Evaluation device, system, and evaluation method

Abstract

Provided is an evaluation device capable of quickly evaluating the amount of oxidized LDL. The present invention is provided with: an acquisition unit that acquires information on the amount of change in absorbance per unit time, said information being obtained by a measurement method in which LDL in a sample is solubilized by a surfactant that acts on LDL; introducing hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase into a chromogenic reaction by causing peroxidase to act on the hydrogen peroxide, and measuring a change in absorbance over time; and an evaluation unit that evaluates the amount of oxidized LDL in the specimen on the basis of the change amount.

Description

Technical Field

[0001] The present invention relates to an evaluation device, a system, and an evaluation method. Background Art

[0002] In the field of clinical examinations, biochemical automatic analyzers that rapidly and automatically measure the amount of a specific component contained in a specimen have been widely spread. Since there are a very large number of inclusions in a specimen, an antibody that selectively binds to a target compound or an enzyme that selectively reacts with the target compound is used to detect the target compound from among them. Detection reagents for detecting the target compound combined with these antibodies or enzymes have been developed by reagent manufacturers. Such reagents are contrived to perform multi-step reactions by a one-pot method in order to measure with high sensitivity and rapidly. For example, in Patent Document 1 or Patent Document 2, a method for quantifying low-density lipoprotein cholesterol (LDL-C) using such a reagent is disclosed.

[0003] Lipids ingested by the small intestine or lipids biosynthesized in the liver (fatty acids or cholesterol) are transported via the blood as spherical molecules such as lipoproteins that are stabilized by covering their surroundings with phospholipids, cholesterol, and apolipoproteins in the state of triglycerides or cholesterol esters (see Non-Patent Document 1).

[0004] Lipoproteins are classified, starting from the lowest density, into chylomicrons (CM), very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) according to differences in size, density, and apolipoprotein composition. Cholesterol encapsulated in these lipoproteins is widely measured in the field of clinical examinations as an indicator of various lipid-related diseases.

[0005] Among them, the value of LDL-C is a risk factor for arteriosclerosis. Therefore, LDL-C lowering therapy using statins and the like has been widely spread (see Non-Patent Document 2). However, since the correlation between the LDL-C value and the onset of a disease is not necessarily high, and the onset risk cannot be completely eliminated even by lowering the LDL-C value, other risk factors have been pointed out.

[0006] Oxygen required for maintaining life changes into reactive oxygen under external stimuli and functions as a cell signaling substance or immune function. However, excessive production can damage cells and become a factor leading to various diseases such as cancer, cardiovascular diseases, and lifestyle-related diseases. Lipoproteins can also cause these diseases due to oxidative stress. Among them, oxidized LDL (hereinafter also referred to as "ox-LDL") is taken up by cells such as macrophages and causes foaming of cells, so it is considered to be one of the factors in the development of arteriosclerosis. The amount of ox-LDL is an important indicator for diseases caused by functional changes in vascular endothelial cells such as arteriosclerosis and coronary syndrome.

[0007] As a method for measuring ox-LDL, a method of colorimetric quantification of ox-LDL by sandwich ELISA (Enzyme Linked ImmunoSorbent Assay) is known. For example, in Patent Document 3, a diagnostic kit for detecting oxidized lipoproteins containing apoB100 in blood by sandwich ELISA is disclosed.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: Japanese Patent Laid-Open No. 10-38888

[0011] Patent Document 2: Japanese Patent Laid-Open No. 9-313200

[0012] Patent Document 3: Japanese Patent No. 3561218

[0013] Non-Patent Documents

[0014] Non-Patent Document 1: Structure and Physiological Functions of Plasma Lipoproteins, Journal of Oleo Science, Vol. 40, No. 10, 1991, pp. 858-868

[0015] Non-Patent Document 2: Oxidized Lipoproteins Involved in the Onset and Progression of Arteriosclerosis, Transactions of the Showa Academy of Sciences, Vol. 81, No. 5, 2021, pp. 370-379 Summary of the Invention

[0016] Problems to be Solved by the Invention

[0017] However, in the above sandwich ELISA method, complicated processing is required and rapid measurement cannot be performed.

[0018] Solutions for Solving the Problems

[0019] One aspect of the evaluation device of the present invention includes:

[0020] An acquisition unit that acquires information on the change amount of absorbance per unit time, and the information is obtained by the following measurement method: solubilize LDL with a surfactant that acts on LDL in the sample, and peroxidase acts on the hydrogen peroxide generated in the oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into the color reaction, and measure the time change of the absorbance; and

[0021] An evaluation unit that evaluates the amount of oxidized LDL in the sample based on the change amount.

[0022] In such an evaluation device, it is possible to evaluate the amount of oxidized LDL in the sample based on the change amount of absorbance per unit time that can be measured by an automatic analyzer. Therefore, in such an evaluation device, the amount of oxidized LDL can be evaluated quickly.

[0023] One aspect of the system of the present invention includes:

[0024] A measurement unit that solubilizes LDL with a surfactant that acts on LDL in the sample, and peroxidase acts on the hydrogen peroxide generated in the oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into the color reaction, measures the time change of the absorbance, and outputs data on the time change of the absorbance;

[0025] A differential operation unit that obtains the change amount of the absorbance per unit time from the data; and

[0026] An evaluation unit that evaluates the amount of oxidized LDL in the sample based on the change amount.

[0027] In such a system, it is possible to evaluate the amount of oxidized LDL in the sample based on the change amount of absorbance per unit time that can be measured by an automatic analyzer. Therefore, in such a system, the amount of oxidized LDL can be evaluated quickly.

[0028] One aspect of the evaluation method of the present invention includes:

[0029] A step of obtaining information on the change amount of absorbance per unit time, and the information is obtained by the following measurement method: solubilize LDL with a surfactant that acts on LDL in the sample, and peroxidase acts on the hydrogen peroxide generated in the oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into the color reaction, and measure the time change of the absorbance; and

[0030] A step of evaluating the amount of oxidized LDL in the specimen based on the change amount.

[0031] In such an evaluation method, the amount of oxidized LDL in the specimen can be evaluated based on the change amount of absorbance per unit time that can be measured by an automatic analyzer. Therefore, in such an evaluation method, the amount of oxidized LDL can be evaluated rapidly.

[0032] One aspect of the evaluation method of the present invention includes:

[0033] A step of reacting a specimen containing a first substance and a second substance with a reagent, and measuring the change amount per unit time of the physical quantity of a reaction product containing a first reaction product generated by a first reaction of the first substance and the reagent and a second reaction product generated by a second reaction of the second substance and the reagent; and

[0034] A step of evaluating the physical quantity of the second substance based on the change amount.

[0035] In such an evaluation method, the physical quantity of the second substance can be evaluated based on the change amount per unit time of the physical quantity of the reaction product. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a diagram for explaining the first step of a method for measuring LDL cholesterol.

[0037] Figure 2 It is a diagram for explaining the second step of a method for measuring LDL cholesterol.

[0038] Figure 3 It is a diagram for explaining an evaluation method of oxidized LDL.

[0039] Figure 4 It is a coordinate diagram showing the result of differential measurement of oxidized LDL.

[0040] Figure 5 It is a coordinate diagram showing the measurement result of the absorbance of an oxidized specimen before dialysis.

[0041] Figure 6 It is a coordinate diagram showing the measurement result of the absorbance of an oxidized specimen after dialysis.

[0042] Figure 7 It is a table showing the production conditions of oxidized specimens A, B, C, D, E, and F.

[0043] Figure 8 It is a table showing the incubation time of an oxidized specimen and a reaction delay index.

[0044] Figure 9It is a histogram showing the reaction delay index for each incubation time.

[0045] Figure 10 It is a table showing the incubation time, reaction delay index, LDL cholesterol concentration, and oxidized LDL ratio of the oxidized specimen.

[0046] Figure 11 It is a histogram showing the reaction delay index and LDL cholesterol concentration for each incubation time.

[0047] Figure 12 It is a histogram showing the oxidized LDL ratio for each incubation time.

[0048] Figure 13 It is a table showing the incubation time and reaction delay index of the oxidized specimen.

[0049] Figure 14 It is a histogram showing the reaction delay index for each incubation time.

[0050] Figure 15 It is a diagram showing the configuration of the evaluation system according to an embodiment of the present invention.

[0051] Figure 16 It is a diagram schematically showing an example of the configuration of the automatic analysis device.

[0052] Figure 17 It is a diagram showing an example of the configuration of the evaluation device.

[0053] Figure 18 It is a flowchart showing an example of the processing of the evaluation device.

[0054] Explanation of Reference Numerals

[0055] 2… Evaluation system, 4… Communication network, 20… Automatic analysis device, 40… Evaluation device, 60… Information processing terminal, 202… Sample turntable, 204… First turntable, 205… Second turntable, 206… Reaction turntable, 207… Specimen dispensing probe, 210… Sample barcode reader, 212… First reagent dispensing probe, 213… Second reagent dispensing probe, 214… First reaction solution stirring mechanism, 215… Second reaction solution stirring mechanism, 216… Multi-wavelength photometer, 217… Thermostatic bath, 218… Reaction vessel cleaning mechanism, 221… Sample container, 224… First reagent container, 225… Second reagent container, 226… Reaction container, 231… Specimen dispensing probe cleaning mechanism, 233… First reagent dispensing probe cleaning mechanism, 234… Second reagent dispensing probe cleaning mechanism, 240… Control unit, 400… Processing unit, 402… Acquisition unit, 404… Evaluation unit, 406… Data generation unit, 410… Operation unit, 420… Display unit, 430… Storage unit, 440… Communication unit. Detailed implementation mode

[0056] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Moreover, the embodiments described below are not intended to unduly limit the content of the present invention described in the claims. In addition, all the components described below are not necessarily essential components of the present invention.

[0057] 1. Evaluation method

[0058] 1.1. Method for measuring LDL cholesterol

[0059] First, a method for measuring LDL cholesterol (quantitative measurement) in a specimen will be described. In addition, the specimen to be measured is blood, or body fluids such as serum, plasma, bone marrow fluid, urine, sweat, puncture fluid, feces, and specimens obtained by preprocessing them (such as dilution). In addition, LDL cholesterol is the cholesterol contained in LDL.

[0060] The method for measuring LDL cholesterol includes: a first step of removing lipoproteins other than LDL in the specimen in the presence of a first surfactant that acts on lipoproteins other than LDL; and a second step of adding a second surfactant to solubilize the remaining LDL, and allowing peroxidase to act on hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into a color reaction and measuring the absorbance.

[0061] In the method for measuring LDL cholesterol, an LDL cholesterol measurement reagent is used for the measurement. The LDL cholesterol measurement reagent contains a first reagent and a second reagent. The first reagent is, for example, an enzyme solution containing 4-aminoantipyrine, cholesterol oxidase, cholesterol esterase, peroxidase, and a first surfactant. The second reagent is, for example, a coloring solution containing a chromogenic agent (DSBmT: N,N-bis(4-sulfobutyl)-m-toluidine) and a second surfactant. The first reagent is added to the sample in the first step, and the second reagent is added to the sample in the second step. In addition, as the cholesterol oxidase, any enzyme having the ability to oxidize cholesterol to generate hydrogen peroxide may be used, and there is no particular limitation. Further, as the cholesterol esterase, any enzyme having the ability to hydrolyze cholesterol may be used, and there is no particular limitation.

[0062] Figure 1 It is a diagram for explaining the first step of the method for measuring LDL cholesterol.

[0063] In the first step, in the presence of the first surfactant, cholesterol esterase and cholesterol oxidase are allowed to act on cholesterol in lipoproteins other than LDL (high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), chylomicron (CM), etc.) to generate hydrogen peroxide. The enzymes used here can be unmodified or modified with a group mainly composed of polyethylene glycol, polypropylene glycol, etc., or a group having a copolymer of polypropylene glycol and polyethylene glycol, a group structurally containing sugar, a sulfopropyl group, or a polyurethane group. The first surfactant dissolves the outer wall of lipoproteins other than LDL and dissolves the cholesterol in lipoproteins other than LDL. Thus, the cholesterol in lipoproteins other than LDL can be introduced into the enzyme reaction using cholesterol esterase and cholesterol oxidase. The generated hydrogen peroxide is scavenged by peroxidase and 4-aminoantipyrine. In addition, the so-called scavenging means decolorizing in such a way that it cannot be detected in the absorbance measurement in the second step.

[0064] The first surfactant has the effect of changing the structure of lipoproteins other than LDL, but does not change the structure of LDL. That is, the first surfactant does not dissolve the outer wall of LDL. As the first surfactant, for example, polyalkylene oxide derivatives with an HLB value of 13 or more and 15 or less can be cited. Examples of the derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercaptan condensates, and alkylphenol condensates. Examples of polyalkylene oxide derivatives with an HLB value of 13 or more and 15 or less include polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, and polyoxyethylene isodecyl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene octylphenyl ether and polyoxyethylene nonylphenyl ether; compounds with an HLB value of 13 or more and 15 or less such as polyoxyethylene tribenzylphenyl ether, but are not limited to them. The first surfactant can also be a surfactant containing a branched alkyl ether structure. The first surfactant can also be a surfactant without double bonds such as polyoxyethylene polyoxypropylene alkyl ether. The first surfactant can also be a combination of a polyoxyethylene polyoxypropylene copolymer and a polyglycerol ether.

[0065] Examples of commercially available products of the first surfactant include EMULGEN B66 (polyoxyethylene tribenzylphenyl ether, HLB = 13.2) manufactured by Kao Corporation, SAFETYCUT ID-1087 (polyoxyethylene isodecyl ether) manufactured by Aoki Yushi Industry Co., Ltd., and the like.

[0066] In addition, as the first surfactant, a cationic surfactant can also be used. The first surfactant can also be allylamine or a polymer compound having a diallylamine unit. The first surfactant can also be a cholic acid derivative and its salt. In the first surfactant, in order to inhibit the reaction with LDL and further improve the clearance of other lipoproteins, divalent metal ions can be included in the reaction solution. As the divalent metal ions, copper ions, iron ions, and magnesium ions can be used, but magnesium ions are particularly preferred. In addition, a lipoprotein-degrading enzyme can also be optionally added to the reaction solution in the first step. By adding this enzyme, cholesterol in VLDL in particular becomes more reactive, so it is preferred.

[0067] Figure 2 It is a diagram for explaining the second step of the method for measuring LDL cholesterol.

[0068] In the second step, a second reagent is added to the specimen that has undergone the first step. The second surfactant contained in the second reagent dissolves the outer wall of the LDL remaining in the first step, causing the cholesterol inside the LDL to dissolve. Cholesterol esterase and cholesterol oxidase are allowed to act on the dissolved cholesterol to produce hydrogen peroxide. In the second step, as a chromogenic agent, a toluidine-based substance such as DSBmT or an aromatic pigment precursor similar thereto is added, and the hydrogen peroxide produced is introduced into a color reaction. In the color reaction, peroxidoreductase is allowed to act on the hydrogen peroxide, and an oxidative coupling reaction is carried out between the aromatic nucleophile as the pigment precursor and the aromatic electrophile to generate a pigment. Specifically, peroxidase is allowed to act on the hydrogen peroxide, and an oxidative coupling reaction is carried out between 4-aminoantipyrine and DSBmT to generate a red-violet pigment. The LDL cholesterol is quantified by measuring the absorbance of the color development of this red-violet pigment.

[0069] The second surfactant may have the effect of changing the structure of all lipoproteins or may have the effect of changing only the structure of LDL. As the second surfactant having the effect of changing the structure of all lipoproteins, for example, polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13 can be cited. As the derivatives, for example, higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercaptan condensates, and alkylphenol condensates can be cited.

[0070] As polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13, for example, compounds such as polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octylphenyl ether, and polyoxyethylene nonylphenyl ether having an HLB value of 11 or more and less than 13 can be cited, but are not limited to them.

[0071] As commercially available products of the second surfactant, EMULGEN A60 (polyoxyethylene stilbenized phenyl ether, HLB = 12.8) manufactured by Kao Corporation, Triton (registered trademark) X-100, etc. can be cited.

[0072] In addition, as the second surfactant having the effect of changing only the structure of LDL, an anionic surfactant can be cited. As the anionic surfactant, a surfactant in which a linear or branched alkyl group having 4 to 18 carbon atoms is bonded to an aromatic ring is preferred. Here, the aromatic ring is preferably an aromatic ring composed only of carbon and hydrogen such as benzene, naphthalene, or biphenyl. Moreover, a surfactant having a hydrophilic group such as a sulfonate bonded to the above aromatic ring is preferred.

[0073] 1.2. Evaluation method for oxidized LDL

[0074] Next, the evaluation method for oxidized LDL of the present embodiment will be described.Figure 3 This is a figure for explaining the evaluation method of oxidized LDL. In addition, oxidized LDL is a substance obtained by oxidatively modifying LDL through the action of reactive oxygen species or the like. The amount of oxidized LDL is an important indicator for diseases caused by functional changes in vascular endothelial cells, such as arteriosclerosis and coronary syndrome.

[0075] In the second step, as Figure 3 shown, when cholesterol oxidase reacts with cholesterol, the 3-hydroxy group of cholesterol is oxidized to a carbonyl group. Furthermore, the carbonyl group undergoes a shift to promote the decomposition reaction. As a result, cholesterol is removed.

[0076] Along with the oxidation of cholesterol by the above-mentioned cholesterol oxidase, hydrogen peroxide is generated. When the peroxide structure is cleaved by peroxidase using the generated hydrogen peroxide as a substrate, two protons are captured from 4-aminoantipyrine and a toluidine compound (DSBmT), and the two are oxidatively combined. Thereby, a pigment is generated.

[0077] Here, lipid peroxide (16:O-18:2+32PC) generated by the oxidation of phospholipid by reactive oxygen species also has a peroxide structure like hydrogen peroxide and becomes a substrate for peroxidase. Therefore, like hydrogen peroxide, when the peroxide structure of lipid peroxide is cleaved by peroxidase, two protons are captured from 4-aminoantipyrine and a toluidine compound (DSBmT), and the two are oxidatively combined to generate a pigment. In addition, like the case of the above-mentioned LDL cholesterol, lipid peroxide is decomposed by the reaction of peroxidase.

[0078] Therefore, if the main reaction of the reagent is set as a color reaction carried out by the reaction of LDL with the reagent, and the side reaction of the reagent is set as a color reaction carried out by the reaction of oxidized LDL with the reagent, then by observing this side reaction, lipid peroxide (oxidized LDL) can be evaluated. In the present embodiment, the side reaction is observed by focusing on the fact that the side reaction is delayed relative to the main reaction.

[0079] As one of the reasons for this delay of the side reaction relative to the main reaction, a difference in the solubilization reaction rate when solubilizing cholesterol by solubilizing LDL with the second surfactant and the solubilization reaction rate when solubilizing lipid peroxide by solubilizing oxidized LDL with the second surfactant can be cited. That is, the solubilization reaction rate when solubilizing oxidized LDL to solubilize lipid peroxide is slower than the solubilization reaction rate when solubilizing LDL to solubilize cholesterol. The difference in the solubilization reaction rate is considered to be due to the following reason: the dissolution ability of the second surfactant for the outer wall of LDL changes due to the oxidative modification of the outer wall of LDL.

[0080] In addition, as one of the reasons for the delay of the side reaction relative to the main reaction, the difference in the reaction rates between hydrogen peroxide, which is a substrate of peroxidase, and lipid peroxide can be cited. Since the reaction rate of lipid peroxide relative to peroxidase is slower than that of hydrogen peroxide relative to peroxidase, the side reaction is delayed relative to the main reaction.

[0081] Furthermore, when LDL undergoes oxidative modification, various phenomena such as the promotion of lipid peroxidation reaction, the modification of apoprotein, and the change in particle state occur. Therefore, it is considered that this delay of the side reaction relative to the main reaction is not caused only by the above two factors, but by the superposition of various factors.

[0082] Oxidized phospholipid (OxPL) is detected by apolipoprotein B (apoB) in plasma, and OxPL-apoB shows a stronger correlation with lipoprotein (a) [Lp(a)], which is a subtype of lipoprotein. That is, this indicates that Lp(a) is the largest carrier of OxPL and is involved in the delay of the side reaction relative to the main reaction.

[0083] The LDL-C value measured by a general clinical examination reagent is the sum of true LDL-C and cholesterol contained in Lp(a) (LDL(a)-C). As a method for measuring LDL(a)-C, a method of extracting and quantifying LDL(a) using an anti-LDL(a) antibody bound to magnetic beads is known. When the reaction delay of the side reaction represents the LDL(a)-C value or it is found that there is some involvement, in this embodiment, such a complicated operation can be omitted, so it is useful.

[0084] In addition, LDL is divided into finer subfractions according to its lipid composition, and the smaller the particle size, the easier it is to be oxidized. These small particles have a higher specific gravity and are called small dense LDL (sd-LDL), while the large particles have a lower specific gravity and are called large buoyant LDL (lb-LDL). It is said that among LDL particles, sd-LDL has a higher arteriosclerosis-inducing property. Both sd-LDL and lb-LDL show a positive correlation with the LDL-C value, but sd-LDL and lb-LDL show a negative correlation with each other. The reaction delay of the side reaction may also be due to the components of sd-LDL.

[0085] Such a delay of the side reaction relative to the main reaction can be observed, for example, by measuring the change in absorbance per unit time in the above-mentioned method for measuring LDL cholesterol. In this embodiment, the amount of oxidized LDL is evaluated based on the change in absorbance per unit time.

[0086] 2. Experimental Examples

[0087] Hereinafter, the evaluation method of oxidized LDL will be specifically described using experimental examples, but the present invention is not limited to these examples.

[0088] 2.1. Determination method

[0089] Measuring device: JCA-BM8040 manufactured by JEOL Ltd.

[0090] Reagent: Cholestest LDL manufactured by Sekisui Medical Co., Ltd.

[0091] <First reagent (enzyme solution)>

[0092] The invention comprises 4-aminoantipyrine, cholesterol oxidase, cholesterol esterase, peroxidase and a first surfactant (EMULGEN B66).

[0093] <Second reagent (coloring solution)>

[0094] Contains DSBmT and a second surfactant (EMULGEN A60).

[0095] (1) Quantitative determination of LDL cholesterol

[0096] 60 μL of the first reagent was added to 3.0 μL of a serum sample diluted 5 times with physiological saline and incubated at 37°C for 5 minutes. Before adding the second reagent, photometry was performed at two wavelengths: a main wavelength of 545 nm and a sub-wavelength of 658 nm. The result was used as the first absorbance data.

[0097] Next, 20 μL of the second reagent was further added, incubated at 37°C for 5 minutes, and photometry was performed at two wavelengths: the main wavelength of 545 nm and the sub-wavelength of 658 nm. The result was used as the second absorbance data. In order to correct the color tone of the specimen, the first absorbance data was subtracted from the second absorbance data. In addition, the first absorbance data to be subtracted is the absorbance after liquid volume correction.

[0098] The LDL cholesterol concentration of the serum sample was determined from the absorbance of the standard solution measured in the same procedure as for the above-mentioned sample.

[0099] (2) Differential measurement

[0100] In order to determine the change in absorbance per unit time, differential measurement is performed simultaneously with the above-mentioned quantitative measurement of LDL cholesterol. Specifically, after the addition of the second reagent, rate photometry (differential measurement) is performed at two wavelengths of 545 nm, a main wavelength, and 658 nm, a sub-wavelength, from 3 minutes before the end point of the reaction to the end point of the reaction (from 45 o'clock (415 seconds) to 64 o'clock (598 seconds)), and the change in absorbance per minute is calculated. The value obtained by multiplying the change in absorbance per minute from 3 minutes before the end point of the reaction to the end point of the reaction by a constant of 10,000 is used as a reaction delay index.

[0101] 2.2 Experimental Example 1

[0102] <Specimens>

[0103] 0.02 mL of copper sulfate solution (Clinimate TP assay reagent manufactured by Sekisui Medical Co., Ltd.) was added to 0.5 mL of volunteer serum, and the mixture was stirred by inversion, and then incubated at 50° C. for 20 days to oxidize LDL in the serum. Thus, an oxidation-treated specimen was prepared.

[0104] In addition, 0.02 mL of ion-exchanged water was added to 0.5 mL of volunteer serum, and the mixture was stirred upside down and then incubated at 50° C. for 20 days to prepare a control sample.

[0105] <Measurement>

[0106] The above-mentioned quantitative measurement of LDL cholesterol was performed on the oxidation-treated specimen and the control specimen. In addition, the above-mentioned differential measurement was performed on the oxidation-treated specimen and the control specimen, and the amount of change in absorbance per minute was determined, and the reaction delay index was calculated from the amount of change.

[0107] <Measurement Results>

[0108] (1) Results of quantitative measurement of LDL cholesterol

[0109] The LDL cholesterol concentration of the oxidation-treated sample was 131 mg / dL, and the LDL cholesterol concentration of the control sample was 138 mg / dL.

[0110] (2) Results of differential measurement

[0111] Figure 4 It is a graph showing the results of differential measurement of an oxidation-treated sample. Figure 4 The horizontal axis of the graph shown is the measurement time (seconds), and the vertical axis is the absorbance (ABS). Figure 4 The graph shown shows measurement results A of the oxidation-treated sample and measurement results B of the control sample.

[0112] As Figure 4 shown, differential analysis of the absorbance curve representing the change in absorbance during the period from 3 minutes before the end of the reaction to the end of the reaction was performed, and the change amount of absorbance per 1 minute was calculated. For the control specimen, the change amount of absorbance per 1 minute (ΔABS / min) was also calculated in the same manner. The value obtained by multiplying the change amount of absorbance per 1 minute (ΔABS / min) by the constant 10,000 (ΔABS / min × 10,000) was used as the reaction delay index. The reaction delay index of the oxidized specimen was 69.5, and the reaction delay index of the control specimen was 18.3. That is, the reaction delay index of the oxidized specimen was 3.8 times that of the control specimen.

[0113] In this way, when the oxidized specimen was compared with the control specimen not subjected to oxidation treatment, a significant reaction delay was observed.

[0114] (3) Influence of copper sulfate on the reagent

[0115] The copper sulfate solution was removed from the oxidized specimen by placing the oxidized specimen in a dialysis tube for dialysis. Differential measurements were performed on the oxidized specimen after dialysis and the oxidized specimen before dialysis.

[0116] Figure 5 is a coordinate diagram showing the measurement results of the absorbance of the oxidized specimen before dialysis. Figure 6 is a coordinate diagram showing the measurement results of the absorbance of the oxidized specimen after dialysis. In Figure 5 and Figure 6 the coordinate diagrams shown, the main wavelength absorbance, the sub-wavelength absorbance, and the arithmetic absorbance obtained by subtracting the sub-wavelength absorbance from the main wavelength absorbance are shown respectively.

[0117] From Figure 5 the coordinate diagram shown and Figure 6 the coordinate diagram shown, it was possible to confirm a reaction delay in the oxidized specimen after dialysis. That is, it was confirmed that the reaction delay of the oxidized specimen was not a reaction delay caused by the absorption from copper ions.

[0118] 2.3. Experimental Example 2

[0119] <Specimen>

[0120] Figure 7This is a table showing the preparation conditions of oxidation-treated specimens A, B, C, D, and E. 0.02 mL (final concentration 3.8%) of copper sulfate solution (Clinimate TP assay reagent manufactured by Sekisui Medical Co., Ltd.) was added to 0.5 mL of basic serum to prepare oxidation-treated specimen A. That is, the incubation time of oxidation-treated specimen A was 0 day. In addition, 0.02 mL (final concentration 3.8%) of copper sulfate solution (Clinimate TP assay reagent manufactured by Sekisui Medical Co., Ltd.) was added to 0.5 mL of basic serum and incubated at 37°C for 1 day to prepare oxidation-treated specimen B. In the same manner as oxidation-treated specimen B, the incubation time was changed to 2 days, 3 days, and 7 days, and oxidation-treated specimens C, oxidation-treated specimen D, oxidation-treated specimen E, and oxidation-treated specimen F were prepared.

[0121] <Measurement>

[0122] Differential measurement was performed on the oxidation treatment specimen A, and the change in absorbance per minute (ΔABS / min) was determined. The reaction delay index (ΔABS / min×10000) was calculated from the change. The reaction delay index (ΔABS / min×10000) was calculated for the oxidation treatment specimens B, C, D, and E in the same manner as for the oxidation treatment specimen A.

[0123] <Measurement Results>

[0124] Figure 8 This is a table showing the incubation time (oxidation treatment time) of the oxidation treatment specimen and the reaction delay index. Figure 9 This is a histogram showing the reaction delay index for each incubation time.

[0125] like Figure 8 and Figure 9 As shown, it can be seen that as the oxidation treatment time for serum increases, the reaction delay index increases. That is, it can be seen that the amount of oxidized LDL can be estimated by the reaction delay index.

[0126] In addition, by comparing the reaction delay index of the actual specimen with Figure 8 and Figure 9 For example, by comparing the reaction delay index of oxidation-treated specimen A with an oxidation treatment time of 0 days as the minimum value of the evaluation value of the oxidized LDL amount and the reaction delay index of oxidation-treated specimen F with an oxidation treatment time of 7 days as the maximum value of the evaluation value of the oxidized LDL amount, it is possible to estimate the oxidized LDL amount of the actual specimen by investigating where the reaction delay index of the actual specimen is within the range.

[0127] 2.4. Experimental Example 3

[0128] <Specimens>

[0129] Oxidized specimens A, B, C, D, and E were prepared in the same manner as in Experimental Example 2.

[0130] <Measurement>

[0131] Quantitative determination of LDL cholesterol was performed on oxidized specimen A, and the LDL cholesterol concentration (LDL-C concentration) was determined. In addition, differential measurement was performed on oxidized specimen A, and the reaction delay index (ΔABS / min × 10,000) was calculated. Furthermore, the reaction delay index (ΔABS / min × 10,000) of oxidized specimen A was divided by the LDL cholesterol concentration (LDL-C concentration) to calculate the oxidized LDL ratio ((ΔABS / min × 10,000) / LDL-C concentration). This oxidized LDL ratio was used as an evaluation value for the amount of oxidized LDL.

[0132] For oxidized specimens B, C, D, and E, the reaction delay index, LDL cholesterol concentration, and evaluation value of the amount of oxidized LDL were also determined in the same manner as for oxidized specimen A.

[0133] <Measurement Results>

[0134] Figure 10 A table showing the incubation time, reaction delay index, LDL cholesterol concentration, and oxidized LDL ratio of each oxidized specimen. Figure 11 A histogram showing the reaction delay index and LDL cholesterol concentration for each incubation time. Figure 12 A histogram showing the oxidized LDL ratio for each incubation time.

[0135] In the oxidized LDL ratio, the reaction delay index is normalized by the LDL cholesterol concentration contained in the specimen, so that the amount of oxidized LDL can be compared even between specimens with different LDL cholesterol concentrations. Therefore, by using the oxidized LDL ratio as an evaluation value for the amount of oxidized LDL, the amount of oxidized LDL can be evaluated more accurately even between specimens with different LDL cholesterol concentrations compared to the case where the reaction delay index is used as an evaluation value for the amount of oxidized LDL.

[0136] 2.5. Experimental Example 4

[0137] <Specimen>

[0138] Oxidation specimen Aˊ was prepared by adding 0.05 mL (final concentration 9.1%) of copper sulfate solution (Clinimate TP assay reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of basic serum. Oxidation specimen Bˊ was prepared by adding 0.05 mL (final concentration 9.1%) of copper sulfate solution (Clinimate TP assay reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of basic serum and incubating at 50°C for 1 day. In the same manner as for oxidation specimen Bˊ, the incubation time was changed to 2 days, 3 days, 4 days, 7 days, and 11 days to prepare oxidation specimens Cˊ, oxidation specimen Dˊ, oxidation specimen Eˊ, oxidation specimen Fˊ, and oxidation specimen Gˊ.

[0139] <Measurement>

[0140] Differential measurement was performed on the oxidation treatment specimen Aˊ, and the change in absorbance per minute (ΔABS / min) was determined. The reaction delay index (ΔABS / min×10000) was calculated from the change. The reaction delay index (ΔABS / min×10000) was also calculated for the oxidation treatment specimens Bˊ, Cˊ, Dˊ, Eˊ, Fˊ, and Gˊ.

[0141] <Measurement Results>

[0142] Figure 13 This is a table showing the incubation time and reaction delay index of the oxidation treatment specimen. Figure 14 This is a histogram showing the reaction delay index for each incubation time.

[0143] like Figure 13 and Figure 14 As shown in the figure, the reaction delay index reached saturation at 2 days of oxidation treatment time. This is considered to be due to the following reasons: the final concentration of the copper sulfate solution was higher and the incubation temperature was higher under the conditions for preparing the oxidation treatment sample of Experimental Example 4 than under the conditions for preparing the oxidation treatment sample of Experimental Example 2, so that more oxidized LDL was generated under the conditions for preparing the oxidation treatment sample of Experimental Example 4 than under the conditions for preparing the oxidation treatment sample of Experimental Example 2.

[0144] 3. System

[0145] 3.1. System composition

[0146] An evaluation system according to an embodiment of the present invention will be described with reference to the drawings. Figure 15 It is a diagram showing the configuration of an evaluation system 2 according to one embodiment of the present invention.

[0147] like Figure 15 As shown, the evaluation system 2 includes an automatic analysis device 20 , an evaluation device 40 , and an information processing terminal 60 .

[0148] As Figure 15 shown, the evaluation system 2 is configured such that the automatic analysis device 20, the evaluation device 40, and the information processing terminal 60 can be connected via a communication network 4 such as the Internet. In addition, the communication network 4 is not limited to the Internet and may be a LAN (Local Area Network), a WAN (Wide Area Network), or the like.

[0149] The automatic analysis device 20 is a biochemical automatic analysis device that automatically measures the amount of a specific component contained in a specimen. In the automatic analysis device 20, the LDL cholesterol concentration can be measured by the above-described quantitative determination of LDL cholesterol. Moreover, in the automatic analysis device 20, the change amount of the absorbance per unit time can be measured by the above-described differential determination.

[0150] The automatic analysis device 20 sends information on the LDL cholesterol concentration and information on the change amount of the absorbance per unit time to the evaluation device 40 via the communication network 4. For example, when the automatic analysis device 20 sends information on the LDL cholesterol concentration and information on the change amount of the absorbance per unit time to the evaluation device 40 via the communication network 4 and requests the evaluation device 40 to execute the evaluation process of the oxidized LDL amount, the evaluation device 40 executes the process of evaluating the oxidized LDL.

[0151] The evaluation device 40 obtains information on the LDL cholesterol concentration and information on the change amount of the absorbance per unit time from the automatic analysis device 20 via the communication network 4. The evaluation device 40 evaluates the amount of oxidized LDL in the specimen based on the change amount of the absorbance per unit time. In addition, the evaluation device 40 evaluates the amount of oxidized LDL in the specimen based on the LDL cholesterol concentration and the change amount of the absorbance per unit time. The evaluation device 40 sends the evaluation result of the oxidized LDL amount to the information processing terminal 60 via the communication network 4.

[0152] The information processing terminal 60 obtains the evaluation result of the oxidized LDL amount from the evaluation device 40 via the communication network 4. The user can obtain the evaluation result from the evaluation device 40 via the communication network 4 by accessing the evaluation device 40 from the information processing terminal 60. The evaluation result is displayed on the display unit of the information processing terminal 60. The information processing terminal 60 is, for example, a portable terminal such as a smart phone or an information processing device such as a personal computer (PC).

[0153] The evaluation system 2 can also be a cloud-based evaluation system. For example, in the cloud-based evaluation system 2, when the automatic analysis device 20 requests the evaluation device 40 to execute the process of evaluating oxidized LDL and sends the information of the measurement result, the evaluation device 40 as the server device performs the process of evaluating the amount of oxidized LDL. In addition, the information processing terminal 60 performs the control of obtaining the result of the evaluation process (the evaluation result of the amount of oxidized LDL) in the evaluation device 40 from the evaluation device 40 and displaying the result of the evaluation process on the display unit.

[0154] 3.2. Automatic analysis device

[0155] Figure 16 FIG. is an example schematically showing the configuration of the automatic analysis device 20.

[0156] The automatic analysis device 20 includes a sample turntable 202, a first turntable 204, a second turntable 205, a reaction turntable 206, a specimen dispensing probe 207, a sample barcode reader 210, a first reagent dispensing probe 212, a second reagent dispensing probe 213, a first reaction solution stirring mechanism 214, a second reaction solution stirring mechanism 215, a multi-wavelength photometer 216, a thermostat 217, a reaction vessel cleaning mechanism 218, a specimen dispensing probe cleaning mechanism 231, a first reagent dispensing probe cleaning mechanism 233, a second reagent dispensing probe cleaning mechanism 234, and a control unit 240.

[0157] The sample turntable 202, the first turntable 204, the second turntable 205, and the reaction turntable 206 are supported by a drive mechanism (not shown) so as to be rotatable along the circumferential direction, and rotate at a predetermined speed in a predetermined angular range in the circumferential direction.

[0158] The sample turntable 202 holds a plurality of sample containers 221. Specimens are stored in the sample containers 221.

[0159] The first turntable 204 holds a plurality of first reagent containers 224, and the second turntable 205 holds a plurality of second reagent containers 225. The first reagent is stored in the first reagent container 224, and the second reagent is stored in the second reagent container 225.

[0160] The reaction turntable 206 holds a plurality of reaction vessels 226. The plurality of reaction vessels 226 are arranged and stored in the circumferential direction of the reaction turntable 206, and the reaction turntable 206 intermittently moves the reaction vessels 226 in the circumferential direction. In the reaction vessel 226, the specimen sampled from the sample container 221, the first reagent sampled from the first reagent container 224, and the second reagent sampled from the second reagent container 225 are injected. In the reaction vessel 226, the specimen reacts with the first reagent and the second reagent by being stirred.

[0161] The specimen dispensing probe 207 aspirates a predetermined amount of specimen from the sample container 221 transported to a preset aspiration position, and ejects the aspirated specimen into the reaction container 226 transported to a preset ejection position. The specimen dispensing probe 207 is cleaned by the specimen dispensing probe cleaning mechanism 231.

[0162] The sample barcode reader 210 reads the specimen ID from the barcode attached to the side of the sample container 221 stored in the sample turntable 202. The identification information read by the sample barcode reader 210 is sent to the control unit 240. Thus, in the control unit 240, the specimen stored in the sample container 221 and dispensed into the reaction container 226 by the specimen dispensing probe 207 can be managed.

[0163] The first reagent dispensing probe 212 aspirates a predetermined amount of the first reagent from the first reagent container 224 transported to a preset aspiration position, and ejects the aspirated first reagent into the reaction container 226 transported to a preset ejection position. The first reagent dispensing probe 212 is cleaned by the first reagent dispensing probe cleaning mechanism 233.

[0164] The second reagent dispensing probe 213 aspirates a predetermined amount of the second reagent from the second reagent container 225 transported to a preset aspiration position, and ejects the aspirated second reagent into the reaction container 226 transported to a preset ejection position. The second reagent dispensing probe 213 is cleaned by the second reagent dispensing probe cleaning mechanism 234.

[0165] The first reaction solution stirring mechanism 214 inserts a stirring rod (not shown) into the reaction container 226 and stirs the mixed solution of the specimen and the first reagent in the reaction container 226. The second reaction solution stirring mechanism 215 inserts a stirring rod (not shown) into the reaction container 226 and stirs the mixed solution of the specimen, the first reagent, and the second reagent in the reaction container 226. The reaction container cleaning mechanism 218 cleans the inside of the reaction container 226 after the analysis is completed.

[0166] The multi-wavelength photometer 216 uses a light source lamp that irradiates light onto the reaction container 226 to perform optical measurement (colorimetric measurement) on the mixed solution of the specimen that has reacted with the first reagent and the second reagent. The multi-wavelength photometer 216 outputs the amounts of various components in the specimen as absorbance, and detects the reaction state of the specimen. The information on the measurement results in the multi-wavelength photometer 216 is sent to the control unit 240. The thermostat 217 always keeps the temperature of the reaction container 226 stored in the reaction turntable 206 constant.

[0167] The control unit 240 acquires information on absorbance output from the multi-wavelength photometer 216 and performs various arithmetic processes. For example, the control unit 240 performs processes such as calculating the arithmetic absorbance obtained by subtracting the absorbance at the sub-wavelength from the absorbance at the main wavelength, differential processing for calculating the change amount of absorbance per unit time, and processing for calculating the concentration of a substance from the absorbance. In addition, the control unit 240 performs processes for controlling each part of the automatic analyzer 20. The control unit 240 includes, for example, a CPU (Central Processing Unit), a storage device (such as a RAM (Random Access Memory) and a ROM (Read Only Memory)). The control unit 240 performs various arithmetic processes and various control processes by executing the programs stored in the storage device by the CPU.

[0168] In the automatic analyzer 20, the above-described LDL cholesterol measurement reagent can be used to measure LDL cholesterol. In the automatic analyzer 20, for example, the LDL cholesterol concentration can be measured by the endpoint method. Moreover, in the automatic analyzer 20, the change amount of absorbance per unit time can be measured by differential measurement. The endpoint method is a method of measuring the absorbance at the end of the reaction and quantifying the concentration of the target substance in the sample based on the concentration and absorbance of the standard solution. In differential measurement, the absorbance is measured repeatedly at a certain time interval, and the change amount of absorbance per unit time is calculated from the obtained data. In differential measurement, the change amount of absorbance per unit time can be measured for the period from the time point obtained by tracing back a specified time from the end point of the reaction to the end point of the reaction. In addition, the specified time and the unit time can be arbitrarily set.

[0169] The above-described sample turntable 202, first turntable 204, second turntable 205, reaction turntable 206, sample dispensing probe 207, sample barcode reader 210, first reagent dispensing probe 212, second reagent dispensing probe 213, first reaction solution stirring mechanism 214, second reaction solution stirring mechanism 215, multi-wavelength photometer 216, thermostat 217, reaction vessel cleaning mechanism 218, sample dispensing probe cleaning mechanism 231, first reagent dispensing probe cleaning mechanism 233, and second reagent dispensing probe cleaning mechanism 234 function as a measurement unit.

[0170] In addition, the control unit 240 functions as a differential operation unit that calculates the change amount of absorbance per unit time from the time change of absorbance.

[0171] 3.3. Evaluation device

[0172] Figure 17This is a diagram showing an example of the configuration of the evaluation device 40.

[0173] As Figure 17 shown, the evaluation device 40 includes a processing unit 400, an operation unit 410, a display unit 420, a storage unit 430, and a communication unit 440.

[0174] The operation unit 410 is used for the user to input operation information and output the input operation information to the processing unit 400. The functions of the operation unit 410 can be implemented by input devices such as a keyboard, a mouse, a button, a touch panel, a touchpad, etc.

[0175] The display unit 420 displays the images generated by the processing unit 400, and its functions can be implemented by displays such as an LCD (Liquid Crystal Display) or a CRT (Cathode Ray Tube).

[0176] The storage unit 430 stores programs and various data for enabling each part of the computer to function as the processing unit 400. In addition, the storage unit 430 also functions as a working area for the processing unit 400 and the communication unit 440. The functions of the storage unit 430 can be implemented by a hard disk, a RAM (Random Access Memory), etc.

[0177] The communication unit 440 performs various controls for communication with the automatic analysis device 20 or the information processing terminal 60, and its functions can be implemented by executing programs with various processors (CPU, DSP, etc.), communication ASICs, etc.

[0178] The processing unit 400 functions as an acquisition unit 402, an evaluation unit 404, and a data generation unit 406 as described below by executing the programs stored in the storage unit 430. The functions of the processing unit 400 can be implemented by executing programs with various processors (CPU, DSP, etc.), ASICs (gate arrays, etc.), etc. The processing unit 400 includes an acquisition unit 402, an evaluation unit 404, and a data generation unit 406.

[0179] The acquisition unit 402 acquires information on the measurement results output from the automatic analysis device 20. The acquisition unit 402 acquires, for example, information on the LDL cholesterol concentration, information on the change amount of absorbance per unit time, etc.

[0180] The evaluation unit 404 evaluates the amount of oxidized LDL in the specimen based on the change amount of absorbance per unit time. The evaluation unit 404 obtains an evaluation value of the amount of oxidized LDL based on the value obtained by multiplying the change amount of absorbance per unit time by a constant. For example, the evaluation unit 404 uses the value (reaction delay index) obtained by multiplying the change amount of absorbance per minute in the period from 3 minutes before the end of the reaction to the end of the reaction by the constant 10,000 as the evaluation value.

[0181] In addition, here, the period from 3 minutes before the end of the reaction to the end of the reaction is taken as the object of evaluation, but the time for evaluation can be appropriately changed. Also, the unit time is set to 1 minute, but this time can be appropriately changed. Additionally, the constant is not limited to 10,000 and can be set to any value.

[0182] Further, for example, the evaluation unit 404 evaluates the amount of oxidized LDL in the specimen based on the value obtained by dividing the change amount of absorbance per unit time by the LDL cholesterol concentration of the specimen. For example, the evaluation unit 404 uses the value (oxidized LDL ratio) obtained by dividing the value obtained by multiplying the change amount of absorbance per minute in the period from 3 minutes before the end of the reaction to the end of the reaction by the constant 10,000 by the LDL cholesterol concentration as the evaluation value.

[0183] The evaluation unit 404 can use both the reaction delay index and the oxidized LDL ratio as the evaluation value of the amount of oxidized LDL, or can use either one of them as the evaluation value.

[0184] The data generation unit 406 generates display data for displaying the LDL cholesterol concentration of the specimen, the change amount of absorbance per unit time, and the evaluation value of the amount of oxidized LDL on the information processing terminal 60. For example, the user can access the evaluation device 40 from the information processing terminal 60 and obtain the display data via the communication network 4. Thereby, the LDL cholesterol concentration, the change amount of absorbance per unit time, and the evaluation value of the amount of oxidized LDL are displayed on the display unit of the information processing terminal 60.

[0185] 4. Processing of the evaluation device

[0186] Figure 18 It is a flowchart showing an example of the evaluation process of the amount of oxidized LDL of the evaluation device 40.

[0187] The acquisition unit 402 acquires information on the LDL cholesterol concentration of the specimen output from the automatic analyzer 20 and information on the change amount of absorbance per unit time (S100).

[0188] In the measurement unit of the automatic analyzer 20, using an LDL cholesterol measurement reagent, measurement is performed by the endpoint method and differential measurement, and the control unit 240 calculates the LDL cholesterol concentration and the change amount of absorbance per unit time. The automatic analyzer 20 transmits the calculated information on the LDL cholesterol concentration and the information on the change amount of absorbance per unit time to the evaluation device 40 via the communication network 4, and requests the evaluation device 40 to execute an evaluation process. The processing unit 400 starts the evaluation process according to this request, and the acquisition unit 402 acquires this information output from the automatic analyzer 20.

[0189] The evaluation unit 404 obtains an evaluation value of the oxidized LDL amount based on the change amount of absorbance per unit time (S102). The evaluation unit 404 calculates, for example, at least one of a delayed reaction index and an oxidized LDL ratio as the evaluation value of the oxidized LDL amount.

[0190] The data generation unit 406 generates display data for displaying the LDL cholesterol concentration, the change amount of absorbance per unit time, and the evaluation value of the oxidized LDL amount on the display unit (S104). After the data generation unit 406 generates the display data, the processing unit 400 ends the evaluation process.

[0191] 5. Effects

[0192] The evaluation device 40 includes: an acquisition unit 402 that acquires information on the change amount of absorbance per unit time, the information being obtained by the following measurement method: solubilizing LDL with a surfactant that acts on LDL in a specimen, causing peroxidase to act on hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into a color development reaction, and measuring the time change of absorbance; and an evaluation unit 404 that evaluates the amount of oxidized LDL in the specimen based on this change amount.

[0193] Therefore, in the evaluation device 40, the amount of oxidized LDL in a specimen can be evaluated based on the change amount of absorbance per unit time that can be measured by the automatic analyzer 20. Therefore, in the evaluation device 40, the amount of oxidized LDL can be evaluated quickly. Accordingly, according to the evaluation device 40, the amount of oxidized LDL in a large number of specimens can be easily evaluated.

[0194] In addition, by evaluating the amount of oxidized LDL in the specimen based on the change in absorbance per unit time, it is possible to comprehensively observe oxidized LDL in one measurement. For example, in the ELISA method, although it is possible to detect oxidized LDL using aldehyde as a target, the functional changes of vascular endothelial cells such as arteriosclerosis and coronary syndrome are not simple enough to be judged by a single biomarker. In contrast, since the amount of oxidized LDL in the specimen is evaluated based on the change in absorbance per unit time, it is possible to comprehensively observe the oxidative modification of LDL. Therefore, it is possible to provide an index that can more accurately evaluate the potential risk of arteriosclerosis or coronary syndrome.

[0195] In the evaluation device 40, an evaluation value of the amount of oxidized LDL is obtained based on the value obtained by multiplying the change in absorbance per unit time by a constant. Therefore, in the evaluation device 40, it is possible to provide an index that can more accurately evaluate the potential risk of arteriosclerosis or coronary syndrome.

[0196] In the evaluation device 40, an evaluation value of the amount of oxidized LDL is obtained based on the value obtained by dividing the change in absorbance per unit time by the LDL cholesterol concentration of the specimen. In this way, in the evaluation device 40, since the change in absorbance per unit time is normalized by the LDL cholesterol concentration of the specimen, it is possible to more accurately compare the amount of oxidized LDL between specimens with different LDL cholesterol concentrations.

[0197] The evaluation device 40 includes a data generation unit 406 that generates data for causing at least one of the LDL cholesterol concentration of the specimen, the change in absorbance per unit time, and the evaluation value of the amount of oxidized LDL to be displayed on the display unit. Therefore, in the evaluation device 40, it is possible to display the LDL cholesterol concentration of the specimen, the change in absorbance per unit time, and the evaluation value of the amount of oxidized LDL on the display unit of the information processing terminal 60.

[0198] In the evaluation device 40, the change in absorbance per unit time is the change in absorbance per unit time during the period from the time point obtained by tracing back a predetermined time from the end point of the reaction to the end point of the reaction. Therefore, in the evaluation device 40, it is possible to observe the delay of the side reaction relative to the main reaction and evaluate the amount of oxidized LDL.

[0199] The evaluation system 2 includes: a measurement unit that solubilizes LDL with a surfactant that acts on LDL in a specimen, causes peroxidase to act on hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into a color reaction, measures the time change in absorbance, and outputs data on the time change in absorbance; a differential operation unit that obtains the change amount of absorbance per unit time from this data; and an evaluation unit 404 that evaluates the amount of oxidized LDL in the specimen based on this change amount.

[0200] Therefore, in the evaluation system 2, the amount of oxidized LDL in a specimen can be evaluated based on the change amount of absorbance per unit time that can be measured by the automatic analyzer 20. Thus, in the evaluation system 2, the amount of oxidized LDL can be evaluated rapidly.

[0201] The evaluation method of the present embodiment includes: a step of obtaining information on the change amount of absorbance per unit time, the information being obtained by the following measurement method: solubilizing LDL with a surfactant that acts on LDL in a specimen, causing peroxidase to act on hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into a color reaction, and measuring the time change in absorbance; and a step of evaluating the amount of oxidized LDL in the specimen based on this change amount.

[0202] Therefore, in the evaluation method of the present embodiment, the amount of oxidized LDL in a specimen can be evaluated based on the change amount of absorbance per unit time that can be measured by the automatic analyzer 20. Thus, in the evaluation method of the present embodiment, the amount of oxidized LDL can be evaluated rapidly.

[0203] 6. Variation

[0204] 6.1. First Variation

[0205] In the above embodiment, the evaluation unit 404 evaluated the amount of oxidized LDL by obtaining a value obtained by multiplying the change amount of absorbance per unit time by a constant (reaction delay index), or a value obtained by dividing this value by the LDL cholesterol concentration of the specimen (oxidized LDL ratio) as an evaluation value.

[0206] In contrast, the evaluation unit 404 may also evaluate the amount of oxidized LDL by determining whether these evaluation values exceed a reference value. For example, the evaluation unit 404 may Figure 12 use the value of the oxidized LDL ratio shown with an incubation time of 0 days as a reference value to determine whether the oxidized LDL ratio of the actual specimen exceeds the reference value. The evaluation unit 404 may, for example, generate a list of specimens whose oxidized LDL ratio exceeds the reference value as an evaluation result.

[0207] 6.2. Second Variation

[0208] In the above-described embodiment, the control unit 240 of the automatic analysis device 20 functions as a differential operation unit that performs differential analysis on the time change of the absorbance, which is the measurement result of the measurement unit, to calculate the change amount of the absorbance per unit time. However, the acquisition unit 402 of the evaluation device 40 may also function as the differential operation unit.

[0209] For example, the automatic analysis device 20 outputs information on the time change of the absorbance, and the acquisition unit 402 acquires the information on the time change of the absorbance. The acquisition unit 402 performs differential operation on the time change of the absorbance to obtain the change amount of the absorbance per unit time. Thus, the acquisition unit 402 can acquire the information on the change amount of the absorbance per unit time.

[0210] 6.3. Third Modification

[0211] In the above-described embodiment, as Figure 15 shown, the evaluation system 2 includes an automatic analysis device 20, an evaluation device 40, and an information processing terminal 60, and is configured such that they can be connected by a communication network 4 such as the Internet, and the evaluation result is displayed on the display unit of the information processing terminal 60.

[0212] In contrast, in the evaluation system 2, the automatic analysis device 20 and the evaluation device 40 may also be connected via a LAN (Local Area Network). In this case, in the evaluation system 2, the information processing terminal 60 may not be included, and the evaluation result may be displayed on the display unit of the control unit 240 of the automatic analysis device 20 or the display unit 420 of the evaluation device 40.

[0213] 7. Other Embodiments

[0214] In the above-described embodiment, the color reaction by the reaction of LDL and the reagent is used as the main reaction (first reaction) of the reagent, and the color reaction by the reaction of oxidized LDL and the reagent is used as the side reaction (second reaction) of the reagent. The change amount per unit time of the absorbance of the pigment generated by the main reaction and the side reaction is measured, and the amount of oxidized LDL is evaluated based on the change amount per unit time of the absorbance.

[0215] This evaluation method can also be applied to specimens other than specimens containing LDL and oxidized LDL. That is, the evaluation method may also include: a step of reacting a specimen containing a first substance and a second substance with a reagent and measuring the change amount per unit time of a physical quantity of a reaction product containing a first reaction product generated by the first reaction of the first substance and the reagent and a second reaction product generated by the second reaction of the second substance and the reagent; and a step of evaluating the physical quantity of the second substance based on the change amount per unit time of the physical quantity of the reaction product.

[0216] Here, in the above-described embodiment, the first substance is LDL, and the first reaction product is a pigment (first pigment) generated by the reaction of LDL with the reagent. Further, the second substance is oxidized LDL, and the second reaction product is a pigment (second pigment) generated by the reaction of oxidized LDL with the reagent. In contrast, the first substance and the second substance only need to generate the first reaction product by the first reaction of the first substance with the reagent, and generate the second reaction product by the second reaction of the second substance with the reagent, and there is no particular limitation.

[0217] In addition, the first reaction and the second reaction are not limited to color development reactions, and may be any one or a combination of modification, oxidation, reduction, addition, binding, decomposition, condensation, solubilization reactions. Therefore, the first reaction product and the second reaction product are not limited to pigments.

[0218] In addition, when the reaction product has a specific absorption wavelength, the physical quantity of the reaction product can also be detected by absorbance. Similarly, when the reaction product has a specific emission wavelength, Raman shift, chemical shift or mass-to-charge ratio, the physical quantity of the reaction product can also be detected by fluorescence, Raman, nuclear magnetic resonance, mass spectrometry and other means respectively.

[0219] For example, when the change in the physical quantity of the reaction product containing the first reaction product and the second reaction product is set to f(x), the side reaction can also be expressed as its derivative fˊ(x), and the physical quantity of the second substance can be expressed as the change amount of the physical quantity of the reaction product per unit time.

[0220] The evaluation value of the physical quantity of the second substance can be obtained in the same manner as the evaluation value of the oxidized LDL amount described above. That is, as the evaluation value of the physical quantity of the second substance, a value obtained by multiplying the change amount of the physical quantity of the reaction product per unit time by a constant (the constant is an arbitrary value), or a value obtained by dividing this value by the amount (concentration) of the first substance can be used.

[0221] In addition, the above-described embodiments and modification examples are just examples and are not limited to them. For example, each embodiment and each modification example can be appropriately combined.

[0222] The present invention is not limited to the above-described embodiments and can be further variously modified. For example, the present invention includes configurations that are substantially the same as those described in the embodiments. The so-called substantially the same configurations refer to, for example, configurations having the same functions, methods, and results, or configurations having the same purposes and effects. In addition, the present invention includes configurations in which non-essential parts among the configurations described in the embodiments are replaced. In addition, the present invention includes configurations that have the same effects as those of the configurations described in the embodiments or can achieve the same purpose. In addition, the present invention includes configurations obtained by adding well-known techniques to the configurations described in the embodiments.

Claims

1. An evaluation device, characterized in that: Include: an acquisition unit that acquires information on the amount of change in absorbance per unit time, the information being obtained by a measurement method in which LDL in a specimen is solubilized with a surfactant that acts on LDL, hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase is allowed to act on peroxidase to introduce hydrogen peroxide into a color development reaction, and the time change in absorbance is measured; and An evaluation unit estimates the amount of oxidized LDL in the sample based on the amount of change.

2. The evaluation device according to claim 1, wherein: The evaluation unit obtains an evaluation value of the amount of oxidized LDL based on a value obtained by multiplying the amount of change by a constant.

3. The evaluation device according to claim 1, wherein: The evaluation unit obtains an estimated value of the amount of oxidized LDL based on a value obtained by dividing the amount of change by the LDL cholesterol concentration of the sample.

4. The evaluation device according to claim 3, wherein: The evaluation unit determines whether the evaluation value exceeds a reference value.

5. The evaluation device according to claim 3 or 4, wherein: The method includes a data generating unit that generates data for causing a display unit to display at least one of the LDL cholesterol concentration of the sample, the amount of change, and the evaluation value.

6. The evaluation device according to claim 1, wherein: The amount of change is the amount of change per unit time of the absorbance from a time point traced back a predetermined time from the end point of the reaction to the end point of the reaction.

7. A system, characterized in that: Include: a measuring unit that solubilizes LDL with a surfactant that acts on LDL in a specimen, causes peroxidase to act on hydrogen peroxide generated in an oxidation reaction by cholesterol oxidase to introduce hydrogen peroxide into a color development reaction, measures a temporal change in absorbance, and outputs data on the temporal change in absorbance; a differential calculation unit for obtaining a change in the absorbance per unit time from the data; as well as An evaluation unit estimates the amount of oxidized LDL in the sample based on the amount of change.

8. An evaluation method, characterized in that Include: A step of obtaining information on the amount of change in absorbance per unit time, the information being obtained by a measurement method comprising solubilizing LDL in a sample with a surfactant that acts on LDL, allowing peroxidase to act on hydrogen peroxide generated in an oxidation reaction performed by cholesterol oxidase to introduce hydrogen peroxide into a color development reaction, and measuring the temporal change in absorbance; as well as and estimating the amount of oxidized LDL in the sample based on the amount of change.

9. An evaluation method, characterized in that Include: a step of causing a sample including a first substance and a second substance to react with a reagent, and measuring a change per unit time in a physical quantity of a reaction product including a first reaction product generated by a first reaction between the first substance and the reagent, and a second reaction product generated by a second reaction between the second substance and the reagent; and A step of estimating a physical quantity of the second substance based on the amount of change.

10. The evaluation method according to claim 9, wherein: The specimen is a body fluid, The first substance is LDL, The second substance is oxidized LDL, The first reaction is a color development reaction performed by the reaction between the LDL and the reagent. The second reaction is a color development reaction caused by the reaction between the oxidized LDL and the reagent.

11. The evaluation method according to claim 10, wherein: The first reaction product is a first pigment generated by the reaction between the LDL and the reagent, The second reaction product is a second dye generated by the reaction between the oxidized LDL and the reagent.

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