JP2007325587A - Method for measuring high-density lipoprotein cholesterol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for readily, rapidly and specifically measuring high-density lipoprotein cholesterol (HDL-C) by using an inexpensive material; and to provide a reagent kit for detecting the HDL-C, and a dry analytic element for detecting the HDL-C. <P>SOLUTION: The method for detecting the HDL-C in a body fluid sample involves selectively measuring the HDL-C by forming hydrogen peroxide from the HDL-C by using a cholesterol esterase originated from Schizophyllum commune or Pseudomonas sp., and a cholesterol oxidase originated from Pseudomonas sp. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for measuring high density lipoprotein cholesterol (HDL-C) in a specimen. It is known that the blood level of high density lipoprotein cholesterol is a useful index for predicting the onset of arteriosclerosis.

  Lipids present in the blood are incorporated into the structure of lipoproteins, except that free fatty acids are bound to albumin, and are composed of chylomicron (CHM), very low density lipoprotein (VLDL), and low density lipoprotein. (LDL), high density lipoprotein (HDL), etc., among which cholesterol is particularly distributed in VLDL, LDL, and HDL. HDL is regarded as a preventive factor for heart disease caused by arteriosclerosis, and therefore, measurement of high density lipoprotein cholesterol (HDL-C) in place of HDL has a clinical significance. Currently, ultracentrifugation, electrophoresis, and precipitation are widely known as methods for measuring HDL cholesterol.

  The ultracentrifugation method requires a long time for the separation operation, and is unsuitable for daily inspections such as the fact that the instrument itself is expensive and inexpensive measurement cannot be expected. The electrophoresis method has a resolution due to the difference in the electrophoresis support medium. The problem remains in terms of quantification, such as differences in use conditions and detection reagents used. Therefore, the precipitation method is currently widely used for daily inspection.

  The precipitation method uses a combination of polyanions and divalent metal ions as a precipitation reagent to precipitate CHM, LDL, and VLDL, and uses cholesterol or HDL-cholesterol in the supernatant, that is, HDL-cholesterol, as a chemical reagent or enzyme. It is a method to measure. Precipitation reagents have been well known since the early 1960s by Nakai Yukihiko, "HDL-Metabolism. Assay Methods and Clinical Application" (Chugai Medical Co., Ltd. 1986), various literatures and teaching materials. A combination system of sulfate polysaccharide-alkaline earth metal ions or divalent metal ions other than alkaline earth, inorganic polyanion salt systems, polyethylene glycol, and the like are widely used. Specific examples of the precipitation reagent include heparin-calcium reagent, dextran sulfate-magnesium reagent, and phosphotungstic acid-magnesium reagent.

  In these precipitation methods, serum and a precipitation reagent are mixed, allowed to stand for a certain period of time, centrifuged at about 3000 rpm, and then a certain amount of the supernatant is separated and subjected to chemical reaction or enzymatic reaction to carry out HDL-cholesterol. Is a method of quantifying

  In the precipitation method, there are problems due to the precipitation reagent and problems due to centrifugation. Therefore, as an improvement of the precipitating agent for increasing the precipitation efficiency, JP-A-55-78254, JP-A-55-93065, JP-A-61-263467, JP-A-62-19768 Various methods are described in JP-B-1-39553.

  In addition, a major drawback of the conventional precipitation method is that in the case of a reagent having a large amount of triglyceride, the precipitate may partially float after centrifugation. For this reason, adjustment of the centrifugal conditions is necessary, which is a serious problem. In the method using phosphotungstate magnesium ions, precipitation may vary depending on the pH of the solution. Therefore, there is a problem that it is necessary to strictly adjust the pH.

  In addition, when the supernatant of the centrifuge solution is separated, especially when the amount of the liquid is small, it is difficult to visually determine the boundary area of the precipitate, which may cause problems in reproducibility and accuracy, and individual differences may occur. The quantitative analysis accuracy may be reduced. There is a need to improve the disadvantages associated with these centrifugation operations.

  In recent years, direct methods that can be used in automatic analyzers without the need for these complicated operation methods have rapidly spread. For example, in JP-A-8-131197, sulfated cyclodextrin is used as an aggregating agent, and after sufficiently reacting with lipoproteins other than HDL, an enzyme modified with polyoxyethylene glycol is allowed to act on the HDL. A method for measuring cholesterol in the blood is disclosed. WO98 / 26090 discloses a method in which lipoproteins other than HDL are eliminated with catalase in the first step and HDL-C is measured using an activator that specifically acts on HDL in the second step. Yes. Further, JP-A-9-96637 describes a method in which an antibody against lipoproteins other than HDL is first acted, and then HDL is dissolved to measure cholesterol in HDL.

  However, in order to suppress reactions from lipoproteins other than HDL, it was necessary to use expensive reagents such as PEG-modified enzymes and antibodies.

  In the field of dry chemistry, the precipitation method has been the mainstream, but recently, a new dry-type test piece using the direct method has been developed and described in Japanese Patent No. 3686326. Japanese Patent Application Laid-Open No. 2005-137360 describes that the selectivity is improved by using a lipase derived from Candida rugosa. However, none of the test pieces can completely remove cholesterol in lipoproteins other than HDL.

Nakai Yukihiko, HDL-Metabolism. Assay Methods and Clinical Application (Chugai Medical. 1986) JP 55-78254 A JP 55-93065 A JP 61-263467 A JP-A-62-19768 JP-B-1-39553 JP-A-8-131197 WO98 / 26090 Publication JP-A-9-96637 Patent No. 3686326 JP 2005-137360 A

  The present invention provides a simple, rapid and specific method for measuring HDL-C in a specimen using an inexpensive material, a reagent kit for detecting HDL-C, and a dry analytical element for detecting HDL-C. Was a problem to be solved.

  As a result of diligent research, the present inventors use Schizophyllum commune-derived or Pseudomonas sp-derived cholesterol esterase and Pseudomonas sp-derived cholesterol oxidase as an enzyme in detecting HDL-C in order to solve the above problems. Thus, it was found that cholesterol in HDL can be selectively measured efficiently. The present invention has been completed based on these findings.

  According to the present invention, in a method for measuring high density lipoprotein cholesterol (HDL-C) in a body fluid sample, HDL- using Schizophyllum commune-derived or Pseudomonas sp.-derived cholesterol esterase and Pseudomonas sp.-derived cholesterol oxidase. There is provided a method for measuring HDL-C, wherein HDL-C is selectively measured by generating hydrogen peroxide from C.

Preferably, a surfactant that preferentially dissolves high density lipoprotein (HDL) is used.
Preferably, the surfactant that preferentially dissolves HDL is polyoxyethylene alkylene phenyl ether or polyoxyethylene alkylene tribenzyl phenyl ether.
Preferably, the polyoxyethylene alkylene phenyl ether is polyoxyethylene styryl phenyl ether, and the polyoxyethylene alkylene tribenzyl phenyl ether is polyoxyethylene tribenzyl phenyl ether.
Preferably, the polyoxyethylene styryl phenyl ether is polyoxyethylene mono or di or tristyryl phenyl ether.
Preferably, a surfactant that inhibits the dissolution of lipoproteins other than high-density lipoprotein (HDL) and / or an aggregating agent that aggregates lipoproteins other than HDL is used.

Preferably, the surfactant that inhibits the dissolution of lipoproteins other than HDL is polyoxyethylene alkyl ether sulfate, alkylbenzene sulfonate, or polyoxyethylene-polyoxypropylene condensate.
Preferably, the aggregating agent that aggregates lipoproteins other than HDL is phosphotungstic acid or a salt thereof and a divalent metal ion, dextran sulfate and a divalent metal ion, heparin and a divalent metal ion, or polyoxyethylene. is there.

Preferably, HDL-C is measured by performing a color reaction by allowing peroxidase and a chromogen to act on hydrogen peroxide generated from HDL-C by cholesterol esterase and cholesterol oxidase.
Preferably, 4-aminoantipyrine or a derivative thereof and a Trinder reagent that couples with the 4-aminoantipyrine or a derivative thereof are used as the chromogen.

  Preferably, HDL-C is measured using a dry analytical element having at least an adhesive layer and a porous spreading layer on a water-impermeable support.

  According to another aspect of the present invention, there is provided an HDL-C detection reagent kit comprising at least cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. And cholesterol oxidase derived from Pseudomonas sp.

  According to still another aspect of the present invention, in a dry analytical element having at least an adhesive layer and a porous spreading layer on a water-impermeable support, a layer on the water-impermeable support is formed on a Schizophyllum commune. A dry analytical element for detecting HDL-C, comprising cholesterol esterase derived from or derived from Pseudomonas sp. And cholesterol oxidase derived from Pseudomonas sp. Is provided.

  According to the HDL-C measurement method of the present invention, it has become possible to measure HDL-C in a sample simply, quickly and selectively.

Hereinafter, embodiments of the present invention will be described in detail.
The method for measuring high density lipoprotein cholesterol (HDL-C) according to the present invention generates hydrogen peroxide from HDL-C using cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. And cholesterol oxidase derived from Pseudomonas sp. To selectively measure HDL-C.

  In the present invention, HDL-C in a body fluid sample can be measured. Blood or urine can be used as the body fluid. As the body fluid sample, blood or urine may be used as it is, or a sample subjected to appropriate pretreatment may be used.

Next, reagents used in the method of the present invention will be described.
The enzymes used in the present invention are cholesterol esterase and cholesterol oxidase. Regarding cholesterol esterase, cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. Is used, and cholesterol esterase derived from Schizophyllum commune is particularly preferred. As for cholesterol oxidase, cholesterol oxidase derived from Pseudomonas sp is used. Any of the enzymes used in the present invention may be an enzyme derived from each microorganism, or may be a recombinant product produced by a well-known method.

  As cholesterol esterase derived from Schizophyllum commune, Toyobo COE-302, Pseudomonas sp derived cholesterol esterase from Toyobo COE-311, LPL-312, LPL-314, Asahi Kasei CEN etc. can give. Examples of cholesterol oxidase derived from Pseudomonas sp include CHO-PEL and CHO-PEWL manufactured by Kikkoman.

In the present invention, it is preferable to use a surfactant that preferentially dissolves HDL. As a surfactant that preferentially dissolves HDL, polyoxyethylene alkylene phenyl ether (general formula 1) and polyoxyethylene alkylene tribenzyl phenyl ether (general formula 2) can be used.

As Y in the general formula, a hydrogen atom, a halogen atom, an alkyl group, an alkene group, a phenyl group, a heterocyclic group, a hydroxyl group, an alkyloxy group, a phenyloxy group, an amino group, an alkylamino group, a cyano group, a carbonyl group, A carbonyloxy group, an alkyloxycarbonyl group, etc. are mentioned. Examples of R include a hydrogen atom and an alkyl group having 1 to 8 carbon chains, which may be the same or different within the same molecule. p represents 2 to 6, m represents 0 to 20, and n represents 5 to 100.

The styryl group addition mole number z of (General Formula 3) is 1 to 5, and polyoxyethylene mono- or di- or tristyryl phenyl ether in which z = 1 to 3 is preferable.
A particularly preferred (general formula 1) combination is a styryl group (general formula 3) and polyoxyethylene styryl phenyl ether in which polyoxyalkylene addition mole number m = 0. The average value n of polyoxyethylene addition moles is preferably 5 to 100, more preferably n = 5 to 50, and particularly preferably n = 10 to 50.
The number of n may be a mixture having a certain width instead of a single number. For example, Neugen EA-157 (Daiichi Kogyo Seiyaku Co., Ltd.) can be mentioned as a commercial product of polyoxyethylene distyryl phenyl ether, and the average number of moles of polyoxyethylene added is 17, but n = about 5 to about 30 It is a mixture with a width.

  Particularly preferred (general formula 2) is polyoxyethylene tribenzylphenyl ether in which polyoxyalkylene addition mole number m = 0. The average value n of polyoxyethylene addition moles is preferably 5 to 100, more preferably n = 5 to 50, and particularly preferably n = 10 to 50. The number of n may be a mixture having a certain width instead of a single number. For example, a commercial product of polyoxyethylene tribenzylphenyl ether is Emulgen B66 (manufactured by Kao Corporation), and the average number of moles of polyoxyethylene added moles is 16, but a mixture having a width of n = about 5 to about 30 It has become.

Examples of such commercially available surfactants include Pionine D-6512 (manufactured by Takemoto Yushi Co., Ltd.) as polyoxyethylene monostyryl phenyl ether, Neugen EA-157 as polyoxyethylene distyryl phenyl ether, and Emulgen A90. (Product of Kao), Solpol T-20 (manufactured by Toho Chemical Co., Ltd.) as polyoxyethylene tristyryl phenyl ether, New Coal 2609 (manufactured by Nippon Emulsifier Co., Ltd.) 005 (manufactured by Toho Chemical Industry Co., Ltd.).
These surfactants can be used alone or as a mixture.

  In the present invention, a surfactant that suppresses dissolution of lipoproteins other than HDL can be used. Examples of the surfactant that suppresses the dissolution of lipoproteins other than HDL include surfactants selected from polyoxyethylene alkyl ether sulfates, alkylbenzene sulfonates, and polyoxyethylene-polyoxypropylene condensates. Examples of the polyoxyethylene alkyl ether sulfate include Emar 20C (manufactured by Kao Corporation), and examples of the polyoxyethylene-polyoxypropylene condensate include the Pluronic series (manufactured by Asahi Denka Co., Ltd.).

  In the present invention, an aggregating agent that aggregates lipoproteins other than HDL can be used. As an aggregating agent that agglutinates lipoproteins, Takehiko Nakai, HDL-Metabolism, Assay Methods and Clinical Application (Chugai Medical Company, 1986), 1960 according to various literatures and textbooks A well-known sulfate polysaccharide-alkaline earth metal ion or a divalent metal ion combination other than alkaline earth, an inorganic polyanion salt system, polyethylene glycol, etc. can be used since the beginning of the ages.

  Among these precipitation reagents, the sulfate polysaccharide-metal ion combination system is the dextran sulfate-calcium (2+) ion complex described in Journal of Laboratory and Clinical Medicine, vol. 82, pp. 473 et seq. (1973). Dextran sulfate-magnesium (2+) ion complex described in “J. Lipid Res.”, Vol. 11, pages 583-595 (1970), “Clin, Chem.”, Vol. 24, pages 931-933 (1978), etc. Heparin alone described in “J. Lipid Res.”, Vol. 11, pp. 583-595 (1970), “Manual of Lipid Operations. Lipid Research Clinics Program. Volume I”, Pub. No. (NIH) 75-628 (1978) Precipitation reagents such as a combination of heparin sodium-manganese ions and a combination of heparin-calcium ions-nickel (2+) ions described in JP-A-55-51359 are preferred. Inorganic polyanion salt-based precipitation reagents include “J. Lipid Res.” 11: 583-595 (1970), Clin, Chem. 23: 882-884 (1977), “Clin, Chem.” 25 939-942 (1979), US Pat. No. 4,226,713, JP-B 63-27659 (US Pat. No. 4,215,993), JP-A-01-39553 (US Pat. No. 4,251,519), etc. The combination of) -magnesium (2+) ions is preferred. More preferred are dextran sulfate and magnesium ion.

  In the present invention, in addition to these reagents, well-known enzyme reagents, chromogens, and pH buffering agents are used as reagents for detecting cholesterol.

  Specifically, the enzyme is peroxidase, and the chromogen is 4-aminoantipyrine (4-AA) and a phenolic or aniline Trinder reagent that develops color by hydrogen-donating coupling. The TRENDER reagent is preferably an aniline reagent such as ADPS, ALPS, TOPS, ADOS, DAOS, HDAOS, MAOS, TOOS manufactured by Dojindo Laboratories.

  Examples of pH buffering agents include carbonates, sulfates, phosphates, and Good's pH buffering agents described in Biochemistry, Vol. 5 (No. 2), pages 467-477 (1966). These pH buffering agents can be selected with reference to descriptions in literatures such as “Basic Experiments for Proteins and Enzymes” (Takeichi Horio et al., Nanedo, 1981), “Biochemistry”, Vol.

  The pH of the buffer is determined according to the indicated pH of the enzyme used, and is preferably adjusted to pH 5.0 to 8.0. More preferably, the pH is adjusted to 6.0 to 7.0.

Next, the measurement method of the present invention when the measurement system is a solution will be described. As a composition of the reagent solution, a solution having a composition including the following (1) to (6) is preferable.
(1) cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. (2) cholesterol oxidase derived from Pseudomonas sp. (3) surfactant that preferentially dissolves HDL (4) peroxidase (5) chromogen (4 -AA and Trinder reagent)
(6) pH buffer

  1 to 1000 μL, preferably 100 to 500 μL, of a reagent solution prepared by adjusting the concentration of these reagents to an optimum concentration at a constant temperature in the range of about 20 ° C. to about 45 ° C., preferably in the range of about 30 ° C. to about 40 ° C. Pre-incubate at constant temperature for 1-10 minutes. A solution sample of 0.5 to 50 μL, preferably 1 to 20 μL is added thereto. While incubating at a constant temperature, the time-dependent change in wavelength according to the color development of the chromogen is measured. The amount of the test substance in the sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance.

  As the necessary enzyme amount, any of cholesterol esterase, cholesterol oxidase and peroxidase is preferably used in the range of 0.2 to 20 U / mL. More preferably, it is used at 1 to 10 U / mL.

  When the measurement system is a solution, the surfactant to be used may be only a surfactant that preferentially dissolves high density lipoprotein (HDL). The concentration of the surfactant is preferably 0.01 to 5%. More preferably, it is used at a concentration of 0.1 to 1%.

  Next, the configuration of a dry analytical element whose measurement system is a dry reagent will be described. The dry analytical element has at least one adhesive layer and a porous spreading layer on a water-impermeable support.

  The porous layer may be fibrous or non-fibrous and functions as a spreading layer for a liquid sample. Therefore, the porous layer is preferably a layer having a liquid metering action. The liquid metering action is an action that spreads the liquid sample spotted and supplied to the surface of the layer at a substantially constant rate per unit area in the surface direction of the layer without substantially uneven distribution of the components contained therein. It is. In the spreading layer, a hydrophilic polymer or a surfactant as described in JP-A-60-222770, JP-A-63-119397, JP-A-62-182652 is used to adjust the development area, development speed, etc. Can be contained.

  The fibrous porous layer is preferably made of polyester fiber, as typified by JP-A-55-164356, JP-A-57-66359, JP-A-60-222769, and the like. The non-fibrous porous layer is preferably an organic polymer such as polysulfonic acid.

  The adhesive layer has a function of adhering the water-impermeable support and the porous layer, and gelatin and derivatives thereof (e.g., phthalated gelatin), cellulose derivatives (e.g., hydroxypropyl cellulose), agarose, Hydrophilic polymers such as acrylamide polymers, methacrylamide polymers, acrylamide or copolymers of methacrylamide and various vinyl monomers can be used.

  An aqueous solution containing a hydrophilic polymer is uniformly applied by a known method, and a known method can be used for the application method. For coating, for example, dip coating, extrusion coating, doctor coating, hopper coating, curtain coating and the like can be appropriately selected and used.

  Although a porous layer can be applied on the adhesive layer, it is preferable to laminate a cloth or a porous film supplied in advance as a knitted fabric. As described in JP-A-55-164356, the laminating method is a method in which the surface of the adhesive layer containing a hydrophilic polymer is uniformly moistened with water, and a cloth or a porous film is layered on the surface to lightly approximately. Bonding is performed by applying pressure uniformly. The thickness of the adhesive layer is preferably 0.5 to 50 μm, more preferably 1 to 20 μm.

  Preferred materials for the light-transmitting support are cellulose ethers such as polyethylene terephthalate, polystyrene, and cellulose triacetate. In order to firmly adhere the water-absorbing layer, the detection layer, the substantially nonporous reagent layer, etc. of the hydrophilic layer to the support, an undercoat layer is usually provided on the support or a hydrophilic treatment is performed. The thickness of the support is not particularly limited, but is preferably 10 to 1000 μm, and more preferably 300 to 800 μm. In the case of a light-transmitting support, the final detection may be on the support side or on the porous layer side, but in the case of light-opacity, detection is performed from the porous layer side.

  Next, the reagent composition for measuring cholesterol used in the dry analytical element used in the measuring method of the present invention and the reagent composition causing an optical change will be described.

  The reagent composition may be included in the first porous layer, may be included in both the adhesive layer and the porous layer, or may be entirely or mostly included in any layer. Alternatively, it may be included in a layer other than the adhesive layer and the porous layer.

  In the dry analytical element for detecting HDL-C, it is preferable to use 0.1 to 20 kU per square meter of any enzyme such as cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. Or cholesterol oxidase derived from Pseudomonas sp. More preferably, 0.5 to 10 kU is used per square meter.

  The two types of surfactants (that is, surfactants that preferentially dissolve HDL and surfactants that inhibit the dissolution of lipoproteins other than HDL) are both supplied in an amount of 0.2 to 20 g per square meter. More preferably, 1-10 g is supplied. The ratio of the surfactant that preferentially dissolves HDL and the surfactant that inhibits the dissolution of lipoproteins other than HDL is preferably 9/1 to 5/5. More preferably, it is used at a ratio of 8/2 to 6/4.

The aggregating agent that agglutinates lipoproteins other than HDL preferably uses dextran sulfate (MW = 5000000) and magnesium ion, 0.05 to 10 g per square meter as dextran sulfate, and per 1 square meter as magnesium chloride hexahydrate. 0.01 to 20 g is preferably used. More preferably, dextran sulfate 0.1-5 g / m ² magnesium chloride hexahydrate 0.5-10 g / m ^{2 is} used.

The origin of the peroxidase is not particularly limited, but is preferably derived from horseradish. The amount used, 1~100kU / m ² is preferred, more preferably 10~50kU / m ² is used.

With respect to the chromogen, a combination of the reagents that develop color by coupling with 4-aminoantipyrine (4-AA) is preferred, and DAOS is particularly preferred. The amount of the chromogen used is preferably 0.1 to 10 g / m ² for both 4-AA and the hydrogen-donating coupling agent. More preferably, 0.3 to 5 g / m ^{2 is} used.

  Other reagent compositions in dry analytical elements for detecting HDL-C contain stabilizers, pH buffering agents, crosslinking agents (hardeners or curing agents), surfactants, polymers, etc. as necessary. Can be made. It can be contained in the adhesive layer or the porous layer of the dry analytical element of the present invention.

  The pH of the buffer is determined according to the indicated pH of the enzyme used, and is preferably adjusted to pH 5.0 to 8.0. More preferably, the pH is adjusted to 6.0 to 7.0.

  The dry analytical element of the present invention is, for example, cut into small pieces such as a square having a side of about 5 mm to about 30 mm or a circle having the same size, and disclosed in Japanese Patent Publication No. 57-283331 (corresponding US Pat. No. 4,169,751), Japanese Unexamined Patent Publication No. 56-142454 (corresponding US Pat. No. 4,387,990), Japanese Unexamined Patent Publication No. 57-63452, Japanese Utility Model Publication No. 58-32350, Japanese Patent Publication No. 58-501144 (corresponding international publication: WO083). / 00391) and the like and can be used as a chemical analysis slide, which is preferable from the viewpoint of manufacturing, packaging, transportation, storage, measurement operation, and the like. Depending on the purpose of use, it is used in a long tape form in a cassette or magazine, or a small piece is placed in a container with an opening, or a small piece is affixed or placed in an open card, or a cut piece is used. It can be used as it is.

  In the dry analytical element of the present invention, for example, an aqueous liquid sample solution in the range of about 2 μL to about 30 μL, preferably 4 μL to 15 μL is spotted on the porous liquid sample developing layer. The spotted dry analytical element is incubated at a constant temperature in the range of about 20 ° C. to about 45 ° C., preferably at a constant temperature in the range of about 30 ° C. to about 40 ° C. for 1 to 10 minutes. The color development or discoloration in the dry analytical element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of the test substance in the sample can be determined based on the principle of the colorimetric measurement method using a calibration curve prepared in advance.

  The measuring operation is disclosed in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding to US Pat. No. 4,424,191). With the described chemical analyzer, highly accurate quantitative analysis can be performed with extremely easy operation. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.

  Since the dry analytical element of the present invention is stored and stored in a dry state until analysis is performed, it is not necessary to prepare the reagent at the time of use, and generally the dry state has higher reagent stability. It is more convenient and quicker than the so-called solution method in which the solution must be prepared at the time of use. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Solution system measurement example using the enzyme of the present invention A reagent solution having the following composition was prepared.
MES buffer (pH7.0) 700mmlo / L
Cholesterol esterase (derived from Schizophyllum commune) 4.5U / mL
Cholesterol oxidase (derived from Pseudomonas sp.) 2.8 U / mL
Peroxidase 4.8U / mL
4-Aminoantipyrine (Wako Pure Chemical Industries, Ltd.) 4.0mmol / L
DAOS (Dojin Laboratories) 4.0mmol / L
Emulgen B66 (Kao Corporation) 0.62mg / mL

  Samples prepared from purified HDL and LDL products with a cholesterol concentration of 100 mg / dL and 7% HSA aqueous solution were used as samples. 245 μL of the reagent solution was previously incubated at 37 ° C. for 3 minutes, 5 μL of the sample was added thereto, and the state of color development at 600 nm was measured. As a result, as shown in FIG. 1, according to the above method, HDL was completely colored in about 5 minutes, but LDL was hardly changed.

Comparative Example 1: When using cholesterol esterase of other origin A reagent prepared with the same formulation as in Example 1 and only cholesterol esterase having the following origin was used.
Cholesterol esterase (derived from Chromobacterium viscosum) 4.5U / mL
The same specimen as in Example 1 was used for measurement.

  As a result, as shown in FIG. 2, the sensitivity was low and specificity was low for both HDL and LDL.

Comparative Example 2: When using cholesterol oxidase of other origin A reagent prepared with the same formulation as in Example 1 and only cholesterol oxidase having the following origin was used.
Cholesterol oxidase (from Microorganism) 2.8U / mL
The same specimen as in Example 1 was used for measurement.

  As a result, as shown in FIG. 3, the sensitivity was low and specificity was low for both HDL and LDL.

Comparative Example 3: When using cholesterol esterase and cholesterol oxidase from other origins Reagents prepared according to the same formulation as in Example 1, and cholesterol esterase and cholesterol oxidase having the following origins were used.
Cholesterol esterase (derived from Chromobacterium viscosum) 4.5U / mL
Cholesterol oxidase (from Microorganism) 2.8U / mL
Measurement was performed using the same specimen as in Example 1.

  As a result, as shown in FIG. 4, the sensitivity was low and specificity was low for both HDL and LDL.

Example 2 Dry Analytical Element Using Enzyme of the Invention A gelatin aqueous solution was coated on a 180 μm polyethylene terephthalate colorless and transparent smooth film coated with gelatin so that the thickness after drying was 14 μm and dried. Next, water is supplied over the entire surface at a supply rate of about 30 g / m ² and moistened, and then a tricot knitted fabric knitted with 36 gauge polyester spun yarn equivalent to 50 denier is laminated with light pressure. , Dried. Next, an aqueous solution having the following composition was applied and dried on the cloth.

MES buffer (pH 6.6) 18g / m ²
Cholesterol esterase (from Schizophyllum commune) 1.9 kU / m ²
Cholesterol oxidase (derived from Pseudomonas sp.) 1.2kU / m ²
Peroxidase 31kU / m ²
4-aminoantipyrine (manufactured by Wako Pure Chemical Industries, Ltd.) 0.4 g / m ²
DAOS (Dojindo Laboratories) 0.4g / m ²
Emulgen B66 (manufactured by Kao Corporation) 2.0g / m ²
Pluronic F-88 (Asahi Denka) 1.3g / m ²
Dextran sulfate (5000,000) (Wako Pure Chemical Industries) 0.7g / m ²
Magnesium chloride hexahydrate (Wako Pure Chemical Industries, Ltd.) 4.6 g / m ²

  Samples prepared by adjusting HDL and LDL purified products to a cholesterol concentration of 100 mg / dL and 7% HSA aqueous solution were used as samples. A 10 μL sample was spotted on the dry analytical element, and then incubated at 37 ° C. for 6 minutes. The state of color development at 600 nm at this time was measured. As a result, as shown in FIG. 5, according to the above method, although HDL developed a complete color in about 5 minutes, the OD of LDL hardly changed.

  For 25 healthy individuals, the multi-analyte correlation was examined for the method of the present invention and the fractionation method using phosphotungstic acid. As a result, as shown in FIG. 6, the present invention was able to obtain a good correlation as compared with the reference method.

Comparative Example 4
A dry analytical element was produced in the same manner as in Example 2, but cholesterol esterase and cholesterol oxidase derived from the following were used.
Cholesterol esterase (from Chromobacterium viscosum) 1.9U / m ²
Cholesterol oxidase (from Microorganism) 1.2U / m ²
A sample similar to that in Example 2 was used, and measurement was performed in the same manner.

  As a result, as shown in FIG. 7, the analytical element produced by the above method has low reactivity to HDL, low reactivity to LDL, and no HDL specificity.

Example 3: Dry analytical element (2) using the enzyme of the present invention
Emulgen B66 of Example 2 was changed to Peganol 005 (manufactured by Toho Chemical Co., Ltd.), Pionin D-6512 (manufactured by Takemoto Yushi Co., Ltd.), Emulgen A90 (manufactured by Kao Corporation), Neugen EA-157 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Solpol. A dry analytical element was produced in the same manner except that it was changed to T20 (manufactured by Toho Chemical Co., Ltd.).

  Samples prepared by adjusting HDL and LDL purified products to a cholesterol concentration of 100 mg / dL and 7% HSA aqueous solution were used as samples. A 10 μL sample was spotted on the dry analytical element, and then incubated at 37 ° C. for 6 minutes. The state of color development at 600 nm at this time was measured. As a result, as shown in FIGS. 8 to 12, according to the above method, HDL was completely colored in about 5 minutes, but the OD of LDL was hardly changed.

FIG. 1 shows the results of a solution system measurement example (Example 1) using the enzyme of the present invention. FIG. 2 shows the results of a solution-based measurement example (Comparative Example 1) when another cholesterol esterase is used. FIG. 3 shows the results of a solution-based measurement example (Comparative Example 2) in the case of using cholesterol oxidase derived from another source. FIG. 4 shows the results of a measurement example (Comparative Example 3) of a solution system using cholesterol esterase and cholesterol oxidase derived from other sources. FIG. 5 shows the results of a measurement example (Example 2) using a dry analytical element using the enzyme of the present invention. FIG. 6 shows the results of examining the multi-sample correlation between the method of the present invention and the fractionation method using phosphotungstic acid. FIG. 7 shows the results of a measurement example (Comparative Example 4) using a dry analytical element using cholesterol esterase and cholesterol oxidase derived from other sources. FIG. 8 shows the results of a measurement example (Example 3, Peganol 005) using a dry analytical element using the enzyme of the present invention. FIG. 9 shows the results of a measurement example using a dry analytical element using the enzyme of the present invention (Example 3, Pionine D-6512). FIG. 10 shows the results of a measurement example (Example 3, Emulgen A90) using a dry analytical element using the enzyme of the present invention. FIG. 11 shows the results of a measurement example (Example 3, Neugen EA-157) using a dry analytical element using the enzyme of the present invention. FIG. 12 shows the results of a measurement example (Example 3, Solpol T20) using a dry analytical element using the enzyme of the present invention.

Claims (13)

In a method for measuring high density lipoprotein cholesterol (HDL-C) in a body fluid sample, hydrogen peroxide from HDL-C is obtained using cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. And cholesterol oxidase derived from Pseudomonas sp. A method for measuring HDL-C, wherein HDL-C is selectively measured by generating the HDL-C. The method for measuring HDL-C according to claim 1, wherein a surfactant that preferentially dissolves high-density lipoprotein (HDL) is used. The method for measuring HDL-C according to claim 2, wherein the surfactant that preferentially dissolves HDL is polyoxyethylene alkylene phenyl ether or polyoxyethylene alkylene tribenzyl phenyl ether. The method for measuring HDL-C according to claim 3, wherein the polyoxyethylene alkylene phenyl ether is polyoxyethylene styryl phenyl ether and the polyoxyethylene alkylene tribenzyl phenyl ether is polyoxyethylene tribenzyl phenyl ether. The method for measuring HDL-C according to claim 4, wherein the polyoxyethylene styryl phenyl ether is polyoxyethylene mono-, di- or tristyryl phenyl ether. The HDL-C according to any one of claims 1 to 5, wherein a surfactant that inhibits dissolution of lipoproteins other than high-density lipoprotein (HDL) and / or an aggregating agent that aggregates lipoproteins other than HDL is used. Measuring method. The HDL-C according to claim 6, wherein the surfactant that inhibits the dissolution of lipoproteins other than HDL is polyoxyethylene alkyl ether sulfate, alkylbenzene sulfonate, or polyoxyethylene-polyoxypropylene condensate. Measuring method. The aggregating agent for aggregating lipoproteins other than HDL is phosphotungstic acid or a salt thereof and a divalent metal ion, dextran sulfate and a divalent metal ion, heparin and a divalent metal ion, or polyoxyethylene. Item 8. The method for measuring HDL-C according to Item 6 or 7. The HDL according to any one of claims 1 to 8, wherein HDL-C is measured by performing a color reaction by allowing peroxidase and a chromogen to act on hydrogen peroxide generated from HDL-C by cholesterol esterase and cholesterol oxidase. -C measurement method. The method for measuring HDL-C according to claim 9, wherein 4-aminoantipyrine or a derivative thereof and a Trinder reagent coupled to the 4-aminoantipyrine or a derivative thereof are used as the chromogen. The HDL-C according to any one of claims 1 to 10, wherein HDL-C is measured using a dry analytical element having at least an adhesive layer and a porous spreading layer on a water-impermeable support. Measuring method. A reagent kit for detecting HDL-C, comprising at least cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. And cholesterol oxidase derived from Pseudomonas sp. In a dry analytical element having at least an adhesive layer and a porous spreading layer on a water-impermeable support, a cholesterol esterase derived from Schizophyllum commune or Pseudomonas sp. A dry analytical element for detecting HDL-C, comprising cholesterol oxidase derived from Pseudomonas sp.