra-low-density lipoprotein and chylomicron in the sample in a reaction solution containing a chemically modified or unmodified cholesterol esterase, a chemically modified or unmodified cholesterol oxidase and a material inhibiting the reaction of these enzymes with the low-density lipoprotein, and a second reaction for adding a material capable of reducing or eliminating the inhibiting actions of both of the enzymes in the first reaction on cholesterol in the low-density lipoprotein into the reaction solution. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

  The main components of lipids in serum are cholesterol, neutral fat, phospholipid and the like. These serum lipids bind to apoproteins to form lipoproteins and circulate in the blood. These lipoproteins are classified into high density lipoprotein (HDL), LDL, very low density lipoprotein (VLDL), chylomicron (CM), etc. according to the difference in density. Among these lipoproteins, HDL has an effect of transporting excess cholesterol deposited in tissues to the liver, and has an anti-atherogenic effect. On the other hand, LDL is the main transporter of cholesterol from the liver to each tissue, and the increase in LDL is considered to be closely related to the occurrence of arteriosclerosis. From this, cholesterol in LDL (LDL-C) is considered to be a risk factor for arteriosclerosis and ischemic heart disease (coronary artery disease), and knowing the content of LDL-C is a diagnosis of these diseases, Important in treatment and prevention.

  Conventionally, as a method for measuring LDL-C, a precipitation method, an ultracentrifugation method and an electrophoresis method, a calculation method using a calculation formula, and the like are known. Among these conventional methods, the precipitation method, ultracentrifugation method and electrophoresis method require a pretreatment step for separating LDL and lipoproteins other than LDL by precipitation / centrifugation treatment, ultracentrifugation treatment, and electrophoresis treatment. Therefore, the operation is complicated, and there is a problem that direct measurement cannot be carried out only with an automatic analyzer that is currently widely used in the clinical laboratory field. Moreover, the calculation method which calculates from the total cholesterol value known by the formula of Friedewald, the cholesterol (HDL-C) value in HDL, and a neutral fat value is also used when a high neutral fat high-value sample is used. Suffers from the influence of meals and the like, and there is a problem that an accurate LDL-C amount cannot be measured and calculated.

  On the other hand, as a method for solving these problems, a method of directly measuring LDL-C with an automatic analyzer without pretreatment such as centrifugation has been developed. As a method using a lipoprotein flocculant containing a polyanion, there is disclosed a method for measuring the reaction turbidity increased by the aggregation of LDL-C in the presence of a water-soluble polymer that aggregates only LDL (for example, patents) Reference 1). Also disclosed is a method of measuring LDL-C by once aggregating LDL, erasing cholesterol in lipoproteins other than LDL, further deaggregating the aggregated LDL, and enzymatically reacting LDL-C. (For example, refer to Patent Document 2). In addition, in the presence of a reagent that inhibits only LDL reaction, the remaining LDL-C is detected after erasing lipoprotein cholesterol other than LDL, or reacted with HDL-C by aggregating lipoprotein cholesterol other than HDL. A method for measuring LDL-C using an enzyme that acts only on LDL after erasing has been disclosed (see, for example, Patent Document 3). In addition, a method using a cyclodextrin derivative that suppresses the enzymatic reaction of lipoprotein cholesterol other than LDL is disclosed (see, for example, Patent Documents 4 and 5). However, the method using the lipoprotein aggregating agent containing these polyanions and divalent metal ions is inferior in specificity and precision because the turbid scattered light of the aggregate is measured as the absorbance. In addition, precipitation occurs due to the reaction between the lipoprotein aggregating agent containing polyanions and divalent metal ions and the alkaline cell washing solution of the automatic analyzer. Adversely affects precision.

  As a method using a surfactant, LDL-C remaining after reacting all lipoprotein cholesterol other than LDL using a surfactant such as a polyalkylene oxide derivative having an HLB value of 13 to 15 in the first step is used. A method of detecting by reacting with another type of surfactant in the second step is disclosed (for example, see Patent Document 6). However, although the surfactant used here works well on HDL-C, it does not have sufficient action on cholesterol in VLDL and CM, and it is necessary to have a large excess of divalent metal ions. Precipitation is generated by the reaction of the analyzer with the alkaline cell washing solution, which causes a problem in applicability to the automatic analyzer. In addition, a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether and an enzyme reagent for measuring cholesterol were added to serum to preferentially react cholesterol in HDL, VLDL, and CM. Thereafter, a method of measuring LDL-C by measuring the subsequent reaction amount is disclosed (for example, see Patent Document 7). Even in this method, the action on cholesterol in VLDL and CM is not sufficient, and a polyanion and a divalent metal are required to enhance the reactivity to VLDL and CM. A precipitate is generated by the reaction with the cell washing solution, which causes a problem in applicability to an automatic analyzer. Also disclosed is a method for detecting LDL-C by selective reaction in the presence of an amphoteric surfactant and an aliphatic amine having a carboxyl group or a sulfonic acid group (see, for example, Patent Document 8). Further, a method for detecting LDL-C by selectively reacting in the presence of an amphoteric surfactant and a polyanion is disclosed (for example, see Patent Document 9). These methods are intended to suppress reactivity to lipoprotein cholesterol other than LDL such as HDL, VLDL, CM, etc. in the first reaction.

  In addition, a method of selectively reacting and detecting LDL-C using a chemical modification enzyme that acts only on LDL is also disclosed (see, for example, Patent Document 10). In addition, a biological sample, pancreatic cholesterol esterase and cholesterol oxidase are reacted under the condition that albumin is present in an amount of 0.01% by weight or more of the whole measurement system and bile acid or a salt thereof, and then the microorganism-derived cholesterol esterase is allowed to act. A measuring method is disclosed (for example, see Patent Document 11). In this method, since different cholesterol esterases derived from the first reagent and the second reagent are allowed to act, the amount of the enzyme used increases and there is a problem in economy.

Japanese Patent No. 2749409 Japanese Patent No. 3107492 Japanese Patent No. 3256241 JP 10-311833 A Japanese Patent Laid-Open No. 11-30617 Japanese Patent No. 30586602 Japanese Patent No. 3193634 Japanese Patent Laid-Open No. 10-84997 Japanese Patent Laid-Open No. 2000-60600 Japanese Patent Laid-Open No. 10-80300 Japanese Patent Laid-Open No. 11-9300

  Therefore, the problem to be solved by the present invention is excellent in simplicity, specificity and precision applicable to various automatic analyzers without the need for pretreatment such as centrifugation and electrophoresis. It is to provide a fractional measurement method for LDL-C.

As a result of intensive studies to achieve the above object, the present inventors have determined that in a means for measuring cholesterol in a low-density lipoprotein in a sample by an enzymatic reaction, lipoproteins in HDL, VLDL, and CM in the first reaction. Chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase, which act on cholesterol, and substances that inhibit their reactivity to low-density lipoproteins are added, and high-density lipoproteins, ultra-low-density lipoproteins are added. After eliminating cholesterol in proteins and chylomicrons, the second reaction reduces the inhibitory effect of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase in the first reaction on cholesterol in low-density lipoproteins Or add a disappearing substance Conveniently the LDL-C in the specificity was found to be able to precisely good fractionation measurement. In addition, the present measurement method has found that it is not necessary to use a known lipoprotein flocculant such as polyanion, and is excellent in applicability to an automatic analyzer, thereby completing the present invention. That is, the present invention has the following configuration.
(1) In means for measuring cholesterol in a low-density lipoprotein in a sample by enzymatic reaction, cholesterol esterase chemically modified or unmodified in the first reaction, cholesterol oxidase chemically modified or unmodified, and these low-density lipoproteins After adding a substance that inhibits the reactivity to and preferentially eradicating cholesterol in high-density lipoprotein, ultra-low-density lipoprotein and chylomicron, chemically modified or unmodified cholesterol in the first reaction in the second reaction A method for measuring cholesterol in low-density lipoprotein, comprising adding a substance that reduces or eliminates the inhibitory action of esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein (2) Chemically modified or unmodified Modified cholesterol esthetics (1) A method for measuring cholesterol in low-density lipoprotein according to (1), wherein the substance that inhibits the reactivity with chemically modified or unmodified cholesterol oxidase and these low-density lipoproteins is a cholic acid derivative (3) Cholic acid The derivatives are 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid, N, N-bis (3 -D-gluconamidopropyl) colamide, N, N-bis (3-D-gluconamidopropyl) deoxycholamide, cholic acid, deoxycholic acid and salts thereof are one or more selected from (2) Method for Measuring Cholesterol in Low Density Lipoprotein (4) The concentration of cholic acid derivative during the first reaction is 0.01 to 0.2 (2) The method for measuring cholesterol in low-density lipoprotein of (3) (5) Chemically modified or unmodified cholesterol esterase in the first reaction and chemically modified or unmodified cholesterol oxidase in low-density lipoprotein (1) The method for measuring cholesterol in low density lipoprotein, wherein the substance that reduces or eliminates the inhibitory action on cholesterol is one or more selected from polyoxypropylene polyoxyethylene alkyl ether and polyoxyethylene styryl phenyl ether (6) A substance that reduces or eliminates the inhibitory action of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein in the first reaction is polyoxyp Method of measuring cholesterol in low density lipoprotein of (5) which is propylene (2 mol) polyoxyethylene decyl ether (7) In the low density lipoprotein of (1) containing a divalent metal ion at least during the first reaction Cholesterol measurement method (8) Divalent metal ions are magnesium ion, calcium ion, manganese ion, nickel ion, copper ion, iron ion (7) Method for measuring cholesterol in low density lipoprotein (9) Polyanion (1) Cholesterol measurement method in low density lipoprotein of (1) not containing (10) polyanion is heparin, phosphotungstic acid, dextran flow acid, sulfated cyclodextrin, heparan sulfate, chondroitin sulfate, hyaluronic acid, sulfated oligosaccharide, Sulfated polyacrylamide, carboxymethylated polyacryl (9) A method for measuring cholesterol in low-density lipoprotein of (9) which is a salt thereof or a salt thereof. (11) In a means for measuring cholesterol in low-density lipoprotein in a sample by an enzymatic reaction, Add unmodified cholesterol esterase and chemically or unmodified cholesterol oxidase and substances that inhibit their reactivity to low-density lipoproteins, giving priority to high-density lipoproteins, ultra-low-density lipoproteins, and cholesterol in chylomicrons In the second reaction, the chemical modification or unmodified cholesterol esterase in the first reaction and the substance that reduces or eliminates the inhibitory effect on the cholesterol in the low-density lipoprotein of the chemically modified or unmodified cholesterol oxidase are added in the second reaction Low density Reagent for measuring cholesterol in protein (12) Chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase and substances that inhibit their reactivity to low density lipoproteins are cholic acid derivatives (11) (13) Cholic acid derivative is 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-colamidopropyl) dimethylammonio] 2-hydroxypropanesulfonic acid, N, N-bis (3-D-gluconamidopropyl) colamide, N, N-bis (3-D-gluconamidopropyl) deoxycolamide, cholic acid, deoxycholic acid and its The low density lipoprotein of (12), which is one or more selected from salts Reagent for measuring cholesterol in white (14) The reagent for measuring cholesterol in low-density lipoprotein of (12) and (13), wherein the concentration of the cholic acid derivative during the first reaction is 0.01 to 0.2% (15 ) A substance that reduces or eliminates the inhibitory effect of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein in the first reaction is polyoxypropylene polyoxyethylene alkyl ether, (11) A reagent for measuring cholesterol in low density lipoprotein of (11) or one or more selected from polyoxyethylene styryl phenyl ether (16) Chemically modified or unmodified cholesterol esterase in the first reaction and chemically modified or unmodified Cholesterol oxy (11) The reagent for measuring cholesterol in low density lipoprotein (17), wherein the substance that reduces or eliminates the inhibitory action on cholesterol in low density lipoprotein is polyoxypropylene (2 mol) polyoxyethylene decyl ether. (11) A reagent for measuring cholesterol in low-density lipoprotein according to (11) containing at least a divalent metal ion in the first reaction. (18) Divalent metal ions are magnesium ions, calcium ions, manganese ions, nickel ions, copper ions. (17) A reagent for measuring cholesterol in low-density lipoprotein of (17) which is an iron ion (19) A reagent for measuring cholesterol in low-density lipoprotein of (11) which does not contain polyanion (20) The polyanion contains heparin, phosphotungstic acid, Dextran flow acid, sulfated cyclodextrin, hepara Sulfate, chondroitin sulfate, hyaluronic acid, sulfated oligosaccharide, sulfated polyacrylamides, carboxymethylated polyacrylamide or reagent for measuring low density lipoprotein cholesterol in a salt thereof (19),

  According to the present invention, there is no need for sample pretreatment such as centrifugation, electrophoresis, etc., and it can be applied to various automatic analyzers. LDL-C fractional measurement method excellent in simplicity, specificity and precision Can be provided.

  In the present invention, “inhibition” means to reduce the reactivity of cholesterol esterase and cholesterol oxidase to LDL without aggregating LDL.

  In one embodiment of the present invention, the first reagent comprises a chemically modified or unmodified cholesterol esterase, a chemically modified or unmodified cholesterol oxidase, a catalase, a chromogen, a chemically modified or unmodified cholesterol esterase and a chemically modified or unmodified A substance that inhibits the reactivity of the modified cholesterol oxidase to low-density lipoproteins, a divalent metal ion, and the like, and the second reagent includes peroxidase, coupler, chemically modified or unmodified cholesterol esterase, and chemically modified or unmodified There are LDL-C measurement reagents that constitute substances that reduce or eliminate the inhibitory action of cholesterol oxidase on LDL-C.

  The first reagent contains low amounts of chemically modified or unmodified cholesterol esterase, chemically modified or unmodified cholesterol oxidase, peroxidase, chromogen, chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase. A substance that inhibits the reactivity to density lipoprotein, a divalent metal ion, etc. are constituted, and the second reagent contains a coupler, a chemically modified or unmodified cholesterol esterase and a chemically modified or unmodified cholesterol oxidase that inhibits LDL-C. There is an LDL-C measurement reagent that constitutes a substance that reduces or eliminates the action.

The origin of cholesterol oxidase (an enzyme having the ability to oxidize cholesterol to produce hydrogen peroxide) used in the present invention is not particularly limited. For example, microorganisms such as Nocardia and Pseudomonas, and animal organs such as bovine pancreas The thing etc. which originate in can be used. These can be obtained commercially.
In the present invention, it is preferable to use an enzyme that has better reactivity with lipoprotein cholesterol in HDL, VLDL, and CM than LDL-C.
In addition, for the purpose of improving the reactivity of these enzymes to lipoprotein cholesterol in HDL, VLDL, and CM, the above-mentioned enzymes may be used with groups mainly composed of polyethylene glycol, monosaccharides, water-soluble oligosaccharide residues, sulfopropyl groups, etc. Those chemically modified are used.
In addition, these genes are taken by genetic manipulation, introduced into another microorganism and expressed, or modified products obtained by chemically modifying these genes, or enzymes expressed by modifying these genes or chemically A modified product modified to have been suitably used.
The lower limit of the amount of enzyme used is usually 0.5 U / mL, preferably 1 U / mL in the first reagent. The upper limit of the amount used is 10 U / mL, preferably 5 U / mL.

The origin of the cholesterol esterase (enzyme having the ability to hydrolyze cholesterol ester) used in the present invention is not particularly limited. For example, microorganisms such as Candida and Pseudomonas, and those derived from animal organs such as bovine pancreas, etc. Can be used. These can be obtained commercially.
In the present invention, it is preferable to use an enzyme that has better reactivity with lipoprotein cholesterol in HDL, VLDL, and CM than LDL-C.
In addition, for the purpose of improving the reactivity of these enzymes to lipoprotein cholesterol in HDL, VLDL, and CM, the above-mentioned enzymes may be used with groups mainly composed of polyethylene glycol, monosaccharides, water-soluble oligosaccharide residues, sulfopropyl groups, etc. Those chemically modified are used.
In addition, these genes are taken by genetic manipulation, introduced into another microorganism and expressed, or modified products obtained by chemically modifying these genes, or enzymes expressed by modifying these genes or chemically A modified product modified to have been suitably used.
The lower limit of the amount of enzyme used is usually 0.01 U / mL, preferably 0.05 U / mL in the first reagent. The upper limit of the amount used is 10 U / mL, preferably 5 U / mL.

Examples of substances that inhibit the reactivity of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase to low-density lipoprotein used in the present invention include reactivity to lipoprotein cholesterol in HDL, VLDL, and CM. There is no particular limitation as long as there is substantially no influence on the reaction and the reactivity to LDL-C is lowered.
An example of such a substance is a cholic acid derivative. The cholic acid derivative is not particularly limited but is preferably 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid or 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropane. Suitable are sulfonic acid, N, N-bis (3-D-gluconamidopropyl) colamide, N, N-bis (3-D-gluconamidopropyl) deoxycolamide, cholic acid, deoxycholic acid and salts thereof. However, since the reactivity with respect to LDL-C can be reduced with a small addition amount, N-bis (3-D-gluconamidopropyl) colamide and N, N-bis (3-D-gluconamidopropyl) are more preferable. Deoxycolamide and its salts are good. Moreover, 1 type selected from these cholic acid derivatives may be used, or you may use combining several. As for the amount used, the total amount of cholic acid derivative can be used in the range of usually 0.001 to 5% in the first reagent, but preferably used near or below the critical micelle concentration (cmc). It is good to do. This is due to the fact that the action of these cholic acid derivatives as an inherent surfactant is warped and weakens the inhibitory effect of cholesterol esterase and / or cholesterol oxidase on the low-density lipoprotein expected by the present invention.
The critical micelle concentration can be measured by measuring sudden change points of physical properties such as electrical conductivity, osmotic pressure, freezing point depression, vapor pressure, viscosity, density, solubilization ability, detergency, light scattering, and dye changes. Is possible. For example, the limiting micelle concentration of 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid is 0.50% (w / v), and 3-[(3-colamidopropyl) dimethylammonio ] -2-hydroxypropanesulfonic acid is 0.51% (w / v), N, N-bis (3-D-gluconamidopropyl) colamide is 0.26% (w / v), N , N-bis (3-D-gluconamidopropyl) deoxycolamide is known to be 0.12% (w / v). Therefore, the preferred amount of use is 0.01 to 0.6%. Similarly, other cholic acid derivatives can be used in an optimized manner by knowing the critical micelle concentration.

On the other hand, flocculants such as polyanions also reduce the reactivity to LDL-C, but since they adversely affect the applicability to automatic analyzers, they are not added or are used in the above-mentioned automatic analyzers. It can be added to the extent that turbidity or precipitation does not occur due to coexistence with the detergent. However, it is preferable not to add them. Such polyanions include heparin, phosphotungstic acid, dextran flow acid, sulfated cyclodextrin, heparan sulfate, chondroitin sulfate, hyaluronic acid, sulfated oligosaccharides, sulfated polyacrylamide, carboxymethylated polyacrylamide, or these Salt.
The amount of these used is 0 to 1% (w / v), preferably 0 to 0.5% (w / v) as the final reaction concentration.

  As the divalent metal ion used in the present invention, magnesium ion, calcium ion, manganese ion, nickel ion, copper ion and iron ion are used for the purpose of improving the reactivity to HDL-C. Preferably, Magnesium ions and calcium ions are preferred. Moreover, these divalent metal ions may be used alone or in combination. Moreover, what is necessary is just to be added to the 1st reagent at least. These use amounts are usually 0.0005 to 0.2 mol / L, preferably 0.001 to 0.1 mol / L, more preferably 0.00 in the first reagent in consideration of applicability to an automatic analyzer. 005 to 0.05 mol / L.

  The substance that reduces or eliminates the inhibitory action on the LDL-C of the chemically modified or unmodified cholesterol esterase and the chemically modified or unmodified cholesterol oxidase of the present invention refers to the inhibition of LDL-C in the first reagent. Is not particularly limited as long as it can be reduced or eliminated by the addition of a second reagent containing, but preferably one or more selected from polyoxypropylene polyoxyethylene alkyl ether and polyoxyethylene styryl phenyl ether are combined. Used together. More preferably, one or more selected from polyoxypropylene (2) polyoxyethylene decyl ethers are used in combination. Examples of polyoxyethylene styryl phenyl ethers include Emulgen A60 (HLB: 12.8), Emulgen A90 (HLB: 14.5), Emulgen B66 (HLB: 13.2), and the like. Examples of polyoxypropylene (2) polyoxyethylene decyl ethers include EMALEX DAPE0207 (HLB: 10), EMALEX DAPE0210 (HLB: 11), EMALEX DAPE0212 (HLB: 12), EMALEX DAPE0215 (HLB: 13) , Emalex DAPE0220 (HLB: 14), Emarex DAPE0220 (HLB: 15), and the like. The HLB of these surfactants is known as “New Surfactant”, Hiroshi Horiuchi, 1986, Sankyo Publishing, but is usually 10-15, preferably 10-13 as the range of HLB. Preferably it is 10-12. These use amounts are usually 0.005 to 1% (w / v), preferably 0.01 to 0.5% (w / v) in the second reagent in consideration of applicability to an automatic analyzer. More preferably, it is 0.05 to 0.3% (w / v).

  Although it does not specifically limit as a color source body used by this invention, A hydrogen donor, a leuco body, a tetrazolium salt, etc. are mentioned.

  Examples of hydrogen donors include N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl. 3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N- (2-hydroxy-3 -Sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy Aniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3 Sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-sulfopropylaniline, N- (2-hydroxy-3-sulfopropyl) ) -2,5-dimethylaniline, N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine, N-ethyl-N- (3-methylphenyl) -N′-acetylethylenediamine and the like. . These hydrogen donors can be used in combination with a coupler. Examples of the coupler include 4-aminoantipyrine, MBTH, NCP and the like.

  The leuco form includes 4,4'-benzylidenebis (N, N-dimethylaniline), 4,4'-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl. ] Methane, 1- (ethylaminothiocarbonyl) -2- (3,5-dimethoxy-4-hydroxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole, 4,4'-bis (dimethylamino) Examples include diphenylamine, N-carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine salt, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine salt, and the like.

  Examples of the tetrazolium salt include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium salt, 3,3 ′-[3,3′-dimethoxy- (1,1′-biphenyl) -4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium] salt, 3,3 ′-[3,3′-dimethoxy -(1,1'-biphenyl) -4,4'-diyl] -bis (2,5-diphenyl-2H-tetrazolium) salt, 2- (4-iodophenyl) -3- (4-nitrophenyl)- 5- (2,4-Disulfophenyl) -2H-tetrazolium salt, 3,3 ′-(1,1′-biphenyl-4,4′-diyl) -bis (2,5-diphenyl-2H-tetrazolium) Salt, 3- (4,5-dimethyl) 2-thiazolyl) -2,5-diphenyl -2H- tetrazolium salts.

  The amount of the color source used is preferably 0.01 to 10 mmol / L as the final reaction concentration in consideration of solubility.

  In the present invention, a surfactant can coexist as long as the specificity and precision for LDL-C are not impaired. As the surfactant, a nonionic surfactant and / or a zwitterionic surfactant is preferably used.

Nonionic surfactants used in the present invention include polyoxyethylene lauryl ethers such as Emulgen 104P, Emulgen 105, Emulgen 106, Emulgen 108, Emulgen 109P, Emulgen 120, Emulgen 123P, Emulgen 147, Emulgen 130K, Nonion K- 204, Nonion K-215, Nonion K-220, Nonion K-230, NIKKOL BL-2, NIKKOL BL-4.2, NIKKOL BL-9EX, NIKKOL BL-21, NIKKOL BL-25, polyoxyethylene cetyl ethers As Emulgen 210, Emulgen 220, NIKKOL BC-2, NIKKOL BC-5.5, NIKKOL BC-7, NIKKOL
BC-10TX, NIKKOL BC-15TX, NIKKOL BC-20TX, NIKKOL BC-23, NIKKOL BC-25TX, NIKKOL BC-30TX, NIKKOL BC-40TX, Nonion P-1013, Nonion P-20813 As oxyethylene stearyl ethers, Emulgen 306P, Emulgen 320P, NIKKOL BS-2, NIKKOL BS-4, NIKKOL
BS-20, Nonion S-206, Nonion S-207, Nonion S-215, Nonion S-220, Polyoxyethylene oleyl ethers include Emulgen 404, Emulgen 408, Emulgen 409P, Emulgen 420, Emulgen 430, NIKKOL BO -2, NIKKOL BO-7, NIKKOL BO-10TX, NIKKOL BO-20, NIKKOL BO-50, nonion E-206, nonion E-215, nonion E-230, and polyoxyethylene behenyl ethers include NIKKOL
BB-5, NIKKOL BB-10, NIKKOL BB-20, NIKKOL BB-30, etc. are mentioned.

Examples of the polyoxyethylene secondary alkyl ethers include Emulgen 707, NIKKOL BT-5, NIKKOL BT-7, NIKKOL BT-9, Adekatol SO-80, Adecator SO-105, Adecator SO-120, Adecator SO-135. , Adekatol SO-145, Adekatol SO-160, Emulgen 705, Emulgen 707, Emulgen 709 and the like. Examples of polyoxyethylene alkylphenyl ethers include Emulgen 810, Emulgen 840S, Emulgen 909, Emulgen 910, Emulgen 930, Emulgen 950, Triton X-100, Triton X-114, NIKKOL NP-5, NIKKOL NP-7.5, NIKKOL NP-10, NIKKOL NP-15, NIKKOL NP-20, NIKKOL OP-10, NIKKOL OP-30, and the like. Examples of the oxyethylene / oxypropylene block copolymers include Emulgen PP-150, Emulgen PP-230, Emulgen PP-250, Emulgen PP-290, NIKKOL PBC-34, NIKKOL PBC-44, and the like. Examples of fatty acid esters include Leodol TW-L120, Leodol TW-L106, Leodol TW-P120, Leodol TW-S120, Leodol TW-O120, Leodol 460, Emanon 1112, Emanon 3115, Emanon 3170, Emanon 3299, Emanon 3130 and the like. Can be mentioned. Examples of the polyoxyethylene sterols include NIKKOL BPS-10, NIKKOL BPS-20, NIKKOL BPS-30, NIKKOL BPSH-25, and NIKKOL DHC-30. In addition, n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, n-octanoyl-N-methylglucoamide, n-nonanoyl-N-methylglucoamide, n-decanoyl-N- Examples include methyl glucoamide, sucrose monocaprate, sucrose monolaurate, sucrose monocholate, digitonin and the like.

  Examples of the zwitterionic surfactant used in the present invention include alkylimidazolium betaine, alkylbetaine, alkylamidobetaine, alkylalanine, alkylamine oxide, and derivatives thereof. These Amphitol 20BS, Amphitol 24B, Amphitol 86B, Amphitol 20Z, etc. are mentioned.

  Conventionally used buffers include Tris buffer, phosphate buffer, citrate buffer, succinate buffer, phthalate buffer, and GOOD buffer. On the other hand, GOOD buffer contains N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -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), N-tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2 -Hydroxy-3-aminopropanesulfonic acid (CAPSO), 3- [N, N-bis (2-hydro) Cyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), 2-hydroxy-3- [4- (2- Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis (2-hydroxy) -3-propanesulfonic acid) (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N- (2-acetamido) iminoniacetic acid (ADA), N, N-bis (2 -Hydroxyethyl) glycine (Bicine), N- [Tris (hydro Shimechiru) methyl] glycine (Tricine), etc. are exemplified. The pH of the buffer is adjusted in the range of 5-9. Furthermore, 6-8 are preferable. Among these, in the first reagent, 7.0 to 7.5 is preferable for the purpose of reducing the reactivity to LDL-C. The second reagent is preferably 6.5 to 7.5 for the purpose of improving the reactivity to LDL-C in consideration of the pH when mixed with the second reagent. These usage-amounts are 0.01-0.2 mol / L normally, Preferably it is 0.02-0.1 mol / L.

  In the present invention, preservatives, salts, enzyme stabilizers, color source stabilizers and the like may be added within a range not affecting the reaction. Examples of antiseptics include azides, chelating agents, antibiotics, and antibacterial agents. Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt. Antibiotics include gentamicin, kanamycin, chloramphenicol and the like. Examples of the antibacterial agent include imidazolidinyl urea. Examples of the salts include sodium chloride, potassium chloride, aluminum chloride and the like. Enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like. Examples of the color source stabilizer include chelating agents such as ethylenediaminetetraacetic acid and salts thereof, and cyclodextrin.

  In the present invention, as a method for preferentially erasing cholesterol in HDL, VLDL and CM in the first reaction, hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase is converted into, for example, catalase or peroxidase and the aforementioned color. Although it can erase | eliminate using a source body or a peroxidase and the above-mentioned coupler, it is not specifically limited.

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.
Example 1
Prepare LDL-C measuring reagents of the following composition 1 to 3 and measure HDL, LDL, VLDL, CM purified from human serum as samples under the following measuring conditions, and determine the cholesterol concentration in each lipoprotein from each measured value. Relative% was calculated as 100%. Moreover, the LDL-C measuring reagent of the following compositions 4-6 was prepared as a comparative example, and examination similar to an Example was performed. The purified lipoprotein fraction was separated and fractionated by ultracentrifugation using three kinds of density solutions according to the method of Hatch and Lees.

(Preparation of reagents)
LDL-C measurement reagents having the following compositions 1 to 3 were prepared.
Composition 1
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycollamide (manufactured by Dojindo) 0.05%
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxypropylene (2) polyoxyethylene decyl ether (Emalex DAPE0207: manufactured by Nippon Emulsion Co., Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

Composition 2
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
3-[(3-Colamidopropyl) dimethylammonio] propanesulfonic acid (manufactured by Dojindo) 0.2%
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxypropylene (2) polyoxyethylene decyl ether (Emalex DAPE0207: manufactured by Nippon Emulsion Co., Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

Composition 3
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycollamide (manufactured by Dojindo) 0.05%
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxyethylene styryl phenyl ethers (Emulgen B66; manufactured by Kao Corporation)
0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

Composition 4
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycollamide (manufactured by Dojindo) 0.05%
Unmodified cholesterol esterase (COE-311 manufactured by Toyobo Co., Ltd.) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxypropylene (2) polyoxyethylene decyl ether (Emalex DAPE0207: manufactured by Nippon Emulsion Co., Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

Composition 5
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxypropylene (2) polyoxyethylene decyl ether (Emalex DAPE0207: manufactured by Nippon Emulsion Co., Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

Composition 6
First reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
N, N-bis (3-D-gluconamidopropyl) deoxycollamide (manufactured by Dojindo) 0.05%
Chemically modified cholesterol esterase (Toyobo COE-313) 0.5 U / mL
Unmodified cholesterol oxidase (Toyobo COO-321) 3 U / mL
Catalase (from Roche microorganisms) 100 U / mL
4-Aminoantipyrine 0.1g / L
Second reagent PIPES-NaOH 50 mM pH 7.5
Ethylenediaminetetraacetic acid disodium salt 0.2 mmol / L
Magnesium chloride 10mmol / L
Calcium chloride 0.1mmol / L
Polyoxyethylene (10) octylphenyl ether (Wako Pure Chemical Industries, Ltd.) 0.3%
Peroxidase (Toyobo PEO-301) 8U / mL
N-ethyl-N-sulfopropyl-3-methoxyaniline (manufactured by Dojin Chemical) 0.4 g / L

(Measurement method)
A Hitachi 7170 automatic analyzer was used. The first reaction was carried out by adding 180 μL of the first reagent to 2.4 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. Absorbance was measured at 600 nm dominant wavelength and 800 nm subwavelength by a two-point end method, which takes the difference between the absorbances obtained by correcting the absorbances of the first reaction and the second reaction. The LDL-C concentration of the sample with unknown LDL-C concentration was calculated from the measured absorbance of purified water and 110 mg / dL LDL-C standard serum.

Results are shown in Table 1. In Examples 1 to 3, the nonspecific reaction to cholesterol in HDL, VLDL, and CM was 5.0% or less, and good specificity was confirmed. On the other hand, the composition 4 of the comparative example does not recover LDL-C at all. As this cause, it is considered that LDL-C was almost eliminated in the first reaction. Composition 5 of the comparative example recovers a part of LDL-C, but has a nonspecific reaction to cholesterol (HDL-C) in high density lipoprotein. As this cause, it is considered that HDL-C is not completely erased in the first reaction. Composition 6 of the comparative example has a nonspecific reaction to cholesterol in HDL, VLDL, and CM of 5.0% or less, but recovery of LDL-C is low. This is considered to be because the reactivity to LDL-C is not sufficiently obtained in the second reaction.

(Example 2)
20 samples of healthy human serum were measured as samples under the measurement conditions shown in Example 1 using the LDL-C measurement reagents having compositions 1 to 3 shown in Example 1. Moreover, the LDL-C measuring reagent of the following compositions 4-6 was prepared as a comparative example, and the same measurement as an Example was performed. The measured value in each reagent is the total cholesterol value of the sample (using Tolesbo's Cores Color Liquid) and the HDL-C value (sampled by the Daiichi Kagaku's fraction reagent solution) Measured with liquid) and neutral fat value (Lipidos Liquid manufactured by Toyobo Co., Ltd.) were measured, and relative% was determined using the LDL-C value calculated using the Friedewald formula as a control.

Results Table 2 shows the measured values, and Table 3 shows the relative% of the measured values of the control. The relative% of each composition was 95.7 to 106.5% in composition 1 of the example, 93.1 to 108.9% in composition 2, and 92.5 to 105.6% in composition 3. . On the other hand, in the comparative example, -58.8 to 105.3% in composition 4, 79.8 to 104.9% in composition 5, and 87.3 to 114.9% in composition 6 are problems in specificity or precision. was there.

  The method for measuring cholesterol in low-density lipoprotein according to the present invention is capable of fractionating and measuring cholesterol in low-density lipoprotein in blood simply, with high specificity and precision, and it is necessary to use a known lipoprotein aggregating agent such as polyanion. Furthermore, since the applicability to an automatic analyzer is excellent, a more accurate and quick diagnosis can be performed particularly in the field of clinical examination such as arteriosclerosis.

Claims (7)

In a means for measuring cholesterol in a low-density lipoprotein in a sample using an enzymatic reaction,
(1) chemically modified cholesterol esterase, and
(2) chemically modified or unmodified cholesterol oxidase, and
(3) Cholic acid derivatives as substances that inhibit the reactivity to these low-density lipoproteins, and
(4) In a reaction solution containing a divalent metal ion as a substance that improves the reactivity of high-density lipoprotein to cholesterol, high-density lipoprotein in samples, ultra-low-density lipoprotein, and cholesterol in chylomicron are given priority. After erasing
In the second reaction,
(5) a substance that reduces or eliminates the inhibitory action of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein in the first reaction;
A method for measuring cholesterol in low-density lipoprotein, which is added to a reaction solution.   Cholic acid derivatives are 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid, 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid, N, N-bis. One or more selected from (3-D-gluconamidopropyl) colamide, N, N-bis (3-D-gluconamidopropyl) deoxycolamide, cholic acid, deoxycholic acid and salts thereof, Item 2. A method for measuring cholesterol in a low-density lipoprotein according to Item 1.   The method for measuring cholesterol in a low density lipoprotein according to claim 1 or 2, wherein the concentration of the cholic acid derivative during the first reaction is 0.01 to 0.2%.   In the second reaction, a substance that reduces or eliminates the inhibitory effect of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein in the first reaction is polyoxypropylene polyoxy The method for measuring cholesterol in low density lipoprotein according to any one of claims 1 to 3, wherein the cholesterol is one or more selected from ethylene alkyl ether and polyoxyethylene styryl phenyl ether.   In the second reaction, a substance that reduces or eliminates the inhibitory action of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase in cholesterol in low-density lipoprotein in the first reaction is polyoxypropylene (2 5. The method for measuring cholesterol in low density lipoprotein according to claim 4, wherein the cholesterol is one or more selected from polyoxyethylene decyl ether and polyoxyethylene styryl phenyl ether.   The method for measuring cholesterol in low density lipoprotein according to claim 1, which does not contain a polyanion. A reagent for measuring cholesterol in a low density lipoprotein in a sample,
In the reagent for the first reaction,
(1) chemically modified cholesterol esterase, and
(2) chemically modified or unmodified cholesterol oxidase, and
(3) Cholic acid derivatives as substances that inhibit the reactivity to these low-density lipoproteins, and
(4) containing a divalent metal ion as a substance that improves the reactivity of high density lipoprotein to cholesterol,
In the reagent for the second reaction,
(5) a substance that reduces or eliminates the inhibitory action of chemically modified or unmodified cholesterol esterase and chemically modified or unmodified cholesterol oxidase on cholesterol in low-density lipoprotein in the first reaction;
A reagent for measuring cholesterol in low-density lipoprotein, comprising: