JP2023067248A - Method and kit for measuring reduced albumin concentration, or proportion of oxidized or reduced albumin in blood serum or plasma - Google Patents

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 The present invention relates to a method and kit for simply measuring the concentration of reduced albumin or the ratio of oxidized or reduced albumin in serum or plasma.

Albumin in blood is the protein with the highest total amount in the living body, and takes an oxidized or reduced form. Therefore, the ratio of oxidized albumin in human serum or plasma is considered to be an index that reflects systemic oxidative stress, 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). There are also reports 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). Therefore, measurement of the ratio of oxidized or reduced albumin is considered useful for diagnosis and risk assessment of these diseases (Patent Document 1 and Non-Patent Document 1).

The ratio 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 are used as standard methods because they can measure the percentage of oxidized or reduced albumin with high accuracy.
However, these methods have a limited throughput, and methods using a mass spectrometer, in particular, require equipment capable of measuring proteins with large molecular weights with high resolution (e.g., EST-TOF MS) among mass spectrometry (e.g., EST-TOF MS). Non-Patent Documents 2 and 5).

On the other hand, a method using a coloring reagent has been developed as a simpler and higher-throughput measurement method, and the BCP method and the improved BCP method are known as typical methods for measuring albumin. 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 metachromatism 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. developed a modified BCP method by reacting with BCP, which is used as a standard method for quantifying total albumin. In the improved BCP method, various oxidizing agents have been investigated, and oxidizing agents such as potassium permanganate, sodium periodide, and sodium metavanadate sufficiently avoid the effects of the SH group of reduced albumin. 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reagent with added sodium dodecyl sulfate (SDS) was reported to circumvent this effect (Non-Patent Document 6). For this reason, modified BCP methods typically use a combination of SDS and DTNB as oxidizing agents (Patent Document 3).

In the method of measuring the reduced albumin concentration by combining the BCP method and the improved BCP method, the total albumin concentration is determined by the improved BCP method, and the value obtained by subtracting the albumin concentration determined by the BCP method from this is used as the reduced albumin concentration. Also in this method, the oxidant used in the improved BCP method is typically a combination of SDS and DTNB (Patent Documents 1 and 2).
This method of combining the BCP method and the modified BCP method to determine reduced albumin concentration or percentage has a low correlation (r= 0.53) has been reported (Non-Patent Document 7), and there is also a report that the redox state of albumin cannot be estimated due to large measurement errors in patient specimens (Non-Patent Document 8). In addition, in the method of determining the concentration or ratio of reduced albumin using BCP, compared to these standard methods, the higher the concentration of total albumin in a sample, the higher the concentration of oxidized albumin tends to be measured. , has the problem of low sensitivity.

A method using 4,4'-bis(dimethylamino)benzhydrol (BDAB) (BDAB method) has also been reported as another method for measuring the concentration of reduced albumin in a chromogenic reagent system (Non-Patent Document 9. and Patent Document 4). This method is characterized by utilizing the absorbance change resulting from the nucleophilic reaction of the thiol group to the cation of BDAB. The reduced albumin concentration is approximately determined by measuring the thiol concentration of (Non-Patent Documents 9, 10, and 11 and Patent Document 4). This BDAB method is different from the combined method of the BCP method and the improved BCP method in that it can measure the concentration of reduced albumin directly. In this method, unlike the previous method in which 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a color-developing reagent for thiol groups in serum, It is said that there is no need to perform pretreatment by column chromatography or the like (Patent Document 4).

However, even with this BDAB method, as a result of investigations by the present inventors, there are some specimens that deviate from the reduced albumin concentration calculated from the measurement results of the standard HPLC method and the improved BCP method. is known and a measurement method that provides more correlated and more reliable results to standard methods 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 the problem that preparation of the calibrator is troublesome. Also, there have been no reports of determining the ratio of oxidized or reduced albumin by a method using BDAB.

JP 2018-025486 JP 2018-194453 Patent No. 3266079 JP-A-1-161151 JP 2017-058278

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 Biochemis try and Nutrition, 2017, Vol. 61, No. 2, p. 79-84 Nagumo K. et al. , “Cys34-Cysteinylated Human Serum Album Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases” PLOS ONE, 2014, Vo. 9, no. 1, p. e85216. 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 Ueno Set. al. , "Nonmercaptalbumin as an oxidative stress marker in Parkinson's and PARK2 disease", Annals of Clinical and Translational Neurology, 2020, Vol. 7, No. 3, P. 307-317 Magzal F. et al. , “In-vivo oxidized albumin-a pro-inflammatory agent in hypoalbuminemia” PLOS ONE, 2017, Vol. 12, No. 5, p. e0177799 Ryozo Muramoto (and 2 others), "Development of serum albumin quantification method by bromcresol purple method with improved accuracy", Clinical Chemistry, 1997, Vol. 26, No. 1, p. 38-43 Yoshihiro S. et al. . "New colorimetric method with bromocresol purple for estimating the redox state of human serum albumin", Journal of Clinical Biochemistry and Nutrition, 2020, Vol. 67, No. 3,257-262 Fumimi Yoshitomi, (6 others), "A novel spectroscopic measurement method for estimating the redox state of human serum albumin", Abstracts of the 59th Annual Meeting of the Japanese Society of Clinical Chemistry, 2019, p. 194 Mitsuo Hama and Mitsunao Tanaka. "Measurement of mercaptoalbumin in blood" Report of Ogata Medical Chemical Laboratory, 1991 issue, p. 7-13, 1991. Colombo G. et. al. , "Redox Albuminomics: Oxidized Albumin in Human Diseases" Antioxidants & Redox Signaling, 2012, Vol. 17, No. 11, p. 1515-1527 Turell L. et al. , "The thiol pool in human plasma: The central contribution of albumin to redox processes" Free Radical Biology and Medicine, 2013, Vol. 65, p. 244-253 Chen X. et al. , "Fluorescence and colorimetric probes for detection of thiols" Chemical Society Review, 2010, Vol. 39, No. 6, p. 2120-2135 Deng L. et al. , "Colorimetric and Ratiometric Fluorescent Chemosensor Based on Diketopyrrolopyrrole for Selective Detection of Thiols: An Experimental and Theoretical Study", The Journal of Organic Chemistry, 2011, Vol. 76, p. 9294-9304 Shen Y. et al. , "A Simple and New Fluorescent and Colorimetric Probe Based on NBD-Maleimide for Detecting Thiols in Living Cells", Analytical Methods, 2015, Vol. 7, No. 15, p. 6419-6425 Ros-LisJ. V. et al. , "Squaraines as Fluoro-Chromogenic Probes for Thiol-Containing Compounds and Their Application to the Detection of Biorelevant Thiols", Journal of America an Chemical Society, 2004, Vol. 126, No. 13, p. 4064-4065 FanJ. et al. , "A fast response square-based colorimetric probe for detection of physical conditions", Sensors and Actuators B: Chemical, 2013, Vol. 80, p. 886-893 Yasukawa K. et al. , "Establishment of a stable sampling method to assay mercaptoalbumin/non-mercaptoalbumin and reference ranges" Practical Laboratory Medicine, Vol. 17, p. e00132, 2019. Hayashi T. et. al. , "The importance of sample preservation for analysis of the redox state of human serum albumin"Clinica Chimica Acta, Vol. 316, No. 1-2, p. 175-178, 2002. FabisiakJ. P. et al. , "Quantification of Oxidative/Nitrosatie Modification of CYS34 in Human Serum Album Using a Fluorescence-Based SDs-PAGE Assay" Antioxidant & Redox Si Gnaling, Vol. 4, No. 5, p. 855-865, 2002. Turell L. et al. , "Oxidation of the albumin thiol to sulfuric acid and its implications in the intravascular compartment," Brazilian Journal of Medical and Biological Research, 2009, Vol. 42, No. 4, p. 305-11

Therefore, the present invention provides a method for easily measuring the concentration of reduced albumin using a chromogenic reagent, which has less deviation from the standard method than the conventional method and enables more accurate measurement, and a kit therefor. intended to provide Another object of the present invention is to provide a method for measuring reduced albumin and a kit therefor, in which an easily prepared sample such as standard serum or plasma can be used as a calibrator. A further object of the present invention is to provide a novel method for determining the proportion of oxidized or reduced albumin using a chromogenic reagent.

The present inventors have confirmed that there are samples in which the concentration of reduced albumin measured by the conventional BDAB method deviates relatively greatly from the standard method. In a measurement system using a substance that reacts with thiol to change the absorbance, the concentration of reduced albumin is measured using the difference between the absorbance of a sample added with a specific oxidizing agent and the absorbance of a sample without addition. , found that the deviation from the standard method can be improved, and completed the present invention.

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 ratio of oxidized or reduced albumin.
[1] A kit for measuring reduced albumin concentration, comprising a chromogenic substance that reacts with thiol to change absorbance and an oxidizing agent that oxidizes free SH groups of reduced albumin,
The kit, wherein said oxidizing agent is selected from the group consisting of alkylating agents, maleimides and derivatives thereof, and substituted or unsubstituted dithiodipyridines.
[2] The coloring substance is a substance that causes a change in absorbance as a result of a nucleophilic reaction of a thiol group. 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-diazole maleimide, and 2,4-bis[4-(N,N-bis(2-methoxyethoxy)methyl)-diphenyl]squaraine according to [1]. kit.
[3] The kit according to [1], wherein the coloring substance is 4-4′-bisdimethylaminobenzhydrol (BDAB).
[4] said alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB), [1 ] The kit according to any one of [3].
[5] The oxidizing agent is iodoacetic acid, iodoacetamide, maleimide, 2,2′-dipyridyl disulfide, 4,4′-dipyridyl disulfide, 2,2′-dithiobis(5-nitropyridine), 6,6′- Any one of [1] to [3] selected from the group consisting of dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB) 1. The kit according to item 1.
[6] The kit according to any one of [1] to [5], which contains a protein denaturant.
[7] When the reagent containing the oxidizing agent and the sample are mixed, the molar concentration of the oxidizing agent becomes 10 times or more the molar concentration of albumin in the mixture, and the mixture is mixed with the reagent containing the coloring substance. The kit according to any one of [1] to [6], wherein the molar concentration is set to be 270 times or less than the molar concentration of the coloring substance.
[8] The kit according to any one of [1] to [7], which contains standard serum or plasma, purified albumin solution, or purified reduced albumin solution as a calibrator.
[9] a first reagent containing the oxidizing agent and a buffer; and a second reagent containing the chromogenic substance and a buffer, wherein one or both of the first reagent and the second reagent contain a protein The kit according to any one of [1] to [8], which contains a denaturant.
[10] A method for measuring reduced albumin concentration, comprising:
An oxidizing agent that oxidizes free SH groups of reduced albumin is added to the sample, then mixed with a reagent containing a coloring substance that reacts with thiol to change absorbance, and absorbance (OD _ox ) is measured at a predetermined wavelength. process and
On the other hand, mixing the sample with a reagent containing the chromogenic substance without adding the oxidizing agent, and measuring the absorbance (OD _non-ox ) at a predetermined wavelength;
determining the absorbance difference (ΔOD) between the absorbance (OD _ox ) and the absorbance (OD _non-ox ), and calculating the reduced albumin concentration in the sample from the absorbance difference (ΔOD);
The method, wherein said oxidizing agent is selected from the group consisting of alkylating agents, maleimides and derivatives thereof, and substituted or unsubstituted dithiodipyridines.
[11] The coloring substance is a substance that causes a change in absorbance as a result of a nucleophilic reaction of a thiol group. -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, the method of [10].
[12] The method of [10], wherein the coloring substance is 4-4'-bisdimethylaminobenzhydrol (BDAB).
[13] said alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB); [10 ] to [12].
[14] The oxidizing agent is iodoacetic acid, iodoacetamide, maleimide, 2,2′-dipyridyl disulfide, 4,4′-dipyridyl disulfide, 2,2′-dithiobis(5-nitropyridine), 6,6′- Any of [10] to [12], selected from the group consisting of dithiodinicotinic acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB) 1. The method according to item 1.
[15] The method according to any one of [10] to [14], further comprising adding a protein denaturant to the sample.
[16] The method according to any one of [10] to [15], wherein a standard curve is prepared using standard serum or plasma, purified albumin solution, or purified reduced albumin solution as a calibrator.
[17] creating a dilution series of the calibrator;
After adding the oxidizing agent to each dilution in the dilution series and then mixing with the reagent containing the coloring substance, the absorbance (OD _ox ) is measured at the predetermined wavelength,
On the other hand, each diluted solution in the dilution series is mixed with the reagent containing the coloring substance without the addition of the oxidizing agent, and then the absorbance (OD _non-ox ) is measured at the predetermined wavelength,
For each of the diluted solutions, the absorbance difference (ΔOD) between the absorbance (OD _ox ) and the absorbance (OD _non-ox ) is obtained,
The method according to [16], wherein the calibration curve is prepared from each absorbance difference (ΔOD) and the known reduced albumin concentration of each diluent.
[18] A method for determining the proportion of reduced albumin, comprising:
determining 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 [10] to [17],
dividing the reduced albumin concentration by the total albumin concentration to calculate the percentage of reduced albumin.
[19] A method of calculating the ratio of oxidized albumin by subtracting the ratio of reduced albumin determined by the method described in [18] from 1.

INDUSTRIAL APPLICABILITY According to the present invention, reduced albumin can be measured more easily and with higher throughput than methods involving measurement by HPLC and mass spectrometry. In addition, since the correlation with the standard method is higher than the BCP/improved BCP method and the conventional BDAB method, and the deviation from the standard method is small, the reduced albumin concentration can be measured more accurately.

Here, some terms used in this specification are defined.
As used herein, "HPLC method" shall refer to a method that uses HPLC to determine the oxidized/reduced albumin ratio or the percentage of oxidized or reduced albumin in total albumin.

As used herein, "mass spectrometry" shall refer to a method of determining the oxidized/reduced albumin ratio or the percentage of oxidized or reduced albumin in total albumin using a mass spectrometer. . In addition, the analysis by "mass spectrometry" shall be performed under the procedures and conditions described in Non-Patent Documents 2 and 5, unless otherwise specified.

As used herein, the “improved BCP method” refers to bromocresol purple (BCP) after converting reduced albumin to oxidized albumin by adding an oxidizing agent to the sample that oxidizes the free SH groups of reduced albumin. is reacted with oxidized albumin to measure the total albumin concentration.

When referring herein to a method for determining the percentage of oxidized or reduced albumin in total albumin, "standard method" refers to "HPLC method" performed according to the procedures and conditions described in Patent Document 5. shall be 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 specimen is injected, and at the same time A linear gradient of Buffer A (100% to 75%) and Buffer B (0% to 25%) was run for 7.5 min at a flow rate of 1.0 mL/min to separate oxidized and reduced albumin. , fluorescence with 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

Also, when referring to a method for determining total albumin concentration herein, the "standard method" shall refer to the "improved BCP method" performed according to the procedure described in US Pat. In this standard method, specifically, 85 μL of first reagent (100 μM DTNB, and 0.03% (w/v) SDS in 25 mM Tris HCl, pH 8.0) was added to 15 μL of sample. After addition and incubation at 37° C. for 5 minutes, 150 μL of the second reagent (150 μM bromocresol purple and 0.3% (v/v) Triton X-100 in 250 mM sodium succinate, pH 5.5 solution). ) is added and further incubated at 37° C. for 20 minutes, and the absorbance 600 nm (major wavelength)/660 (minor wavelength) is measured to determine the total albumin concentration.

When referring to a method for determining the concentration of reduced albumin herein, the “standard method” refers to determining the ratio of reduced albumin in total albumin by the above “standard method”, It refers to a method of determining the albumin concentration and multiplying the obtained total albumin concentration by the ratio of the reduced albumin to determine the reduced albumin concentration.

As used herein, the “BDAB method” means a method for measuring the concentration of reduced albumin using 4-4′-bisdimethylaminobenzhydrol (BDAB) as a chromogenic substance.
Further, as used herein, the term "improved BDAB method" refers to a sample prepared by adding a solution containing an oxidizing agent that oxidizes free SH groups of reduced albumin to a sample in the "BDAB method". A reagent containing BDAB is added, and the absorbance (OD _ox ) measured at a predetermined wavelength. A method of measuring the absorbance (OD _non-ox ) measured in , and calculating the concentration of reduced albumin in the sample from the difference between the two absorbances (ΔOD).
In addition, as used herein, the “conventional BDAB method” refers to the “BDAB method” in which a reagent containing BDAB is added without treating the specimen with an oxidizing agent that oxidizes free SH groups of reduced albumin. and a method of calculating the concentration of reduced albumin in a sample from the absorbance (OD _non-ox ) measured at a predetermined wavelength.

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 lower left table shows the area and area ratio of the reduced albumin peak and the oxidized albumin peak in the upper chromatogram. The table on the lower right shows the total albumin concentration measured by the modified BCP method (standard method) for one plasma sample according to the procedure described in Patent Document 3, and multiplied by the ratio of reduced albumin determined by the HPLC method (standard method). shows the concentration of reduced albumin calculated by The chromatograms analyzed by the HPLC method (standard method) before treating the standard serum with maleimide and the chromatograms analyzed by the HPLC method (standard method) after treating the standard serum with maleimide are shown. It can be confirmed that by treating with maleimide, the peak of reduced albumin completely disappears and the peak of oxidized albumin increases. 3 is a calibration curve for determining the concentration of reduced albumin prepared using the data shown in Table 10. FIG. FIGS. 3A and B are calibration curves for determining reduced albumin concentration using the difference in absorbance (ΔOD), FIG. 3A being a linear calibration curve and FIG. 3B being a non-linear calibration curve. FIG. 3C is a calibration curve for determining reduced albumin concentration using OD _non-ox , which is the same calibration curve as the conventional "BDAB method". Specimens (8 serum, 4 plasma) from 12 donors were diluted with buffer to 183.5 μM based on reduced albumin concentrations determined by standard methods (HPLC method & modified BCP method). The reduced albumin concentration of the sample adjusted to the concentration was determined using the calibration curve shown in FIG. HPLC method & improved BCP method) shows the results of evaluating the deviation from the standard value of 183.5 μM. FIG. 4A shows the deviation from the reference value for each specimen, and FIG. 4B shows the distribution of the deviation from the reference value. The top and bottom ends of the bars in FIG. 4B indicate the maximum (absolute value) and minimum (absolute value) differences, respectively, from the standard method for each measurement method. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. Specimens (8 serum, 4 plasma) from 12 donors were diluted with buffer based on reduced albumin concentrations determined by standard methods (HPLC method & modified BCP method) for a total of 183. Using a sample adjusted to a concentration of 5 μM, the percentage of oxidized albumin was calculated 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 (standard method). , and shows the results of evaluating the deviation from the ratio of oxidized albumin by a standard method (HPLC method). The upper and lower ends of the bars in the figure indicate the maximum difference (absolute value) and minimum difference (absolute value) from the standard method for each measurement method, respectively. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. (Since Patent Document 5 and Non-Patent Documents 1 and 2 generally describe the ratio of oxidized albumin, the ratio of oxidized albumin is also preferentially described in this specification.) Regarding the test results shown in Table 13, 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. Reduced albumin concentrations were determined in samples (21 serum, 11 plasma) from 32 donors by a modified BDAB method (with correction) or a conventional BDAB method (without correction) using maleimide as the oxidizing agent; Fig. 2 shows the test results of evaluating the deviation from the concentration of reduced albumin determined by the standard method (HPLC method & 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) from the standard method for each measurement method, respectively. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. In the modified BDAB method (with correction), a linear calibration curve and a non-linear calibration curve were prepared to determine the concentration of reduced albumin. Specimens (21 serum, 11 plasma) from 32 donors were tested using modified BDAB (with correction) or conventional BDAB (without correction) and modified BCP (standard) using maleimide as an oxidizing agent. Shown are test results in which the percentage of oxidized albumin was determined and the deviation from the concentration of oxidized albumin determined by a 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) from the standard method for each measurement method, respectively. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. In the modified BDAB method (with correction), a linear calibration curve and a non-linear calibration curve were prepared to determine the concentration of reduced albumin. Regarding the test results shown in Table 23, the correlation between the percentage of oxidized albumin determined by the standard method (HPLC method) and the percentage of oxidized albumin determined by each method is shown. Reduced albumin concentrations were determined in samples (21 serum, 11 plasma) from 32 donors by the modified BDAB method (with correction) or the conventional BDAB method (without correction) using iodoacetamide as the oxidizing agent. , shows the results of evaluating the deviation from the reduced albumin concentration determined by the standard method (HPLC method & 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) from the standard method for each measurement method, respectively. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. In the modified BDAB method (with correction), a linear calibration curve and a non-linear calibration curve were prepared to determine the concentration of reduced albumin. Specimens from 32 donors (21 serum, 11 plasma) modified BDAB (corrected) or conventional BDAB (uncorrected) and modified BCP (standard) with iodoacetamide as oxidizing agent The ratio of oxidized albumin was determined by the standard method (HPLC method), and the results of evaluating the deviation from the ratio of oxidized albumin determined by the standard method (HPLC 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) from the standard method for each measurement method, respectively. The top and bottom ends of the boxes indicate the 75% and 25% percentile values, respectively. X indicates the median difference to the standard method. In the modified BDAB method (with correction), a linear calibration curve and a non-linear calibration curve were prepared to determine the concentration of reduced albumin. Regarding the test results shown in Table 26, the correlation between the percentage of oxidized albumin determined by the standard method (HPLC method) and the percentage of oxidized albumin determined by each method is shown. FIG. 1 shows a standard curve of reduced albumin concentration prepared by a conventional BDAB method (without correction) or an improved BDAB method (with correction) using standard serum, purified albumin solution, or purified reduced albumin solution. FIG. 1 shows a calibration curve of reduced albumin concentration prepared by a conventional BDAB method (without correction) or an improved BDAB method (with correction) using standard serum or cysteine solution. FIG. 1 shows calibration curves for reduced albumin concentrations prepared by a conventional BDAB method (without correction) or an improved BDAB method (with correction) using various standard sera. FIG. 1 shows calibration curves of reduced albumin concentration prepared by conventional BDAB method (without correction) or improved BDAB method (with correction) using various standard sera or plasma.

Embodiments of the present invention will be described in detail. However, the present invention should not be understood as being limited to the following embodiments.

1. Method for Measuring Reduced Albumin Concentration One embodiment of the present invention relates to a method for measuring reduced albumin concentration in a sample using a substance that reacts with thiol to change absorbance.
Human blood albumin has 35 cysteine residues (SH groups) in its molecule, 17 pairs of which form intramolecular disulfide bonds, but the SH group of the 34th cysteine from the N-terminus is free. In some cases, it is oxidized. The former is called reduced albumin and the latter is called oxidized albumin. Since human serum albumin is the protein with the highest total amount in the body, the ratio of oxidized albumin in human serum or plasma is considered to be an index that reflects the oxidative stress of the whole body. It has been reported that it increases with disease progression (Non-Patent Documents 1 and 2). It has also been reported to 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). Therefore, measurements of reduced albumin concentration, oxidized or reduced albumin ratio, and oxidized/reduced albumin ratio are used in the diagnosis and risk assessment of these conditions and diseases.

を有する化合物であり、好ましいスクアライン系色素は、式中のＲが、それぞれ独立して

（式中の左端が上記化合物のＮと結合する）の化合物であり、より好ましい化合物は、式中のＲが、上記の構造の何れかである化合物である。
上述した各発色物質については、非特許文献１２～１６に詳細に記載されている。 As the color-developing substance that changes absorbance by reacting with thiol, a substance that causes a change in absorbance as a result of a nucleophilic reaction of a thiol group is preferable, and a thiol-reacting substance that does not have a disulfide structure is more preferable. Such substances are, 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, Squaraine dyes such as N-bis(2-methoxyethoxy)methyl)-diphenyl]squaraine and 2,4-bis[4-(N,N-bis(butylcarbamate))-diphenyl]squaraine can be mentioned. A squaraine dye has the following structure:

A preferred squaraine dye is a compound having R in the formula, each independently

(the left end in the formula is bonded to N of the above compound), and more preferred compounds are compounds in which R in the formula has any of the above structures.
Each of the coloring substances mentioned above is described in detail in Non-Patent Documents 12-16.

の構造を有する化合物であり、６１２ｎｍに最大吸光度波長を有する。ＢＤＡＢと還元型アルブミンとの反応は、以下に示すカチオンに対するチオール基の求核反応であり、この反応の結果生じる複合体では、６１２ｎｍでの吸光度が減少し、この吸光度変化によって、チオール基の量を直接測定することができる。

測定波長は、試料中に存在する夾雑物質の吸収スペクトル等を考慮して、ＢＤＡＢの発色を特異的に検出できる波長を選択すればよいが、通常は、最大吸収波長である６１２ｎｍ付近、具体的には、５７０～６６０ｎｍ、好ましくは６００ｎｍ～６２５ｎｍの範囲から選択すればよく、最大吸収波長である６１２ｎｍが好ましい。また、ＢＤＡＢに対するチオール基の求核反応は、ＢＤＡＢが酸性下で上記のカチオンとなることで生じるため、酸性下、典型的にはｐＨ５～６の溶液中でＢＤＡＢと還元型アルブミンを反応させることが好ましい。従って、ＢＤＡＢを含む発色試薬は、通常緩衝液を含み、酸性下、典型的にはｐＨ５～６に維持されている。 4-4'-Bisdimethylaminobenzhydrol (BDAB) is particularly preferred as the coloring substance.
4,4′-bis(dimethylamino)benzhydrol (BDAB) is

and has a maximum absorbance wavelength at 612 nm. The reaction between BDAB and reduced albumin is a nucleophilic reaction of a thiol group to a cation shown below, and the resulting complex exhibits a decrease in absorbance at 612 nm, and this absorbance change determines the amount of thiol groups. can be measured directly.

As for the measurement wavelength, a wavelength that can specifically detect the color development of BDAB may be selected in consideration of the absorption spectrum of contaminants present in the sample. can be selected from the range of 570 to 660 nm, preferably 600 nm to 625 nm, preferably 612 nm, which is the maximum absorption wavelength. In addition, since the nucleophilic reaction of the thiol group to BDAB occurs when BDAB becomes the above cation under acidic conditions, BDAB and reduced albumin can be reacted in an acidic solution, typically at pH 5-6. is preferred. Therefore, a BDAB-containing chromogenic reagent usually contains a buffer and is maintained in an acidic environment, typically at pH 5-6.

The concentration of BDAB is preferably such that when mixed with the sample, all the reduced albumin in the sample reacts with BDAB and is not affected by the oxidant's color development. In this regard, the upper limit of the normal albumin concentration in human serum is about 5 g/dL, and this includes the percentage of reduced albumin, the dilution due to mixing with the reagent, and the complete conversion of reduced albumin to oxidized albumin. Considering the amount of oxidizing agent required for the analysis, 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 that of the sample containing albumin, preferably 15 to 40 times. It should be doubled. From the same point of view, the concentration of BDAB is 1.5 to 6 times, preferably 1.5 to 3 times, the molar concentration of reduced albumin in the reaction mixture. It is preferable to adjust the amount to 0.05 to 0.5 times the molar concentration.

In the present invention, in the method of measuring the concentration of reduced albumin in a sample by a coloring reaction using a coloring substance that reacts with a thiol such as BDAB to change the absorbance, the specimen is divided into two, and one of the specimens to which a solution containing an oxidizing agent that oxidizes the free SH groups of reduced albumin was added (correction sample), and to the other sample was added the same amount of solution containing no oxidizing agent (for measurement of reduced albumin). sample). Next, each mixture is mixed with a reagent containing a coloring substance (eg, BDAB), absorbance (OD _ox ) and absorbance (OD _non-ox ) are measured at a predetermined wavelength, and the absorbance difference (ΔOD) between the two is obtained. The concentration of reduced albumin in the sample is calculated from (ΔOD).

When an oxidizing agent that oxidizes free SH groups is added, reduced albumin is completely converted to oxidized albumin. In some specimens, a decrease in absorbance is observed in the sample, and the absorbance at the measurement wavelength is considered to be affected by the chromogenic substance reacting with a substance other than reduced albumin or by a substance other than reduced albumin. In such specimens, a 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 discrepancy with the HPLC method. Based on such a phenomenon, in the method of the present invention, the absorbance difference (ΔOD) between the absorbance (OD _ox ) of a sample treated with an oxidizing agent and the absorbance (OD _non-ox ) of a sample not treated with an oxidizing agent is By using , a correction is performed to eliminate absorbance changes that do not originate from reduced albumin, and reduced albumin is accurately measured.
The problems of the conventional BDAB method and their causes were recognized for the first time by the present inventors, and the principle of solution is a method of measuring reduced albumin by combining the BCP method and the improved BCP method. is quite different.

The method of the present invention also uses certain compounds as oxidizing agents to oxidize free SH groups. As a result of investigation by the present inventors, although the cause is unknown, in the improved BCP method, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) used for converting reduced albumin to oxidized albumin ), although the conversion occurs, it is difficult to reduce the deviation from the standard HPLC method by the above correction.

The present inventors verified the suitability of various oxidizing agents that oxidize free SH groups to the method of the present invention, and by selecting specific compounds, the above correction reduces the deviation from the standard HPLC method. I have confirmed that it can be done. Specifically, in preferred embodiments of the present invention, the oxidizing agent that oxidizes free SH groups is selected from the group consisting of alkylating agents, maleimide and its derivatives, and substituted or unsubstituted dithiodipyridines.

Alkylating agents include, for example, acetyl halides, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). Halogenated acetyl includes, for example, halogenated acetic acid, halogenated acetamide and halogenated acetophenone, more specifically iodoacetic acid, iodoacetamide and iodoacetophenone.
Derivatives of maleimide include, for example, N-alkyl (preferably N—C _1-5 alkyl) maleimide such as N-methylmaleimide and N-ethylmaleimide, N,N '-C _5-12 aromatic ring dimaleimides. Substituents of the substituted dithiodipyridine include nitro group, carboxyl group, amino group, hydroxy group, etc. Specific compounds include 2,2′-dipyridyl disulfide, 4,4′-dipyridyl disulfide, 2 , 2′-dithiobis(5-nitropyridine), 6,6′-dithiodinicotinic acid, 2,2′-dithiobis(5-aminopyridine), 2,2′-dithiobis(5-hydroxypyridine) .
Among these, substituted or unsubstituted dithiodipyridine is advantageous in terms of analysis in that oxidation reaction can be performed at pH 5 to 6, which is the reaction pH of BDAB, and is preferable in that correction has a large effect, particularly 2,2'- Dipyridyl disulfide is preferred.

The concentration of the oxidizing agent is 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 mixed, the coloring of BDAB is adversely affected. It is preferable to use a concentration that does not affect 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. When mixed, the molar concentration of albumin in the mixture is 10 times or more, and when this mixture is further mixed with a reagent containing BDAB, the molar concentration is 270 times or less than the molar concentration of BDAB.
In this regard, the upper limit of the normal albumin concentration in human serum is about 5 g/dL, plus the ratio of reduced albumin, dilution by mixing with reagents, and the amount of BDAB required for reaction with reduced albumin. Considering the amount and the like, 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 used for the reaction, it is preferable to determine the concentration of the oxidizing agent in consideration of the dilution ratio.

According to a preferred embodiment, in the method of the invention a protein denaturing agent is added to the sample.

The protein denaturant used in the present invention is preferably one that denatures protein and increases the coloration of BDAB (=increases sensitivity), such as 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; ketones such as acetone; can be used alone or in combination as appropriate.
Of these, guanidine salts, particularly guanidine hydrochloride, are preferred.
In addition, surfactants such as anionic surfactants, nonionic surfactants, and amphoteric surfactants having protein denaturing action may be included, and may be used together with the above protein denaturants.

Examples of such surfactants include surfactants described in Patent Document 3, and among them, anionic surfactants are preferable. As the anionic surfactant, an alkyl sulfate surfactant is preferable, and sodium lauryl sulfate (SDS) is particularly preferable.
The protein denaturant is preferably added before or during the addition of the reagent containing the coloring substance.

The concentration of the protein denaturant may vary depending on the type of denaturant, but it is preferably a concentration that improves the coloration of BDAB without affecting the measured value of reduced albumin of BDAB.
Generally, the concentration in the reagent may be set so as to be 0.5 to 80% by mass in the reaction mixture, preferably 10 to 70% by mass, more preferably 20 to 60% by mass. should be set.

The sample used for measuring reduced albumin is typically human serum or plasma, but blood products such as albumin preparations for blood transfusion can also be used as samples.

Although the sample can be used for measurement as it is, it is preferable to prepare a sample for analysis by adding a stabilizing agent because reduced albumin is relatively unstable. Examples of stabilizers include citrate, acetate, succinate and the like, with citrate being preferred. In addition, since reduced albumin is stable at pH 5-6, it is preferable to adjust the pH of the obtained sample to pH 5-6 by adding these stabilizing agents to the sample.

The amount of sample to be analyzed may vary depending on the concentration of BDAB and oxidizing agent in the reagent and the amount of reagent dispensed, but it is usually 1-40 μL, preferably 2-15 μL.
The reaction time when the reagent containing the oxidizing agent is added to the sample can be 10 to 120 minutes, preferably 10 to 20 minutes. Further, the reaction temperature when adding the coloring reagent containing BDAB to the mixed solution of the reagent containing the sample and the oxidizing agent is 15 to 37°C, preferably 37°C. The reaction time for adding the coloring reagent containing BDAB to the mixture of the reagent containing the sample and the oxidizing agent can be 10 to 30 minutes, preferably 15 to 20 minutes. Also, the reaction temperature can be generally 15 to 37°C, preferably 37°C.
Absorbance is measured, for example, near 612 nm, which is the maximum absorption wavelength of BDAB. good. Further, since the nucleophilic reaction of the thiol group to BDAB occurs under acidic conditions, it is preferable to react BDAB and reduced albumin in an acidic solution. It is also possible to measure the absorbance from the dispensing of the sample and reagent using an automatic analyzer.

In the improved BDAB method, the conventional BDAB method is used to measure reduced albumin using the difference in absorbance (ΔOD) between a sample treated with an oxidizing agent and a sample not treated with an oxidizing agent. Unlike , it is possible to create a calibration curve using not only purified albumin and purified reduced albumin, but also standard serum or standard plasma. In this case, the standard curve is prepared by determining the reduced albumin concentration in the standard serum or standard plasma by the standard method (HPLC method & improved BCP method), and diluting the standard serum or standard plasma with an oxidizing agent. The absorbance difference (ΔOD) between the treated sample and the sample not treated with oxidizing agent can be determined and a plot of reduced albumin concentration versus absorbance difference (ΔOD) can be generated.

More specifically, in one embodiment of the present invention, a dilution series is prepared using standard serum or standard plasma as a calibrator, and a solution containing an oxidizing agent is added to each diluted serum or plasma in the dilution series. and then mixed with a reagent containing BDAB, the absorbance (OD _ox ) was measured at a given wavelength, while each diluted serum or plasma in the same dilution series was added in an equal volume of oxidant-free solution. Then, after mixing with a reagent containing BDAB, the absorbance (OD _non-ox ) is measured at a predetermined wavelength, and for each diluted serum or plasma, the absorbance difference between the absorbance (OD _ox ) and the absorbance (OD _non-ox ) (ΔOD) is determined, 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 a standard method (HPLC method & improved BCP method)). The calibration curve corrected in this manner has a high correlation coefficient with the standard method and a slope close to 1 because the influence of substances other than reduced albumin on the color development of BDAB is eliminated. Therefore, the reduced albumin concentration can be measured more accurately than the conventional BDAB method.

The improved BDAB method according to the present invention can be combined with the BCP method or the improved BCP method to easily determine the ratio of oxidized or reduced albumin in total albumin.
For example, the reduced albumin concentration is determined by the modified BDAB method described above, the total albumin concentration is determined by the modified BCP method, and the reduced albumin concentration is divided by the total albumin concentration to determine the percentage of reduced albumin in total albumin. You can also Also, the ratio of oxidized albumin can be calculated by subtracting the ratio of reduced albumin thus obtained from 1.

2. Kit for Measuring Reduced Albumin Another embodiment of the present invention relates to a kit for carrying out the method for measuring reduced albumin described above.
Thus, the kit according to this embodiment includes a chromogenic substance that reacts with thiols to change absorbance, and an oxidizing agent that oxidizes free SH groups of reduced albumin. Specific examples of the chromogenic substance and oxidizing agent are as described above. In a preferred embodiment, the chromogenic material is 4-4'-bisdimethylaminobenzhydrol (BDAB) and the oxidizing agent is the group consisting of alkylating agents, maleimide and its derivatives, and substituted or unsubstituted dithiodipyridines. is selected from Further, the oxidizing agent is more preferably substituted or unsubstituted dithiodipyridine, particularly preferably 2,2'-dipyridyl disulfide.

In a preferred embodiment, the kit also contains a protein denaturant, specific examples of which are also described above.
Also, the concentrations of the oxidant, BDAB and protein denaturant in the reagent can be set as described above.

In addition, as described above, the kit for the improved BDAB method can contain a serum or plasma standard, purified albumin, or purified reduced albumin as a calibrator. is convenient.
In the conventional BDAB method, reduced albumin purified using Thiol Sepharose is used as a calibrator, and preparation of the calibrator is very time-consuming. The kit for the modified BDAB method does not require the use of such laborious preparation standards, which is a great practical advantage.

Each reagent that constitutes the kit may contain components commonly used in biochemical or clinical chemical test reagents. Such components include, for example, buffers, preservatives, surfactants that do not fall under protein denaturants, and the like. These components may be in the concentration ranges normally selected when used in biochemical or clinical chemistry test reagents.
Examples of surfactants include Triton X-405 and Triton X-100.
Buffers include, for example, acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris(hydroxymethyl)aminomethane (Tris), Good buffer (eg, 3- (N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), etc.). It is preferable to appropriately select a buffer containing these buffers so as to maintain the pH of the sample and reagents at a suitable pH for performing the improved BDAB method. In particular, since reduced albumin is stable at pH 5-6, reagents to be added to a sample, particularly reagents containing color-developing substances, are preferably adjusted to pH 5-6 with a buffer solution. On the other hand, the reagent containing the oxidizing agent can have a pH of 5-8, preferably pH 5-6.

According to one embodiment of the present invention, the kit comprises a first reagent containing an oxidizing agent and a buffer, and a second reagent containing BDAB and a buffer, wherein said first reagent and said second Either one or both of the two reagents contain a protein denaturant.

Examples of more specific kits are shown below.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.

I. Investigation of improved BDAB method by comparison with conventional BDAB method In this test, the effect of correction of the improved BDAB method was evaluated in comparison with the conventional BDAB method.
1. Materials and measurement methods 1-1. Specimens Human serum and plasma prepared from blood individually collected from donors were used, and samples were prepared by diluting them with a stabilizer, citrate buffer, so that the concentration of reduced albumin was uniformly 183 μM. The reduced albumin concentration was determined by the standard method described in "1-3" below.

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

ＨＰＬＣ法による酸化型または還元型アルブミンの割合の決定は、特許文献５に従って実施した。具体的には、イオン交換基としてＤＥＡＥ（Ｄｉｅｔｈｙｌａｍｉｎｏｅｔｈｙｌ）を有するイオン交換樹脂が充填された陰イオン交換カラムを使用し、以下の緩衝液Ａにて平衡化した。その後、検体を注入し、同時に、緩衝液Ａ（１００％から７５％）と以下の緩衝液Ｂ（０％から２５％）によるリニアグラジエントに、７．５分間１．０ｍＬ／ｍｉｎの流速でかけて、酸化型アルブミンおよび還元型アルブミンを分離し、励起波長２８０ｎｍ、検出波長３４０ｎｍの蛍光での検出を行った。
緩衝液Ａ：６０ｍＭ Ｎａ_２ＳＯ_４を含む２５ｍＭリン酸緩衝液（ｐＨ６．０）
緩衝液Ｂ：１Ｍ ＭｇＣｌ_２
改良ＢＣＰ法による総アルブミン濃度の決定は、特許文献３の記載に従って実施をした。１５μＬの検体に対して８５μＬの第一試薬（２５ｍＭ Ｔｒｉｓ ＨＣｌ、ｐＨ８．０中に、１００μＭ ＤＴＮＢ、および０．０３％（ｗ／ｖ）ＳＤＳを含有）を添加、３７℃で５分間インキュベーション後、１５０μＬの第二試薬（２５０ｍコハク酸ナトリウム、ｐＨ５．５中に、１５０μＭブロモクレソールパープル、および０．３％（ｖ／ｖ）Ｔｒｉｔｏｎ Ｘ－１００を含有）を添加して、さらに３７℃で２０分間インキュベーションし、６００ｎｍ（主波長）／６６０（副波長）で吸光度を測定した
一例として、血漿１検体をＨＰＬＣ法（標準法）により分析したクロマトグラムを図１に示す。還元型アルブミンと酸化型アルブミンを示すピークの面積比は、それぞれ、７９．７％および２０．３％である。また、改良ＢＣＰ法（標準法）により同じ検体の総アルブミン濃度は、５５０μＭと決定され、これにＨＰＬＣ法により決定された還元型アルブミンの割合（７９．７％）をかけることで還元型アルブミン濃度が、４３９μＭと算出された。 1-3. Determination of concentration of reduced albumin and percentage of oxidized or reduced albumin (reference value) by standard method % means peak area %), reference value) was determined by HPLC method (standard method). In addition, the reduced albumin concentration (reference value) in the sample was determined by determining the ratio of reduced albumin by the HPLC method (standard method) and determining the total albumin concentration by the improved BCP method (standard method). , was calculated by multiplying the percentage of reduced albumin determined by the HPLC method by the total albumin concentration determined by the modified BCP method.

Determination of the percentage of oxidized or reduced albumin by HPLC method was carried out according to US Pat. Specifically, an anion exchange column packed with an ion exchange resin having DEAE (Diethylaminoethyl) as an ion exchange group was used and equilibrated with the following buffer solution A. Then, the sample was injected and simultaneously subjected to a linear gradient of Buffer A (100% to 75%) and the following Buffer B (0% to 25%) at a flow rate of 1.0 mL/min for 7.5 minutes. , oxidized albumin and reduced albumin were separated and detected with 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
Determination of total albumin concentration by the modified BCP method was performed as described in US Pat. After adding 85 μL of the first reagent (containing 100 μM DTNB and 0.03% (w/v) SDS in 25 mM Tris HCl, pH 8.0) to 15 μL of the sample, incubation at 37° C. for 5 minutes, 150 μL of the second reagent (containing 150 μM bromocresol purple and 0.3% (v/v) Triton X-100 in 250 mM sodium succinate, pH 5.5) was added and further incubated at 37° C. for 20 minutes. After incubating for 1 minute, absorbance was measured at 600 nm (dominant wavelength)/660 nm (secondary wavelength). As an example, FIG. 1 shows a chromatogram of one plasma sample analyzed by the HPLC method (standard method). The area ratios of the peaks representing reduced albumin and oxidized albumin are 79.7% and 20.3%, respectively. In addition, the total albumin concentration of the same sample was determined to be 550 μM by the improved BCP method (standard method), and the reduced albumin concentration (79.7%) determined by the HPLC method was multiplied by was calculated to be 439 μM.

1-4. Pretreatment and Absorbance Measurement 5 μL of all specimens and standard dilutions were dispensed into each well of a 96-well plate for correction and reduced albumin measurement, respectively. 10 μL of 2 mM maleimide (manufactured by Sigma-Aldrich, dissolved in 500 mM Tris buffer, pH 7.2) for correction sample and standard diluent, 500 mM Tris buffer for sample and standard diluent for measurement of reduced albumin The same amount of pH 7.2 solution was added, mixed, and incubated at 37° C. for 15 minutes. Then, 200 μL of a coloring reagent (15 μM BDAB (manufactured by Tokyo Kasei Kogyo) and 6 M guanidine hydrochloride (manufactured by Tokyo Kasei Kogyo) dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes. After that, the solution was transferred to a cuvette (10 mm path) and the absorbance was measured at a wavelength of 612 nm. Pretreatment and absorbance measurement were performed by preparing 3 sets (n=3) of samples for correction and measurement of reduced albumin for each specimen and each standard dilution. The average value of 3 sets (n=3) was used as the measured value for each specimen and each standard dilution, and used for correction, calibration curve preparation, and concentration calculation described below. )

1-5. Confirmation of disappearance of reduced albumin due to oxidizing agent treatment Analyze the correction sample before and after adding the oxidizing agent solution to the standard diluent by HPLC, and confirm whether the peak of reduced albumin has disappeared due to the oxidizing agent. bottom. FIG. 2 shows an example of a chromatogram obtained by analyzing a correction sample by HPLC method before and after adding a 2 mM maleimide solution to a standard (dilution factor: 1). It was confirmed that the peak of reduced albumin disappeared due to the oxidizing agent.

次いで、各標準品希釈液のΔＯＤと、上記１－３より算出した還元型アルブミン濃度をプロットし、検量線を作成した。作成した検量線は、図３に示す。検量線は、最小二乗法による線形検量線（図３Ａ）と、４パラメータロジスティクスモデルによる非線形検量線（図３Ｂ）を作成した。
比較対象として、特許文献４に記載する従来のＢＤＡＢ法に従って、還元型アルブミン濃度を決定する検量線を作成した（図３Ｃ）。ここでは各標準品希釈液の補正用試料の６１２ｎｍでの吸光度（ＯＤ_ｏｘ）は使用せずに（補正無し）、各標準品希釈液の還元型アルブミン測定用試料の同じ波長での吸光度（ＯＤ_{ｎｏｎ－ｏｘ}）のみを使い、各標準品希釈液の吸光度（ＯＤ_{ｎｏｎ－ｏｘ}）と上記１－３より算出した還元型アルブミン濃度をプロットし、検量線を作成した。 1-6. Preparation of calibration curve In the improved BDAB method, as shown in the table below, the absorbance at 612 nm (OD _ox ) of the correction sample of each standard dilution solution was converted to the absorbance at the same wavelength of the corresponding reduced albumin measurement sample ( OD _non-ox ) was subtracted to calculate ΔOD.

Then, the ΔOD of each standard dilution and the reduced albumin concentration calculated from 1-3 above were plotted to prepare a calibration curve. The prepared calibration curve is shown in FIG. As calibration curves, a linear calibration curve (FIG. 3A) by the method of least squares and a nonlinear calibration curve (FIG. 3B) by a 4-parameter logistics model were created.
For comparison, a calibration curve for determining reduced albumin concentration was prepared according to the conventional BDAB method described in Patent Document 4 (Fig. 3C). Here, instead of using the absorbance (OD ox ) at 612 nm of the correction sample of each standard dilution (no correction), the absorbance (OD _ox ) of each standard dilution at the same wavelength of the reduced albumin measurement sample _Non-ox ) alone was used, and the absorbance (OD _non-ox ) of each standard dilution and the reduced albumin concentration calculated from 1-3 above were plotted to prepare a calibration curve.

次いで、各検体のΔＯＤを、上述の各標準品希釈液のΔＯＤと還元型アルブミン濃度をプロットした検量線（線形および非線形）に当てはめ、還元型アルブミン濃度を算出した。
従来のＢＤＡＢ法に従った方法では、各検体の還元型アルブミン測定用試料の６１２ｎｍでの吸光度（ＯＤ_{ｎｏｎ－ｏｘ}）を、上述の各標準品希釈液の吸光度（ＯＤ_{ｎｏｎ－ｏｘ}）と還元型アルブミン濃度をプロットした検量線に当てはめ、還元型アルブミン濃度を算出した。
また、何れの方法でも、算出した還元型アルブミン濃度を、改良ＢＣＰ法（標準法）で決定した同じ検体の総アルブミン濃度で割り、還元型アルブミンの割合（％）を算出した。さらに、そこで算出した還元型アルブミンの割合（％）を１から差し引き、酸化型アルブミンの割合を算出した（近年の論文等では酸化型アルブミンの割合での表示が一般的になりつつあるため、以下では酸化型アルブミンの割合を記載する）。得られた還元型アルブミン濃度および酸化型アルブミンの割合を以下の表に示す。

1-7. Determination of Reduced Albumin Concentration and Percentage of Oxidized Albumin in Each Sample In the improved BDAB method, the absorbance (OD _ox ) of the correction sample for each sample at 612 nm is converted to the absorbance at the same wavelength of the corresponding reduced albumin measurement sample. (OD _non-ox ) was subtracted to calculate ΔOD. Each data is shown in the table below.

Next, the ΔOD of each sample was applied to the calibration curve (linear and non-linear) in which the ΔOD of each standard dilution and the concentration of reduced albumin were plotted to calculate the concentration of reduced albumin.
In the method according to the conventional BDAB method, the absorbance (OD _non-ox ) at 612 nm of the sample for measuring reduced albumin of each specimen is compared with the absorbance (OD _non-ox ) of each standard dilution solution described above and the reduced albumin The reduced albumin concentration was calculated by applying the albumin concentration to the plotted calibration curve.
In either method, the ratio (%) of reduced albumin was calculated by dividing the calculated reduced albumin concentration by the total albumin concentration of the same sample determined by the improved BCP method (standard method). Furthermore, the ratio (%) of reduced albumin calculated there was subtracted from 1 to calculate the ratio of oxidized albumin. describes the ratio of oxidized albumin). The resulting reduced albumin concentrations and oxidized albumin ratios are shown in the table below.

1-8. Evaluation method Concentration of reduced albumin determined by conventional BDAB method (without correction), improved BDAB method (with correction, linear calibration curve) and improved BDAB method (with correction, nonlinear calibration curve), and those methods and improved BCP Each reference value determined by the standard method was subtracted from the ratio of oxidized albumin determined by the method (1-(reduced albumin concentration/total albumin concentration), and the absolute value of the difference was taken as the deviation from the standard method. The correction effect of each oxidizing agent was evaluated based on whether the improved BDAB method (with correction) reduced the divergence from the reference value compared to the conventional BDAB method (without correction).

2. Results The table below and FIG. 4 show the deviation of the concentration of reduced albumin determined by each method from the reference value.

The table below and FIGS. 5 and 6 show the deviation of the percentage of oxidized albumin determined by each method from the reference value. FIG. 6 shows the correlation of the reference values for the percentage of oxidized albumin determined by each method.

As shown in Table 14 and FIG. 4, in the conventional BDAB method, there were samples that greatly deviated from 183 μM, but in the improved BDAB method, it was confirmed that the deviation decreased in all samples except one case.
In addition, as shown in Table 15 and FIG. 5, the ratio (%) of oxidized primary albumin determined by the conventional BDAB method and the improved BCP method (standard method) was significantly different from that of the HPLC method (standard method) and the improved BCP method (standard method). There were samples that deviated greatly from the percentage (%) of oxidized albumin determined by the method), but the percentage (%) of oxidized albumin determined by the improved BDAB method and the improved BCP method (standard method) was the same for all samples. It was confirmed that the divergence decreased in In addition, as shown in FIG. 6, compared with the ratio (%) of oxidized albumin determined by the conventional BDAB method and the improved BCP method (standard method), the oxidized albumin determined by the improved BDAB method and the improved BCP method (standard method) The percentage of albumin has improved correlation with the percentage of oxidized albumin determined by the HPLC method (standard method) and the modified BCP method (standard method).

II. Comparative study of various oxidizing agents (1)
This study builds on the results of Study I and evaluates the improved BDAB method compared to the conventional BDAB method for various oxidizing agents.

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

1-2. Determination of reduced albumin concentration and percentage of oxidized or reduced albumin (standard value) by standard method As in Test I, the percentage of oxidized or reduced albumin in a sample (%, standard value) was determined by HPLC method ( standard method). In addition, the reduced albumin concentration (reference value) in the sample was determined by determining the ratio of reduced albumin by the HPLC method (standard method), determining the total albumin concentration by the improved BCP method (standard method), and determining by the HPLC method. The ratio (%) of reduced albumin obtained was multiplied by the total albumin concentration determined by the modified BCP method.

1-3. Oxidant A solution of the following eight thiol reactants was prepared as an oxidant solution.
・ 200 mM 4-vinylpyridine (manufactured by Tokyo Kasei Kogyo, dissolved in 250 mM Tris buffer, pH 8.0)
· 200 mM iodoacetamide (manufactured by Tokyo Kasei Kogyo, dissolved in 250 mM Tris buffer, pH 8.0)
・ 200 mM iodoacetic acid (manufactured by Fuji Film Wako Pure Chemical, dissolved in 250 mM Tris buffer, pH 8.0)
・ 1 mM 2,2'-dithiobis(5-nitropyridine) (manufactured by Tokyo Kasei Kogyo, dissolved in 250 mM Tris buffer, pH 8.0)
・ 1 mM 6,6'-dithiodinicotinic acid (manufactured by Tokyo Kasei Kogyo, dissolved in 250 mM Tris buffer, pH 8.0)
· 4 mM maleimide (manufactured by Sigma-Aldrich, dissolved in 250 mM Tris buffer, pH 7.2)
· 4 mM 2,2'-dipyridyl disulfide (manufactured by Tokyo Chemical Industry, 50 mM citrate buffer, diluted with pH 5.0)
· 4 mM 4,4'-dipyridyl disulfide (manufactured by Tokyo Kasei Kogyo, 50 mM citrate buffer, diluted with pH 5.0)

1-4. Pretreatment and Absorbance Measurement Pretreatment and absorbance measurement were basically the same as in Test I above, but a plate reader was used to accommodate more samples. Accordingly, in order to ensure sensitivity, the addition amount of the sample and each reagent, the concentration of the oxidizing agent and other reagents, etc. were slightly changed.
Specifically, 15 μL of each sample and standard diluent was dispensed to each well of a 96-well plate for correction and reduced albumin measurement. 10 μL of each oxidizing agent was added to the sample for correction and the diluent of the standard, and the same amount of 250 mM Tris buffer, pH 7.2 was added to the sample and the diluent of the standard for measurement of reduced albumin. for 20 minutes. Then, 225 μL of a coloring reagent (35 μM DBAB (manufactured by Tokyo Kasei Kogyo) and 6 M guanidine hydrochloride (manufactured by Tokyo Kasei Kogyo) dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, and incubated at room temperature for 20 minutes. After that, absorbance was measured at a wavelength of 612 nm using a plate reader. Pretreatment and absorbance measurement were performed by preparing 3 sets (n=3) of samples for correction and measurement of reduced albumin for each specimen and each standard dilution. The average value of 3 sets (n=3) was used as the measured value for each specimen and each standard dilution, and used for correction, calibration curve preparation, and concentration calculation described below.

1-5. Preparation of standard curve, determination of concentration of reduced albumin and percentage of oxidized albumin standard curve, and standard curve without correction), the modified BDAB method fits the ΔOD of each analyte to a linear standard curve with correction or a non-linear standard curve with correction, and the conventional BDAB method involves treating each analyte with an oxidizing agent. The absorbance of untreated samples (OD _non-ox ) was fitted to the uncorrected calibration curve to calculate reduced albumin concentration. In either method, the calculated reduced albumin concentration was divided by the total albumin concentration of the same sample determined by the modified BCP method (standard method) to calculate the ratio (%) of reduced albumin, and was subtracted from 1 to calculate the ratio (%) of oxidized albumin.

1-6. Evaluation method Similar to Test I, subtract the standard value determined by the standard method from the concentration of reduced albumin or the ratio of oxidized albumin determined by each method, and take the absolute value of the difference as the deviation from the standard method. bottom. The correction effect of each oxidizing agent was evaluated based on whether the improved BDAB method (with correction) reduced the divergence from the reference value compared to the conventional BDAB method (without correction).

上記表に示す通り、８種の酸化剤の総てにおいて、改良ＢＤＡＢ法（補正あり）での補正効果が認められ、従来のＢＤＡＢ法（補正なし）と比べて還元型アルブミンの濃度および酸化型アルブミンの割合は標準法による基準値に対して乖離が減少した。 2. Results In Table 16 below, the reduced albumin concentration was determined by the modified BDAB method (with correction) or the conventional BDAB method (without correction) using maleimide as an oxidizing agent, and the standard method (HPLC method & modified BCP method). ) along with the reduced albumin concentration determined by ). In addition, Table 17 shows the results of determination of the ratio of oxidized albumin by the improved BDAB method (with correction) using maleimide as an oxidizing agent or by the conventional BDAB method (without correction) and the improved BCP method. ) along with the reduced albumin concentration determined by ). When the improved BDAB method (with correction) is used, the concentration of reduced albumin determined by creating a linear calibration curve and a nonlinear calibration curve and the ratio of oxidized albumin calculated using the concentration of reduced albumin are shown.
Tables 18 and 19 list 4-vinylpyridine, iodoacetamide, iodoacetic acid, 2,2′-dithiobis(5-nitropyridine), 6,6′-dithiodinicotinic acid, maleimide, 2,2 as oxidizing agents. Reduced albumin concentrations determined by the modified BDAB method (with correction) or the conventional BDAB method (without correction) using '-dipyridyl disulfide, 4,4'-dipyridyl disulfide, or maleimide and combined with the modified BCP method The ratio of oxidized albumin determined by the standard method (HPLC method & improved BCP method) shows the degree of divergence from the concentration of reduced albumin determined by the standard method (HPLC method & improved BCP method) and the ratio of oxidized albumin determined by the standard method (HPLC method). When the improved BDAB method (with correction) is used, the concentration of reduced albumin determined by creating a linear calibration curve and a nonlinear calibration curve and the ratio of oxidized albumin calculated using the concentration of reduced albumin are shown.

As shown in the above table, the improved BDAB method (with correction) was found to have a correction effect for all of the eight oxidizing agents, and compared to the conventional BDAB method (without correction), the concentration of reduced albumin and oxidized form The ratio of albumin decreased in deviation from the standard value by the standard method.

III. Comparative study of various oxidizing agents (2)
This study builds on the results of Study I and further evaluates the modified BDAB process in comparison to the conventional BDAB process for various oxidizing agents.

1. Materials and Measurements 1-1. Samples and Reference Standards The samples and standards are the same as in Study II. Also, the sample was mixed with a citrate buffer at a volume ratio of 1:1 and diluted to 1/2.

1-2. Determination of reduced albumin concentration and percentage of oxidized or reduced albumin (standard value) by standard method As in Test I, the percentage of oxidized or reduced albumin in a sample (%, standard value) was determined by HPLC method ( standard method). In addition, the reduced albumin concentration (reference value) in the sample was determined by determining the ratio of reduced albumin by the HPLC method (standard method), determining the total albumin concentration by the improved BCP method (standard method), and determining by the HPLC method. The ratio (%) of reduced albumin obtained was multiplied by the total albumin concentration determined by the modified BCP method.

1-3. Oxidizing agent, pretreatment and absorbance measurement Four types of thiol reactants were used as oxidizing agents, and pretreatment and absorbance measurement were basically performed in the same manner as in Test II. Various reagent preparation buffers, reagents, and mixing ratios were performed. Each thiol reactant, pretreatment conditions and absorbance measurements are as follows.

5,5'-dithiobis(2-nitrobenzoic acid)
An oxidant solution was prepared by diluting 4 mM 5,5′-dithiobis(2-nitrobenzoic acid) (manufactured by Sigma-Aldrich) into 250 mM Tris buffer, pH 8.0. Next, 15 μL of each diluted sample and each standard diluted solution were dispensed into each well of a 96-well plate for correction and measurement of reduced albumin. 10 μL of oxidizing agent 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. After that, 200 μL of a coloring reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, incubated at room temperature for 20 minutes, and the absorbance was measured at a wavelength of 612 nm using a plate reader. It was measured. The average value of 3 sets (n=3) was used as the measured value for each sample and each standard dilution.

2,2'-dithiodibenzoic acid 0.3 mM 2,2'-dithiodibenzoic acid (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was diluted with 50 mM Tris buffer containing 6 M guanidine hydrochloride (manufactured by Tokyo Kasei Kogyo Co., Ltd.), pH 8.0. to prepare an oxidant solution. Next, 15 μL of each diluted sample and each standard diluted solution were dispensed into each well of a 96-well plate for correction and measurement of reduced albumin. 35 μL of the oxidizing agent 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. After that, 200 μL of a coloring reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, incubated at room temperature for 20 minutes, and the absorbance was measured at a wavelength of 612 nm using a plate reader. It was measured. The average value of 3 sets (n=3) was used as the measured value for each sample and each standard dilution.

Bis(4-nitrophenyl) disulfide 0.4 mM bis(4-nitrophenyl) disulfide (manufactured by Tokyo Kasei Kogyo) was added to 50 mM Tris buffer containing 6 M guanidine hydrochloride (manufactured by Tokyo Kasei Kogyo) and 20% (v/v) DMSO. was diluted to pH 8.0 to prepare an oxidant solution. Next, 15 μL of each diluted sample and each standard diluted solution were dispensed into each well of a 96-well plate for correction and measurement of reduced albumin. 35 μL of the oxidizing agent 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. After that, 200 μL of a coloring reagent (39 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, incubated at room temperature for 20 minutes, and the absorbance was measured at a wavelength of 612 nm using a plate reader. It was measured. The average value of 3 sets (n=3) was used as the measured value for each sample and each standard dilution.

Hydrogen peroxide 50 mM hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries) was diluted to 250 mM Tris buffer, pH 7.2 to prepare an oxidant solution. Next, 15 μL of each diluted sample and each standard diluted solution were dispensed into each well of a 96-well plate for correction and measurement of reduced albumin. 10 μL of oxidizing agent solution was added to the correction sample, and the same amount of Tris buffer, pH 7.2 was added to the reduced albumin measurement sample, mixed, and incubated at 37° C. for 20 minutes. After that, 225 μL of a coloring reagent (35 μM DBAB and 6 M guanidine hydrochloride dissolved in 250 mM acetate buffer, pH 5.0) was added, mixed, incubated at room temperature for 20 minutes, and then absorbance was measured at a wavelength of 612 nm using a plate reader. It was measured. The average value of 3 sets (n=3) was used as the measured value for each sample and each standard dilution.

1-4. Preparation of standard curve, determination of concentration of reduced albumin and percentage of oxidized albumin standard curve, and standard curve without correction), the modified BDAB method fits the ΔOD of each analyte to a linear standard curve with correction or a non-linear standard curve with correction, and the conventional BDAB method involves treating each analyte with an oxidizing agent. The absorbance of untreated samples (OD _non-ox ) was fitted to the uncorrected calibration curve to calculate reduced albumin concentration. In either method, the calculated reduced albumin concentration was divided by the total albumin concentration of the same sample determined by the modified BCP method (standard method) to calculate the ratio (%) of reduced albumin. The ratio (%) of oxidized albumin was calculated by subtracting the ratio from 1.

1-5. Evaluation method Similar to Test I, subtract the standard value determined by the standard method from the concentration of reduced albumin or the ratio of oxidized albumin determined by each method, and take the absolute value of the difference as the deviation from the standard method. bottom. The correction effect of each oxidizing agent was evaluated based on whether the improved BDAB method (with correction) reduced the divergence from the reference value compared to the conventional BDAB method (without correction).

上記表に示す通り、本試験で用いた４種類の酸化剤を使った改良ＢＤＡＢ法では、従来のＢＤＡＢ法（補正なし）と比較して、基準値に対する乖離が縮小している検体もあったが、基準値に対する乖離が縮小しなかった検体、むしろ基準値に対する乖離が大きくなった検体も多くあり、全体として明らかな補正の効果が見られなかった。 2. Results In the table below, 5,5'-dithiobis(2-nitrobenzoic acid), 2,2'-dithiodibenzoic acid, bis(4-nitrophenyl) disulfide, or hydrogen peroxide were used as oxidizing agents. Reduced albumin concentration determined by the improved BDAB method (with correction) or conventional BDAB method (without correction), and oxidized albumin calculated from the reduced albumin concentration and the total albumin concentration determined by the improved BCP method (standard method) The percentage of albumin deviates from the concentration of reduced albumin determined by the standard method (HPLC method and modified BCP method) and the percentage of oxidized albumin determined by the standard method (HPLC method). In the improved BDAB method (with correction), the concentration of reduced albumin determined by creating a linear calibration curve and a nonlinear calibration curve is shown, and the concentration of reduced albumin determined from these calibration curves was used as the percentage of oxidized albumin. indicate a value.

As shown in the table above, in the improved BDAB method using the four types of oxidizing agents used in this test, some specimens showed a smaller deviation from the reference value than the conventional BDAB method (without correction). However, there were many samples in which the deviation from the standard value did not decrease, and in fact there were many samples in which the deviation from the standard value increased.

IV. Extended test using maleimide as oxidizing agent1 . Materials and measurement methods 1-1. Specimens In addition to the specimens (serum and plasma) used in Test I, the specimens were collected by mixing whole blood with a stabilizing citrate buffer in a 1:1 volume ratio immediately after blood collection from a donor, and centrifuging. Plasma was also used (preparation method for preventing oxidation and stably storing specimens assumed in practical use. See Non-Patent Document 17). The sample used in Test I was diluted to 1/2 by mixing with citrate buffer at a volume ratio of 1:1 immediately before measurement.
The standard used in Test I was used as the standard.
1-2. Oxidizing agent 4 mM maleimide (manufactured by Sigma-Aldrich, dissolved in Tris buffer, pH 7.2) was used.
1-3. Miscellaneous Other points were carried out in the same manner as Test II.

上記表ならびに図７および８に示す通り、還元型アルブミンの濃度および酸化型アルブミンの割合が、標準法による基準値から乖離する検体があったが、全体として補正により基準値からの乖離が減少し、乖離度合いのばらつきも収束した。
また、図９に示す通り、酸化型アルブミンの割合について、標準法との相関係数が従来のＢＤＡＢ法（補正なし）では０．８８であったのに対し、改良ＢＤＡＢ法（補正あり、線形）では０．９７になり、相関直線の傾きもＢＤＡＢ法（補正なし）では０．６８であったのに対して、改良ＢＤＡＢ法（補正あり）では、０．９７（線形検量線）および０．９３（非線形検量線）となり、傾きがより１に近づいた。従って、改良ＢＤＡＢ法では標準法に近似するより正確な数値が得られることが確認された。 2. Results Tables 22 and 23 and Figures 7 and 8 below show the concentration of reduced albumin and the percentage of oxidized albumin determined by each method and the deviation of these values from the reference values. In addition, Table 24 below shows statistical analysis of the deviation of the ratio of oxidized albumin shown in Table 23 from the reference value, and FIG. 9 shows the correlation between the reference values of the ratio of oxidized albumin determined by each method. show.

As shown in the above table and FIGS. 7 and 8, the concentration of reduced albumin and the ratio of oxidized albumin in some specimens deviated from the reference values determined by the standard method, but overall the deviations from the reference values were reduced by correction. , the dispersion of the degree of divergence also converged.
In addition, as shown in FIG. 9, the correlation coefficient with the standard method for the ratio of oxidized albumin was 0.88 with the conventional BDAB method (without correction), whereas the improved BDAB method (with correction, linear ), and the slope of the correlation line was 0.68 for the BDAB method (without correction), while the improved BDAB method (with correction) gave 0.97 (linear calibration curve) and 0 .93 (non-linear calibration curve), and the slope is closer to 1. Therefore, it was confirmed that the modified BDAB method gave more accurate values that approximated the standard method.

V. Extended test using iodoacetamide as oxidizing agent1 . Materials and measurement methods 1-1. Specimens The same specimens as in Test IV were used after being treated in the same manner.
1-2. Oxidizing agent 200 mM iodoacetamide (manufactured by Tokyo Kasei Kogyo, dissolved in Tris buffer, pH 8.0) was used.
1-3. Miscellaneous Other points were carried out in the same manner as Test II.

上記表ならびに図１０および１１に示す通り、還元型アルブミン濃度および酸化型アルブミン割合について、検体によっては標準法による基準値から大きく乖離する検体があったが、全体として補正により乖離が減少し、乖離度合いのばらつきも収束した。より具体的には、還元型アルブミンの濃度について、改良ＢＤＡＢ法（補正あり、線形）では３２検体中２５検体、改良ＢＤＡＢ法（補正あり、非線形）では２４検体で乖離が減少した。酸化型アルブミンの割合について、改良ＢＤＡＢ法（補正あり、線形）では３２検体中２６検体、改良ＢＤＡＢ法（補正あり、非線形）では２４検体で乖離が減少した（図１１）。中には、従来のＢＤＡＢ法（補正なし）で、標準法による基準値からの乖離が小さく、改良ＢＤＡＢ法（補正あり）で若干乖離が大きくなったものもあったが、そのような検体でも酸化型アルブミン割合の基準法による基準値からの乖離は７％以下であり、全体として改良ＢＤＡＢ法（補正あり）の標準法からの乖離は小さく、平均で２．２％であり、最も乖離が大きな数値でも６－７％に収まることが確認された。
また、図１２に示す通り、酸化型アルブミンの割合について、標準法との相関係数が従来のＢＤＡＢ法（補正なし）では０．８８であったのに対し、改良ＢＤＡＢ法（補正あり）では０．９７になり、相関直線の傾きもＢＤＡＢ法（補正なし）では０．６８であったのに対して、改良ＢＤＡＢ法（補正あり）では、１．０３（線形検量線）および０．９８（非線形検量線）となり、傾きがより１に近づいた。従って、改良ＢＤＡＢ法では標準法に近似するより正確な数値が得られることが確認された。 2. Results Tables 25 and 26 and Figures 10 and 11 below show the concentration of reduced albumin and the percentage of oxidized albumin determined by each method and the deviation of these values from the reference values. Table 27 below shows the statistical analysis of the deviation of the ratio of oxidized albumin shown in Table 26 from the reference value, and FIG. 12 shows the correlation between the reference values of the ratio of oxidized albumin determined by each method. .

As shown in the above table and FIGS. 10 and 11, the concentration of reduced albumin and the percentage of oxidized albumin in some samples greatly deviated from the standard values determined by the standard method. The degree of variability also converged. More specifically, with respect to the concentration of reduced albumin, the improved BDAB method (with correction, linear) reduced the divergence in 25 out of 32 samples, and the improved BDAB method (with correction, nonlinear) in 24 samples. Regarding the ratio of oxidized albumin, the deviation decreased in 26 of 32 specimens by the improved BDAB method (with correction, linear) and in 24 specimens by the improved BDAB method (with correction, nonlinear) (Fig. 11). Among them, the conventional BDAB method (without correction) showed a small deviation from the standard value by the standard method, and the improved BDAB method (with correction) showed a slightly larger deviation, but even such samples The deviation from the standard value of the oxidized albumin ratio by the standard method is 7% or less, and the deviation from the standard method of the improved BDAB method (with correction) is small overall, 2.2% on average, and the deviation is the largest. It was confirmed that even a large number falls within 6-7%.
In addition, as shown in FIG. 12, the correlation coefficient with the standard method for the proportion of oxidized albumin was 0.88 in the conventional BDAB method (without correction), whereas in the improved BDAB method (with correction) The slope of the correlation line was 0.68 for the BDAB method (without correction), while the improved BDAB method (with correction) was 1.03 (linear calibration curve) and 0.98 (non-linear calibration curve), and the slope became closer to 1. Therefore, it was confirmed that the modified BDAB method gave more accurate values that approximated the standard method.

VI. Investigation on the use of purified albumin and reduced albumin as standard products In this test, various standard products were compared and studied.

1. Materials and measurement methods 1-1. Preparation of Human Serum Albumin Standards Albumin was purified from standard serum (Consela No. 1) used in previous studies. A plastic column with an inner diameter of 15 mm was filled with 10 mL (bed volume) of Affigel Blue Gel (manufactured by Bio-Rad) as a carrier, equilibrated with phosphate buffer pH 7.1, the column was capped, and 6 mL of standard serum was added. Add and mix 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. After elution, the sample was dialyzed against a citrate buffer containing 100 mM NaCl, pH 5.0, using a cellulose dialysis membrane with a molecular weight cutoff of 3500, and then concentrated with an ultrafiltration membrane to obtain a purified albumin solution. rice field. The percentage of oxidized or reduced albumin (reference value) in the obtained purified albumin solution 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 determined by the HPLC method (standard method) as in Test I, and the total albumin concentration was determined by the modified BCP method (standard method). and calculated by multiplying the percentage 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 serially diluted with a citrate buffer to prepare a dilution series and used as a calibrator.

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

1-3. Reference standard for comparison As a reference standard for comparison, the standard serum (Consela No. 1) used in previous tests and a solution of cysteine as a low-molecular-weight thiol compound dissolved in citrate buffer were used. A dilution series was prepared by serially diluting with liquid and used as a calibrator.

1-4. Oxidant As an oxidant solution, 4 mM maleimide (manufactured by Sigma-Aldrich, dissolved in Tris buffer, pH 7.2) was used.
1-5. Pretreatment and Absorbance Measurement Pretreatment and absorbance measurement were performed as in Test II.
1-6. Preparation of calibration curve Using the dilution series of each standard and the reference standard for comparison, in the same manner as Test I, the calibration curve (linear) by the improved BDAB method (with correction) and the conventional BDAB method (without correction) A calibration curve was created by
1-7. Evaluation Using the calibration curve prepared with the standard serum (Consela No. 1) as a reference, the gradients and intercepts of the calibration curves prepared with each standard product and the reference standard product examined this time were compared.

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

各標準血清または血漿中の還元型アルブミンの割合（基準値）を、試験Ｉと同様に、ＨＰＬＣ法（標準法）によって決定した。また、各標準血清または血漿中の総アルブミン濃度は改良ＢＣＰ法（標準法）で決定した。また、各標準血清または血漿中の還元型アルブミン濃度（基準値）は、試験Ｉと同様に、ＨＰＬＣ法（標準法）で決定した還元型アルブミンの割合を、改良ＢＣＰ法で決定した総アルブミン濃度を掛けて算出した。
得られた精製アルブミン溶液を、クエン酸緩衝液で段階的に希釈して希釈系列を調製し、キャリブレーターとして使用した。
各標準血清または血漿は、クエン酸緩衝液で段階的に希釈し希釈系列を調製し、キャリブレーターとして使用した。
１－２．酸化剤
酸化剤溶液として、４ｍＭマレイミド（トリス緩衝液、ｐＨ７．２に溶解）を使用した。
１－３．前処理と吸光度測定
前処理および吸光度測定は、試験ＩＩと同様に行った。
１－４．検量線の作成
各標準血清または血漿の希釈系列を使用して、試験Ｉと同様にして、改良ＢＤＡＢ法（補正あり）による検量線（線形）と、従来のＢＤＡＢ法（補正無し）による検量線を作成した（図１５および図１６）。
１－５．評価
標準血清（コンセーラ１号）により作成した検量線を基準として、今回検討した各標準血清または血漿により作成した検量線の傾き、切片を比較した。 VII. Evaluation of Multiple Types of Standard Serum/Plasma as Calibrator Candidates In this test, a dilution series of multiple types of standard serum or plasma was prepared to evaluate suitability as a calibrator.
1. Materials and measurement methods 1-1. Preparation of Standard Serum or Plasma and Dilution Series The following standard serum or plasma was used as a candidate calibrator.

The percentage of reduced albumin (reference value) in each standard serum or plasma was determined by the HPLC method (standard method) as in Test I. In addition, the total albumin concentration in each standard serum or plasma was determined by the modified BCP method (standard method). In addition, the reduced albumin concentration (reference value) in each standard serum or plasma was determined by the HPLC method (standard method), the ratio of reduced albumin determined by the HPLC method (standard method), and the total albumin concentration determined by the improved BCP method. calculated by multiplying
The resulting purified albumin solution was serially diluted with a citrate buffer to prepare a dilution series and used as a calibrator.
Each standard serum or plasma was serially diluted with a citrate buffer to prepare a dilution series and used as a calibrator.
1-2. Oxidant As an oxidant 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 as in Test II.
1-4. Preparation of standard curve Using each standard serum or plasma dilution series, standard curve (linear) by improved BDAB method (with correction) and standard curve by conventional BDAB method (without correction) in the same manner as Test I was created (FIGS. 15 and 16).
1-5. Evaluation Using the calibration curve prepared with the standard serum (Consela No. 1) as a reference, the slopes and intercepts of the calibration curves prepared with each standard serum or plasma examined this time were compared.

2. Results In the case of the conventional BDAB method (without correction), the calibration curve prepared using some standard plasmas deviated from the standard calibration curve, but in the improved BDAB method (with correction), There was no deviation from the reference standard curve for any standard serum or plasma, and similar standard curves were obtained (Figs. 15 and 16). This suggests that serum or plasma can be used as a calibrator in the method of the present invention as long as the reduced albumin concentration is sufficient to cover the measurement range.

Claims (19)

A kit for measuring reduced albumin concentration, comprising a chromogenic substance that reacts with thiol to change absorbance and an oxidizing agent that oxidizes free SH groups of reduced albumin,
The kit, wherein said oxidizing agent is selected from the group consisting of alkylating agents, maleimides and derivatives thereof, and substituted or unsubstituted dithiodipyridines. 2. The kit according to claim 1, wherein the chromogenic substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of a thiol group. The kit of claim 1, wherein the chromogenic substance is 4-4'-bisdimethylaminobenzhydrol (BDAB). Claims 1-3, wherein said alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). The kit according to any one of the above. The oxidizing agent is iodoacetic acid, iodoacetamide, maleimide, 2,2′-dipyridyl disulfide, 4,4′-dipyridyl disulfide, 2,2′-dithiobis(5-nitropyridine), 6,6′-dithiodinicotine Acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB), according to any one of claims 1-3. kit. The kit according to any one of claims 1 to 5, comprising a protein denaturant. The molar concentration of the oxidizing agent is 10 times or more the molar concentration of albumin when the reagent containing the oxidizing agent and the sample or standard are mixed, and the mixture containing the oxidizing agent and albumin contains the BDAB. The kit according to any one of claims 1 to 6, which is set to be 270 times or less the molar concentration of the coloring substance when mixed. The kit according to any one of claims 1 to 7, which contains standard serum or plasma, purified albumin solution, or purified reduced albumin solution as a calibrator. A first reagent containing the oxidizing agent and a buffer solution, and a second reagent containing the chromogenic substance and a buffer solution, wherein either or both of the first reagent and the second reagent contain a protein denaturant. The kit of any one of claims 1-8, comprising: A method for measuring reduced albumin concentration, comprising:
An oxidizing agent that oxidizes free SH groups of reduced albumin is added to the sample, then mixed with a reagent containing a coloring substance that reacts with thiol to change absorbance, and absorbance (OD _ox ) is measured at a predetermined wavelength. process and
On the other hand, a step of mixing the sample with a reagent containing the coloring substance without adding an oxidizing agent and measuring the absorbance (OD _non-ox ) at a predetermined wavelength;
determining the absorbance difference (ΔOD) between the absorbance (OD _ox ) and the absorbance (OD _non-ox ), and calculating the reduced albumin concentration in the sample from the absorbance difference (ΔOD);
The method, wherein said oxidizing agent is selected from the group consisting of alkylating agents, maleimides and derivatives thereof, and substituted or unsubstituted dithiodipyridines. 11. The method of claim 10, wherein the chromogenic substance is a substance that undergoes a change in absorbance as a result of a nucleophilic reaction of thiol groups. 12. The method of claim 10 or 11, wherein the chromogenic substance is 4-4'-bisdimethylaminobenzhydrol (BDAB). Claims 10-12, wherein said alkylating agent is selected from the group consisting of acetyl halide, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB). The method according to any one of . The oxidizing agent is iodoacetic acid, iodoacetamide, maleimide, 2,2′-dipyridyl disulfide, 4,4′-dipyridyl disulfide, 2,2′-dithiobis(5-nitropyridine), 6,6′-dithiodinicotine Acid, 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), and bromoacetonylquinolinium bromide (BQB), according to any one of claims 10-12. Method. The method according to any one of claims 10 to 14, further comprising adding a protein denaturant to the sample. The method according to any one of claims 10 to 15, wherein a standard curve is prepared using standard serum or plasma, purified albumin solution, or purified reduced albumin solution as a calibrator. preparing a dilution series of the standard serum or plasma, purified albumin solution, or purified reduced albumin solution;
After adding the oxidizing agent to each dilution in the dilution series and then mixing with the reagent containing the coloring substance, the absorbance (OD _ox ) is measured at the predetermined wavelength,
On the other hand, each diluted solution in the dilution series is mixed with the reagent containing the coloring substance without the addition of the oxidizing agent, and then the absorbance (OD _non-ox ) is measured at the predetermined wavelength,
For each of the diluted solutions, the absorbance difference (ΔOD) between the absorbance (OD _ox ) and the absorbance (OD _non-ox ) is obtained,
17. The method of claim 16, wherein the calibration curve is generated from the absorbance difference ([Delta]OD) for each diluent and the known reduced albumin concentration of each diluent. A method for determining the proportion of reduced albumin, comprising:
determining 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;
dividing the reduced albumin concentration by the total albumin concentration to calculate the percentage of reduced albumin. A method of calculating the ratio of oxidized albumin by subtracting the ratio of reduced albumin determined by the method according to claim 18 from 1.

Images