Method and kit for measuring reduced albumin concentration or the proportion of oxidized or reduced albumin in serum or plasma

Abstract

To provide a method of easily measuring reduced albumin using a chromogenic regent, which enables more accurate measurement with less deviation from the standard method compared with conventional methods, and to provide a kit therefor.SOLUTION: Reduced albumin concentration is measured using a difference in absorbance between a sample added with a specific oxidizer and the sample with no additives.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method and a kit for simply measuring the concentration of reduced albumin or the proportion of oxidized or reduced albumin in serum or plasma. [Background technology]

[0002] Albumin in blood is the most abundant protein in the body, and is in the form of oxidized or reduced form. For this reason, the proportion of oxidized albumin in human serum or plasma is considered to be an index reflecting oxidative stress in the whole body, and it has been reported that it increases with the progression of chronic diseases such as chronic kidney disease and diabetes (Non-Patent Documents 1 and 2). It has also been reported that it can be used as a biomarker for auxiliary diagnosis of central nervous system diseases associated with aging, such as Parkinson's disease and Alzheimer's disease (Non-Patent Documents 3 and 4). For this reason, it is considered that the measurement of the proportion of oxidized or reduced albumin is useful for the diagnosis and risk assessment of these diseases (Patent Document 1 and Non-Patent Document 1).

[0003] The proportion of oxidized or reduced albumin is measured by a method using HPLC (Non-Patent Documents 1 and 2 and Patent Document 5) or a method using a mass spectrometer (Non-Patent Documents 2 and 5). These methods are well established and can measure the proportion of oxidized or reduced albumin with high accuracy, and therefore are used as standard methods. However, these methods have limitations in throughput. In particular, methods using mass spectrometers require instruments (e.g., EST-TOF MS) capable of measuring large molecular weight proteins with high resolution (Non-Patent Documents 2 and 5).

[0004] On the other hand, a method using a color-developing reagent has been developed as a simpler and higher throughput measurement method, and the BCP method and the improved BCP method are known as representative methods for measuring albumin. In addition, a method for quantifying reduced albumin by combining the BCP method and the improved BCP method has also been reported (Patent Documents 1 and 2). The BCP method is a method for quantifying albumin by utilizing metachromasia, in which the absorption spectrum changes when bromocresol purple (BCP) binds to albumin. However, since BCP has different reactivity to reduced albumin and oxidized albumin, the total albumin value is affected by the ratio of the two, so an improved BCP method has been developed in which reduced albumin is converted to oxidized albumin using an oxidizing agent and then reacted with BCP, and this method is used as a standard method for quantifying total albumin. In addition, various oxidizing agents have been investigated for the improved BCP method, and it has been reported that oxidizing agents such as potassium permanganate, sodium periodide, and sodium metavanadate cannot fully avoid the influence of the SH group of reduced albumin, and that this influence can be avoided by using a 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reagent with added sodium dodecyl sulfate (SDS) (Non-Patent Document 6). For this reason, a combination of SDS and DTNB is typically used as an oxidizing agent in the improved BCP method (Patent Document 3).

[0005] The method of measuring the reduced albumin concentration by combining the BCP method and the improved BCP method is a method in which the total albumin concentration is determined by the improved BCP method, and the reduced albumin concentration is calculated by subtracting the albumin concentration determined by the BCP method from this total albumin concentration. In this method, the oxidizing agents used in the improved BCP method are typically a combination of SDS and DTNB (Patent Documents 1 and 2). In a method of determining the reduced albumin concentration or ratio by combining the BCP method and the improved BCP method, a low correlation (r=0.53) has been reported with respect to the ratio of oxidized or reduced albumin measured by the standard HPLC method (Non-Patent Document 7), and it has also been reported that the measurement error is large in patient samples, making it impossible to estimate the redox state of albumin (Non-Patent Document 8). Furthermore, in a method of determining the reduced albumin concentration or ratio using such BCP, the concentration of oxidized albumin tends to be higher in samples with higher total albumin concentrations than in these standard methods, and there is also a problem of low sensitivity.

[0006] As another method for measuring the reduced albumin concentration in a color-developing reagent system, a method using 4,4'-bis(dimethylamino)benzhydrol (BDAB) (BDAB method) has also been reported (Non-Patent Document 9 and Patent Document 4). This method is characterized by utilizing the change in absorbance resulting from the nucleophilic reaction of thiol groups with the BDAB cation, and since most of the thiols in serum or plasma are derived from reduced albumin, the reduced albumin concentration is approximately determined by measuring the thiol concentration in serum or plasma (Non-Patent Documents 9, 10, and 11 and Patent Document 4). This BDAB method is different from the method of combining the BCP method and the improved BCP method in that it can directly measure the reduced albumin concentration. In addition, this method is different from the previous method in which 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a color-developing reagent for thiol groups in serum, and it is said that pretreatment by column chromatography or the like is not required to remove the influence of coexisting substances (Patent Document 4).

[0007] However, as a result of the study by the present inventors, it has been found that even with this BDAB method, there are some samples where the reduced albumin concentration calculated from the measurement results of the standard HPLC method and the improved BCP method is deviated, and a measurement method that is more correlated with the standard method and can obtain more reliable results is desired. In addition, the reduced albumin used as a calibrator in this method is purified using a special column called Thiol Sepharose (Non-Patent Document 9), and there is a problem that the preparation of the calibrator is time-consuming. In addition, there has been no report so far on the determination of the ratio of oxidized or reduced albumin using a method using BDAB. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 2018-025486 [Patent Document 2] Patent Publication 2018-194453

Patent Document 3

Patent document 4

Patent document 5

Non-licensed literature

[0009] [Non-licensed document 1] Masudo R.et al., "Evaluation of human nonmercaptalbumin as a marker for oxidative stress and its association with various parameters in blood" Journal of Clinical Biochemistry and Nutrition, 2017, Vol.61, No.2, p.79-84 [Non-licensed document 2] Nagumo K.et al., "Cys34-Cysteinylated Human Serum Albumin Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases" PLOS ONE, 2014, Vo.9, No.1, p. e85216. [Non-licensed document 3] Costa M et. al., "Increased Albumin Oxidation in Cerebrospinal Fluid and Plasma from Alzheimer's Disease Patients", Journal of Alzheimer's Disease, 2018, Vol.63, p.1395-1404

Non-licensed Document 4

[0010] Therefore, the present invention aims to provide a method for easily measuring reduced albumin concentration using a color-developing reagent, which is less likely to deviate from standard methods and allows more accurate measurement than conventional methods, and a kit therefor.The present invention also aims to provide a method for measuring reduced albumin that allows the use of easily prepared samples such as standard serum or plasma as a calibrator, and a kit therefor.A further object of the present invention is to provide a novel method for determining the proportion of oxidized or reduced albumin using a color-developing reagent. [Means for solving the problem]

[0011] The present inventors confirmed that when the reduced albumin concentration was measured by the conventional BDAB method, there existed samples in which the results deviated relatively greatly from the standard method. They then conducted extensive research to improve this problem, and discovered that in a measurement system using a substance such as BDAB whose absorbance changes upon reacting with thiols, the deviation from the standard method could be improved by measuring the reduced albumin concentration using the difference in absorbance between a sample to which a specific oxidizing agent had been added and a sample to which no specific oxidizing agent had been added, thereby completing the present invention.

[0012] That is, the present invention provides the following kit for measuring reduced albumin, a method for measuring the concentration of reduced albumin in a sample, and a method for determining the proportion of oxidized or reduced albumin. [1] A kit for measuring a concentration of reduced albumin, comprising: a color-developing substance that changes absorbance upon reacting with thiol; and an oxidizing agent that oxidizes a free SH group of reduced albumin, The oxidizing agent is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines. [2] The kit described in [1], wherein the color-developing substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of a thiol group, and is selected from the group consisting of, for example, 4-4'-bisdimethylaminobenzhydrol (BDAB), 7-nitro-2,3-dihydro-1H-cyclopenta[b]chromen-1-one, diketopyrrolopyrrole, 1,3-bis(4-N,N-dimethylamino-2-hydroxy-phenol)croconine, 7-nitrobenz-2-oxa-1,3-diazolemaleimide, and 2,4-bis[4-(N,N-bis(2-methoxyethoxy)methyl)-diphenyl]squaraine. [3] The kit described in [1], wherein the color-developing substance is 4-4'-bisdimethylaminobenzhydrol (BDAB). [4] The kit according to any one of [1] to [3], wherein the alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). [5] The kit according to any one of [1] to [3], wherein the oxidizing agent is selected from the group consisting of iodoacetic acid, iodoacetamide, maleimide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, 2,2'-dithiobis(5-nitropyridine), 6,6'-dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). [6] The kit according to any one of [1] to [5], further comprising a protein denaturing agent. [7] The kit described in any one of [1] to [6], wherein the molar concentration of the oxidizing agent is set so that, when a reagent containing the oxidizing agent is mixed with a sample, the molar concentration is 10 times or more the molar concentration of albumin in the mixture, and, when a reagent containing the chromogenic substance is mixed with the mixture, the molar concentration is 270 times or less the molar concentration of the chromogenic substance. [8] The kit according to any one of [1] to [7], comprising standard serum or plasma, a purified albumin solution, or a purified reduced albumin solution as a calibrator. [9] The kit described in any one of [1] to [8], comprising a first reagent containing the oxidizing agent and a buffer solution, and a second reagent containing the color-developing substance and a buffer solution, wherein either or both of the first reagent and the second reagent contain a protein denaturant.

[10] A method for measuring a reduced albumin concentration, comprising: An oxidizing agent that oxidizes the free SH groups of reduced albumin is added to the sample, which is then mixed with a reagent containing a color-developing substance that reacts with thiols to change its absorbance, and the absorbance (OD) is measured at a specified wavelength. ox ) measuring the On the other hand, the sample is mixed with a reagent containing the color-developing substance without adding the oxidizing agent, and the absorbance (OD non-ox ) measuring the The absorbance (OD ox ) and the absorbance (OD non-ox ) and calculating the absorbance difference (ΔOD) between the sample and the reduced albumin concentration in the sample from the absorbance difference (ΔOD), The method, wherein the oxidizing agent is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines.

[11] The method according to

[10] , wherein the color-developing substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of a thiol group, and is selected from the group consisting of, for example, 4-4'-bisdimethylaminobenzhydrol (BDAB), 7-nitro-2,3-dihydro-1H-cyclopenta[b]chromen-1-one, diketopyrrolopyrrole, 1,3-bis(4-N,N-dimethylamino-2-hydroxy-phenol)croconine, 7-nitrobenz-2-oxa-1,3-diazolemaleimide, 2,4-bis[4-(N,N-bis(butylcarbamate))-diphenyl]squaraine, and 2,4-bis[4-(N,N-bis(2-methoxyethoxy)methyl)-diphenyl]squaraine.

[12] The method according to

[10] , wherein the color-developing substance is 4-4'-bisdimethylaminobenzhydrol (BDAB).

[13] The method according to any one of

[10] to

[12] , wherein the alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

[14] The method according to any one of

[10] to

[12] , wherein the oxidizing agent is selected from the group consisting of iodoacetic acid, iodoacetamide, maleimide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, 2,2'-dithiobis(5-nitropyridine), 6,6'-dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

[15] The method according to any one of items

[10] to

[14] , further comprising adding a protein denaturing agent to the sample.

[16] The method according to any one of

[10] to

[15] , wherein a calibration curve is created using standard serum or plasma, a purified albumin solution, or a purified reduced albumin solution as a calibrator.

[17] preparing a dilution series of the calibrator; The oxidizing agent is added to each dilution in the dilution series, and then the mixture is mixed with the reagent containing the color-developing substance, and the absorbance (OD ox ) is measured, On the other hand, each dilution in the dilution series is mixed with the reagent containing the color-developing substance without adding the oxidizing agent, and then the absorbance (OD non-ox ) is measured, For each of the dilutions, the absorbance (OD ox ) and the absorbance (OD non-ox ) and calculate the absorbance difference (ΔOD) The method according to

[16] , wherein the calibration curve is created from each of the absorbance differences (ΔOD) and the known reduced albumin concentrations of each of the dilutions.

[18] A method for determining a proportion of reduced albumin, comprising the steps of: Determine the total albumin concentration in the sample by the improved BCP method. determining a reduced albumin concentration in the sample by the method according to any one of

[10] to

[17] ; The reduced albumin concentration is divided by the total albumin concentration to calculate the percentage of reduced albumin.

[19] A method of calculating the percentage of oxidized albumin by subtracting the percentage of reduced albumin determined by the method described in

[18] from 1.

[0013] The present invention enables the measurement of reduced albumin with a higher throughput and more easily than methods involving measurements by HPLC and mass spectrometry. In addition, the present invention has a higher correlation with the standard method and a smaller deviation from the standard method than the BCP / improved BCP method and the conventional BDAB method, and therefore allows the measurement of reduced albumin concentration more accurately.

[0014] Here, some terms used in this specification are defined. As used herein, "HPLC method" refers to a method in which HPLC is used to determine the oxidized / reduced albumin ratio or the percentage of oxidized or reduced albumin in total albumin.

[0015] As used herein, "mass spectrometry" refers to a method for determining the oxidized / reduced albumin ratio or the proportion of oxidized or reduced albumin in total albumin using a mass spectrometer. Furthermore, unless otherwise specified, analysis by "mass spectrometry" is performed according to the procedures and conditions described in Non-Patent Documents 2 and 5.

[0016] As used herein, the "improved BCP method" refers to a method in which an oxidizing agent that oxidizes the free SH groups of reduced albumin is added to a sample to convert reduced albumin to oxidized albumin, and then bromocresol purple (BCP) is reacted with the oxidized albumin to measure the total albumin concentration.

[0017] In the present specification, when referring to a method for determining the proportion of oxidized or reduced albumin in total albumin, the "standard method" refers to the "HPLC method" carried out according to the procedure and conditions described in Patent Document 5. Specifically, in this standard method, an anion exchange column packed with an ion exchange resin having DEAE (Diethylaminoethyl) as an ion exchange group is used, and after equilibration with buffer A, a sample is injected, and at the same time, a linear gradient of buffer A (100% to 75%) and buffer B (0% to 25%) is applied at a flow rate of 1.0 mL / min for 7.5 minutes to separate oxidized albumin and reduced albumin, and detection is performed by fluorescence at an excitation wavelength of 280 nm and a detection wavelength of 340 nm. Buffer A: 25 mM phosphate buffer (pH 6.0) containing 60 mM Na2SO4 Buffer B: 1M MgCl2

[0018] In addition, when referring to a method for determining total albumin concentration in this specification, the "standard method" refers to the "improved BCP method" performed according to the procedure described in Patent Document 3. Specifically, in this standard method, 85 μL of a first reagent (containing 100 μM DTNB and 0.03% (w / v) SDS in 25 mM Tris HCl, pH 8.0) is added to 15 μL of a sample, and the mixture is incubated at 37° C. for 5 minutes, followed by adding 150 μL of a second reagent (containing 150 μM bromocresol purple and 0.3% (v / v) Triton X-100 in 250 mM sodium succinate, pH 5.5 solution), and further incubating at 37° C. for 20 minutes, and measuring the absorbance at 600 nm (main wavelength) / 660 (secondary wavelength) to determine the total albumin concentration.

[0019] When referring to a method for determining the concentration of reduced albumin in this specification, the term "standard method" refers to a method in which the proportion of reduced albumin in total albumin is determined by the above-mentioned "standard method", the total albumin concentration is determined by the above-mentioned "standard method", and the reduced albumin concentration is determined by multiplying the obtained total albumin concentration by the proportion of reduced albumin.

[0020] As used herein, the term "BDAB method" refers to a method for measuring reduced albumin concentration using 4-4'-bisdimethylaminobenzhydrol (BDAB) as a color developing substance. In addition, as used herein, the term "improved BDAB method" refers to a method in which, in the "BDAB method," a solution containing an oxidizing agent that oxidizes the free SH groups of reduced albumin is added to a specimen to prepare a sample, to which a reagent containing BDAB is added, and the absorbance (OD ox ) and a sample prepared by adding a solution without an oxidizing agent to the same specimen, and adding a reagent containing BDAB to the sample, and measuring the absorbance (OD non-ox ) and calculate the reduced albumin concentration in the sample from the absorbance difference (ΔOD) between the two. In addition, as used herein, the term "conventional BDAB method" refers to a method in which a sample is not treated with an oxidizing agent that oxidizes the free SH group of reduced albumin, but is instead added with a reagent containing BDAB, and the absorbance (OD ) is measured at a predetermined wavelength. non-ox ) to calculate the reduced albumin concentration in a sample. [Brief description of the drawings]

[0021] [Figure 1] The upper graph shows a chromatogram obtained by analyzing one plasma sample by the HPLC method (standard method) according to the procedure described in Patent Document 5. The table on the lower left shows the areas and area ratios of the reduced albumin peak and oxidized albumin peak in the chromatogram shown above. The table on the lower right shows the total albumin concentration measured for one plasma sample by the improved BCP method (standard method) according to the procedure described in Patent Document 3, and the reduced albumin concentration calculated by multiplying this by the proportion of reduced albumin determined by the HPLC method (standard method). [Diagram 2] The chromatograms shown are of the standard serum analyzed by the HPLC method (standard method) before and after treatment with maleimide. It can be seen that the reduced albumin peak completely disappears and the oxidized albumin peak becomes larger by treatment with maleimide. [Diagram 3] FIG. 3 is a calibration curve for determining the reduced albumin concentration created using the data shown in Table 10. FIG. 3A and B are calibration curves for determining the reduced albumin concentration using the absorbance difference (ΔOD), FIG. 3A is a linear calibration curve, and FIG. 3B is a nonlinear calibration curve. FIG. 3C is a calibration curve for determining the reduced albumin concentration using ODnon-ox, and is the same as the conventional "BDAB method". [Figure 4]Samples (8 serum samples, 4 plasma samples) obtained from 12 donors were diluted with a buffer solution to a concentration of 183.5 μM based on the reduced albumin concentration determined by the standard method (HPLC method & improved BCP method). The reduced albumin concentration was determined using the calibration curve shown in Figure 3 by the improved BDAB method (with correction) using maleimide as an oxidizing agent or the conventional BDAB method (without correction), and the deviation from the standard value of 183.5 μM of the standard method (HPLC method & improved BCP method) was evaluated. Figure 4A shows the deviation from the standard value of each sample, and Figure 4B shows the distribution of deviation from the standard value. The upper and lower ends of the bars in Figure 4B indicate the maximum difference (absolute value) and minimum difference (absolute value) for each measurement method relative to the standard method, respectively. The upper and lower ends of the boxes indicate the 75% percentile value and the 25% percentile value, respectively. × indicates the median difference from the standard method. [Diagram 5] Samples (8 serum samples, 4 plasma samples) obtained from 12 donors were diluted with buffer to a total concentration of 183.5 μM based on the reduced albumin concentration determined by the standard method (HPLC method & improved BCP method). The percentage of oxidized albumin was calculated using the improved BDAB method (with correction) using maleimide as an oxidizing agent or the conventional BDAB method (without correction) and the improved BCP method (standard method), and the deviation from the percentage of oxidized albumin obtained by the standard method (HPLC method) is shown in the figure. The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) for each measurement method compared to the standard method, respectively. The upper and lower ends of the boxes indicate the 75% percentile value and the 25% percentile value, respectively. × indicates the median difference compared to the standard method. (Since the ratio of oxidized albumin is generally described in Patent Document 5 and Non-Patent Documents 1 and 2, the ratio of oxidized albumin will be preferentially described in this specification.) [Figure 6] The test results shown in Table 13 show the correlation between the percentage of oxidized albumin determined by each method and the percentage of oxidized albumin determined by the standard method (HPLC method). [Figure 7]The reduced albumin concentrations of samples (21 serum samples, 11 plasma samples) obtained from 32 donors were determined by the improved BDAB method (with correction) using maleimide as an oxidizing agent or the conventional BDAB method (without correction), and the results of the test were evaluated to evaluate the deviation from the reduced albumin concentration determined by the standard method (HPLC method and improved BCP method). The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) for each measurement method against the standard method, respectively. The upper and lower ends of the boxes indicate the 75th and 25th percentile values, respectively. × indicates the median difference against the standard method. For the improved BDAB method (with correction), linear and nonlinear calibration curves were created to determine the reduced albumin concentration. [Figure 8] The results of the test are shown below. The percentage of oxidized albumin was determined for samples (21 serum samples, 11 plasma samples) obtained from 32 donors by the improved BDAB method (with correction) using maleimide as an oxidizing agent, the conventional BDAB method (without correction), and the improved BCP method (standard method), and the deviation from the oxidized albumin concentration determined by the standard method (HPLC method) was evaluated. The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) for each measurement method compared to the standard method, respectively. The upper and lower ends of the boxes indicate the 75th and 25th percentile values, respectively. × indicates the median difference compared to the standard method. For the improved BDAB method (with correction), linear and nonlinear calibration curves were created to determine the reduced albumin concentration. [Figure 9] For the test results shown in Table 23, the correlation between the percentage of oxidized albumin determined by each method and the percentage of oxidized albumin determined by the standard method (HPLC method) is shown. [Figure 10]The reduced albumin concentrations of samples (21 serum samples, 11 plasma samples) obtained from 32 donors were determined by the improved BDAB method (with correction) using iodoacetamide as an oxidizing agent or the conventional BDAB method (without correction), and the results of evaluating the deviation from the reduced albumin concentration determined by the standard method (HPLC method and improved BCP method) are shown. The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) of each measurement method against the standard method, respectively. The upper and lower ends of the boxes indicate the 75th and 25th percentile values, respectively. × indicates the median difference against the standard method. For the improved BDAB method (with correction), linear and nonlinear calibration curves were created to determine the reduced albumin concentration. [Figure 11] The percentage of oxidized albumin was determined for samples obtained from 32 donors (21 serum samples, 11 plasma samples) by the improved BDAB method (with correction) using iodoacetamide as an oxidizing agent, the conventional BDAB method (without correction), and the improved BCP method (standard method), and the deviation from the percentage of oxidized albumin determined by the standard method (HPLC method) was evaluated. The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) for each measurement method compared to the standard method, respectively. The upper and lower ends of the boxes indicate the 75th and 25th percentile values, respectively. × indicates the median difference compared to the standard method. For the improved BDAB method (with correction), linear and nonlinear calibration curves were created to determine the reduced albumin concentration. [Figure 12] For the test results shown in Table 26, the correlation between the percentage of oxidized albumin determined by each method and the percentage of oxidized albumin determined by the standard method (HPLC method) is shown. [Figure 13] The calibration curves of reduced albumin concentration prepared by the conventional BDAB method (without correction) or the improved BDAB method (with correction) using standard serum, purified albumin solution, or purified reduced albumin solution are shown. [Figure 14] The calibration curves of reduced albumin concentration prepared by the conventional BDAB method (without correction) or the improved BDAB method (with correction) using standard serum or cysteine ​​solution are shown. [Figure 15] The following shows calibration curves of reduced albumin concentration prepared by the conventional BDAB method (without correction) or the improved BDAB method (with correction) using various standard sera. [Figure 16] The following shows calibration curves of reduced albumin concentration prepared by the conventional BDAB method (without correction) or the improved BDAB method (with correction) using various standard serum or plasma samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be described in detail below with reference to the preferred embodiments. However, the present invention should not be understood as being limited to the following embodiments.

[0023] 1. How to measure reduced albumin concentration One embodiment of the present invention relates to a method for measuring the concentration of reduced albumin in a sample using a substance that changes its absorbance upon reaction with a thiol. Human blood albumin has 35 cysteine ​​residues (SH groups) in its molecule, of which 17 pairs form intramolecular disulfide bonds, but the SH group of the 34th cysteine ​​from the N-terminus may be free or oxidized. The former is called reduced albumin, and the latter is called oxidized albumin. Since human serum albumin is the protein with the largest total amount in the body, the proportion of oxidized albumin in human serum or plasma is considered to be an indicator of oxidative stress throughout the body, and it has been reported that it increases with the progression of chronic diseases such as chronic kidney disease and diabetes (Non-Patent Documents 1 and 2). It has also been reported that it can be used as a biomarker for auxiliary diagnosis of central nervous system diseases associated with aging, such as Parkinson's disease and Alzheimer's disease (Non-Patent Documents 3 and 4). For this reason, the measurement of reduced albumin concentration, the proportion of oxidized or reduced albumin, and the oxidized / reduced albumin ratio are used to diagnose and determine the risk of these conditions and diseases.

[0024] As a color-developing substance whose absorbance changes upon reaction with thiol, a substance whose absorbance changes as a result of a nucleophilic reaction of a thiol group is preferred, and a thiol-reactive substance that does not have a disulfide structure is more preferred. Examples of such substances include squaraine dyes such as 4-4'-bisdimethylaminobenzhydrol (BDAB), 7-nitro-2,3-dihydro-1H-cyclopenta[b]chromen-1-one, diketopyrrolopyrrole, 1,3-bis(4-N,N-dimethylamino-2-hydroxy-phenol)croconine, 7-nitrobenz-2-oxa-1,3-diazolemaleimide, 2,4-bis[4-(N,N-bis(2-methoxyethoxy)methyl)-diphenyl]squaraine, and 2,4-bis[4-(N,N-bis(butylcarbamate))-diphenyl]squaraine. Squaraine dyes have the following structure: [ka] In the preferred squaraine dye, each R is independently [ka] (the left end of the formula is bonded to N of the above compound), and more preferred compounds are compounds in which R in the formula is any of the above structures. The above-mentioned color-forming substances are described in detail in Non-Patent Documents 12-16.

[0025] As the color-developing substance, 4-4'-bisdimethylaminobenzhydrol (BDAB) is particularly preferable. 4,4'-Bis(dimethylamino)benzhydrol (BDAB) is [ka] It has a maximum absorbance wavelength at 612 nm. The reaction of BDAB with reduced albumin is a nucleophilic reaction of the thiol group with the cation shown below, and the resulting complex has a reduced absorbance at 612 nm, and the amount of thiol groups can be directly measured by this change in absorbance. [ka] The measurement wavelength may be selected so that the color development of BDAB can be specifically detected, taking into consideration the absorption spectrum of impurities present in the sample, and is usually selected from the vicinity of the maximum absorption wavelength of 612 nm, specifically, 570 to 660 nm, preferably 600 to 625 nm, and the maximum absorption wavelength of 612 nm is preferred. In addition, since the nucleophilic reaction of the thiol group on BDAB occurs when BDAB becomes the above-mentioned cation under acidic conditions, it is preferable to react BDAB with reduced albumin under acidic conditions, typically in a solution of pH 5 to 6. Therefore, the color development reagent containing BDAB usually contains a buffer solution and is maintained under acidic conditions, typically pH 5 to 6.

[0026] The concentration of BDAB is preferably such that when mixed with a sample, all reduced albumin in the sample reacts with BDAB and is not affected by color development by the oxidizing agent. In this regard, the upper limit of the normal value of albumin concentration in human serum is about 5 g / dL, and considering the ratio of reduced albumin, dilution by mixing with the reagent, and the amount of oxidizing agent required to completely convert reduced albumin to oxidized albumin, the concentration of the reagent containing BDAB is usually adjusted to 15 to 80 μM, and the volume of the reagent is 15 to 45 times, preferably 15 to 40 times, the volume of the sample containing albumin. From the same point of view, the concentration of BDAB is preferably adjusted to 1.5 to 6 times, preferably 1.5 to 3 times, the molar concentration of reduced albumin in the reaction mixture, and 0.05 to 0.5 times the molar concentration of the oxidizing agent in the reaction mixture.

[0027] In the present invention, in a method for measuring the concentration of reduced albumin in a sample by a color reaction using a color-forming substance whose absorbance changes upon reaction with a thiol such as BDAB, the specimen is divided into two, and one specimen is prepared by adding a solution containing an oxidizing agent that oxidizes the free SH groups of reduced albumin (a correction sample), and the other specimen is prepared by adding an equal amount of a solution that does not contain an oxidizing agent (a sample for measuring reduced albumin). Next, each specimen is mixed with a reagent containing a color-forming substance (e.g., BDAB), and the absorbance (OD ox ) and absorbance (OD non-ox ) is measured, the difference in absorbance (ΔOD) between the two is calculated, and the reduced albumin concentration in the sample is calculated from this difference in absorbance (ΔOD).

[0028] When an oxidizing agent that oxidizes free SH groups is added, reduced albumin is completely converted to oxidized albumin, but even when a sample that has been treated with an oxidizing agent is mixed with a reagent containing a color-developing substance, a decrease in absorbance is observed compared to the blank, and there are specimens in which the color-developing substance is thought to have reacted with a substance other than reduced albumin or the absorbance at the measurement wavelength is affected by a substance other than reduced albumin. In such specimens, the decrease in absorbance due to substances other than reduced albumin is measured as a decrease due to reduced albumin, which is understood to be the cause of the deviation from the HPLC method. Taking such phenomena into consideration, in the method of the present invention, the absorbance (OD ox ) and the absorbance (OD non-ox The absorbance difference (ΔOD) of the reduced albumin is used to perform correction to eliminate absorbance changes not attributable to reduced albumin, thereby accurately measuring reduced albumin. It should be understood that the problems with the conventional BDAB method and their causes were first recognized by the present inventors, and the principle of solving the problem is completely different from the method of measuring reduced albumin by combining the BCP method and the improved BCP method.

[0029] The method of the present invention also uses a specific compound as an oxidizing agent that oxidizes free SH groups. As a result of the investigations by the present inventors, it has been found that, although the conversion occurs with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which is used to convert reduced albumin to oxidized albumin in the improved BCP method, it is difficult to reduce the deviation from the standard HPLC method by the above correction, although the cause is unknown.

[0030] The present inventors have verified the suitability of various oxidizing agents that oxidize free SH groups for the method of the present invention, and have confirmed that the above correction can reduce the deviation from the standard HPLC method by selecting a specific compound. Specifically, in a preferred embodiment of the present invention, the oxidizing agent that oxidizes free SH groups is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines.

[0031] Examples of the alkylating agent include acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). Examples of the acetyl halide include acetic halide, acetamide halide, and acetophenone halide, more specifically, iodoacetic acid, iodoacetamide, and iodoacetophenone. Examples of the maleimide derivatives include N-alkyl (preferably N-C- 1-5 N,N'-C alkyl)maleimide, N,N'-p-phenylenedimaleimide, etc. 5-12 Examples of the substituent of the substituted dithiodipyridine include a nitro group, a carboxyl group, an amino group, and a hydroxyl group, and specific examples of the compound include 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, 2,2'-dithiobis(5-nitropyridine), 6,6'-dithiodinicotinic acid, 2,2'-dithiobis(5-aminopyridine), and 2,2'-dithiobis(5-hydroxypyridine). Among these, substituted or unsubstituted dithiodipyridines are preferred because they are analytically advantageous in that they can undergo oxidation reaction at pH 5 to 6, which is the reaction pH of BDAB, and because they also have a large correction effect, and 2,2'-dipyridyl disulfide is particularly preferred.

[0032] The concentration of the oxidizing agent is preferably such that when the reagent containing the oxidizing agent and the sample are mixed, all of the reduced albumin in the mixture is converted to oxidized albumin, and when the reagent containing BDAB is further mixed in, the color development of BDAB is not adversely affected. The concentration of such an oxidizing agent may vary depending on the type of oxidizing agent as well as the reaction temperature and reaction time, but is usually set so that the molar concentration of the oxidizing agent in the reaction reagent is 10 times or more the molar concentration of albumin in the mixture when the reagent containing the oxidizing agent and the sample are mixed, and is 270 times or less the molar concentration of BDAB when the mixture is further mixed with a reagent containing BDAB. In this regard, the upper limit of the normal value of albumin concentration in human serum is about 5 g / dL, and taking into consideration the ratio of reduced albumin, dilution by mixing with the reagent, and the amount of BDAB required for reaction with reduced albumin, the concentration of the oxidizing agent in the reagent is usually set to 1 to 500 mM, preferably 2 to 200 mM. When serum or plasma is diluted and subjected to the reaction, it is preferable to determine the concentration of the oxidizing agent taking into consideration the dilution rate.

[0033] According to a preferred embodiment, the method of the present invention comprises adding a protein denaturing agent to the sample.

[0034] The protein denaturant used in the present invention is preferably one that denatures proteins and increases the color development of BDAB (=increases the sensitivity), and examples thereof include urea; guanidine salts such as guanidine hydrochloride and guanidine sulfate; inorganic salts such as sodium fluoride, sodium azide, sodium chloride and potassium chloride; organic salts such as ammonium thiocyanate, potassium thiocyanate and sodium thiocyanate; alcohols such as ethanol; and ketones such as acetone, which may be used alone or in appropriate combination. Among these, guanidine salts, particularly guanidine hydrochloride, are preferred. Furthermore, surfactants having a protein denaturing effect, such as anionic surfactants, nonionic surfactants, and amphoteric surfactants, may be contained, and may be used together with the above protein denaturing agents.

[0035] Examples of such surfactants include those described in Patent Document 3, and among them, anionic surfactants are preferred. Furthermore, as the anionic surfactant, alkyl sulfate ester surfactants are preferred, and sodium lauryl sulfate (SDS) is particularly preferred. The protein denaturant is preferably added before or at the same time as the addition of the reagent containing the color-developing substance.

[0036] The concentration of the protein denaturing agent may vary depending on the type of denaturing agent, but is preferably a concentration that enhances the color development of BDAB without affecting the measured value of reduced albumin of BDAB. Usually, the concentration in the reagent may be set to 0.5 to 80% by mass in the reaction mixture, preferably 10 to 70% by mass, and more preferably 20 to 60% by mass.

[0037] The sample used in the measurement of reduced albumin is typically human serum or plasma, but blood products such as albumin preparations for transfusion can also be used as the sample.

[0038] Although the specimen can be directly subjected to the measurement, since reduced albumin is relatively unstable, it is preferable to prepare the sample to be subjected to the analysis by adding a stabilizer. Examples of the stabilizer include citrate, acetate, succinate, etc., and citrate is preferable. Furthermore, since reduced albumin is stable at pH 5 to 6, it is preferable to add such a stabilizer to the specimen so that the obtained sample has a pH of 5 to 6.

[0039] The amount of sample to be analyzed varies depending on the concentrations of BDAB and the oxidizing agent in the reagent and the amount of the reagent dispensed, but is usually 1 to 40 μL, and preferably 2 to 15 μL. The reaction time when the reagent containing an oxidizing agent is added to the sample can be 10 to 120 minutes, and is preferably 10 to 20 minutes. The reaction temperature when the color-developing reagent containing BDAB is added to the mixed solution of the sample and the reagent containing an oxidizing agent is 15 to 37°C, and is preferably 37°C. The reaction time when the color-developing reagent containing BDAB is added to the mixed solution of the sample and the reagent containing an oxidizing agent can be 10 to 30 minutes, and is preferably 15 to 20 minutes. The reaction temperature can usually be 15 to 37°C, and is preferably 37°C. The absorbance may be measured, for example, at a wavelength near 612 nm, which is the maximum absorption wavelength of BDAB, specifically 570 to 660 nm, preferably 600 to 625 nm, and particularly preferably 612 nm, which is the maximum absorption wavelength. In addition, since the nucleophilic reaction of the thiol group with BDAB occurs under acidic conditions, it is preferable to react BDAB with reduced albumin in an acidic solution. It is also possible to use an automatic analyzer to perform the process from dispensing the sample and reagent to measuring the absorbance.

[0040] In the improved BDAB method, reduced albumin is measured using the absorbance difference (ΔOD) between a sample treated with an oxidizing agent and a sample not treated with an oxidizing agent, so unlike the conventional BDAB method, a calibration curve can be created using not only purified albumin and purified reduced albumin, but also standard serum or standard plasma. In this case, the reduced albumin concentration in the standard serum or standard plasma is determined by a standard method (HPLC method & improved BCP method), while the absorbance difference (ΔOD) between a sample treated with an oxidizing agent and a sample not treated with an oxidizing agent is determined for a dilution series of the standard serum or standard plasma, and the reduced albumin concentration and the absorbance difference (ΔOD) are plotted.

[0041] More specifically, in one embodiment of the present invention, a dilution series is prepared using a standard serum or standard plasma as a calibrator, a solution containing an oxidizing agent is added to each diluted serum or plasma in the dilution series, and then the solution is mixed with a reagent containing BDAB, and the absorbance (OD) at a predetermined wavelength is measured. ox On the other hand, the same amount of a solution not containing an oxidizing agent was added to each diluted serum or plasma in the same dilution series, and then mixed with a reagent containing BDAB, and the absorbance (OD) was measured at a predetermined wavelength. non-ox ) and the absorbance (OD) of each diluted serum or plasma was measured. ox ) and absorbance (OD non-ox ) is calculated, and a calibration curve is created from each absorbance difference (ΔOD) and the known reduced albumin concentration of each diluted serum or plasma (determined by the standard method (HPLC method & improved BCP method)). This type of corrected calibration curve has a high correlation coefficient with the standard method and a slope close to 1, because the effects of substances other than reduced albumin on the color development of BDAB are eliminated. This allows for more accurate measurement of reduced albumin concentration than the conventional BDAB method.

[0042] The improved BDAB method of the present invention can be combined with the BCP method or the improved BCP method to easily determine the proportion of oxidized or reduced albumin in total albumin. For example, the reduced albumin concentration can be determined by the above-mentioned improved BDAB method, the total albumin concentration can be determined by the improved BCP method, and the reduced albumin concentration can be divided by the total albumin concentration to determine the proportion of reduced albumin in total albumin. The proportion of oxidized albumin can also be calculated by subtracting the proportion of reduced albumin obtained from 1.

[0043] 2. Kit for measuring reduced albumin Another embodiment of the present invention relates to a kit for carrying out the above-mentioned method for measuring reduced albumin. Therefore, the kit according to this embodiment includes a color-forming substance whose absorbance changes upon reaction with thiols, and an oxidizing agent that oxidizes free SH groups of reduced albumin. Specific examples of the color-forming substance and the oxidizing agent are as described above. In a preferred embodiment, the color-forming substance is 4-4'-bisdimethylaminobenzhydrol (BDAB), and the oxidizing agent is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines. Moreover, the oxidizing agent is more preferably substituted or unsubstituted dithiodipyridines, and particularly preferably 2,2'-dipyridyl disulfide.

[0044] In a preferred embodiment, the kit further comprises a protein denaturing agent, specific examples of which are as described above. In addition, the concentrations of the oxidizing agent, BDAB, and protein denaturing agent in the reagent can also be set as described above.

[0045] As described above, the kit for the improved BDAB method can contain, as a calibrator, a serum or plasma standard, purified albumin, or purified reduced albumin, and a serum or plasma standard, which is easy to produce, is particularly convenient. In the conventional BDAB method, reduced albumin purified using Thiol Sepharose was used as a calibrator, and the preparation of the calibrator was very time-consuming. The improved BDAB method kit does not require the use of such a time-consuming standard material, which is a great advantage in practice.

[0046] Each reagent constituting the kit may contain components that are typically used in biochemical or clinical chemical test reagents. Examples of such components include buffers, preservatives, and surfactants that do not fall under the category of protein denaturants. These components may be present in a concentration range that is typically selected when used in biochemical or clinical chemical test reagents. Examples of the surfactant include Triton X-405 and Triton X-100. Examples of the buffer include acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris(hydroxymethyl)aminomethane (Tris), and Good's buffer (e.g., 3-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), etc.). It is preferable to select an appropriate buffer containing these buffers in order to maintain a suitable pH for the sample and reagents in performing the improved BDAB method. In particular, since reduced albumin is stable at pH 5 to 6, it is preferable to prepare the reagents added to the sample, particularly the reagents containing a color-developing substance, so that the pH is 5 to 6 with the buffer. On the other hand, the reagent containing an oxidizing agent can be adjusted to pH 5 to 8, but is preferably adjusted to pH 5 to 6.

[0047] According to one embodiment of the present invention, the kit includes a first reagent containing an oxidizing agent and a buffer solution, and a second reagent containing BDAB and a buffer solution, and either or both of the first and second reagents contain a protein denaturant.

[0048] More specific examples of the kit are shown below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] EXAMPLES

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0050] I. Comparison of the improved BDAB method with the conventional BDAB method In this study, we evaluated the effect of correction by the improved BDAB method in comparison with the conventional BDAB method. 1. Materials and Measurement Methods 1-1. Sample Human serum and plasma prepared from blood collected from individual donors were used and diluted with a stabilizer, citrate buffer, to a uniform reduced albumin concentration of 183 μM to prepare samples. The reduced albumin concentration was determined by the standard method described in "1-3" below.

[0051] 1-2.Standard product Consela No. 1 (Nissui Pharmaceutical) was used as the standard serum, and a dilution series was prepared with citrate buffer, pH 5.0 to prepare a calibrator (dilution ratio: 1 to 10 times; reduced albumin concentration: 10 to 300 μM). The reduced albumin concentration was determined by the standard method described in "1-3" below.

[0052] 1-3. Determination of reduced albumin concentration and the proportion of oxidized or reduced albumin (reference value) by standard method The proportion of oxidized or reduced albumin in the sample (peak area % (hereinafter, for the proportion of oxidized or reduced albumin, % means peak area %), reference value) was determined by the HPLC method (standard method). The reduced albumin concentration in the sample (reference value) was calculated by determining the proportion of reduced albumin by the HPLC method (standard method) and the total albumin concentration by the improved BCP method (standard method), and multiplying the proportion (%) of reduced albumin determined by the HPLC method by the total albumin concentration determined by the improved BCP method according to the following formula.

number

[0053] 1-4. Pretreatment and absorbance measurement All specimens and standard dilutions were dispensed into each well of a 96-well plate in 5 μL portions for correction and reduced albumin measurement, respectively. 10 μL of 2 mM maleimide (Sigma-Aldrich, dissolved in 500 mM Tris buffer, pH 7.2) was added to the specimens and standard dilutions for correction, and the same amount of 500 mM Tris buffer, pH 7.2 was added to the specimens and standard dilutions for reduced albumin measurement, mixed, and incubated at 37°C for 15 minutes. Then, 200 μL of color-developing reagent (15 μM BDAB (Tokyo Chemical Industry Co., Ltd.) and 6 M guanidine hydrochloride (Tokyo Chemical Industry Co., Ltd.) dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the solution was transferred to a cuvette (10 mm path) and the absorbance was measured at a wavelength of 612 nm. Pretreatment and absorbance measurements were performed by preparing three sets (n=3) of samples for correction and reduced albumin measurement for each specimen and each standard dilution. The average value of the three sets (n=3) was used as the measurement value for each specimen and each standard dilution, and was used for correction, calibration curve creation, and concentration calculation described below.

[0054] 1-5. Confirmation of disappearance of reduced albumin by treatment with an oxidizing agent The standard dilution solution was analyzed by HPLC for the correction sample before and after the addition of an oxidizing agent solution to confirm whether the reduced albumin peak had disappeared due to the oxidizing agent. Figure 2 shows an example of a chromatogram obtained by HPLC analysis of the correction sample before and after the addition of 2 mM maleimide solution to the standard (dilution ratio: 1x). It was confirmed that the reduced albumin peak had disappeared due to the oxidizing agent.

[0055] 1-6. Creating a calibration curve In the modified BDAB method, the absorbance (OD) at 612 nm of the correction sample for each standard dilution solution is ox ) to the absorbance (OD ) of the corresponding reduced albumin measurement sample at the same wavelength. non-ox ) was subtracted to calculate ΔOD. [Table 10] Next, the ΔOD of each standard dilution solution was plotted against the reduced albumin concentration calculated from 1-3 above to create a calibration curve. The created calibration curve is shown in Figure 3. A linear calibration curve based on the least squares method (Figure 3A) and a nonlinear calibration curve based on a four-parameter logistic model (Figure 3B) were created. For comparison, a calibration curve for determining the reduced albumin concentration was created according to the conventional BDAB method described in Patent Document 4 (FIG. 3C). ox ) was not used (no correction), and the absorbance (OD non-ox ) and measure the absorbance (OD non-ox ) and the reduced albumin concentration calculated from 1-3 above were plotted to create a calibration curve.

[0056] 1-7. Determination of reduced albumin concentration and oxidized albumin ratio in each sample In the improved BDAB method, the absorbance at 612 nm (OD ox ) to the absorbance (OD ) of the corresponding reduced albumin measurement sample at the same wavelength. non-ox The data is shown in the table below. [Table 11] Next, the ΔOD of each sample was fitted to a calibration curve (linear and nonlinear) plotting the ΔOD of each of the above-mentioned standard dilutions versus reduced albumin concentration, to calculate the reduced albumin concentration. In the conventional BDAB method, the absorbance at 612 nm (OD non-ox ) to the absorbance (OD non-ox The reduced albumin concentration was calculated by fitting the results to a calibration curve plotting the concentration of reduced albumin. In both methods, the calculated reduced albumin concentration was divided by the total albumin concentration of the same sample determined by the improved BCP method (standard method) to calculate the percentage of reduced albumin (%). Furthermore, the calculated percentage of reduced albumin (%) was subtracted from 1 to calculate the percentage of oxidized albumin (since the percentage of oxidized albumin is becoming more common in recent papers, the percentage of oxidized albumin will be shown below). The obtained reduced albumin concentrations and percentages of oxidized albumin are shown in the table below. [Table 12] [Table 13]

[0057] 1-8.Evaluation method The respective reference values ​​determined by the standard method were subtracted from the concentrations of reduced albumin determined by the conventional BDAB method (uncorrected), the improved BDAB method (corrected, linear calibration curve), and the improved BDAB method (corrected, nonlinear calibration curve), as well as the proportions of oxidized albumin determined by these methods and the improved BCP method (1-(reduced albumin concentration / total albumin concentration), and the absolute value of this difference was taken as the deviation from the reference method. The correction effect of each oxidizing agent was evaluated based on whether the deviation from the reference value was smaller for the improved BDAB method (corrected) compared to the conventional BDAB method (uncorrected).

[0058] 2.Results The following table and FIG. 4 show the deviation of the reduced albumin concentration determined by each method from the reference value. [Table 14]

[0059] The deviation of the percentage of oxidized albumin determined by each method from the reference value is shown in the table below and in Figures 5 and 6. Figure 6 shows the correlation of the percentage of oxidized albumin determined by each method with the reference value. [Table 15]

[0060] As shown in Table 14 and Figure 4, there were samples in which the conventional BDAB method gave values ​​that deviated significantly from 183 µM, but it was confirmed that the improved BDAB method reduced the deviation in all samples except for one. In addition, as shown in Table 15 and Figure 5, the percentage of oxidized albumin (%) determined by the conventional BDAB method and the improved BCP method (standard method) deviated significantly from the percentage of oxidized albumin (%) determined by the HPLC method (standard method) and the improved BCP method (standard method) in some samples, but the percentage of oxidized albumin (%) determined by the improved BDAB method and the improved BCP method (standard method) was confirmed to decrease the deviation in all samples. In addition, as shown in Figure 6, the correlation between the percentage of oxidized albumin (%) determined by the improved BDAB method and the improved BCP method (standard method) and the percentage of oxidized albumin (%) determined by the HPLC method (standard method) and the improved BCP method (standard method) was improved compared to the percentage of oxidized albumin (%) determined by the conventional BDAB method and the improved BCP method (standard method).

[0061] II. Comparative study of various oxidizing agents (1) Based on the results of Test I, this test evaluated the improved BDAB method in comparison with the conventional BDAB method for various oxidizing agents.

[0062] 1. Materials and Measurements 1-1. Samples and standards The serum samples used in Test I were used as samples, and the standard samples used in Test I were used as standards. However, in this test, all samples were uniformly mixed with citrate buffer in a 1:1 volume ratio and diluted to 1 / 2. Therefore, the reduced albumin concentrations of the samples used in this test are different.

[0063] 1-2. Determination of reduced albumin concentration and the proportion of oxidized or reduced albumin (reference value) by standard method As in Test I, the percentages of oxidized or reduced albumin in the samples (%, reference value) were determined by the HPLC method (standard method). The reduced albumin concentration (reference value) in the samples was calculated by determining the percentage of reduced albumin by the HPLC method (standard method), determining the total albumin concentration by the modified BCP method (standard method), and multiplying the percentage of reduced albumin (%) determined by the HPLC method by the total albumin concentration determined by the modified BCP method.

[0064] 1-3. Oxidizing agents As oxidizing agent solutions, solutions of the following eight types of thiol reactants were prepared. 200 mM 4-vinylpyridine (Tokyo Chemical Industry Co., Ltd., dissolved in 250 mM Tris buffer, pH 8.0) 200 mM iodoacetamide (Tokyo Chemical Industry Co., Ltd., dissolved in 250 mM Tris buffer, pH 8.0) 200mM iodoacetic acid (manufactured by Fujifilm Wako Pure Chemical Industries, dissolved in 250mM Tris buffer, pH 8.0) 1 mM 2,2'-dithiobis(5-nitropyridine) (Tokyo Chemical Industry Co., Ltd., dissolved in 250 mM Tris buffer, pH 8.0) 1mM 6,6'-dithiodinicotinic acid (Tokyo Chemical Industry Co., Ltd., dissolved in 250mM Tris buffer, pH 8.0) 4mM maleimide (Sigma-Aldrich, dissolved in 250mM Tris buffer, pH 7.2) 4mM 2,2'-dipyridyl disulfide (Tokyo Chemical Industry Co., Ltd., diluted with 50mM citrate buffer, pH 5.0) 4mM 4,4'-dipyridyl disulfide (Tokyo Chemical Industry Co., Ltd., diluted with 50mM citrate buffer, pH 5.0)

[0065] 1-4. Pretreatment and absorbance measurement The pretreatment and absorbance measurement were basically the same as in Test I above, but a plate reader was used to accommodate a larger number of samples. To ensure sensitivity, some changes were made to the amounts of samples and each reagent added, and the concentrations of oxidizing agents and other reagents. Specifically, all specimens and standard dilutions were dispensed into each well of a 96-well plate in 15 μL each for correction and reduced albumin measurement. 10 μL of each oxidizing agent was added to the specimens and standard dilutions for correction, and the same amount of 250 mM Tris buffer, pH 7.2 was added to the specimens and standard dilutions for reduced albumin measurement, mixed, and incubated at 37°C for 20 minutes. Then, 225 μL of color development reagent (35 μM DBAB (Tokyo Chemical Industry Co., Ltd.) and 6 M guanidine hydrochloride (Tokyo Chemical Industry Co., Ltd.) dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the absorbance was measured at a wavelength of 612 nm using a plate reader. Pretreatment and absorbance measurement were performed by preparing three sets (n = 3) of samples for correction and reduced albumin measurement for each specimen and each standard dilution. The average value of the three sets (n=3) was used as the measurement value for each sample and each standard dilution solution, and was used for the correction, preparation of a calibration curve, and calculation of concentration described below.

[0066] 1-5. Preparation of a calibration curve and determination of the concentration of reduced albumin and the proportion of oxidized albumin Using the standard dilution series in the same manner as in Test I, calibration curves were created (linear calibration curve with correction, nonlinear calibration curve with correction, and calibration curve without correction). In the improved BDAB method, the ΔOD of each sample was fitted to the linear calibration curve with correction or the nonlinear calibration curve with correction. In the conventional BDAB method, each sample was fitted to the absorbance (OD non-ox) was fitted to an uncorrected calibration curve to calculate the reduced albumin concentration. In both methods, the calculated reduced albumin concentration was divided by the total albumin concentration of the same sample determined by the improved BCP method (standard method) to calculate the percentage of reduced albumin, and the percentage of oxidized albumin was calculated by subtracting the percentage of reduced albumin from 1.

[0067] 1-6.Evaluation method The respective standard values ​​determined by the standard method were subtracted from the concentration of reduced albumin or the proportion of oxidized albumin determined by each method in the same manner as in Test I, and the absolute value of the difference was regarded as the deviation from the standard method. The correction effect of each oxidizing agent was evaluated based on whether the deviation from the standard value was reduced by the improved BDAB method (with correction) compared with the conventional BDAB method (without correction).

[0068] 2.Results Table 16 below shows the results of the reduced albumin concentration determined by the improved BDAB method (with correction) using maleimide as an oxidizing agent or the conventional BDAB method (without correction), together with the reduced albumin concentration determined by the standard method (HPLC method & improved BCP method). Table 17 also shows the results of the percentage of oxidized albumin determined by the improved BDAB method (with correction) using maleimide as an oxidizing agent or the conventional BDAB method (without correction) and the improved BCP method, together with the reduced albumin concentration determined by the standard method (HPLC method). When the improved BDAB method (with correction) is used, the reduced albumin concentration determined by creating a linear calibration curve and a nonlinear calibration curve and the percentage of oxidized albumin calculated using the reduced albumin concentration are shown. Tables 18 and 19 show the degree of deviation of the reduced albumin concentration determined by the improved BDAB method (with correction) or the conventional BDAB method (without correction) using 4-vinylpyridine, iodoacetamide, iodoacetic acid, 2,2'-dithiobis (5-nitropyridine), 6,6'-dithiodinicotinic acid, maleimide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, or maleimide as an oxidizing agent, and the oxidized albumin ratio determined by combining this with the improved BCP method, from the reduced albumin concentration determined by the standard method (HPLC method & improved BCP method) and the oxidized albumin ratio determined by the standard method (HPLC method). When the improved BDAB method (with correction) is used, the reduced albumin concentration determined by creating a linear calibration curve and a nonlinear calibration curve and the oxidized albumin ratio calculated using the reduced albumin concentration are shown. [Table 16] [Table 17] [Table 18] [Table 19] As shown in the table above, a correction effect was observed for all eight oxidizing agents using the improved BDAB method (with correction), and the deviation of the reduced albumin concentration and the oxidized albumin ratio from the reference values ​​obtained by the standard method was reduced compared to the conventional BDAB method (without correction).

[0069] III. Comparative study of various oxidizing agents (2) Based on the results of Test I, this test further evaluated the improved BDAB method in comparison with the conventional BDAB method for various oxidizing agents.

[0070] 1. Materials and Measurements 1-1. Samples and standards The samples and standards were the same as those in Test II. Also, the samples were mixed with citrate buffer in a 1:1 volume ratio and diluted to 1 / 2.

[0071] 1-2. Determination of reduced albumin concentration and the proportion of oxidized or reduced albumin (reference value) by standard method As in Test I, the percentages of oxidized or reduced albumin in the samples (%, reference value) were determined by the HPLC method (standard method). The reduced albumin concentration (reference value) in the samples was calculated by determining the percentage of reduced albumin by the HPLC method (standard method), determining the total albumin concentration by the modified BCP method (standard method), and multiplying the percentage of reduced albumin (%) determined by the HPLC method by the total albumin concentration determined by the modified BCP method.

[0072] 1-3. Oxidizing agent, pretreatment and absorbance measurement Four types of thiol reactants were used as oxidizing agents, and pretreatment and absorbance measurements were basically performed in the same manner as in Test II, but the reagent preparation buffer, reagent, and mixing ratio were changed according to the reactivity and solubility of the reagent. Each thiol reactant, pretreatment conditions, and absorbance measurements are as follows.

[0073] 5,5'-Dithiobis(2-nitrobenzoic acid) An oxidant solution was prepared by diluting 4 mM 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma-Aldrich) in 250 mM Tris buffer, pH 8.0. Next, 15 μL of each diluted specimen and each standard dilution solution was dispensed into each well of a 96-well plate for correction and reduced albumin measurement. 10 μL of oxidant solution was added to the correction sample, and the same amount of 250 mM Tris buffer, pH 8.0 was added to the test sample for reduced albumin measurement, mixed, and incubated at 37°C for 20 minutes. Then, 200 μL of color development reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the absorbance was measured at a wavelength of 612 nm using a plate reader. The average value of three sets (n=3) was used as the measured value for each sample and each standard dilution.

[0074] 2,2'-Dithiodibenzoic Acid An oxidant solution was prepared by diluting 0.3 mM 2,2'-dithiodibenzoic acid (Tokyo Chemical Industry Co., Ltd.) in 50 mM Tris buffer, pH 8.0, containing 6 M guanidine hydrochloride (Tokyo Chemical Industry Co., Ltd.). Next, 15 μL of each diluted specimen and each standard dilution solution was dispensed into each well of a 96-well plate for correction and reduced albumin measurement. 35 μL of oxidant solution was added to the correction sample, and the same amount of the above Tris buffer was added to the reduced albumin measurement sample, mixed, and incubated at 37°C for 20 minutes. Then, 200 μL of color development reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the absorbance was measured at a wavelength of 612 nm using a plate reader. The average value of three sets (n=3) was used as the measured value for each sample and each standard dilution.

[0075] Bis(4-nitrophenyl) disulfide An oxidant solution was prepared by diluting 0.4 mM bis(4-nitrophenyl) disulfide (Tokyo Chemical Industry Co., Ltd.) in 50 mM Tris buffer, pH 8.0, containing 6 M guanidine hydrochloride (Tokyo Chemical Industry Co., Ltd.) and 20% (v / v) DMSO. Next, 15 μL of each diluted specimen and each standard dilution solution was dispensed into each well of a 96-well plate for correction and reduced albumin measurement. 35 μL of oxidant solution was added to the correction sample, and the same amount of the above Tris buffer was added to the reduced albumin measurement sample, mixed, and incubated at 37°C for 20 minutes. Then, 200 μL of color development reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the absorbance was measured at a wavelength of 612 nm using a plate reader. The average value of three sets (n=3) was used as the measured value for each sample and each standard dilution.

[0076] hydrogen peroxide An oxidant solution was prepared by diluting 50 mM hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) in 250 mM Tris buffer, pH 7.2. Next, 15 μL of each diluted specimen and each standard dilution was dispensed into each well of a 96-well plate for correction and reduced albumin measurement. 10 μL of oxidant solution was added to the specimen for correction, and the same amount of Tris buffer, pH 7.2 was added to the specimen for reduced albumin measurement, mixed, and incubated at 37°C for 20 minutes. Then, 225 μL of color reagent (35 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes, after which the absorbance was measured at a wavelength of 612 nm using a plate reader. The average value of three sets (n=3) was used as the measurement value for each specimen and each standard dilution.

[0077] 1-4. Preparation of a calibration curve and determination of the concentration of reduced albumin and the proportion of oxidized albumin Using the standard dilution series in the same manner as in Test I, calibration curves were created (linear calibration curve with correction, nonlinear calibration curve with correction, and calibration curve without correction). In the improved BDAB method, the ΔOD of each sample was fitted to the linear calibration curve with correction or the nonlinear calibration curve with correction. In the conventional BDAB method, each sample was fitted to the absorbance (OD non-ox ) was fitted to an uncorrected calibration curve to calculate the reduced albumin concentration. In both methods, the calculated reduced albumin concentration was divided by the total albumin concentration of the same sample determined by the improved BCP method (standard method) to calculate the percentage of reduced albumin, and the percentage of oxidized albumin was calculated by subtracting the percentage of reduced albumin from 1.

[0078] 1-5.Evaluation method The respective standard values ​​determined by the standard method were subtracted from the concentration of reduced albumin or the proportion of oxidized albumin determined by each method in the same manner as in Test I, and the absolute value of the difference was regarded as the deviation from the standard method. The correction effect of each oxidizing agent was evaluated based on whether the deviation from the standard value was reduced by the improved BDAB method (with correction) compared with the conventional BDAB method (without correction).

[0079] 2.Results The following table shows the degree of deviation of the reduced albumin concentration determined by the improved BDAB method (with correction) or the conventional BDAB method (without correction) using 5,5'-dithiobis(2-nitrobenzoic acid), 2,2'-dithiodibenzoic acid, bis(4-nitrophenyl) disulfide, or hydrogen peroxide as an oxidizing agent, and the oxidized albumin ratio calculated from the reduced albumin concentration and the total albumin concentration determined by the improved BCP method (standard method), from the reduced albumin concentration determined by the standard method (HPLC method & improved BCP method) and the oxidized albumin ratio determined by the standard method (HPLC method). For the improved BDAB method (with correction), the reduced albumin concentration determined by creating a linear calibration curve and a nonlinear calibration curve is shown, and the oxidized albumin ratio is shown as the value using the reduced albumin concentration determined from these calibration curves. [Table 20] [Table 21] As shown in the table above, with the improved BDAB method using the four types of oxidizing agents used in this test, the deviation from the standard value was reduced for some samples compared to the conventional BDAB method (no correction). However, there were also many samples where the deviation from the standard value did not decrease, or even increased. Overall, no clear effect of correction was observed.

[0080] IV. Extended Studies Using Maleimide as an Oxidizing Agent 1. Materials and Measurement Methods 1-1. Sample In addition to the samples (serum and plasma) used in Test I, the samples were prepared by mixing whole blood with a stabilizing citrate buffer solution in a 1:1 volume ratio immediately after collection from a donor, centrifuging the mixture, and then collecting plasma (this is a preparation method for preventing oxidation of samples and stably preserving them, as envisioned in practical use. See Non-Patent Document 17). The samples used in Test I were diluted to 1 / 2 by mixing with a 1:1 volume ratio citrate buffer solution immediately before measurement. The standard used in Test I was used. 1-2. Oxidizing agents 4 mM maleimide (Sigma-Aldrich, dissolved in Tris buffer, pH 7.2) was used. 1-3.Other Other than that, the test was carried out in the same manner as in Test II.

[0081] 2.Results The following Tables 22 and 23 and Figures 7 and 8 show the concentrations of reduced albumin and the proportion of oxidized albumin determined by each method, as well as the deviations of these values ​​from the standard values. Table 24 shows a statistical analysis of the deviations of the values ​​of the proportion of oxidized albumin shown in Table 23 from the standard values, and Figure 9 shows the correlation of the proportion of oxidized albumin determined by each method with the standard values. [Table 22] [Table 23] [Table 24] As shown in the table above and in Figures 7 and 8, there were samples in which the reduced albumin concentration and the oxidized albumin ratio deviated from the reference values ​​obtained by the standard method, but overall the deviation from the reference value decreased after correction, and the variability in the degree of deviation also converged. 9, the correlation coefficient for the proportion of oxidized albumin with the standard method was 0.88 for the conventional BDAB method (uncorrected), whereas it was 0.97 for the improved BDAB method (with correction, linear), and the slope of the correlation line was 0.68 for the BDAB method (uncorrected), whereas it was 0.97 (linear calibration curve) and 0.93 (nonlinear calibration curve) for the improved BDAB method (with correction), with the slope being closer to 1. Therefore, it was confirmed that the improved BDAB method can obtain more accurate values ​​that are closer to those of the standard method.

[0082] V. Extended Testing Using Iodoacetamide as an Oxidizing Agent 1. Materials and Measurement Methods 1-1. Sample The same specimens as those used in Test IV were treated in the same manner. 1-2. Oxidizing agents 200 mM iodoacetamide (Tokyo Chemical Industry Co., Ltd., dissolved in Tris buffer, pH 8.0) was used. 1-3.Other Other than that, the test was carried out in the same manner as in Test II.

[0083] 2.Results The following Tables 25 and 26 and Figures 10 and 11 show the concentrations of reduced albumin and the proportion of oxidized albumin determined by each method, as well as the deviations of these values ​​from the standard values. Table 27 shows a statistical analysis of the deviations of the values ​​of the proportion of oxidized albumin shown in Table 26 from the standard values, and Figure 12 shows the correlation of the proportion of oxidized albumin determined by each method with the standard values. [Table 25] [Table 26] [Table 27] As shown in the above table and Figures 10 and 11, some samples showed a large deviation in reduced albumin concentration and oxidized albumin ratio from the reference values ​​obtained by the standard method, but overall the deviation decreased with correction, and the variability in the degree of deviation also converged. More specifically, for reduced albumin concentration, the deviation decreased in 25 of 32 samples using the improved BDAB method (with correction, linear) and in 24 samples using the improved BDAB method (with correction, nonlinear). For oxidized albumin ratio, the deviation decreased in 26 of 32 samples using the improved BDAB method (with correction, linear) and in 24 samples using the improved BDAB method (with correction, nonlinear) (Figure 11). Among the samples, the conventional BDAB method (uncorrected) showed a small deviation from the reference value obtained by the standard method, while the improved BDAB method (with correction) showed a slightly larger deviation. However, even in such samples, the deviation of the oxidized albumin percentage from the reference value obtained by the standard method was less than 7%. Overall, it was confirmed that the deviation of the improved BDAB method (with correction) from the standard method was small, averaging 2.2%, and even the largest deviation was within 6-7%. 12, for the proportion of oxidized albumin, the correlation coefficient with the standard method was 0.88 for the conventional BDAB method (without correction), whereas it was 0.97 for the improved BDAB method (with correction), and the slope of the correlation line was 0.68 for the BDAB method (without correction), whereas it was 1.03 (linear calibration curve) and 0.98 (nonlinear calibration curve) for the improved BDAB method (with correction), with the slope being closer to 1. Therefore, it was confirmed that the improved BDAB method can obtain more accurate values ​​that are closer to those of the standard method.

[0084] VI. Consideration of the use of purified albumin and reduced albumin as standards In this study, a comparison was made with various standard products.

[0085] 1. Materials and Measurement Methods 1-1. Preparation of human serum albumin standard Albumin was purified from the standard serum (Consela No. 1) used in the previous tests. 10 mL (bed volume) of Affigel Blue Gel (Bio-Rad) was packed into a plastic column with an inner diameter of 15 mm, and after equilibration with phosphate buffer pH 7.1, the column was capped, 6 mL of standard serum was added, and mixed for 1 hour. The column was then washed with a total of 30 mL of phosphate buffer, and eluted with phosphate buffer containing 1.2 M NaCl. The eluted sample was dialyzed using a cellulose dialysis membrane with a molecular weight cutoff of 3500 against citrate buffer containing 100 mM NaCl, pH 5.0, and then concentrated with an ultrafiltration membrane to obtain a purified albumin solution. The percentage of oxidized or reduced albumin in the obtained purified albumin solution (reference value) was determined by the HPLC method (standard method) as in Test I. In addition, the reduced albumin concentration (reference value) in the purified albumin solution was calculated in the same manner as in Test I, by determining the proportion of reduced albumin by the HPLC method (standard method) and the total albumin concentration by the modified BCP method (standard method), and multiplying the proportion (%) of reduced albumin determined by the HPLC method by the total albumin concentration determined by the modified BCP method. The resulting purified albumin solution was diluted stepwise with citrate buffer to prepare a dilution series, which was used as a calibrator.

[0086] 1-2. Preparation of reduced human serum albumin standard Reduced albumin was purified using isopropyl-thiol-agarose (manufactured by Creative Biomart) as a carrier (see Non-Patent Document 9). 4 mL (bed volume) of the carrier was packed into a plastic column with an inner diameter of 15 mm, and after equilibration with Tris buffer containing 500 mM NaCl, pH 7.5, the column was capped, and 5 mL of purified albumin solution purified with Affigel Blue gel as described above was added and mixed for 1 hour. Thereafter, the column was washed with a total of 15 mL of Tris buffer containing 500 mM NaCl, and eluted with Tris buffer containing 25 mM DTT and 500 mM NaCl. The sample after elution was dialyzed using a cellulose dialysis membrane with a molecular weight cutoff of 3500 against citrate buffer containing 100 mM NaCl, pH 5.0, and then concentrated using an ultrafiltration membrane to obtain a purified reduced human serum albumin solution. The thiol concentration in the solution was calculated based on the absorbance at 412 nm when reacted with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) according to Non-Patent Document 9. The resulting purified reduced human serum albumin solution was serially diluted with citrate buffer to prepare a dilution series, which was used as a calibrator.

[0087] 1-3. Standard product for comparison As comparative standards, the standard serum used in previous tests (Consela No. 1) and a solution of cysteine ​​as a low molecular weight thiol compound dissolved in citrate buffer were used, and similarly, a dilution series was prepared by stepwise dilution with citrate buffer to be used as calibrators.

[0088] 1-4. Oxidizing agents As the oxidizing agent solution, 4 mM maleimide (Sigma-Aldrich, dissolved in Tris buffer, pH 7.2) was used. 1-5. Pretreatment and absorbance measurement Pretreatment and absorbance measurement were performed in the same manner as in Test II. 1-6. Creating a calibration curve Using a dilution series of each standard and comparison standard, calibration curves (linear) were prepared using the improved BDAB method (with correction) and the conventional BDAB method (without correction) in the same manner as in Test I. 1-7.Evaluation Using the calibration curve prepared using standard serum (Consela No. 1) as a standard, the slopes and intercepts of the calibration curves prepared using each of the standards and the comparative standard products examined in this study were compared.

[0089] 2.Results As shown in Figure 13, with the conventional BDAB method (without correction), the calibration curves prepared with purified albumin and purified reduced albumin were slightly deviated from the calibration curve prepared with standard serum, whereas with the improved BDAB method (with correction), almost the same calibration curves were obtained. On the other hand, as shown in Figure 14, the standard curve prepared with the cysteine ​​solution showed a significant deviation from the standard curve prepared with the standard serum, even in the improved BDAB method (with correction), and the deviation was not eliminated by correction. This shows that the standard serum, purified albumin, and purified reduced albumin can all be used as standards in the improved BDAB method (with correction).

[0090] VII. Evaluation of Several Standard Serum and Plasma as Calibrator Candidates In this test, a dilution series of multiple types of standard serum or plasma was prepared to evaluate their suitability as calibrators. 1. Materials and Measurement Methods 1-1. Preparation of standard serum or plasma and dilution series The following standard serum or plasma samples were used as calibrator candidates. [Table 28] The proportion of reduced albumin in each standard serum or plasma (reference value) was determined by the HPLC method (standard method) as in Test I. The total albumin concentration in each standard serum or plasma was determined by the modified BCP method (standard method). The reduced albumin concentration in each standard serum or plasma (reference value) was calculated by multiplying the proportion of reduced albumin determined by the HPLC method (standard method) by the total albumin concentration determined by the modified BCP method as in Test I. The resulting purified albumin solution was diluted stepwise with citrate buffer to prepare a dilution series, which was used as a calibrator. Each standard serum or plasma was serially diluted with citrate buffer to prepare a dilution series, which were used as calibrators. 1-2. Oxidizing agents As the oxidizing agent solution, 4 mM maleimide (dissolved in Tris buffer, pH 7.2) was used. 1-3.Pretreatment and absorbance measurement Pretreatment and absorbance measurement were performed in the same manner as in Test II. 1-4. Creating a calibration curve Using a dilution series of each standard serum or plasma, calibration curves (linear) using the improved BDAB method (with correction) and the conventional BDAB method (without correction) were prepared in the same manner as in Test I (Figures 15 and 16). 1-5.Evaluation Using the calibration curve prepared using standard serum (Consela No. 1) as the standard, the slopes and intercepts of the calibration curves prepared using each standard serum or plasma examined in this study were compared.

[0091] 2.Results In the case of the conventional BDAB method (without correction), the calibration curves prepared using some standard plasmas deviated from the standard calibration curve, but in the case of the improved BDAB method (with correction), none of the standard serum or plasma deviated from the standard calibration curve, and similar calibration curves were obtained (Figures 15 and 16). This suggests that in the method of the present invention, serum or plasma can be used as a calibrator if a reduced albumin concentration sufficient to cover the measurement range can be obtained.

Claims

1. A kit for measuring a concentration of reduced albumin, comprising: a coloring substance that changes its absorbance upon reacting with a thiol; and an oxidizing agent that oxidizes a free SH group of reduced albumin, The oxidizing agent is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines.

2. The kit according to claim 1 , wherein the color-developing substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of a thiol group.

3. The kit according to claim 1, wherein the color-developing substance is 4-4'-bisdimethylaminobenzhydrol (BDAB).

4. The kit according to any one of claims 1 to 3, wherein the alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

5. The kit according to any one of claims 1 to 3, wherein the oxidizing agent is selected from the group consisting of iodoacetic acid, iodoacetamide, maleimide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, 2,2'-dithiobis(5-nitropyridine), 6,6'-dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

6. The kit according to any one of claims 1 to 5, further comprising a protein denaturing agent.

7. The kit according to any one of claims 1 to 6, wherein a molar concentration of the oxidizing agent is set to be 10 times or more the molar concentration of albumin when a reagent containing the oxidizing agent is mixed with a specimen or a standard, and is set to be 270 times or less the molar concentration of the color-forming substance when a reagent containing the color-forming substance is mixed with a mixture containing the oxidizing agent and the albumin.

8. The kit according to any one of claims 1 to 7, comprising standard serum or plasma, a purified albumin solution, or a purified reduced albumin solution as a calibrator.

9. The kit according to any one of claims 1 to 8, comprising a first reagent containing the oxidizing agent and a buffer solution, and a second reagent containing the color-developing substance and a buffer solution, wherein either or both of the first reagent and the second reagent contain a protein denaturing agent.

10. A method for measuring a reduced albumin concentration, comprising: An oxidizing agent that oxidizes the free SH group of reduced albumin is added to the sample, which is then mixed with a reagent containing a color-developing substance that reacts with thiols to change its absorbance, and the absorbance (OD) is measured at a specified wavelength. ｏｘ ) measuring the On the other hand, the sample is mixed with a reagent containing the color-developing substance without adding an oxidizing agent, and the absorbance (OD) is measured at a predetermined wavelength. ｎｏｎ－ｏｘ ) measuring the The absorbance (OD ｏｘ ) and the absorbance (OD ｎｏｎ－ｏｘ ) and calculating the absorbance difference (ΔOD) between the sample and the reduced albumin concentration in the sample from the absorbance difference (ΔOD), The method, wherein the oxidizing agent is selected from the group consisting of alkylating agents, maleimides and their derivatives, and substituted or unsubstituted dithiodipyridines.

11. The method according to claim 10 , wherein the color-developing substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of a thiol group.

12. The method according to claim 10 or 11, wherein the color-developing substance is 4-4'-bisdimethylaminobenzhydrol (BDAB).

13. 13. The method of any one of claims 10 to 12, wherein the alkylating agent is selected from the group consisting of acetyl halides, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

14. 13. The method of any one of claims 10 to 12, wherein the oxidizing agent is selected from the group consisting of iodoacetic acid, iodoacetamide, maleimide, 2,2'-dipyridyl disulfide, 4,4'-dipyridyl disulfide, 2,2'-dithiobis(5-nitropyridine), 6,6'-dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB).

15. The method according to any one of claims 10 to 14, further comprising adding a protein denaturing agent to the sample.

16. The method according to any one of claims 10 to 15, wherein a calibration curve is prepared using standard serum or plasma, a purified albumin solution, or a purified reduced albumin solution as a calibrator.

17. A dilution series of the standard serum or plasma, purified albumin solution, or purified reduced albumin solution is prepared, The oxidizing agent is added to each dilution in the dilution series, and then the mixture is mixed with the reagent containing the color-developing substance, and the absorbance (OD) is measured at the predetermined wavelength. ｏｘ ) is measured, On the other hand, each dilution in the dilution series is mixed with the reagent containing the color-developing substance without adding the oxidizing agent, and then the absorbance (OD ｎｏｎ－ｏｘ ) is measured, For each of the dilutions, the absorbance (OD ｏｘ ) and the absorbance (OD ｎｏｎ－ｏｘ ) and calculate the absorbance difference (ΔOD) The method of claim 16, wherein the calibration curve is generated from the absorbance difference (ΔOD) for each of the dilutions and the known reduced albumin concentration of each of the dilutions.

18. A method for determining a proportion of reduced albumin, comprising the steps of: Determine the total albumin concentration in the sample by the modified BCP method; Determining the concentration of reduced albumin in the sample by the method according to any one of claims 10 to 17, The reduced albumin concentration is divided by the total albumin concentration to calculate the percentage of reduced albumin.

19. 19. A method for calculating a percentage of oxidized albumin by subtracting the percentage of reduced albumin determined by the method according to claim 18 from 1.

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