Evaluation device, system and evaluation method

Abstract

To provide an evaluation device that can quickly evaluate an amount of oxidized LDL.SOLUTION: An evaluation device includes: an acquisition unit that acquires information on an amount of change in absorbance per unit time obtained by a measurement method of solubilizing LDL by use of a surface active agent acting on the LDL in a sample, causing peroxidase to act on hydrogen peroxide generated based on an oxidation reaction by a cholesterol oxidase to thereby guide the hydrogen peroxide to a coloring reaction, and measuring a time change in absorbance; and an evaluation unit that evaluates an amount of oxidized LDL in the sample on the basis of the amount of change.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to an evaluation apparatus, 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 become widespread. Since there are a very large number of contaminants in a specimen, in order to detect a target compound from among them, an antibody that selectively binds to the target compound or an enzyme that selectively reacts with the target compound is used. Detection reagents for detecting a target compound combining these antibodies and enzymes have been developed by reagent manufacturers. Such reagents are devised to perform multi-step reactions in one pot for highly sensitive and rapid measurement. For example, Patent Document 1 and Patent Document 2 disclose methods for quantifying low-density lipoprotein cholesterol (LDL-C) using such reagents.

[0003] Lipids ingested in the small intestine and lipids biosynthesized in the liver (fatty acids and cholesterol) are transported to other tissues via the blood as spherical molecules called lipoproteins, which are stabilized by covering them with phospholipids, cholesterol, and apolipoproteins in the form of triacylglycerols and cholesteryl esters (see Non-Patent Document 1).

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

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

[0006] Oxygen necessary for maintaining life changes into active oxygen by external stimuli, while acting as a cell signaling substance and an immune function. On the other hand, excessive production damages cells and is a factor leading to various diseases such as cancer, cardiovascular diseases, and lifestyle-related diseases. Lipoproteins also cause these diseases by undergoing oxidative stress. Among them, oxidized LDL (hereinafter also referred to as "ox-LDL") is considered to be one of the factors causing arteriosclerosis because it is taken up by cells such as macrophages and causes cell foaming. 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 Immuno Sorbent Assay) is known. For example, Patent Document 3 discloses a diagnostic kit for detecting oxidized lipoproteins containing apoB100 in blood by sandwich ELISA.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, in the sandwich ELISA method described above, complicated procedures are required and rapid measurement cannot be achieved.

Means for Solving the Problems

[0011] One aspect of the evaluation apparatus according to the present invention is an acquisition unit that acquires information on the amount of change in the absorbance per unit time obtained by a measurement method of solubilizing LDL with a surfactant that acts on LDL in a sample, causing peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and measuring the change in absorbance over time; an evaluation unit that evaluates the amount of oxidized LDL in the sample based on the amount of change; and includes.

[0012] In such an evaluation apparatus, the amount of oxidized LDL in a sample can be evaluated based on the amount of change in absorbance per unit time that can be measured by an automatic analyzer. Therefore, with such an evaluation apparatus, the amount of oxidized LDL can be evaluated rapidly.

[0013] One aspect of the system according to the present invention is a measurement unit that solubilizes LDL with a surfactant that acts on LDL in a sample, causes peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, measures the change in absorbance over time, and outputs data on the change in absorbance over time; A differential calculation unit that obtains the amount of change in the absorbance per unit time from the data, An evaluation unit that evaluates the amount of oxidized LDL in the specimen based on the amount of change, and includes.

[0014] In such a system, the amount of oxidized LDL in a specimen can be evaluated based on the amount of change in 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.

[0015] One aspect of the evaluation method according to the present invention is Solubilizing LDL with a surfactant that acts on LDL in a specimen, allowing peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and obtaining information on the amount of change in the absorbance per unit time obtained by a measurement method for measuring the time change in absorbance, Based on the amount of change, a step of evaluating the amount of oxidized LDL in the specimen, and includes.

[0016] In such an evaluation method, the amount of oxidized LDL in a specimen can be evaluated based on the amount of change in 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 quickly.

[0017] One aspect of the evaluation method according to the present invention is Reacting a specimen containing a first substance and a second substance with a reagent, and measuring the amount of change per unit time of the physical quantity of a reaction product containing 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, Based on the amount of change, a step of evaluating the physical quantity of the second substance, and includes.

[0018] In such an evaluation method, the physical quantity of the second substance can be evaluated based on the amount of change per unit time of the physical quantity of the reaction product.

Brief Description of the Drawings

[0019]

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Embodiments for Carrying Out the Invention

[0020] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. It should be noted that the embodiments described below do not unduly limit the content of the present invention described in the claims. Also, not all of the configurations described below are essential constituent elements of the present invention.

[0021] 1. Evaluation Method 1.1. Method for Measuring LDL Cholesterol First, a method for measuring LDL cholesterol (quantitative measurement) in a specimen will be described. The specimen to be measured is blood, serum, plasma, cerebrospinal fluid, urine, sweat, puncture fluid, feces, and other body fluids, as well as those obtained by pretreatment (such as dilution) of these. Also, LDL cholesterol is the cholesterol contained in LDL.

[0022] The method for measuring LDL cholesterol includes a first step of eliminating 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, allowing peroxidase to act on hydrogen peroxide generated by the oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and measuring the absorbance.

[0023] In the method for measuring LDL cholesterol, the measurement is performed using a reagent for measuring LDL cholesterol. The reagent for measuring LDL cholesterol 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 chromogen (DSBmT: N,N-bis(4-sulfobutyl)-m-toluidine) and a second surfactant. The first reagent is added to the specimen in the first step, and the second reagent is added to the specimen in the second step. Note that the cholesterol oxidase is not particularly limited as long as it is an enzyme having the ability to oxidize cholesterol to produce hydrogen peroxide. Also, the cholesterol esterase is not particularly limited as long as it is an enzyme having the ability to hydrolyze cholesterol.

[0024] Figure 1 is a diagram for explaining the first step of the method for measuring LDL cholesterol.

[0025] In the first step, in the presence of the first surfactant, cholesterol esterase and cholesterol oxidase are allowed to act on the cholesterol in lipoproteins other than LDL (high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL), chylomicron (CM), etc.) to generate hydrogen peroxide. The enzyme used here may be unmodified, or may be 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 containing sugar in its structure, a sulfopropyl group, or a polyurethane group. The first surfactant dissolves the outer wall of lipoproteins other than LDL and elutes the cholesterol in lipoproteins other than LDL. Thereby, the cholesterol in lipoproteins other than LDL can be led to an enzymatic reaction using cholesterol esterase and cholesterol oxidase. The generated hydrogen peroxide is eliminated by peroxidase and 4-aminoantipyrine. Note that elimination means to be made colorless so as not to be detected in the measurement of absorbance in the second step.

[0026] The first surfactant has the effect of changing only the structure of lipoproteins other than LDL and does not change the structure of LDL. That is, the first surfactant does not dissolve the outer wall of LDL. Examples of the first surfactant include polyalkylene oxide derivatives having an HLB value of 13 or more and 15 or less. 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 having 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; and compounds having an HLB value of 13 or more and 15 or less such as polyoxyethylene tribenzylphenyl ether, but are not limited thereto. The first surfactant may contain a branched alkyl ether structure. The first surfactant may be one that does not contain a double bond, such as polyoxyethylene polyoxypropylene alkyl ether. The first surfactant may be a combination of a polyoxyethylene polyoxypropylene copolymer and a polyglyceryl ether.

[0027] Commercially available products of the first surfactant include Emulgen B66 (polyoxyethylene tribenzylphenyl ether, HLB = 13.2) manufactured by Kao Corporation, and Soft Cut ID-1087 (polyoxyethylene isodecyl ether) manufactured by Aoki Yushi Industry Co., Ltd.

[0028] Also, a cationic surfactant may be used as the first surfactant. The first surfactant may be an allylamine or a polymer compound having a diallylamine unit. The first surfactant may also be a cholic acid derivative and its salt. In the first surfactant, divalent metal ions may be included in the reaction solution in order to suppress the reaction with LDL and further enhance the elimination of other lipoproteins. As the divalent metal ions, copper ions, iron ions, and magnesium ions can be used, and magnesium ions are particularly preferred. In addition, a lipoprotein-degrading enzyme can optionally be added to the reaction solution in the first step. Adding this enzyme is preferable because it particularly facilitates the reaction of cholesterol in VLDL.

[0029] Figure 2 is a diagram for explaining the second step of the method for measuring LDL cholesterol.

[0030] In the second step, a second reagent is added to the specimen after the first step has been performed. The second surfactant contained in the second reagent dissolves the outer wall of the LDL remaining in the first step and elutes the cholesterol inside the LDL. Cholesterol esterase and cholesterol oxidase are allowed to act on the eluted cholesterol to generate hydrogen peroxide. In the second step, a toluidine-based or similar aromatic dye precursor such as DSBmT is added as a chromogenic agent to lead the generated hydrogen peroxide to a color reaction. In the color reaction, a peroxidase is allowed to act on the hydrogen peroxide, and an oxidative coupling reaction is performed between an aromatic nucleophile and an aromatic electrophile, which are dye precursors, to generate a dye. Specifically, peroxidase is allowed to act on the hydrogen peroxide, and an oxidative coupling reaction is performed between 4-aminoantipyrine and DSBmT to generate a red-violet dye. By measuring the absorbance of the color development of this red-violet dye, LDL cholesterol is quantified.

[0031] The second surfactant may have an effect of changing the structures of all lipoproteins or may have an effect of changing only the structure of LDL. Examples of the second surfactant having an effect of changing the structures of all lipoproteins include polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13. 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.

[0032] Examples of the polyalkylene oxide derivative having an HLB value of 11 or more and less than 13 include compounds having an HLB value of 11 or more and less than 13 such as polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octylphenyl ether, and polyoxyethylene nonylphenyl ether, but are not limited thereto.

[0033] Examples of commercially available products of the second surfactant include Emulgen A60 (polyoxyethylene distyrenated phenyl ether, HLB = 12.8) manufactured by Kao Corporation, Triton (registered trademark) X-100, and the like.

[0034] Examples of the second surfactant having an effect of changing only the structure of LDL include anionic surfactants. As the anionic surfactant, those having a linear or branched alkyl group having 4 to 18 carbon atoms bonded to an aromatic ring are preferable. Here, the aromatic ring is preferably composed of only carbon and hydrogen such as benzene, naphthalene, diphenyl, etc. Further, those having a hydrophilic group such as a sulfonate bonded to the aromatic ring are preferable.

[0035] 1.2. Evaluation method of oxidized LDL Next, the method for evaluating oxidized LDL according to this embodiment will be described. FIG. 3 is a diagram for explaining the method for evaluating oxidized LDL. Note that oxidized LDL is LDL that has been oxidatively modified by the action of active oxygen 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.

[0036] In the second step, as shown in FIG. 3, when cholesterol is reacted with cholesterol oxidase, the 3-hydroxy group of cholesterol is oxidized to a carbonyl group. Furthermore, a decomposition reaction proceeds due to the transfer of this carbonyl group. As a result, cholesterol is eliminated.

[0037] Along with the oxidation of cholesterol by the above-described cholesterol oxidase, hydrogen peroxide is generated. When the generated hydrogen peroxide is used as a substrate and peroxidase cleaves the peroxide structure, two protons are captured from 4-aminoantipyrine and a toluidine compound (DSBmT), and the two are oxidatively bonded. Thereby, a dye is generated.

[0038] Here, lipid peroxide (16:O-18:2+32PC) generated by the oxidation of phospholipid by active oxygen also has a peroxide structure and serves as a substrate for peroxidase, similar to hydrogen peroxide. Therefore, similar to hydrogen peroxide, when lipid peroxide cleaves the peroxide structure with peroxidase, two protons are captured from 4-aminoantipyrine and a toluidine compound (DSBmT), and the two are oxidatively bonded to generate a dye. In addition, lipid peroxide is decomposed by receiving the reaction of peroxidase, similar to the case of the above-described LDL cholesterol.

[0039] Therefore, if the main reaction of the reagent is defined as a color reaction due to the reaction between LDL and the reagent, and the side reaction of the reagent is defined as a color reaction due to the reaction between oxidized LDL and the reagent, then by observing this side reaction, lipid peroxide (oxidized LDL) can be evaluated. In this embodiment, attention is paid to the fact that the side reaction is delayed with respect to the main reaction, and the side reaction is observed.

[0040] Among the reasons for the delay of side reactions with respect to this main reaction, one is the difference in the rate of solubilization reaction when solubilizing LDL with the second surfactant to elute cholesterol and the rate of solubilization reaction when solubilizing oxidized LDL with the second surfactant to elute lipid peroxides. That is, the rate of solubilization reaction when solubilizing oxidized LDL to elute lipid peroxides is slower than the rate of solubilization reaction when solubilizing LDL to elute cholesterol. This difference in the rate of solubilization reaction is considered to be because the dissolving ability of the second surfactant with respect to the outer wall of LDL changes due to the oxidative modification of the outer wall of LDL.

[0041] Also, among the reasons for the delay of side reactions with respect to the main reaction, one is the difference in the reaction rate between hydrogen peroxide and lipid peroxide, which is the substrate of peroxidase. The side reaction is delayed with respect to the main reaction because the reaction rate of lipid peroxide with respect to peroxidase is slower than the reaction rate of hydrogen peroxide with respect to peroxidase.

[0042] In addition, when LDL is oxidatively modified, various phenomena such as the progress of lipid peroxidation reaction, modification of apolipoprotein, and change in particle state occur. Therefore, it is considered that the delay of the side reaction with respect to this main reaction occurs due to the overlap of various factors, not just the two factors described above.

[0043] 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, suggesting its involvement in the delay of side reactions with respect to the main reaction.

[0044] The LDL-C value measured by a general clinical test reagent is the sum of the true LDL-C and the cholesterol contained in Lp(a) (LDL(a)-C). As a method for measuring LDL(a)-C, a method is known in which LDL(a) is extracted and quantified using an anti-LDL(a) antibody bound to magnetic beads. If the reaction delay of the side reaction represents the LDL(a)-C value or if any involvement is found, in this embodiment, it is useful because such complicated operations do not have to be performed. Or when any involvement is found, in this embodiment, it is useful because such complicated operations do not have to be performed.

[0045] In addition, LDL is further 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 high specific gravity and are called small dense LDL (sd-LDL), and the large particles have a low specific gravity and are called large buoyant LDL (lb-LDL). It has been pointed out that sd-LDL among LDL particles 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 derived from the sd-LDL component.

[0046] The delay of the side reaction with respect to such a main reaction can be observed, for example, by measuring the change amount of the absorbance per unit time in the above-described method for measuring LDL cholesterol. In this embodiment, the amount of oxidized LDL is evaluated based on the change amount of the absorbance per unit time.

[0047] 2. Experimental Examples Hereinafter, the method for evaluating oxidized LDL will be specifically described by experimental examples, but the present invention is not limited to these examples.

[0048] 2.1. Measurement Method Measuring device: JCA-BM8040 manufactured by JEOL Ltd. Reagent: Cholestest LDL manufactured by Sekisui Medical Co., Ltd. <First Reagent (Enzyme Solution)> It contains 4 - aminoantipyrine, cholesterol oxidase, cholesterol esterase, peroxidase, and a first surfactant (Emulgen B66).

[0049] <Second reagent (color - developing solution)> It contains DSBmT and a second surfactant (Emulgen A60).

[0050] (1) Quantitative measurement of LDL cholesterol To 3.0 μL of a serum specimen diluted 5 - fold with physiological saline, add 60 μL of the first reagent and incubate at 37°C for 5 minutes. Before adding the second reagent, measure the absorbance at two wavelengths with a main wavelength of 545 nm and a sub - wavelength of 658 nm. Take this result as the first absorbance data.

[0051] Next, add another 20 μL of the second reagent, incubate at 37°C for 5 minutes, and measure the absorbance at two wavelengths with a main wavelength of 545 nm and a sub - wavelength of 658 nm. Take this result as the second absorbance data. For color correction of the specimen, subtract the first absorbance data from the second absorbance data. Note that the first absorbance data to be subtracted is the absorbance after volume correction.

[0052] Determine the LDL cholesterol concentration of the serum specimen from the absorbance of the standard solution measured in the same procedure as the above specimen.

[0053] (2) Differential measurement For differential measurement to measure the change in absorbance per unit time, it is carried out simultaneously with the above quantitative measurement of LDL cholesterol. Specifically, after adding the second reagent, from 3 minutes before the end of the reaction to the end of the reaction (between 45 points (415 seconds) and 64 points (598 seconds)), perform rate measurement (differential measurement) at two wavelengths with a main wavelength of 545 nm and a sub - wavelength of 658 nm, and determine the change in absorbance per minute. Multiply the change in absorbance per minute during the period from 3 minutes before the end of the reaction to the end of the reaction by a constant of 10000, and take this value as the reaction delay index.

[0054] 2.2. Experimental example 1 <Specimen> 0.02 mL of copper sulfate solution (Clinimate TP measurement reagent manufactured by Sekisui Medical Co., Ltd.) was added to 0.5 mL of volunteer serum, and after stirring by inversion, the serum was incubated at 50°C for 20 days to oxidize the LDL in the serum. Thus, an oxidation-treated sample was prepared.

[0055] In addition, 0.02 mL of ion-exchanged water 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 prepare a control sample.

[0056] <Measurement> The above-mentioned quantitative measurement of LDL cholesterol was performed on the oxidation-treated samples and the control samples. In addition, the above-mentioned differential measurement was performed on the oxidation-treated samples and the control samples to determine the change in absorbance per minute, and the reaction delay index was calculated from the change.

[0057] <Measurement results> (1) Quantitative measurement results of LDL cholesterol The LDL cholesterol concentration in the oxidation-treated samples was 131 mg / dL, and in the control samples was 138 mg / dL.

[0058] (2) Differential measurement results Figure 4 is a graph showing the results of differential measurement of the oxidation treated sample. The horizontal axis of the graph shown in Figure 4 is measurement time (seconds), and the vertical axis is absorbance (ABS). The graph shown in Figure 4 also shows measurement result A of the oxidation treated sample and measurement result B of the control sample.

[0059] As shown in Fig. 4, differential analysis of the absorbance curve showing the change in absorbance from 3 minutes before the end of the reaction to the end of the reaction was performed, and the change in absorbance per minute was calculated. Similarly, for the control specimen, the change in absorbance per minute (ΔABS / min) was calculated. The value obtained by multiplying the change in absorbance per 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.

[0060] Thus, a clear delay in the reaction was observed in the oxidized specimen as compared with the control specimen that had not been subjected to the oxidation treatment.

[0061] (3) Influence of Copper Sulfate on the Reagent The copper sulfate solution was removed from the oxidized specimen by putting the oxidized specimen into a dialysis tube and performing dialysis. Differential measurement was performed on the oxidized specimen after this dialysis and the oxidized specimen before dialysis.

[0062] Fig. 5 is a graph showing the measurement results of the absorbance of the oxidized specimen before dialysis. Fig. 6 is a graph showing the measurement results of the absorbance of the oxidized specimen after dialysis. The graphs shown in Fig. 5 and Fig. 6 respectively show the main wavelength absorbance, the sub-wavelength absorbance, and the calculated absorbance obtained by subtracting the sub-wavelength absorbance from the main wavelength absorbance.

[0063] From the graphs shown in Fig. 5 and Fig. 6, a reaction delay was confirmed in the oxidized specimen after dialysis. That is, it was confirmed that the reaction delay of the oxidized specimen was not due to the absorption derived from copper ions.

[0064] 2.3. Experimental Example 2 <Specimen> FIG. 7 is a table showing the conditions for preparing oxidation-treated specimens A, B, C, D, and E. Oxidation-treated specimen A was prepared by adding 0.02 mL (final concentration 3.8%) of copper sulfate solution (Clinimate TP measurement reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of base serum. In other words, the incubation time for oxidation-treated specimen A was 0 days. Oxidation-treated specimen B was prepared by adding 0.02 mL (final concentration 3.8%) of copper sulfate solution (Clinimate TP measurement reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of base serum and incubating at 37° C. for 1 day. Similar to oxidation-treated specimen B, oxidation-treated specimens C, D, E, and F were prepared by changing the incubation time to 2, 3, and 7 days.

[0065] <Measurement> A differential measurement was performed on the oxidized specimen A to determine the change in absorbance per minute (ΔABS / min), and the reaction delay index (ΔABS / min×10000) was calculated from this change. The reaction delay index (ΔABS / min×10000) was also calculated for the oxidized specimens B, C, D, and E in the same manner as for the oxidized specimen A.

[0066] <Measurement results> Fig. 8 is a table showing the incubation time (oxidation treatment time) of the oxidation treatment sample and the reaction delay index, and Fig. 9 is a histogram showing the reaction delay index for each incubation time.

[0067] 8 and 9, it was found that the reaction delay index increases as the oxidation treatment time for serum increases. In other words, it was found that the amount of oxidized LDL can be evaluated by the reaction delay index.

[0068] In addition, by comparing the reaction delay index of the test sample with the results shown in FIGS. 8 and 9, the amount of oxidized LDL in the test sample can be evaluated. For example, in the range where the reaction delay index of the oxidized sample A with an oxidation treatment time of 0 days is set as the minimum value of the evaluation value of the oxidized LDL amount, and the reaction delay index of the oxidized sample F with an oxidation treatment time of 7 days is set as the maximum value of the evaluation value of the oxidized LDL amount, by examining where the reaction delay index of the test sample is located within this range, the amount of oxidized LDL in the test sample can be evaluated.

[0069] 2.4. Experimental Example 3 <Specimen> In the same manner as in Experimental Example 2, oxidized specimens A, B, C, D, and E were prepared.

[0070] <Measurement> Quantitative measurement of LDL cholesterol was performed on the oxidized specimen A to determine the LDL cholesterol concentration (LDL-C concentration). In addition, differential measurement was performed on the oxidized specimen A to calculate the reaction delay index (ΔABS / min × 10000). Furthermore, the reaction delay index (ΔABS / min × 10000) of the oxidized specimen A was divided by the LDL cholesterol concentration (LDL-C concentration) to calculate the oxidized LDL ratio ((ΔABS / min × 10000) / LDL-C concentration). This oxidized LDL ratio was used as the evaluation value of the amount of oxidized LDL.

[0071] For the oxidized specimens B, C, D, and E, in the same manner as for the oxidized specimen A, the reaction delay index, LDL cholesterol concentration, and evaluation value of the amount of oxidized LDL were determined.

[0072] <Measurement Results> FIG. 10 is a table showing the incubation time, reaction delay index, LDL cholesterol concentration, and oxidized LDL ratio of each oxidized specimen. FIG. 11 is a histogram showing the reaction delay index and LDL cholesterol concentration for each incubation time. FIG. 12 is a histogram showing the oxidized LDL ratio for each incubation time.

[0073] ​In the oxidized LDL ratio, the reaction delay index is normalized by the LDL cholesterol concentration contained in the sample, so the amount of oxidized LDL can be compared even between samples with different LDL cholesterol concentrations. Therefore, by using the oxidized LDL ratio as the evaluation value of the amount of oxidized LDL, the amount of oxidized LDL can be evaluated more accurately even between samples with different LDL cholesterol concentrations, compared to when the reaction delay index is used as the evaluation value of the amount of oxidized LDL.

[0074] 2.5. Experimental Example 4 <Sample> Oxidation-treated specimen A' was prepared by adding 0.05 mL (final concentration 9.1%) of copper sulfate solution (Clinimate TP measurement reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of base serum. Oxidation-treated specimen B' was also prepared by adding 0.05 mL (final concentration 9.1%) of copper sulfate solution (Clinimate TP measurement reagent manufactured by Sekisui Medical Co., Ltd.) to 0.5 mL of base serum and incubating at 50°C for one day. As with oxidation-treated specimen B', the incubation time was changed to 2, 3, 4, 7, and 11 days to prepare oxidation-treated specimens C', D', E', F', and G'.

[0075] <Measurement> The reaction delay index (ΔABS / min×10000) was calculated from the amount of change in absorbance per minute (ΔABS / min) of the oxidized specimen A'. The reaction delay index (ΔABS / min×10000) was calculated similarly for the oxidized specimens B', C', D', E', F', and G'.

[0076] <Measurement results> Fig. 13 is a table showing the incubation time and reaction delay index of the oxidation-treated sample, and Fig. 14 is a histogram showing the reaction delay index for each incubation time.

[0077] As shown in FIGS. 13 and 14, the reaction delay index saturated at an oxidation treatment time of 2 days. This is considered to be because, compared with the preparation conditions of the oxidized specimen in Experimental Example 2, the final concentration of the copper sulfate solution was high and the incubation temperature was high in the preparation conditions of the oxidized specimen in Experimental Example 4, so more oxidized LDL was generated under the preparation conditions of the oxidized specimen in Experimental Example 4 than in the preparation conditions of the oxidized specimen in Experimental Example 2.

[0078] 3. System 3.1. System Configuration An evaluation system according to an embodiment of the present invention will be described with reference to the drawings. FIG. 15 is a diagram showing the configuration of an evaluation system 2 according to an embodiment of the present invention.

[0079] As shown in FIG. 15, the evaluation system 2 includes an automatic analyzer 20, an evaluation device 40, and an information processing terminal 60.

[0080] As shown in FIG. 15, the evaluation system 2 is configured such that the automatic analyzer 20, the evaluation device 40, and the information processing terminal 60 can be connected via a communication network 4 such as the Internet. Note that 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.

[0081] The automatic analyzer 20 is a biochemical automatic analyzer that automatically measures the amount of a specific component contained in a specimen. In the automatic analyzer 20, the LDL cholesterol concentration can be measured by the quantitative measurement of LDL cholesterol described above. Further, in the automatic analyzer 20, the change amount of the absorbance per unit time can be measured by the differential measurement described above. In the automatic analyzer 20, the LDL cholesterol concentration can be measured by the quantitative measurement of LDL cholesterol described above. Further, in the automatic analyzer 20, the change amount of the absorbance per unit time can be measured by the differential measurement described above.

[0082] The automatic analyzer 20 sends information on the LDL cholesterol concentration and the amount of change in absorbance per unit time to the evaluation device 40 via the communication network 4. For example, when the automatic analyzer 20 sends information on the LDL cholesterol concentration and the amount of change in 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 amount of oxidized LDL, the evaluation device 40 executes the process of evaluating the oxidized LDL.

[0083] The evaluation device 40 acquires information on the LDL cholesterol concentration and the amount of change in absorbance per unit time from the automatic analyzer 20 via the communication network 4. The evaluation device 40 evaluates the amount of oxidized LDL in the sample based on the amount of change in absorbance per unit time. In addition, the evaluation device 40 evaluates the amount of oxidized LDL in the sample based on the LDL cholesterol concentration and the amount of change in absorbance per unit time. The evaluation device 40 sends the evaluation result of the amount of oxidized LDL to the information processing terminal 60 via the communication network 4.

[0084] The information processing terminal 60 acquires the evaluation result of the amount of oxidized LDL from the evaluation device 40 via the communication network 4. When the user accesses the evaluation device 40 from the information processing terminal 60, the evaluation result can be acquired from the evaluation device 40 via the communication network 4. The evaluation result is displayed on the display unit of the information processing terminal 60. The information processing terminal 60 is, for example, a mobile terminal such as a smartphone or an information processing device such as a personal computer (PC).

[0085] The evaluation system 2 may be a cloud-based evaluation system. For example, in a cloud-based evaluation system 2, when the automatic analyzer 20 requests the evaluation device 40 to execute the process of evaluating the oxidized LDL and sends the measurement result information, the evaluation device 40 as a server device performs the process of evaluating the amount of oxidized LDL. In addition, the information processing terminal 60 acquires the result of the evaluation process in the evaluation device 40 (the evaluation result of the amount of oxidized LDL) from the evaluation device 40 and controls to display the result of the evaluation process on the display unit.

[0086] 3.2. Automatic Analyzer FIG. 16 is a diagram schematically showing an example of the configuration of the automatic analyzer 20.

[0087] The automatic analyzer 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.

[0088] The sample turntable 202, the first turntable 204, the second turntable 205, and the reaction turntable 206 are rotatably supported along the circumferential direction by a drive mechanism (not shown), and rotate at a predetermined speed for each predetermined angular range in the circumferential direction.

[0089] The sample turntable 202 holds a plurality of sample containers 221. Specimens are accommodated in the sample containers 221.

[0090] 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. A first reagent is accommodated in the first reagent container 224, and a second reagent is accommodated in the second reagent container 225.

[0091] The reaction turntable 206 holds a plurality of reaction vessels 226. The plurality of reaction vessels 226 are accommodated side by side in the circumferential direction of the reaction turntable 206, and the reaction turntable 206 intermittently moves the reaction vessels 226 in the circumferential direction. A specimen sampled from the sample container 221, a first reagent sampled from the first reagent container 224, and a second reagent sampled from the second reagent container 225 are injected into the reaction vessel 226. In the reaction vessel 226, the specimen, the first reagent, and the second reagent are stirred and a reaction is carried out.

[0092] The specimen dispensing probe 207 sucks a predetermined amount of specimen from the sample container 221 conveyed to a preset suction position, and discharges the sucked specimen into the reaction vessel 226 conveyed to a preset discharge position. The specimen dispensing probe 207 is cleaned by the specimen dispensing probe cleaning mechanism 231.

[0093] The sample barcode reader 210 reads the specimen ID from the barcode attached to the side of the sample container 221 accommodated in the sample turntable 202. The identification information read by the sample barcode reader 210 is sent to the control unit 240. Thereby, in the control unit 240, the specimen accommodated in the sample container 221 and dispensed into the reaction vessel 226 by the specimen dispensing probe 207 can be managed.

[0094] The first reagent dispensing probe 212 sucks a predetermined amount of the first reagent from the first reagent container 224 conveyed to a preset suction position, and discharges the sucked first reagent into the reaction vessel 226 conveyed to a preset discharge position. The first reagent dispensing probe 212 is cleaned by the first reagent dispensing probe cleaning mechanism 233.

[0095] The second reagent dispensing probe 213 sucks a predetermined amount of the second reagent from the second reagent container 225 conveyed to a preset suction position, and discharges the sucked second reagent into the reaction vessel 226 conveyed to a preset discharge position. The second reagent dispensing probe 213 is cleaned by the second reagent dispensing probe cleaning mechanism 234.

[0096] The first reaction solution stirring mechanism 214 inserts a stirring rod (not shown) into the reaction vessel 226 and stirs the mixed solution of the specimen and the first reagent in the reaction vessel 226. The second reaction solution stirring mechanism 215 inserts a stirring rod (not shown) into the reaction vessel 226 and stirs the mixed solution of the specimen, the first reagent, and the second reagent in the reaction vessel 226. The reaction vessel cleaning mechanism 218 cleans the inside of the reaction vessel 226 after the analysis is completed.

[0097] The multi-wavelength photometer 216 performs optical measurement (colorimetric measurement) on the mixed solution of the specimen reacted with the first reagent and the second reagent using a light source lamp that irradiates the reaction vessel 226 with light rays. 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 by the multi-wavelength photometer 216 is sent to the control unit 240. The thermostat 217 constantly maintains the temperature of the reaction vessel 226 housed in the reaction turntable 206 at a constant level.

[0098] The control unit 240 acquires the absorbance information output from the multi-wavelength photometer 216 and performs various arithmetic processes. The control unit 240 performs processes such as a process of calculating an arithmetic absorbance obtained by subtracting the sub-wavelength absorbance from the main-wavelength absorbance, a differential process of calculating the change amount of absorbance per unit time, and a process for calculating the concentration of a substance from the absorbance. In addition, the control unit 240 performs a process of controlling each part of the automatic analyzer 20. The control unit 240 includes, for example, a CPU (Central Processing Unit) and 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.

[0099] In the automatic analyzer 20, LDL cholesterol can be measured using the above-described reagent for measuring LDL cholesterol. In the automatic analyzer 20, for example, the LDL cholesterol concentration can be measured by the endpoint method. Further, 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 specimen based on the concentration of the standard solution and the absorbance of the standard solution. In differential measurement, the measurement of absorbance is repeated at regular time intervals, 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 from the time point retrogressed by a predetermined time from the end point of the reaction to the end point of the reaction. Note that the predetermined time and the unit time can be arbitrarily set.

[0100] The above-described sample turntable 202, first turntable 204, second turntable 205, reaction turntable 206, specimen 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, specimen 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.

[0101] Further, the control unit 240 functions as a differential calculation unit that calculates the change amount of absorbance per unit time from the time change of absorbance.

[0102] 3.3. Evaluation Device FIG. 17 is a diagram showing an example of the configuration of the evaluation device 40.

[0103] As shown in FIG. 17, 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.

[0104] The operation unit 410 is for the user to input operation information and outputs the input operation information to the processing unit 400. The functions of the operation unit 410 can be realized by input devices such as a keyboard, a mouse, buttons, a touch panel, and a touch pad.

[0105] The display unit 420 displays the image generated by the processing unit 400, and its functions can be realized by displays such as an LCD (Liquid Crystal Display) and a CRT (Cathode Ray Tube).

[0106] The storage unit 430 stores programs and various data for making the computer function as each part of the processing unit 400. Also, the storage unit 430 also functions as a work area for the processing unit 400 and the communication unit 440. The functions of the storage unit 430 can be realized by a hard disk, a RAM (Random Access Memory), etc.

[0107] The communication unit 440 performs various controls for communication with the automatic analyzer 20 and the information processing terminal 60, and its functions can be realized by hardware such as various processors (CPU, DSP, etc.) and communication ASICs by executing a program.

[0108] The processing unit 400 functions as an acquisition unit 402, an evaluation unit 404, and a data generation unit 406, which will be described below, by executing the programs stored in the storage unit 430. The functions of the processing unit 400 can be realized by hardware such as various processors (CPU, DSP, etc.) and ASICs (gate arrays, etc.) by executing a program. The processing unit 400 includes an acquisition unit 402, an evaluation unit 404, and a data generation unit 406.

[0109] The acquisition unit 402 acquires the information of the measurement results output from the automatic analyzer 20. The acquisition unit 402 acquires, for example, information on LDL cholesterol concentration and information on the change amount of absorbance per unit time.

[0110] The evaluation unit 404 evaluates the amount of oxidized LDL in the sample based on the change in absorbance per unit time. The evaluation unit 404 obtains an evaluation value of the amount of oxidized LDL based on a value obtained by multiplying the change in absorbance per unit time by a constant. For example, the evaluation unit 404 uses, as the evaluation value, a value (reaction delay index) obtained by multiplying the change in absorbance per minute between 3 minutes before the end of the reaction and the end of the reaction by a constant 10,000.

[0111] Here, the period from 3 minutes before the end of the reaction to the end of the reaction is taken as the evaluation target, but the evaluation target time can be changed as appropriate. Also, although the unit time is set to 1 minute, that time can be changed as appropriate. Further, the constant is not limited to 10,000 and can be set to any number.

[0112] Also, for example, the evaluation unit 404 evaluates the amount of oxidized LDL in the sample based on a value obtained by dividing the change in absorbance per unit time by the LDL cholesterol concentration of the sample. For example, the evaluation unit 404 uses, as the evaluation value, a value (oxidized LDL ratio) obtained by dividing a value obtained by multiplying the change in absorbance per minute between 3 minutes before the end of the reaction and the end of the reaction by a constant 10,000 by the LDL cholesterol concentration.

[0113] The evaluation unit 404 may use both the reaction delay index and the oxidized LDL ratio as the evaluation value of the amount of oxidized LDL, or may use either one as the evaluation value.

[0114] The data generation unit 406 generates display data for causing the information processing terminal 60 to display the LDL cholesterol concentration of the sample, the change in absorbance per unit time, and the evaluation value of the amount of oxidized LDL. For example, when a user accesses the evaluation device 40 from the information processing terminal 60, the display data can be acquired via the communication network 4. As a result, the LDL cholesterol concentration, the change in 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.

[0115] 4. Processing of the Evaluation Device FIG. 18 is a flowchart showing an example of the evaluation process of the amount of oxidized LDL in the evaluation device 40.

[0116] The acquisition unit 402 acquires information on the LDL cholesterol concentration of the specimen output from the automatic analyzer 20 and information on the amount of change in absorbance per unit time (S100).

[0117] In the measurement unit of the automatic analyzer 20, measurement is performed by the endpoint method and differential measurement using an LDL cholesterol measurement reagent, and the control unit 240 calculates the LDL cholesterol concentration and the amount of change in absorbance per unit time. The automatic analyzer 20 sends information on the LDL cholesterol concentration calculated via the communication network 4 and information on the amount of change in absorbance per unit time to the evaluation device 40 and requests the evaluation device 40 to execute an evaluation process. The processing unit 400 starts the evaluation process in response to this request, and the acquisition unit 402 acquires this information output from the automatic analyzer 20.

[0118] The evaluation unit 404 obtains an evaluation value of the amount of oxidized LDL based on the amount of change in absorbance per unit time (S102). The evaluation unit 404 calculates at least one of, for example, a delay reaction index and an oxidized LDL ratio as an evaluation value of the amount of oxidized LDL. at least one of them as an evaluation value of the amount of oxidized LDL.

[0119] The data generation unit 406 generates display data for displaying the LDL cholesterol concentration, the amount of change in absorbance per unit time, and the evaluation value of the amount of oxidized LDL on the display unit (S104). After the data generation unit 406 generates the display data, the processing unit 400 ends the evaluation process.

[0120] 5. Effects The evaluation device 40 includes an acquisition unit 402 that acquires information on the change amount of absorbance per unit time obtained by a measurement method of solubilizing LDL with a surfactant that acts on LDL in a sample, causing peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and measuring the change in absorbance over time, and an evaluation unit 404 that evaluates the amount of oxidized LDL in the sample based on the change amount.

[0121] Therefore, in the evaluation device 40, the amount of oxidized LDL in the sample can be evaluated based on the change amount of absorbance per unit time measurable by the automatic analyzer 20. Therefore, in the evaluation device 40, the amount of oxidized LDL can be evaluated quickly. Thus, according to the evaluation device 40, the amount of oxidized LDL in a large number of samples can be easily evaluated.

[0122] In addition, by evaluating the amount of oxidized LDL in the sample from the change amount of absorbance per unit time, oxidized LDL can be comprehensively observed in one measurement. For example, in the ELISA method, oxidized LDL can be detected targeting aldehyde, but functional changes in vascular endothelial cells such as arteriosclerosis and coronary syndrome are not simple enough to be judged by a single biomarker. In contrast, by evaluating the amount of oxidized LDL in the sample from the change amount of absorbance per unit time, the oxidative modification of LDL can be comprehensively observed, so that an index that can more accurately evaluate the potential risks of arteriosclerosis and coronary syndrome can be provided.

[0123] In the evaluation device 40, an evaluation value of the amount of oxidized LDL is obtained based on a value obtained by multiplying the change amount of absorbance per unit time by a constant. Thus, the evaluation device 40 can provide an index that can more accurately evaluate the potential risks of arteriosclerosis and coronary syndrome.

[0124] In the evaluation device 40, an evaluation value of the amount of oxidized LDL is obtained based on a value obtained by dividing the change amount of absorbance per unit time by the LDL cholesterol concentration of the specimen. Thus, in the evaluation device 40, since the change amount of absorbance per unit time is normalized by the LDL cholesterol concentration of the specimen, the amount of oxidized LDL can be more accurately compared even among specimens with different LDL cholesterol concentrations.

[0125] 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 amount of 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, 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 can be displayed on the display unit of the information processing terminal 60.

[0126] In the evaluation device 40, the change amount of absorbance per unit time is the change amount of absorbance per unit time from a point in time that is a predetermined time back from the end point of the reaction to the end point of the reaction. Therefore, in the evaluation device 40, the delay of the side reaction with respect to the main reaction can be observed, and the amount of oxidized LDL can be evaluated.

[0127] The evaluation system 2 solubilizes LDL with a surfactant that acts on LDL in the specimen, and peroxidase is allowed to act on hydrogen peroxide generated by the oxidation reaction by cholesterol oxidase to guide hydrogen peroxide to a color reaction, measures the time change of absorbance, and outputs data on the time change of absorbance, a differential calculation unit that obtains the change amount of absorbance per unit time from the data, and an evaluation unit 404 that evaluates the amount of oxidized LDL in the specimen based on the change amount.

[0128] Therefore, in the evaluation system 2, 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 the automatic analyzer 20. Therefore, in the evaluation system 2, the amount of oxidized LDL can be evaluated quickly.

[0129] The evaluation method according to this embodiment includes a step of obtaining information on the amount of change in absorbance per unit time, which is obtained by a measurement method of solubilizing LDL with a surfactant acting on LDL in a sample, causing peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and measuring the temporal change in absorbance, and a step of evaluating the amount of oxidized LDL in the sample based on the amount of change.

[0130] Therefore, in the evaluation method according to this embodiment, the amount of oxidized LDL in the sample can be evaluated based on the amount of change in absorbance per unit time measurable by the automatic analyzer 20. Therefore, in the evaluation method according to this embodiment, the amount of oxidized LDL can be evaluated quickly.

[0131] 6. Modification 6.1. First Modification In the above-described embodiment, the evaluation unit 404 evaluates the amount of oxidized LDL by obtaining, as an evaluation value, a value obtained by multiplying the amount of change in absorbance per unit time by a constant (reaction delay index) or a value obtained by dividing the value by the LDL cholesterol concentration of the sample (oxidized LDL ratio).

[0132] In contrast, the evaluation unit 404 may evaluate the amount of oxidized LDL by determining whether or not these evaluation values exceed a reference value. For example, the evaluation unit 404 may determine whether or not the oxidized LDL ratio of the actual sample exceeds the reference value, using the value at day 0 of the incubation time of the oxidized LDL ratio shown in FIG. 12 as the reference value. The evaluation unit 404 may generate, for example, a list of samples for which the reference value has been exceeded as an evaluation result.

[0133] 6.2. Second Modification In the above-described embodiment, the control unit 240 of the automatic analyzer 20 functions as a differential calculation unit that performs differential analysis on the temporal change in absorbance, which is the measurement result of the measurement unit, to calculate the amount of change in absorbance per unit time. However, the acquisition unit 402 of the evaluation device 40 may function as the differential calculation unit.

[0134] For example, the automatic analyzer 20 outputs information on the temporal change in absorbance, and the acquisition unit 402 acquires the information on the temporal change in absorbance. The acquisition unit 402 performs a differential operation on the temporal change in absorbance to obtain the amount of change in absorbance per unit time. Thereby, the acquisition unit 402 can acquire information on the amount of change in absorbance per unit time.

[0135] 6.3. Third Variant Example In the above-described embodiment, as shown in FIG. 15, the evaluation system 2 includes the automatic analyzer 20, the evaluation device 40, and the information processing terminal 60, and these are configured to be connectable via a communication network 4 such as the Internet, and the evaluation result is displayed on the display unit of the information processing terminal 60.

[0136] On the other hand, in the evaluation system 2, the automatic analyzer 20 and the evaluation device 40 may be connected via a LAN (Local Area Network). In this case, the evaluation system 2 does not include the information processing terminal 60, and the evaluation result may be displayed on the display unit of the control unit 240 of the automatic analyzer 20 or the display unit 420 of the evaluation device 40.

[0137] 7. Other Embodiments In the above-described embodiment, the color reaction due to the reaction between LDL and the reagent is defined as the main reaction (first reaction) of the reagent, and the color reaction due to the reaction between oxidized LDL and the reagent is defined as the side reaction (second reaction) of the reagent. The amount of change per unit time in the absorbance of the dye generated from the main reaction and the side reaction is measured, and the amount of oxidized LDL is evaluated based on the amount of change per unit time in the absorbance.

[0138] This evaluation method is also applicable to specimens other than specimens containing LDL and oxidized LDL. That is, the evaluation method may include a step of reacting a specimen containing a first substance and a second substance with a reagent and measuring the amount of 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 evaluating the physical quantity of the second substance based on the amount of change per unit time in the physical quantity of the reaction product.

[0139] Here, in the above embodiment, the first substance was LDL, and the first reaction product was a dye (first dye) produced by the reaction of LDL and the reagent. Also, the second substance was oxidized LDL, and the second reaction product was a dye (second dye) produced by the reaction of oxidized LDL and the reagent. In contrast, the first substance and the second substance are not particularly limited as long as the first reaction product is produced by the first reaction of the first substance and the reagent, and the second reaction product is produced by the second reaction of the second substance and the reagent.

[0140] Also, the first reaction and the second reaction are not limited to color development reactions, and may be any of denaturation, oxidation, reduction, addition, binding, decomposition, condensation, solubilization reactions, or a combination thereof. Therefore, the first reaction product and the second reaction product are not limited to dyes.

[0141] Also, when the reaction product has a specific absorption wavelength, the physical quantity of the reaction product may 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 may be detected by means such as fluorescence, Raman, nuclear magnetic resonance, and mass spectrometry, respectively.

[0142] For example, when the change in the physical quantity of the reaction product including the first reaction product and the second reaction product is represented as f(x), the side reaction may be represented as its derivative f´(x), and the physical quantity of the second substance may be represented as the change amount of the physical quantity of the reaction product per unit time.

[0143] The evaluation value of the physical quantity of the second substance can be obtained in the same manner as the evaluation value of the amount of oxidized LDL 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 number) or a value obtained by dividing the value by the amount (concentration) of the first substance can be used.

[0144] Note that the above-described embodiments and modifications are examples, and are not limited thereto. For example, each embodiment and each modification can be combined as appropriate.

[0145] The present invention is not limited to the above-described embodiments, and various modifications are further possible. For example, the present invention includes a configuration that is substantially the same as the configuration described in the embodiments. The substantially same configuration means, for example, a configuration having the same functions, methods, and results, or a configuration having the same purpose and effects. Further, the present invention includes a configuration in which a non-essential part of the configuration described in the embodiments is replaced. Further, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiments or a configuration that can achieve the same purpose. Further, the present invention includes a configuration in which a known technique is added to the configuration described in the embodiments.

Explanation of Reference Numerals

[0146] 2… Evaluation system, 4… Communication network, 20… Automatic analyzer, 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… Multiwavelength photometer, 217… Thermostatic bath, 218… Reaction vessel cleaning mechanism, 221… Sample container, 224… First reagent container, 225… Second reagent container, 226… Reaction vessel, 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

Claims

1. An acquisition unit that acquires information on the amount of change in the absorbance per unit time, obtained by a measurement method in which LDL is solubilized with a surfactant that acts on LDL in a sample, peroxidase is allowed to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and the time change in absorbance is measured; An evaluation unit that evaluates the amount of oxidized LDL in the sample based on the amount of change; An evaluation apparatus comprising the above.

2. In Claim 1, 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. An evaluation apparatus.

3. In Claim 1, The evaluation unit obtains an evaluation 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. An evaluation apparatus.

4. In Claim 3, The evaluation unit determines whether or not the evaluation value exceeds a reference value. An evaluation apparatus.

5. In Claim 3 or 4, An evaluation apparatus including a data generation unit that generates data for causing at least one of the LDL cholesterol concentration of the sample, the amount of change, and the evaluation value to be displayed on a display unit.

6. In Claim 1, The amount of change is the amount of change in absorbance per unit time from a point in time retroactively a predetermined time from the end point of the reaction to the end point of the reaction. An evaluation apparatus.

7. A measurement unit that solubilizes LDL with a surfactant that acts on LDL in a sample, allows peroxidase to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, measures the time change in absorbance, and outputs data on the time change in absorbance; A differential calculation unit that obtains the amount of change in the absorbance per unit time from the data; An evaluation unit that evaluates the amount of oxidized LDL in the sample based on the amount of change; A system comprising the above.

8. A step of acquiring information on the amount of change in the absorbance per unit time, obtained by a measurement method in which LDL is solubilized with a surfactant that acts on LDL in a sample, peroxidase is allowed to act on hydrogen peroxide generated by an oxidation reaction with cholesterol oxidase to lead the hydrogen peroxide to a color reaction, and the time change in absorbance is measured; A step of evaluating the amount of oxidized LDL in the sample based on the amount of change; An evaluation method comprising the above.

9. A step of reacting a specimen containing a first substance and a second substance with a reagent and measuring a change amount per unit time of a physical quantity of a reaction product containing 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; A step of evaluating a physical quantity of the second substance based on the change amount; An evaluation method comprising the steps.

10. In claim 9, The specimen is a body fluid, The first substance is LDL, The second substance is oxidized LDL, The first reaction is a color reaction by a reaction between the LDL and the reagent, The second reaction is a color reaction by a reaction between the oxidized LDL and the reagent. An evaluation method.

11. In claim 10, The first reaction product is a first dye generated by a reaction between the LDL and the reagent, The second reaction product is a second dye generated by a reaction between the oxidized LDL and the reagent. An evaluation method.

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