Method and reagent for measuring HMGB1 in a sample, method for suppressing nonspecific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized, and method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample

Abstract

To provide a measuring method of HMGB1 in a specimen suppressing non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized and thereby suppressing rise of a reagent blank caused by the aggregation, measurement reagent, etc.SOLUTION: A measuring method of HMGB1 in a specimen, etc. measures the concentration of HMGB1 in a specimen by bringing HMGB1 in the specimen into contact with an anti-HMGB1 antibody-immobilized carrier and measuring aggregation of the anti-HMGB1 antibody-immobilized carrier bound via HMGB1. The measuring method is characterized in that when HMGB1 in the specimen and the anti-HMGB1 antibody-immobilized carrier come into contact, 0.001% (w / v)-0.01% (w / v) of glycerin / propylene oxide / ethylene oxide adduct or 0.0054% (w / v)-0.022% (w / v) of glycerin / propylene oxide adduct coexists therein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method and a measurement reagent for measuring HMGB1 (high mobility group protein-1; HMG-1) in a sample, which can be a marker for diseases such as sepsis, a method for suppressing nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody is immobilized, and a method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample. The present invention is useful in fields of life science such as clinical testing, clinical pathology and medicine, and fields of chemistry such as analytical chemistry. [Background technology]

[0002] High mobility group proteins (HGM) were discovered in 1964 as abundant non-histone proteins contained in the chromatin structure. They are proteins that are universally found in all higher plants and animals, and their primary structure is highly conserved across species. It has also been found that it is abundant not only in the nucleus but also in the cytoplasm. Although its physiological function is not clearly understood, HMGB1 is thought to function as a very broad-spectrum transcription-promoting factor and nucleosome-relaxing factor, as it relaxes the double helix structure when it binds to DNA, thereby optimizing the higher-order structure of DNA during the transcription reaction and enhancing transcriptional activity.

[0003] There are several types of high mobility group proteins, including high mobility group protein-1 (HMGB1), high mobility group protein-2 (HMGB2), high mobility group protein-3 (HMGB3), high mobility group protein-8 (HMGB8), high mobility group protein-17 (HMGB17), high mobility group protein-I (HMGBI), high mobility group protein-Y (HMGBY), high mobility group protein-I(Y) (HMGBI(Y)), and high mobility group protein IC (HMGB IC).

[0004] In 1999, Wang et al. performed the first quantitative measurement of HMGB1 in serum (blood) by Western blotting using a polyclonal antibody prepared using HMGB1 itself as an immunogen. As a result, Wang et al. showed that HMGB1 could be a marker for sepsis. They also demonstrated that precise measurement of HMGB1 in the blood makes it possible to distinguish between surviving and dying sepsis patients.

[0005] In other words, the usefulness of not only simply confirming the presence of HMGB1 in blood but also accurately quantifying it has been demonstrated (see Non-Patent Document 1).

[0006] It has previously been shown by Parkinen et al. and Repp et al. that antibodies used to measure HMGB1, i.e., antibodies that specifically bind to HMGB1 (anti-HMGB1 antibodies), can be prepared (see Non-Patent Documents 2 and 3). Using this antibody, Repp et al. have stated that a solid-phase enzyme immunoassay for HMGB1 is possible. (Note that this solid-phase enzyme immunoassay involves immobilizing purified HMGB1 to the wells of a microplate (microtiter plate), contacting this with an antibody that binds to enzyme-labeled HMGB1, and allowing it to react, confirming that the antibody that binds to HMGB1 binds to the purified HMGB1.) Furthermore, in 2000, Ruuhiainen et al. measured HMGB1 in human blood by sandwich ELISA using two polyclonal antibodies: one prepared using rat HMGB1 itself, which had been prepared from recombinant DNA by genetic engineering, as an immunogen; and the other prepared using a peptide consisting of part of the HMGB1 amino acid sequence, "Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala," as an immunogen (see Non-Patent Document 4).

[0007] Yamada et al. also disclosed an antibody that binds to HMGB1 but not to HMGB2, as well as a method and reagent for measuring HMGB1 that uses this antibody to measure only HMGB1 but not HMGB2 (Patent Document 1).

[0008] Furthermore, Yamada et al. disclosed a method and reagent for measuring HMGB1 and / or HMGB2 contained in a sample, characterized in that cations and anions are present at concentrations such that the molar concentration of each ion multiplied by the absolute value of the ion's valence is 150 mM or more (Patent Document 2). This measurement method and measurement reagent can increase the sensitivity of the measurement.

[0009] Furthermore, Yamada et al. disclosed an avian anti-human HMGB1 polyclonal antibody, which consists of an avian antibody that specifically binds to the amino acid sequence of human HMGB1 represented by the following formula (I): Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu Lys Ser (I) (Patent Document 3). This avian-derived anti-human HMGB1 polyclonal antibody has a high productivity, making it possible to obtain high-titer antibodies with high binding ability to human HMGB1 with a high probability.

[0010] The present inventors have also disclosed a method for measuring HMGB1 in a sample using a carrier on which anti-HMGB1 antibodies are immobilized, characterized in that two monoclonal antibodies produced by specific hybridomas are combined and immobilized on the same carrier (Patent Document 4). This method for measuring HMGB1 in a sample allows for highly sensitive measurements. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-96099 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-144728 [Patent Document 3] WO2008 / 075788 publication [Patent Document 4] Japanese Patent Publication No. 2020-148557 [Non-patent literature]

[0012] [Non-Patent Document 1] H. Wang et al., SCIENCE, Vol. 285, No. 9, pp. 248-251, 1999 [Non-patent document 2] J. Parkkinen et al., The Journal of Biological Chemistry, Vol. 268, No. 26, pp. 19726-19738, 1993 [Non-patent document 3] W.A. Lepp et al., Journal of Immunoassay, Vol. 10, No. 4, pp. 449-465, 1989 [Non-patent document 4] A. Rouhiainen et al., Thrombosis Haemost, Vol. 84, pp. 1087-1094, 2000 Summary of the Invention [Problem to be solved by the invention]

[0013] The inventors of the present application have discovered that in a method and reagent for measuring HMGB1 in a sample, in which the concentration of HMGB1 in the sample is measured by contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized bound via HMGB1, the carrier on which the anti-HMGB1 antibody has been immobilized will agglutinate nonspecifically when shaken during transportation or for other reasons, resulting in an increase in the reagent blank (reagent blank) caused by this agglutination.

[0014] Specifically, the inventors of the present application discovered that the degree of nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized can be evaluated by measuring the two-wavelength ratio of the absorbance of a measurement reagent containing a carrier onto which an anti-HMGB1 antibody has been immobilized. More specifically, the absorbance at 570 nm and the absorbance at 800 nm of a measurement reagent containing a carrier onto which anti-HMGB1 antibody has been immobilized are measured, and the absorbance ratio is calculated by dividing the absorbance value at 570 nm by the absorbance value at 800 nm. When the carrier on which anti-HMGB1 antibody is immobilized aggregates nonspecifically, the absorbance value at longer wavelengths increases, and the absorbance ratio obtained by dividing the absorbance value at 570 nm by the absorbance value at 800 nm decreases. Therefore, the degree of nonspecific aggregation of the carrier on which anti-HMGB1 antibody is immobilized can be evaluated by calculating the absorbance ratio obtained by dividing the absorbance value at 570 nm of a measurement reagent containing a carrier on which anti-HMGB1 antibody is immobilized by the absorbance value at 800 nm.

[0015] Furthermore, the carrier on which the anti-HMGB1 antibody is immobilized aggregates nonspecifically, increasing the apparent particle size of the carrier on which the anti-HMGB1 antibody is immobilized, and thus increasing the reagent blank. Furthermore, nonspecific aggregation of the carrier onto which anti-HMGB1 antibodies are immobilized reduces the effective number of "carriers onto which anti-HMGB1 antibodies are immobilized" used in the antigen-antibody reaction between "HMGB1 in the sample" and "anti-HMGB1 antibodies on the carrier onto which anti-HMGB1 antibodies are immobilized," thereby reducing the slope of the calibration curve when measuring HMGB1 in a sample. Due to the increase in the reagent blank and the decrease in the slope of the calibration curve, the calibration curve for a measurement reagent containing a carrier on which an anti-HMGB1 antibody that has been shaken is immobilized will differ from the calibration curve for a measurement reagent containing a carrier on which an anti-HMGB1 antibody that has not been shaken is immobilized.

[0016] The object of the present invention is to provide a method and a measurement reagent for measuring HMGB1 in a sample, which measures the concentration of HMGB1 in a sample by contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized bound via HMGB1, and which prevents the carrier on which the anti-HMGB1 antibody has been immobilized from causing nonspecific aggregation due to shaking during transportation or for other reasons, thereby suppressing an increase in the reagent blank caused by such aggregation. Another object of the present invention is to provide a method for inhibiting nonspecific aggregation of a carrier on which an anti-HMGB1 antibody has been immobilized, which can inhibit the carrier from causing nonspecific aggregation due to shaking during transportation or for other reasons. Furthermore, the present invention provides a method for suppressing an increase in reagent blank when measuring HMGB1 in a sample, which can suppress an increase in reagent blank caused by nonspecific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized due to shaking during transportation or for other reasons. [Means for solving the problem]

[0017] The inventors of the present application conducted extensive research into the suppression of nonspecific aggregation of carriers onto which anti-HMGB1 antibodies have been immobilized, and the suppression of increases in reagent blanks due to such aggregation. As a result, they discovered that the above problems can be solved by allowing a specific concentration range of glycerin-propylene oxide-ethylene oxide adduct or glycerin-propylene oxide adduct to coexist when the HMGB1 in the sample comes into contact with the carrier onto which anti-HMGB1 antibodies have been immobilized, and thus completed the present invention.

[0018] That is, the present invention comprises the following inventions. (1) A method for measuring HMGB1 in a sample, which involves contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized, thereby measuring the concentration of HMGB1 in the sample, characterized in that 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is co-present when the HMGB1 in the sample is contacted with the carrier on which the anti-HMGB1 antibody has been immobilized. (2) A method for measuring HMGB1 in a sample described in (1), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting the HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0054% (w / v). (3) A method for measuring HMGB1 in a sample described in (1) or (2), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting the HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0022% (w / v). (4) A method for measuring HMGB1 in a sample described in (1), characterized in that the concentration of glycerin propylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which anti-HMGB1 antibody is immobilized is 0.0076% (w / v) to 0.022% (w / v). (5) The method for measuring HMGB1 in a sample according to any one of (1) to (4) above, wherein the carrier is a particle. (6) The method for measuring HMGB1 in a sample according to any one of (1) to (5) above, wherein the carrier is latex particles. (7) A method for measuring HMGB1 in a sample according to any one of (1) to (6), wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0019] [ka]

[0020] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0021] (8) A method for measuring HMGB1 in a sample according to any one of (1) to (6), wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2):

[0022] [ka]

[0023] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

[0024] (9) A reagent for measuring HMGB1 in a sample, which measures the concentration of HMGB1 in a sample by contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized that have bound via HMGB1, characterized in that the reagent contains a concentration of a glycerin-propylene oxide-ethylene oxide adduct or a glycerin-propylene oxide adduct that allows the coexistence of 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct when the HMGB1 in the sample is contacted with the carrier on which the anti-HMGB1 antibody has been immobilized. (10) A reagent for measuring HMGB1 in a sample described in (9), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0054% (w / v). (11) A reagent for measuring HMGB1 in a sample described in (9) or (10), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0022% (w / v). (12) A reagent for measuring HMGB1 in a sample described in (9), characterized in that the concentration of glycerin propylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0076% (w / v) to 0.022% (w / v). (13) The reagent for measuring HMGB1 in a sample according to any one of (9) to (12) above, wherein the carrier is a particle. (14) The reagent for measuring HMGB1 in a sample according to any one of (9) to (13) above, wherein the carrier is latex particles. (15) A reagent for measuring HMGB1 in a sample according to any one of (9) to (14), wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0025] [ka]

[0026] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0027] (16) A reagent for measuring HMGB1 in a sample according to any one of (9) to (14), wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2):

[0028] [ka]

[0029] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

[0030] (17) A method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized, which involves contacting HMGB1 in the sample with a carrier onto which an anti-HMGB1 antibody has been immobilized, and measuring the aggregates of the carrier onto which the anti-HMGB1 antibody has been immobilized bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample, characterized in that, in the measurement of HMGB1 in the sample, 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is coexistent when the HMGB1 in the sample comes into contact with the carrier onto which the anti-HMGB1 antibody has been immobilized. (18) A method for inhibiting nonspecific aggregation of a carrier having immobilized anti-HMGB1 antibodies described in (17) above, characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist during contact between the HMGB1 in the sample and the carrier having immobilized anti-HMGB1 antibodies is 0.0016% (w / v) to 0.0054% (w / v). (19) A method for inhibiting nonspecific aggregation of a carrier having immobilized anti-HMGB1 antibodies described in (17) or (18), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist during contact between the HMGB1 in the sample and the carrier having immobilized anti-HMGB1 antibodies is 0.0016% (w / v) to 0.0022% (w / v). (20) A method for inhibiting nonspecific aggregation of a carrier having immobilized anti-HMGB1 antibodies described in (17) above, characterized in that the concentration of glycerin propylene oxide adduct that is allowed to coexist when the HMGB1 in the sample comes into contact with the carrier having immobilized anti-HMGB1 antibodies is 0.0076% (w / v) to 0.022% (w / v). (21) The method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized according to any one of (17) to (20) above, wherein the carrier is a particle. (22) The method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized according to any one of (17) to (20) above, wherein the carrier is a latex particle. (23) A method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized, according to any one of (17) to (22), wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0031] [ka]

[0032] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0033] (24) A method for inhibiting nonspecific aggregation of a carrier having an immobilized anti-HMGB1 antibody described in any one of (17) to (22), wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2):

[0034] [ka]

[0035] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

[0036] (25) A method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample, which involves contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized that have bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample, characterized in that 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is co-present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody has been immobilized. (26) A method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample described in (25) above, characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0054% (w / v). (27) A method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample described in (25) or (26), characterized in that the concentration of glycerin-propylene oxide-ethylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0016% (w / v) to 0.0022% (w / v). (28) A method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample described in (25), characterized in that the concentration of glycerin propylene oxide adduct that is allowed to coexist when contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody is immobilized is 0.0076% (w / v) to 0.022% (w / v). (29) The method for suppressing an increase in the reagent blank during measurement of HMGB1 in a sample according to any one of (25) to (28) above, wherein the carrier is a particle. (30) The method for suppressing an increase in the reagent blank during measurement of HMGB1 in a sample according to any one of (25) to (29) above, wherein the carrier is latex particles. (31) A method for suppressing an increase in the reagent blank during measurement of HMGB1 in a sample according to any one of (25) to (30), wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0037] [ka]

[0038] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0039] (32) A method for suppressing an increase in the reagent blank during measurement of HMGB1 in a sample according to any one of (25) to (30), wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2):

[0040] [ka]

[0041] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)] [Effects of the Invention]

[0042] In the method and reagent for measuring HMGB1 in a sample, the carrier on which the anti-HMGB1 antibody is immobilized is prevented from undergoing nonspecific agglutination due to shaking during transportation or for other reasons, thereby preventing an increase in the reagent blank caused by such agglutination.

[0043] Furthermore, it is possible to prevent nonspecific aggregation of the carrier onto which the anti-HMGB1 antibody is immobilized, which may occur when the carrier is shaken during transportation or for other reasons.

[0044] Furthermore, it is possible to suppress an increase in reagent blanks caused by nonspecific aggregation of the carrier on which the anti-HMGB1 antibody is immobilized due to shaking during transportation or for other reasons. [Brief explanation of the drawings]

[0045] [Figure 1] FIG. 10 shows a graph of the calibration curve when HMGB1 in a sample was measured using the second reagent (0% FA-195) (with shaking) and the second reagent (0% FA-195) (without shaking) as the second reagent.

[0046] [Figure 2]FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0004% FA-195) (with shaking) and the second reagent (0.0004% FA-195) (without shaking) as the second reagent.

[0047] [Figure 3] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.001% FA-195) (with shaking) and the second reagent (0.001% FA-195) (without shaking) as the second reagent.

[0048] [Figure 4] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0016% FA-195) (with shaking) and the second reagent (0.0016% FA-195) (without shaking) as the second reagent.

[0049] [Figure 5] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0022% FA-195) (with shaking) and the second reagent (0.0022% FA-195) (without shaking) as the second reagent.

[0050] [Figure 6] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0054% FA-195) (with shaking) and the second reagent (0.0054% FA-195) (without shaking) as the second reagent.

[0051] [Figure 7] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.01% FA-195) (with shaking) and the second reagent (0.01% FA-195) (without shaking) as the second reagent.

[0052] [Figure 8] FIG. 10 shows a graph of the calibration curve when HMGB1 in a sample was measured using the second reagent (0% GP-400) (with shaking) and the second reagent (0% GP-400) (without shaking) as the second reagent.

[0053] [Figure 9] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0022% GP-400) (with shaking) and the second reagent (0.0022% GP-400) (without shaking) as the second reagent.

[0054] [Figure 10] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0054% GP-400) (with shaking) and the second reagent (0.0054% GP-400) (without shaking) as the second reagent.

[0055] [Figure 11] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.0076% GP-400) (with shaking) and the second reagent (0.0076% GP-400) (without shaking) as the second reagent.

[0056] [Figure 12] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.01% GP-400) (with shaking) and the second reagent (0.01% GP-400) (without shaking) as the second reagent.

[0057] [Figure 13] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.016% GP-400) (with shaking) and the second reagent (0.016% GP-400) (without shaking) as the second reagent.

[0058] [Figure 14] FIG. 10 shows a graph of the calibration curves obtained when measuring HMGB1 in samples using the second reagent (0.022% GP-400) (with shaking) and the second reagent (0.022% GP-400) (without shaking) as the second reagent. DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention will be described in detail below. 1. Anti-HMGB1 antibody (1) Antibody The anti-HMGB1 antibody of the present invention is an antibody that specifically binds to HMGB1.

[0060] In the present invention, the anti-HMGB1 antibody is not particularly limited as long as it is an antibody that can specifically bind to HMGB1.

[0061] Examples of this anti-HMGB1 antibody include monoclonal antibodies, polyclonal antibodies, antisera, antibody fragments (such as Fab and F(ab')2), and single-chain antibodies (scFv) that can bind to HMGB1.

[0062] Furthermore, this anti-HMGB1 antibody may be an antibody whose amino acid sequence has been altered by genetic recombination technology or the like to that of an animal species different from that of the animal immunized with the immunogen (such as a chimeric antibody, humanized antibody, or fully humanized antibody).

[0063] The anti-HMGB1 antibody is preferably a monoclonal antibody.

[0064] Furthermore, in the present invention, two or more types of anti-HMGB1 antibodies may be used.

[0065] (2) Immunogen The immunogen used to produce anti-HMGB1 antibodies of the present invention is described below. In the present invention, all or part of HMGB1 can be used as an immunogen for producing anti-HMGB1 antibodies. That is, all or part of HMGB1 can be used, such as HMGB1 derived from humans or mammals such as cows, pigs, dogs, cats, mice, or rats, or birds such as chickens, or HMGB1 obtained by genetic engineering.

[0066] The anti-HMGB1 antibodies of the present invention can be obtained by using all or part of the above-mentioned HMGB1 as an immunogen. Furthermore, the immunogen for producing this anti-HMGB1 antibody may be a peptide or protein containing an amino acid sequence obtained by deleting, substituting, inserting, adding, or modifying one or several (usually 1 to 8, preferably 1 to 6, more preferably 1 to 4, and particularly preferably 1 to 2) amino acid residues in all or part of the amino acid sequence of HMGB1.

[0067] It has also been reported that antibodies can recognize amino acid sequences consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, Vol. 147, pp. 201-210, published 1992). Therefore, the smallest unit of the amino acid sequence of the immunogen for the anti-HMGB1 antibody of the present invention can be considered to be an amino acid sequence consisting of three consecutive amino acid residues from all or part of the amino acid sequence of HMGB1, or from an amino acid sequence obtained by deleting, substituting, inserting, adding or modifying one or several (usually 1 to 8, preferably 1 to 6, more preferably 1 to 4, and particularly preferably 1 to 2) amino acid residues from all or part of this amino acid sequence.Therefore, a tripeptide consisting of an amino acid sequence consisting of these three consecutive amino acid residues, or one to which other amino acids or peptides have been added, can be considered to be the smallest unit of the immunogen for the anti-HMGB1 antibody of the present invention.

[0068] The immunogens described above, such as peptides or proteins containing all or part of the amino acid sequence of HMGB1, or peptides or proteins containing an amino acid sequence obtained by deleting, substituting, inserting, adding, or modifying one or several (usually 1 to 8, preferably 1 to 6, more preferably 1 to 4, and particularly preferably 1 to 2) amino acid residues in all or part of the amino acid sequence of HMGB1, can be obtained by extraction, purification, etc. from body fluids, cells, tissues, organs, etc. of humans, etc., using known methods.

[0069] In the present invention, there are no particular limitations on the method for obtaining a peptide or protein containing all or part of the amino acid sequence of HMGB1, and any method may be used, including, for example, known methods.

[0070] The immunogen can be synthesized by peptide synthesis methods such as the liquid phase method and the solid phase method, or an automatic peptide synthesizer may be used, according to the methods described in "Lectures on Biochemical Experiments 1: Chemistry of Proteins IV" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1975; "Fundamentals and Experiments of Peptide Synthesis" by Izumiya et al., Maruzen, 1985; and "Continued Lectures on Biochemical Experiments 2: Chemistry of Proteins, Part 2" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1987. It is also easy to prepare mutants by subjecting the amino acid sequence to deletions, substitutions, insertions, or additions. Furthermore, modifications such as the introduction of non-natural amino acids, chemical modification of each amino acid residue, or the introduction of cysteine ​​residues to cyclize the molecule and stabilize the structure may also be carried out.

[0071] Furthermore, the immunogen may be prepared using genetic engineering techniques from DNA or RNA having the corresponding nucleic acid base sequence, and may be prepared by referring to "Continued Lectures on Biochemical Experiments 1: Genetic Research Methods I" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1986; "Continued Lectures on Biochemical Experiments 1: Genetic Research Methods II" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1986; "Continued Lectures on Biochemical Experiments 1: Genetic Research Methods III" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1987, etc.

[0072] When the immunogen is a low molecular weight substance, it is common to bind the immunogen to a carrier and then immunize animals with the resulting immunogen. However, there have been reports of the production of specific antibodies against a peptide containing five amino acids as an immunogen (Kiyama et al., "Abstracts of the 112th Annual Meeting of the Pharmaceutical Society of Japan, Vol. 3," p. 122, published in 1992), so the use of a carrier for binding the immunogen is not essential.

[0073] When a carrier for binding an immunogen is used to produce an antibody, the carrier may be any known carrier for binding an immunogen, such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, tetanus toxoid, or a polysaccharide.

[0074] The immunogen can be bound to a carrier by any known method, such as the glutaraldehyde method, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide method, the maleimidobenzoyl-N-hydroxysuccinimide ester method, the bisdiazotized benzidine method, or the N-succinimide-3-(2-pyridyldithio)propionic acid method. Alternatively, the immunogen may be adsorbed onto a carrier for binding the immunogen, such as nitrocellulose particles, polyvinylpyrrolidone, or liposomes, and used as the immunogen.

[0075] (3) Method for preparing polyclonal anti-HMGB1 antibodies Polyclonal antibodies capable of specifically binding to HMGB1, ie, polyclonal anti-HMGB1 antibodies, can be prepared by the following procedure.

[0076] The immunogens described above can be used as immunogens for producing this polyclonal anti-HMGB1 antibody.

[0077] The immunogen or a conjugate of the immunogen and a carrier for binding the immunogen is used to immunize a mammal (such as a mouse, guinea pig, hamster, rabbit, rat, sheep, goat, cow, horse, donkey, or camel) or a bird (such as a chicken, duck, or ostrich).

[0078] The immunizing dose of the immunogen or the conjugate of the immunogen and the carrier for binding the immunogen is determined depending on the immunogen, the carrier for binding the immunogen, the type of animal to be immunized, the site of immunization injection, etc., but in the case of mice, it is preferable to administer 0.1 μg to 5 mg of the immunogen or the conjugate of the immunogen and the carrier for binding the immunogen per animal per immunization injection.

[0079] It is preferable that the immunogen or the conjugate of the immunogen and the carrier for binding the immunogen is added and mixed with an adjuvant before being injected for immunization. As the adjuvant, known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, chemically synthesized adjuvant, or Bordetella pertussis adjuvant can be used. The immunization injection may be carried out subcutaneously, intravenously, intraperitoneally, on the back, or at other sites.

[0080] After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and a carrier for binding the immunogen are given subcutaneously, intravenously, intraperitoneally, on the back, or other sites at intervals of 1 to 2 weeks. The number of booster injections is generally 2 to 6. In this case, too, it is preferable that the immunogen or the conjugate of the immunogen and the carrier for binding the immunogen is mixed with an adjuvant and then injected for booster immunization. After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA or the like, and when the antibody titer reaches a plateau, whole blood is collected and the serum is separated to obtain an antiserum containing the antibody.

[0081] The antiserum is purified by salting out with ammonium sulfate, sodium sulfate, or the like, ion exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods to obtain polyclonal antibodies.

[0082] By the above procedures, a polyclonal antibody capable of binding to HMGB1 (polyclonal anti-HMGB1 antibody) can be obtained.

[0083] When an animal or the like is immunized with a conjugate of an immunogen and a carrier for binding the immunogen, the resulting polyclonal antibodies contain antibodies against the carrier for binding the immunogen, and it is therefore preferable to carry out a treatment to remove such antibodies against the carrier for binding the immunogen.

[0084] This removal treatment method can be performed by adding a carrier for binding the immunogen to a solution of the obtained polyclonal antibody and removing the resulting aggregates, or by immobilizing the carrier for binding the immunogen on an insolubilized solid phase and removing it by affinity chromatography.

[0085] (4) Method for preparing monoclonal anti-HMGB1 antibodies A method for obtaining a monoclonal antibody that can specifically bind to HMGB1, i.e., an anti-HMGB1 antibody, can be obtained using antibody-producing cells such as hybridomas produced by the cell fusion method of Keller et al. (G. Koehler et al., Nature, Vol. 256, pp. 495-497, published 1975), or tumorigenic cells caused by viruses such as Epstein-Barr virus. The preparation of a monoclonal antibody by the cell fusion method can be carried out by the following procedure.

[0086] First, a mammal (mouse, nude mouse, rat, etc., for example, an inbred mouse strain BALB / c) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier (carrier) for binding the immunogen. The immunizing dose of the immunogen or the conjugate of the immunogen and a carrier (carrier) for binding the immunogen is determined appropriately depending on the type of animal to be immunized, the injection site for immunization, etc., but for example, in the case of a mouse, it is preferable to immunize 0.1 μg to 5 mg of the immunogen or the conjugate of the immunogen and a carrier (carrier) for binding the immunogen per animal per injection.

[0087] The immunogen or the conjugate of the immunogen and the carrier for binding the immunogen is preferably mixed with an adjuvant before immunization injection. Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and Bordetella pertussis adjuvant can be used. The immunization injection may be carried out subcutaneously, intravenously, intraperitoneally, on the back, or at other sites.

[0088] After the initial immunization, the immunogen or the conjugate of the immunogen and a carrier for binding the immunogen is booster-injected subcutaneously, intravenously, intraperitoneally, or into the back at intervals of 1 to 2 weeks. The number of booster injections is generally 2 to 6. In this case, it is also preferable to add and mix an adjuvant to the immunogen or the conjugate of the immunogen and a carrier for binding the immunogen before booster injection.

[0089] After the initial immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or a conjugate of the immunogen and a carrier for binding the immunogen is dissolved in physiological saline (0.9% sodium chloride aqueous solution) and injected intravenously or intraperitoneally as the final immunization. Three to five days after this final immunization, antibody-producing cells, such as spleen cells, lymph node cells, or peripheral lymphocytes, from the immunized animal are harvested.

[0090] Antibody-producing cells obtained from these immunized animals are then fused with myeloma cells from mammals (e.g., mice, nude mice, or rats). Myeloma cells are preferably from cell lines deficient in enzymes such as hypoxanthine guanine phosphoribosyltransferase (HGPRT) or thymidine kinase (TK). Examples of HGPRT-deficient cell lines derived from BALB / c mice include P3-X63-Ag8 (ATCC TIB9), P3-X63-Ag8-U1 (JCRB Cancer Research Resources Bank (JCRB) 9085), P3-NS1-1-Ag4-1 (JCRB 0009), P3-X63-Ag8·653 (JCRB 0028), and SP2 / O-Ag-14 (JCRB 0029).

[0091] Cell fusion can be carried out using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposome, or Sendai virus (HVJ), or by electrofusion. When myeloma cells are HGPRT-deficient or TK-deficient, a selection medium containing hypoxanthine, aminopterin, and thymidine (HAT medium) can be used to selectively culture and grow only hybridomas, which are fusion cells of antibody-producing cells and myeloma cells.

[0092] Hybridomas that produce antibodies that bind to HMGB1 can be selected by measuring the culture supernatant of the hybridomas obtained in this manner using the immunogen, a conjugate of the immunogen and a carrier for binding the immunogen, or HMGB1 using immunological assays such as ELISA or Western blotting.

[0093] By combining this hybridoma selection method with known cloning methods such as limiting dilution, cell lines producing antibodies that bind to HMGB1 can be isolated and obtained.

[0094] This monoclonal antibody-producing cell line can be cultured in an appropriate medium, and the monoclonal antibody (anti-HMGB1 antibody, which is a monoclonal antibody) can be obtained from the culture supernatant. The medium may be serum-free or low-serum medium, which is preferable because it makes antibody purification easier. Media such as DMEM medium, RPMI1640 medium, or ASF medium 103 can be used. Alternatively, a monoclonal antibody-producing cell line can be injected into the peritoneal cavity of a compatible mammal that has been stimulated in advance with pristane or other agents, and after a certain period of time, a monoclonal antibody (anti-HMGB1 antibody, which is a monoclonal antibody) can be obtained from the ascites that accumulates in the peritoneal cavity.

[0095] The monoclonal antibody obtained in this manner can be purified to obtain a purified monoclonal antibody (anti-HMGB1 antibody, which is a monoclonal antibody) by methods such as salting out using ammonium sulfate or sodium sulfate, ion exchange chromatography, gel filtration, or affinity chromatography, or by combining these methods.

[0096] 2. Carrier The "carrier" in "carrier on which anti-HMGB1 antibody is immobilized" can be, for example, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid polymer, latex, gelatin, liposomes, microcapsules, red blood cells, silica, alumina, carbon black, metal compounds, metals, ceramics, or particles made of materials such as magnetic materials.

[0097] In the present invention, the carrier is preferably in the form of particles.

[0098] In the present invention, latex particles are more preferred as the carrier.

[0099] 3. Immobilization In the present invention, a "carrier on which an anti-HMGB1 antibody is immobilized" can be prepared by "immobilizing" the anti-HMGB1 antibody on a carrier by adsorbing or binding the anti-HMGB1 antibody to the carrier using known methods such as physical adsorption, chemical bonding, or a combination of these.

[0100] When physical adsorption is used, the anti-HMGB1 antibody can be immobilized on the carrier according to known methods, such as by mixing and contacting the anti-HMGB1 antibody with the carrier in a solution such as a buffer solution, or by contacting the anti-HMGB1 antibody dissolved in a buffer solution with the carrier.

[0101] Alternatively, when using chemical binding, the anti-HMGB1 antibody can be immobilized on the carrier by mixing and contacting the anti-HMGB1 antibody and carrier with a bifunctional cross-linking reagent such as glutaraldehyde, carbodiimide, imidoester, or maleimide, and reacting the anti-HMGB1 antibody with the amino groups, carboxyl groups, thiol groups, aldehyde groups, or hydroxyl groups of the anti-HMGB1 antibody and carrier, respectively, according to known methods described in, for example, "Clinical Pathology Extra Special Issue No. 53: Immunoassays for Clinical Testing - Technology and Applications" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; and "New Biochemical Experiment Course 1: Protein IV" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, published in 1991.

[0102] Two or more types of anti-HMGB1 antibodies may be immobilized on a carrier.

[0103] Furthermore, if further treatment is required to suppress nonspecific reactions or spontaneous aggregation of the carrier, the surface of the carrier on which the anti-HMGB1 antibody has been immobilized may be contacted and coated with a protein such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, a surfactant, or skim milk powder, to perform a blocking treatment (masking treatment) of the carrier using a known method.

[0104] 4. Sample The samples of the present invention include any biological sample that may contain HMGB1, such as body fluids such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, sweat, tears, ascites, or amniotic fluid; feces; organs such as blood vessels or the liver; tissues; cells; or extracts of feces, organs, tissues, or cells.

[0105] The sample used for the measurement is preferably in the form of a liquid. If the sample is not a liquid, it may be subjected to a known method of extraction, solubilization, or the like to prepare a liquid sample.

[0106] If necessary, the sample may be subjected to a concentration treatment.

[0107] Furthermore, the sample may be diluted by adding a diluent before measurement. For example, a dilution treatment may be performed by adding a diluent to the sample before contacting the sample with an anti-HMGB1 antibody and allowing it to bind. As the diluent, various aqueous solvents can be used. For example, aqueous solvents such as water, physiological saline, or various buffer solutions such as tris(hydroxymethyl)aminomethane buffer solution (Tris buffer solution), phosphate buffer solution, or phosphate buffered physiological saline solution can be used. The pH of this buffer solution is preferably in the range of pH5 to pH10.

[0108] 5. Measurement of aggregates on the carrier immobilized with anti-HMGB1 antibodies In the present invention, "measurement of aggregates on a carrier on which anti-HMGB1 antibodies have been immobilized" involves contacting "HMGB1 in a sample" with "a carrier on which anti-HMGB1 antibodies have been immobilized" and measuring the aggregates of "a carrier on which anti-HMGB1 antibodies have been immobilized" bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample.

[0109] That is, "HMGB1 in a sample" is brought into contact with "a carrier on which an anti-HMGB1 antibody has been immobilized," and the formation of immune complex aggregates of the "carrier on which an anti-HMGB1 antibody has been immobilized," which are bound via HMGB1, is measured by optically measuring the transmitted light or scattered light, or by visual observation. In other words, the formation of aggregates of "complexes resulting from antigen-antibody reactions" is measured (agglutination reaction method).

[0110] In addition, the present invention is characterized in that when "HMGB1 in a sample" is brought into contact with "a carrier having an immobilized anti-HMGB1 antibody," 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is coexistent.

[0111] Examples of methods for measuring aggregates of carriers onto which anti-HMGB1 antibodies have been immobilized include latex turbidimetry, latex agglutination, hemagglutination, and particle agglutination.

[0112] When measuring aggregates on a carrier onto which anti-HMGB1 antibodies have been immobilized using a measurement method such as latex turbidimetry, latex agglutination, hemagglutination, or particle agglutination, in which the formation of immune complex aggregates is measured by optical methods or visually by measuring the transmitted or scattered light, phosphate buffer, glycine buffer, Tris buffer, Good's buffer, or the like can be used as the solvent.

[0113] When latex turbidimetry is used as the measurement principle, there are no particular restrictions on the particle size of the latex particles used as a carrier. However, due to factors such as the extent to which latex particles bind to the substance to be measured (HMGB1) and form aggregates, and the ease of measuring the formed aggregates, it is preferable that the average particle size of the latex particles be 0.04 to 1 μm.

[0114] Furthermore, when latex turbidimetry is used as the measurement principle, the optimal concentration of latex particles onto which anti-HMGB1 antibodies are immobilized cannot be generalized, as it varies depending on various conditions such as the HMGB1 concentration in the sample, the distribution density of the antibodies of the present invention on the surface of the latex particles, the particle size of the latex particles, and the mixing ratio of the sample and measurement reagent.

[0115] However, during the measurement reaction in which the sample and measurement reagent are mixed and an antigen-antibody reaction occurs between the "anti-HMGB1 antibody" immobilized on the latex particles and the "HMGB1" contained in the sample, it is common to adjust the concentration of the "latex particles on which anti-HMGB1 antibodies are immobilized" to 0.005 to 1% (w / v) when the "HMGB1 in the sample" comes into contact with the "latex particles on which anti-HMGB1 antibodies are immobilized."In this case, the measurement reagent for HMGB1 in the sample contains a concentration of "latex particles on which anti-HMGB1 antibodies are immobilized" that will result in this concentration when the "HMGB1 in the sample" comes into contact with the "latex particles on which anti-HMGB1 antibodies are immobilized."

[0116] When an indirect agglutination reaction such as latex agglutination, hemagglutination, or particle agglutination is used as the measurement principle, the particle size of the particles used as the carrier is not particularly limited, but the average particle size is preferably in the range of 0.01 to 100 μm, more preferably in the range of 0.3 to 10 μm. The specific gravity of these particles is preferably in the range of 1 to 10, more preferably in the range of 1 to 2.

[0117] Examples of containers used in measurements using an indirect agglutination reaction such as latex agglutination, hemagglutination, or particle agglutination as the measurement principle include test tubes, microplates (microtiter plates), trays, etc. made of glass, polystyrene, polyvinyl chloride, polymethacrylate, etc. The bottom of the solution-receiving portion of these containers (such as the wells of a microplate) preferably has a U-, V-, or UV-shaped shape that slopes from the center to the periphery of the bottom.

[0118] Measurement can be performed using known methods, but for example, when measuring by optical methods, the sample is reacted with a "carrier on which anti-HMGB1 antibody is immobilized," and transmitted light and scattered light are measured using the endpoint method or rate method. Alternatively, when measuring visually, the sample is reacted with a "carrier on which an anti-HMGB1 antibody is immobilized" in a container such as a plate or microplate, and the state of aggregation is determined visually. Alternatively, measurement may be performed using an instrument such as a microplate reader.

[0119] An example of the measurement procedure is given below. For example, first, a measurement reagent containing a "carrier on which an anti-HMGB1 antibody has been immobilized" and a measurement reagent containing a buffer solution (aqueous solvent) containing a glycerin-propylene oxide-ethylene oxide adduct or a glycerin-propylene oxide adduct at a concentration that allows the coexistence of 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct when the "HMGB1 in the sample" and the "carrier (latex particles) on which an anti-HMGB1 antibody has been immobilized" are prepared and ready.

[0120] Next, for example, the sample is mixed with a measurement reagent containing a buffer solution (aqueous solvent) containing a glycerin-propylene oxide-ethylene oxide adduct or a glycerin-propylene oxide adduct at a concentration such that 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct can coexist when the "HMGB1 in the sample" and the "carrier (latex particles) on which the anti-HMGB1 antibody is immobilized" are contacted, and this mixture is then mixed with a measurement reagent containing the "carrier on which the anti-HMGB1 antibody is immobilized," thereby bringing the "HMGB1 in the sample" and the "carrier on which the anti-HMGB1 antibody is immobilized." In the present invention, when the "HMGB1 in the sample" is brought into contact with the "carrier on which anti-HMGB1 antibody is immobilized," 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is allowed to coexist.

[0121] By contacting the "HMGB1 in the sample" with the "carrier on which anti-HMGB1 antibody is immobilized," an antigen-antibody reaction occurs between the "anti-HMGB1 antibody" on the "carrier on which anti-HMGB1 antibody is immobilized" and the "HMGB1" contained in the sample. The resulting aggregates of "carrier with immobilized anti-HMGB1 antibody" bound via "HMGB1" (···-[HMGB1]-[carrier with immobilized anti-HMGB1 antibody]-[HMGB1]-[carrier with immobilized anti-HMGB1 antibody]-[HMGB1]-···) are measured.

[0122] The generated aggregates are measured by measuring the absorbance of transmitted light or scattered light of the reaction mixture during the measurement reaction in which the aggregates are present, using an endpoint method, a rate method, or the like.

[0123] The measured values ​​of absorbance, etc. obtained by measuring the sample are then compared with the measured values ​​of absorbance, etc. obtained by measuring a standard substance (a sample with a known HMGB1 concentration) to calculate the concentration (quantitative value) of HMGB1 contained in the sample.

[0124] The absorbance of transmitted light or scattered light may be measured by measuring transmitted light or scattered light, and may be measured by one wavelength or two wavelengths (the difference or ratio between two wavelengths).

[0125] The measurement wavelength is generally selected from the range of 340 nm to 1,000 nm.

[0126] In the present invention, HMGB1 may be measured manually or using a device such as a measuring device.

[0127] The measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (dedicated machine).

[0128] Furthermore, HMGB1 measurement in the present invention may be carried out by a one-step method (one-reagent method), or may be carried out by a method involving multiple operational steps, such as a two-step method (two-reagent method).

[0129] The following provides a more specific explanation using as an example the case where HMGB1 in a sample is measured by a method based on latex turbidimetry.

[0130] [1] First, prepare and prepare the following as a reagent for measuring HMGB1: First reagent: A buffer solution (aqueous solvent) containing a glycerin-propylene oxide-ethylene oxide adduct or a glycerin-propylene oxide adduct at a concentration that allows the coexistence of 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct when the "HMGB1 in the sample" and the "carrier (latex particles) on which anti-HMGB1 antibodies are immobilized" are in contact. Second reagent: Buffer solution containing "latex particles immobilized with anti-HMGB1 antibodies"

[0131] [2] A certain amount of a sample such as serum and a certain amount of the first reagent are mixed and allowed to stand at a certain temperature for a certain period of time. The mixing ratio (volume ratio) of the sample and the first reagent may be selected appropriately. The temperature during the standing is preferably a constant temperature within the range of room temperature (1°C to 30°C) or lukewarm (30°C to 40°C) (e.g., 37°C).

[0132] [3] After a certain period of time, a certain amount of the second reagent is added to and mixed with the mixture of the sample and the first reagent to form a reaction mixture, which is then allowed to stand at a certain temperature for a certain period of time. This brings "HMGB1 in the sample" into contact with "latex particles with immobilized anti-HMGB1 antibodies." In the present invention, when the "HMGB1 in the sample" comes into contact with the "latex particles with immobilized anti-HMGB1 antibodies," 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is allowed to coexist. The amount of the second reagent added may be selected appropriately. The temperature during the standing is preferably a constant temperature within the range of room temperature (1°C to 30°C) or lukewarm (30°C to 40°C) (for example, 37°C). Furthermore, the time for leaving the mixture to stand is preferably a fixed time of 1 minute or more and 10 minutes or less, and more preferably a fixed time of 3 minutes or more and 5 minutes or less.

[0133] By adding and mixing the second reagent to a mixture of the sample and the first reagent, an antigen-antibody reaction occurs between the "anti-HMGB1 antibody immobilized on latex particles" and the "HMGB1 contained in the sample."

[0134] This antigen-antibody reaction then forms crosslinks such as "···-[HMGB1]-[latex particle with immobilized anti-HMGB1 antibody]-[HMGB1]-[latex particle with immobilized anti-HMGB1 antibody]-[HMGB1]-···", resulting in the formation of aggregates of "latex particles with immobilized anti-HMGB1 antibody" bound via HMGB1.

[0135] [4] Then, using an analytical device or spectrophotometer, the reaction mixture is irradiated with light, and the amount of the complex aggregates formed, i.e., the amount of HMGB1 contained in the sample, is determined by measuring the decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity at an appropriate wavelength, which is a signal generated by the formed aggregates of latex particles.

[0136] [5] Then, the amount (concentration) of HMGB1 contained in the sample is calculated by comparing the "measurement value obtained by measuring the sample [value of the decrease in transmitted light intensity (increase in absorbance) or the increase in scattered light intensity]" with the "measurement value obtained by measuring a standard substance such as a standard solution or standard serum [a sample containing HMGB1 at a known concentration] [value of the decrease in transmitted light intensity (increase in absorbance) or the increase in scattered light intensity]."

[0137] 6. Glycerin-propylene oxide-ethylene oxide adduct (1) Method for measuring HMGB1 in a sample, method for suppressing nonspecific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized, and method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample In the method of the present invention for measuring HMGB1 in a sample, the method for suppressing nonspecific agglutination of a carrier on which an anti-HMGB1 antibody has been immobilized, and the method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample, 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is co-present when the HMGB1 in the sample comes into contact with the carrier on which an anti-HMGB1 antibody has been immobilized.

[0138] Specifically, when the "HMGB1 in the sample" and the "carrier on which the anti-HMGB1 antibody is immobilized" are brought into contact, 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is allowed to coexist with the "HMGB1 in the sample" and the "carrier on which the anti-HMGB1 antibody is immobilized."

[0139] In the present invention, the glycerin-propylene oxide-ethylene oxide adduct is not particularly limited, and any glycerin-propylene oxide-ethylene oxide adduct can be used.

[0140] The concentration of the glycerin-propylene oxide-ethylene oxide adduct at the time of contact between the "HMGB1 in the sample" and the "anti-HMGB1 antibody-immobilized carrier" is preferably 0.0016% (w / v) to 0.0054% (w / v), and more preferably 0.0016% (w / v) to 0.0022% (w / v), in order to prevent nonspecific aggregation of the "anti-HMGB1 antibody-immobilized carrier" caused by shaking during transportation or for other reasons, and to prevent an increase in reagent blank due to such aggregation.

[0141] In the present invention, the glycerin-propylene oxide-ethylene oxide adduct is preferably a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0142] [ka]

[0143] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0144] In the present invention, in order to prevent nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and to prevent an increase in reagent blank caused by such aggregation, it is preferable that in general formula (1), a+b+c is 5 to 18 moles, d+e+f is 7 to 21 moles, and g+h+i is 25 to 65 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.

[0145] In terms of the above-mentioned effects, it is more preferable that in general formula (1), a+b+c is 7 to 16 moles, d+e+f is 10 to 19 moles, and g+h+i is 35 to 55 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0146] Furthermore, in terms of the above-mentioned effects, it is more preferable that in general formula (1), a+b+c is 10 to 13 moles, d+e+f is 12 to 17 moles, and g+h+i is 40 to 50 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0147] In view of the above-mentioned effects, it is particularly preferable that in general formula (1), a+b+c is 11 to 12 moles, d+e+f is 14 to 15 moles, and g+h+i is 44 to 45 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0148] In view of the above-mentioned effects, it is particularly preferable that in general formula (1), a+b+c is 11.5 moles, d+e+f is 14.3 moles, and g+h+i is 44.1 moles. (All mole numbers are average mole numbers added.) The numbers in {} indicate random addition. In general formula (1), a+b+c is 11.5 moles, d+e+f is 14.3 moles, and g+h+i is 44.1 moles [(All mole numbers are average mole numbers added.) The numbers in {} indicate random addition.] An example of the glycerin-propylene oxide-ethylene oxide adduct is "Sannyx FA-195" (molecular weight: 3,370, CAS No. 9082-00-2) sold by Sanyo Chemical Industries, Ltd.

[0149] (2) Reagent for measuring HMGB1 in samples In the reagent for measuring HMGB1 in a sample of the present invention, the glycerin-propylene oxide-ethylene oxide adduct or glycerin-propylene oxide adduct is contained in the "reagent for measuring HMGB1 in a sample" at a concentration that allows the coexistence of 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized.

[0150] That is, the "reagent for measuring HMGB1 in a sample" contains 0.001% (w / v) to 0.01% (w / v) (glycerin-propylene oxide-ethylene oxide adduct) or 0.0054% (w / v) to 0.022% (w / v) (glycerin-propylene oxide-ethylene oxide adduct) in a concentration that allows the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized" to coexist at the time of contact between the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized."

[0151] In the present invention, the glycerin-propylene oxide-ethylene oxide adduct is not particularly limited, and any glycerin-propylene oxide-ethylene oxide adduct can be used.

[0152] The concentration of the glycerin-propylene oxide-ethylene oxide adduct at the time of contact between the "HMGB1 in the sample" and the "anti-HMGB1 antibody-immobilized carrier" is preferably 0.0016% (w / v) to 0.0054% (w / v), and more preferably 0.0016% (w / v) to 0.0022% (w / v), in order to prevent nonspecific aggregation of the "anti-HMGB1 antibody-immobilized carrier" caused by shaking during transportation or for other reasons, and to prevent an increase in reagent blank due to such aggregation. In the reagent for measuring HMGB1 in a sample of the present invention, the glycerin-propylene oxide-ethylene oxide adduct is contained in a concentration that allows the glycerin-propylene oxide-ethylene oxide adduct to coexist at the above concentration when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized.

[0153] In the present invention, the glycerin-propylene oxide-ethylene oxide adduct is preferably a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1):

[0154] [ka]

[0155] [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} are random additions.]

[0156] In the present invention, in order to prevent nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and to prevent an increase in reagent blank caused by such aggregation, it is preferable that in general formula (1), a+b+c is 5 to 18 moles, d+e+f is 7 to 21 moles, and g+h+i is 25 to 65 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.

[0157] In terms of the above-mentioned effects, it is more preferable that in general formula (1), a+b+c is 7 to 16 moles, d+e+f is 10 to 19 moles, and g+h+i is 35 to 55 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0158] Furthermore, in terms of the above-mentioned effects, it is more preferable that in general formula (1), a+b+c is 10 to 13 moles, d+e+f is 12 to 17 moles, and g+h+i is 40 to 50 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0159] In view of the above-mentioned effects, it is particularly preferable that in general formula (1), a+b+c is 11 to 12 moles, d+e+f is 14 to 15 moles, and g+h+i is 44 to 45 moles (all mole numbers are average numbers of moles added). The numbers in {} indicate random addition.

[0160] In view of the above-mentioned effects, it is particularly preferable that in general formula (1), a+b+c is 11.5 moles, d+e+f is 14.3 moles, and g+h+i is 44.1 moles. (All mole numbers are average mole numbers added.) The numbers in {} indicate random addition. In general formula (1), a+b+c is 11.5 moles, d+e+f is 14.3 moles, and g+h+i is 44.1 moles [(All mole numbers are average mole numbers added.) The numbers in {} indicate random addition.] An example of the glycerin-propylene oxide-ethylene oxide adduct is "Sannyx FA-195" (molecular weight: 3,370, CAS No. 9082-00-2) sold by Sanyo Chemical Industries, Ltd.

[0161] The following is an explanation of the fact that, in the reagent for measuring HMGB1 in a sample of the present invention, "the glycerin-propylene oxide-ethylene oxide adduct is contained in the "reagent for measuring HMGB1 in a sample" at a concentration that allows the coexistence of 0.001% (w / v) to 0.01% (w / v), preferably 0.0016% (w / v) to 0.0054% (w / v), and more preferably 0.0016% (w / v) to 0.0022% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized."

[0162] If the HMGB1 measurement reagent in the sample is a single-reagent system, then if the concentration of the glycerin-propylene oxide-ethylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is a% (w / v), the weight of the glycerin-propylene oxide-ethylene oxide adduct at that time is bg (grams), the volume of the HMGB1 measurement reagent in the sample at that time is cmL, and the volume of the sample at that time is dmL, then a% (w / v) = (bg (grams) × 100) ÷ (cmL + dmL). Converting this formula gives bg (grams) x 100 = a% (w / v) x (cmL + dmL). Dividing both sides by cmL gives us (bg (grams) × 100) ÷ cmL = {a% (w / v) × (cmL + dmL)} ÷ cmL. Furthermore, when HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibody is immobilized, 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the HMGB1 measurement reagent in a sample is e% (w / v), then e% (w / v) = (bg (grams) × 100) ÷ cmL, and therefore, from the above formula, e% (w / v) = (bg (grams) × 100) ÷ cmL = {a% (w / v) × (cmL + dmL)} ÷ cmL.

[0163] Furthermore, when the assay reagent for HMGB1 in a sample is a two-reagent system and a carrier with immobilized anti-HMGB1 antibody is contained in the second reagent of the assay reagent for HMGB1 in a sample, if the concentration of the glycerin-propylene oxide-ethylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier with immobilized anti-HMGB1 antibody is a% (w / v), the weight of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the assay reagent for HMGB1 in the sample at that time is b2 g (grams), the volume of the first reagent of the assay reagent for HMGB1 in the sample at that time is c1 mL, the volume of the second reagent of the assay reagent for HMGB1 in the sample at that time is c2 mL, and the volume of the sample at that time is d mL, then a% (w / v) = (b2 g (grams) × 100) ÷ (c1 mL + c2 mL + d mL). Furthermore, when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, the glycerin-propylene oxide-ethylene oxide adduct can coexist at a concentration of 0.001% (w / v) to 0.01% (w / v). If the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the measurement reagent for HMGB1 in the sample is e2% (w / v), then b2g (grams) = (e2% (w / v) × c2mL) ÷ 100. Substituting the b2g (grams) in this formula for the b2g (grams) in the previous formula gives a%(w / v) = (e2%(w / v) × c2mL) ÷ (c1mL + c2mL + dmL). Converting this formula, e2%(w / v) = {a%(w / v) × (c1mL + c2mL + dmL)} ÷ c2mL.

[0164] When the measurement reagent for HMGB1 in a sample is a two-reagent system and a carrier with immobilized anti-HMGB1 antibody is contained in the second reagent of the measurement reagent for HMGB1 in a sample, the concentration of the glycerin-propylene oxide-ethylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier with immobilized anti-HMGB1 antibody is defined as a% (w / v), and the weight of the glycerin-propylene oxide-ethylene oxide adduct in the first reagent of the measurement reagent for HMGB1 in a sample at that time is defined as b1g (grams). Let b2g (grams) be the weight of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the HMGB1 measurement reagent in the sample at that time, c1mL be the volume of the first reagent of the HMGB1 measurement reagent in the sample at that time, c2mL be the volume of the second reagent of the HMGB1 measurement reagent in the sample at that time, and dmL be the volume of the sample at that time, then a%(w / v) = {(b1g (grams) + b2g (grams)) × 100} ÷ (c1mL + c2mL + dmL). Converting this formula, we get (b1g (grams) + b2g (grams)) x 100 = a% (w / v) x (c1mL + c2mL + dmL). Converting this formula, we get b1g (grams) x 100 + b2g (grams) x 100 = a% (w / v) x (c1mL + c2mL + dmL). Converting this formula, we get b1g (grams) × 100 = {a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100). Dividing both sides by c1mL gives us (b1g (grams) × 100) ÷ c1mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100)] ÷ c1mL. Furthermore, when HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibody is immobilized, 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the first reagent of the measurement reagent for HMGB1 in a sample is e1% (w / v), then e1% (w / v) = (b1g (grams) × 100) ÷ c1mL, and therefore, from the above formula, e1% (w / v) = (b1g (grams) × 100) ÷ c1mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100)] ÷ c1mL.

[0165] Furthermore, converting the above formula b1g (grams) × 100 + b2g (grams) × 100 = a% (w / v) × (c1mL + c2mL + dmL) gives b2g (grams) × 100 = {a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100). Dividing both sides by c2mL gives us (b2g (grams) × 100) ÷ c2mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100)] ÷ c2mL. Furthermore, when HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibody is immobilized, 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the measurement reagent for HMGB1 in a sample is e2% (w / v), then e2% (w / v) = (b2g (grams) × 100) ÷ c2mL, and therefore, from the above formula, e2% (w / v) = (b2g (grams) × 100) ÷ c2mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100)] ÷ c2mL.

[0166] 7. Glycerin-propylene oxide adduct (1) Method for measuring HMGB1 in a sample, method for suppressing nonspecific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized, and method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample In the method of the present invention for measuring HMGB1 in a sample, the method for suppressing nonspecific agglutination of a carrier on which an anti-HMGB1 antibody has been immobilized, and the method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample, 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is co-present when the HMGB1 in the sample comes into contact with the carrier on which an anti-HMGB1 antibody has been immobilized.

[0167] Specifically, when the "HMGB1 in the sample" and the "carrier on which the anti-HMGB1 antibody is immobilized" are brought into contact, 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is allowed to coexist with the "HMGB1 in the sample" and the "carrier on which the anti-HMGB1 antibody is immobilized."

[0168] In the present invention, the glycerin-propylene oxide adduct is not particularly limited, and any glycerin-propylene oxide adduct can be used.

[0169] The concentration of the glycerin-propylene oxide adduct at the time of contact between the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized" is preferably 0.0076% (w / v) to 0.022% (w / v), from the standpoint of the effect of suppressing nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and suppressing an increase in reagent blanks caused by such aggregation.

[0170] In the present invention, the glycerin-propylene oxide adduct is preferably a glycerin-propylene oxide adduct represented by the following general formula (2):

[0171] [ka]

[0172] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

[0173] In the present invention, in order to prevent nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and to prevent an increase in the reagent blank caused by such aggregation, it is preferable that a+b+c in general formula (2) is 4 to 10 moles (the number of moles is the average number of moles added).

[0174] In view of the above-mentioned effects, it is more preferable that a+b+c in general formula (2) is 4.5 to 8 moles (the number of moles is the average number of moles added).

[0175] Furthermore, in terms of the above-mentioned effects, it is more preferable that a+b+c in general formula (2) is 5 to 6 moles (the number of moles is the average number of moles added).

[0176] In view of the above-mentioned effects, it is particularly preferable that a+b+c in general formula (2) is 5.6 moles (the number of moles is the average number of moles added). In addition, in this general formula (2), an example of a glycerin-propylene oxide adduct in which a + b + c is 5.6 moles (the number of moles is the average number of moles added) is "Sannyx GP-400" (molecular weight: 420, CAS No. 25791-96-2) sold by Sanyo Chemical Industries, Ltd.

[0177] (2) Reagent for measuring HMGB1 in samples In the reagent for measuring HMGB1 in a sample of the present invention, the glycerin-propylene oxide-ethylene oxide adduct or glycerin-propylene oxide adduct is contained in the "reagent for measuring HMGB1 in a sample" at a concentration that allows the coexistence of 0.001% (w / v) to 0.01% (w / v) of the glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized.

[0178] That is, the "reagent for measuring HMGB1 in a sample" contains 0.001% (w / v) to 0.01% (w / v) (glycerin-propylene oxide-ethylene oxide adduct) or 0.0054% (w / v) to 0.022% (w / v) (glycerin-propylene oxide-ethylene oxide adduct) in a concentration that allows the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized" to coexist at the time of contact between the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized."

[0179] In the present invention, the glycerin-propylene oxide adduct is not particularly limited, and any glycerin-propylene oxide adduct can be used.

[0180] The concentration of the glycerin-propylene oxide adduct at the time of contact between the "HMGB1 in the sample" and the "carrier on which an anti-HMGB1 antibody is immobilized" is preferably 0.0076% (w / v) to 0.022% (w / v), from the standpoint of the effect of suppressing nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and suppressing an increase in reagent blanks caused by such aggregation. In the measurement reagent for HMGB1 in a sample of the present invention, the glycerin-propylene oxide adduct is contained in a concentration that allows the glycerin-propylene oxide adduct to coexist at the above concentration when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized.

[0181] In the present invention, the glycerin-propylene oxide adduct is preferably a glycerin-propylene oxide adduct represented by the following general formula (2):

[0182] [ka]

[0183] [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

[0184] In the present invention, in order to prevent nonspecific aggregation of the "carrier on which an anti-HMGB1 antibody is immobilized" due to shaking during transportation or for other reasons, and to prevent an increase in the reagent blank caused by such aggregation, it is preferable that a+b+c in general formula (2) is 4 to 10 moles (the number of moles is the average number of moles added).

[0185] In view of the above-mentioned effects, it is more preferable that a+b+c in general formula (2) is 4.5 to 8 moles (the number of moles is the average number of moles added).

[0186] Furthermore, in terms of the above-mentioned effects, it is more preferable that a+b+c in general formula (2) is 5 to 6 moles (the number of moles is the average number of moles added).

[0187] In view of the above-mentioned effects, it is particularly preferable that a+b+c in general formula (2) is 5.6 moles (the number of moles is the average number of moles added). In addition, in this general formula (2), an example of a glycerin-propylene oxide adduct in which a + b + c is 5.6 moles (the number of moles is the average number of moles added) is "Sannyx GP-400" (molecular weight: 420, CAS No. 25791-96-2) sold by Sanyo Chemical Industries, Ltd.

[0188] The following is an explanation of the fact that, in the reagent for measuring HMGB1 in a sample of the present invention, the glycerin-propylene oxide adduct is contained in the "reagent for measuring HMGB1 in a sample" at a concentration that allows the coexistence of 0.0054% (w / v) to 0.022% (w / v), preferably 0.0076% (w / v) to 0.022% (w / v), of the glycerin-propylene oxide adduct when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized.

[0189] If the HMGB1 measurement reagent in the sample is a single-reagent system, then if the concentration of the glycerin-propylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is a% (w / v), the weight of the glycerin-propylene oxide adduct at that time is bg (grams), the volume of the HMGB1 measurement reagent in the sample at that time is cmL, and the volume of the sample at that time is dmL, then a% (w / v) = (bg (grams) × 100) ÷ (cmL + dmL). Converting this formula gives bg (grams) x 100 = a% (w / v) x (cmL + dmL). Dividing both sides by cmL gives us (bg (grams) × 100) ÷ cmL = {a% (w / v) × (cmL + dmL)} ÷ cmL. When HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibody is immobilized, 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide adduct in the HMGB1 measurement reagent in a sample is e% (w / v), then e% (w / v) = (bg (grams) × 100) ÷ cmL, and therefore, from the above formula, e% (w / v) = (bg (grams) × 100) ÷ cmL = {a% (w / v) × (cmL + dmL)} ÷ cmL.

[0190] Furthermore, when the measurement reagent for HMGB1 in a sample is a two-reagent system and a carrier with immobilized anti-HMGB1 antibody is contained in the second reagent of the measurement reagent for HMGB1 in a sample, if the concentration of the glycerol propylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier with immobilized anti-HMGB1 antibody is a% (w / v), the weight of the glycerol propylene oxide adduct in the second reagent of the measurement reagent for HMGB1 in the sample at that time is b2 g (grams), the volume of the first reagent of the measurement reagent for HMGB1 in the sample at that time is c1 mL, the volume of the second reagent of the measurement reagent for HMGB1 in the sample at that time is c2 mL, and the volume of the sample at that time is d mL, then a% (w / v) = (b2 g (grams) × 100) ÷ (c1 mL + c2 mL + d mL). When the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, the glycerin-propylene oxide adduct can coexist at a concentration of 0.0054% (w / v) to 0.022% (w / v). If the concentration of the glycerin-propylene oxide adduct in the second reagent of the measurement reagent for HMGB1 in the sample is e2% (w / v), then b2g (grams) = (e2% (w / v) × c2mL) ÷ 100. Substituting the b2g (grams) in this formula for the b2g (grams) in the previous formula gives a%(w / v) = (e2%(w / v) × c2mL) ÷ (c1mL + c2mL + dmL). Converting this formula, e2%(w / v) = {a%(w / v) × (c1mL + c2mL + dmL)} ÷ c2mL.

[0191] When the measurement reagent for HMGB1 in a sample is a two-reagent system and a carrier with immobilized anti-HMGB1 antibody is contained in the second reagent of the measurement reagent for HMGB1 in a sample, the concentration of the glycerin-propylene oxide adduct at the time of contact between the HMGB1 in the sample and the carrier with immobilized anti-HMGB1 antibody is defined as a% (w / v), and the weight of the glycerin-propylene oxide adduct in the first reagent of the measurement reagent for HMGB1 in a sample at that time is defined as b1g (gram). If the weight of the glycerin-propylene oxide adduct in the second reagent of the HMGB1 measurement reagent in the sample at that time is b2 g (grams), the volume of the first reagent of the HMGB1 measurement reagent in the sample at that time is c1 mL, the volume of the second reagent of the HMGB1 measurement reagent in the sample at that time is c2 mL, and the volume of the sample at that time is d mL, then a% (w / v) = {(b1 ​​g (grams) + b2 g (grams)) × 100} ÷ (c1 mL + c2 mL + d mL). Converting this formula, we get (b1g (grams) + b2g (grams)) x 100 = a% (w / v) x (c1mL + c2mL + dmL). Converting this formula, we get b1g (grams) x 100 + b2g (grams) x 100 = a% (w / v) x (c1mL + c2mL + dmL). Converting this formula, we get b1g (grams) × 100 = {a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100). Dividing both sides by c1mL gives us (b1g (grams) × 100) ÷ c1mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100)] ÷ c1mL. Furthermore, when HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibody is immobilized, 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide adduct in the first reagent of the measurement reagent for HMGB1 in a sample is e1% (w / v), then e1% (w / v) = (b1g (grams) × 100) ÷ c1mL, and therefore, from the above formula, e1% (w / v) = (b1g (grams) × 100) ÷ c1mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b2g (grams) × 100)] ÷ c1mL.

[0192] Furthermore, converting the above formula b1g (grams) × 100 + b2g (grams) × 100 = a% (w / v) × (c1mL + c2mL + dmL) gives b2g (grams) × 100 = {a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100). Dividing both sides by c2mL gives us (b2g (grams) × 100) ÷ c2mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100)] ÷ c2mL. Furthermore, when HMGB1 in a sample comes into contact with the carrier on which anti-HMGB1 antibodies are immobilized, 0.0054% (w / v) to 0.022% (w / v) of the glycerin-propylene oxide adduct can coexist. If the concentration of the glycerin-propylene oxide adduct in the second reagent of the measurement reagent for HMGB1 in a sample is e2% (w / v), then e2% (w / v) = (b2g (grams) × 100) ÷ c2mL, and therefore, from the above formula, e2% (w / v) = (b2g (grams) × 100) ÷ c2mL = [{a% (w / v) × (c1mL + c2mL + dmL)} - (b1g (grams) × 100)] ÷ c2mL.

[0193] 8. Method for suppressing nonspecific aggregation of carriers onto which anti-HMGB1 antibodies are immobilized The "method for inhibiting nonspecific aggregation of a carrier having immobilized anti-HMGB1 antibodies" of the present invention involves contacting HMGB1 in a sample with a carrier having immobilized anti-HMGB1 antibodies and measuring the aggregates of the carrier having immobilized anti-HMGB1 antibodies bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample.The measurement of HMGB1 in the sample is characterized in that 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is coexistent when the HMGB1 in the sample comes into contact with the carrier having immobilized anti-HMGB1 antibodies.

[0194] This makes it possible to suppress nonspecific aggregation of the carrier onto which the anti-HMGB1 antibody is immobilized.

[0195] The terms "sample," "carrier with immobilized anti-HMGB1 antibody," "measurement of HMGB1 concentration in the sample by measuring aggregates of carrier with immobilized anti-HMGB1 antibody bound via HMGB1," "coexistence of glycerin-propylene oxide-ethylene oxide adduct," and "coexistence of glycerin-propylene oxide adduct" are as described above.

[0196] 9. Method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample The "method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample" of the present invention involves contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized, and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample.The measurement of HMGB1 in the sample is characterized in that 0.001% (w / v) to 0.01% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w / v) to 0.022% (w / v) of a glycerin-propylene oxide adduct is co-present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody has been immobilized.

[0197] This makes it possible to suppress an increase in the reagent blank caused by nonspecific aggregation occurring on the carrier on which the anti-HMGB1 antibody is immobilized.

[0198] The terms "sample," "carrier with immobilized anti-HMGB1 antibody," "measurement of HMGB1 concentration in the sample by measuring aggregates of carrier with immobilized anti-HMGB1 antibody bound via HMGB1," "coexistence of glycerin-propylene oxide-ethylene oxide adduct," and "coexistence of glycerin-propylene oxide adduct" are as described above. [Example]

[0199] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0200] Example 1 (Preparation of antibody (2H6)) (1) Bovine HMGB1 (full-length) was used as the immunogen.

[0201] Furthermore, this bovine HMGB1 (full length) has 99.5% amino acid sequence homology with human HMGB1 (full length), and can be used in place of human HMGB1 without any problems.

[0202] This immunogen, bovine HMGB1 (full-length), was mixed with Freund adjuvant (DIFCO LABORATORIES) as a chemically synthesized adjuvant in a ratio of 1 volume to 1 volume.

[0203] (2) Next, a mixture of the HMGB1 solution and Freund's complete adjuvant was injected intraperitoneally into mice (BALB / c) at 300 to 500 μg / mouse / injection as an immunogen, and two and four weeks later, the mice were intraperitoneally injected with a mixture of the HMGB1 solution and Freund's incomplete adjuvant.

[0204] (3) Four weeks after the final immunization, a booster was administered with 300 μg / mouse of undiluted bovine HMGB1 (full-length). The following day, spleen cells from the immunized mice were mixed with myeloma cells (P3U1) in a ratio of 1:1 to 10:1. Polyethylene glycol (PEG1500; Roche, Switzerland) was added to allow cell fusion using standard methods, and the grown hybridoma colonies were selected.

[0205] Specifically, cell fusion was carried out as follows. The mixture of spleen cells and myeloma cells (P3U1) was centrifuged to remove the supernatant, and the mixture was suspended in 1 mL of polyethylene glycol (PEG1500; Roche, Switzerland) at room temperature for 1 minute, followed by stirring at 37°C for 1 minute.

[0206] 1 mL of serum-free medium was added twice over 1 minute, and then 7 mL of serum-free medium was added over 2 minutes.

[0207] After washing the cells several times, the cells were suspended in a medium containing hypoxanthine, aminopterin, and thymidine, dispensed into a 96-well microtiter plate, and cultured at 37°C in the presence of 5% CO2.

[0208] To select the grown monoclonal antibody-producing cell lines (fusion cell lines), bovine HMGB1 (full-length) was immobilized 7 to 14 days after cell fusion, and an ELISA system was used in which the fusion cell culture supernatant was used as the primary antibody.

[0209] Specifically, this ELISA was carried out as follows. (i) Bovine HMGB1 and bovine HMGB2 (100.0% amino acid sequence homology with human HMGB2) prepared in Reference Example 1 above were each adjusted to a concentration of 1 μg / mL in phosphate-buffered saline (0.9% sodium chloride aqueous solution), or phosphate-buffered saline (0.9% sodium chloride aqueous solution) as a control was added. 100 μL of each was poured into wells of a 96-well microtiter plate (Thermo Fisher Scientific Inc., Illinois, USA) and allowed to stand at 5°C for 16 to 24 hours (or 37°C for 2 hours). HMGB1 and HMGB2 were then immobilized to the wells of the microtiter plate, which was then blocked by further allowing the plate to stand in phosphate-buffered saline containing 1% BSA at 5°C for 16 to 24 hours (or 37°C for 2 hours).

[0210] (ii) Next, each well of the microtiter plate from (i) above was washed three times with a washing solution (phosphate buffered saline containing 0.05% Tween 20).

[0211] (iii) Next, 100 μL of each of these fusion cell culture supernatant solutions was injected as a sample into the wells of the microtiter plate washed in (ii) above, and left to stand at 37°C for 2 hours to allow the HMGB1 and HMGB2 immobilized in the wells of the microtiter plate to react with the monoclonal antibodies contained in each of the fusion cell culture supernatant solutions.

[0212] (iv) Next, each well of the microtiter plate (iii) was washed three times with the washing solution.

[0213] (v) Next, 100 μL of a POD-labeled anti-mouse IgG antibody (DakoCytomation, Denmark) diluted 1000-fold with phosphate-buffered saline containing 1% bovine serum albumin (BSA) was injected into each well of the microtiter plate washed in (iv) above, and the plate was left to stand at 37°C for 2 hours to allow the reaction to occur.

[0214] (vi) Next, each well of the microtiter plate from (v) above was washed three times with the washing solution.

[0215] (vii) Next, a color-developing solution consisting of a 0.045% aqueous solution of 3,3',5,5'-tetramethylbenzidine hydrochloride (pH 2.0) containing 0.2 mM disodium EDTA was mixed 1:1 with a substrate solution consisting of a 60 mM disodium phosphate (pH 4.3) containing 5 mM hydrogen peroxide, 41 mM citric acid, and 0.2 mM disodium EDTA. 100 μL of this color-developing substrate solution was poured into each well of the microtiter plate washed in (vi) above, and the plate was left to stand at room temperature for 30 minutes to allow the reaction and color development.

[0216] Thereafter, 100 μL of a reaction stop solution consisting of 0.7 N sulfuric acid was dispensed into each well to stop the color reaction.

[0217] (viii) Next, the absorbance of each well of the microtiter plate (vii) was measured at a dominant wavelength of 450 nm and a subordinate wavelength of 550 nm using a spectrophotometer.

[0218] Based on the above measurements, fusion cell lines producing antibodies that bind to HMGB1 were selected, and one clone was established from the grown fusion cell lines and designated 181208 2H6.

[0219] (4) IgG (immunoglobulin G) was purified from this selected monoclonal antibody-producing cell line as follows.

[0220] This monoclonal antibody-producing cell line was cultured in PFHM-II (GIBCO) at 37°C in a CO 2 incubator.

[0221] After the culture, IgG in the supernatant was bound to a protein A column (GE Healthcare Bio-Sciences, Sweden).

[0222] The bound IgG was eluted with 100 mM aqueous citric acid (pH 3.0).

[0223] One volume of 0.5 M phosphate buffer (pH 7.5) was added to one volume of the eluate, and an antibody that binds to HMGB1 was obtained as purified IgG from the monoclonal antibody-producing cell line (181208 2H6).

[0224] This monoclonal antibody was named "Antibody (2H6)."

[0225] The 181208 2H6 strain, a monoclonal antibody-producing cell line for antibody (2H6), was deposited at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary [NPMD] (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-02843 on December 20, 2018.

[0226] Example 2 (Preparation of antibody (2D4)) At a time separate from that of Example 1, anti-HMGB1 monoclonal antibodies were prepared again by carrying out the procedures described in (1) to (4) of Example 1.

[0227] As a result, one clone was established from the grown fused cell lines and named 181212 2D4.

[0228] We were then able to obtain an anti-HMGB1 monoclonal antibody from the monoclonal antibody-producing cell line 181212 2D4.

[0229] This monoclonal antibody was named "Antibody (2D4)."

[0230] The 181212 2D4 strain, a monoclonal antibody-producing cell line for the antibody (2D4), was deposited at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary [NPMD] (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-02842 on December 20, 2018.

[0231] Example 3 (Preparation of anti-HMGB1,2 monoclonal antibodies) At a time separate from Examples 1 and 2, monoclonal antibodies that bind to HMGB1 and HMGB2 were prepared according to the procedures described in (1) to (4) of Example 1.

[0232] As a result, one clone was established from the grown fused cell lines and named 181212 5D1.

[0233] We were able to obtain monoclonal antibodies that bind to HMGB1 and HMGB2 from the monoclonal antibody-producing cell line 181212 5D1.

[0234] This monoclonal antibody was named "anti-HMGB1,2 monoclonal antibody."

[0235] The 181212 5D1 cell line, a monoclonal antibody-producing cell line for anti-HMGB1,2 monoclonal antibodies, was deposited on December 20, 2018, at the National Institute of Technology and Evaluation's Patent Microorganisms Depositary [NPMD] (5-8 Kazusa Kamatari 2-chome, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-02844.

[0236] Example 4 (Confirmation of the effect when glycerin-propylene oxide-ethylene oxide adduct is present) When the HMGB1 in the sample was brought into contact with a carrier on which anti-HMGB1 antibody was immobilized, a glycerin-propylene oxide-ethylene oxide adduct was co-present, and the HMGB1 in the sample was measured, and a calibration curve was created to confirm the effectiveness of the present invention.

[0237] [1] Reagent for measuring HMGB1 in samples 1. Reagent 1 A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.

[0238] 2. Second Reagent (1) Second reagent (0.0004% FA-195) (a) An anti-HMGB1 monoclonal antibody mixture was prepared by mixing equal amounts of the antibody (2H6) prepared in Example 1 and the antibody (2D4) prepared in Example 2. This was designated the "anti-HMGB1 monoclonal antibody mixture."

[0239] (b) The anti-HMGB1 monoclonal antibody mixture from (a) above was diluted with buffer to a concentration of 1 to 2 mg / mL, and 1 mL of this antibody solution was mixed with 1 mL of latex particles (Fujikura Kasei Co., Ltd., Japan) and allowed to stand at 2 to 8°C for 16 hours to immobilize the anti-HMGB1 monoclonal antibody mixture onto the latex particles.

[0240] (c) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which the anti-HMGB1 monoclonal antibody mixture was immobilized, and a blocking treatment was performed to obtain latex particles onto which the blocked anti-HMGB1 monoclonal antibody mixture was immobilized.

[0241] (d) The anti-HMGB1,2 monoclonal antibody prepared in Example 3 was diluted with buffer to a concentration of 1 to 2 mg / mL, and 1 mL of this antibody solution was mixed with 1 mL of latex particles (Fujikura Kasei Co., Ltd., Japan) and allowed to stand at 2 to 8°C for 16 hours to immobilize the anti-HMGB1,2 monoclonal antibody onto the latex particles.

[0242] (e) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which anti-HMGB1,2 monoclonal antibodies had been immobilized, and a blocking treatment was performed to obtain blocked latex particles onto which anti-HMGB1,2 monoclonal antibodies had been immobilized.

[0243] (f) Equal amounts of latex particles immobilized with the blocked anti-HMGB1 monoclonal antibody mixture prepared in (c) above and latex particles immobilized with blocked anti-HMGB1,2 monoclonal antibodies prepared in (e) above were mixed to prepare a mixture of latex particles immobilized with anti-HMGB1 monoclonal antibodies, which was designated the "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture."

[0244] (g) A glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1): a + b + c = 11.5 moles, d + e + f = 14.3 moles, and g + h + i = 44.1 moles. [(All moles are average mole numbers added.) The numbers in {} represent random additions.] A glycerin-propylene oxide-ethylene oxide adduct, "Sannyx FA-195" (molecular weight: 3,370, CAS No. 9082-00-2) available from Sanyo Chemical Industries, Ltd., was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture (f) to a concentration of 0.002% (w / v) after mixing to prepare a second reagent. This reagent was designated "Second Reagent (0.0004% FA-195)."

[0245] [ka]

[0246] The "Sanix FA-195" contained in this second reagent (0.0004% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0004% (w / v), which is the concentration in the second reagent (0.0004% FA-195) of 0.002% (w / v) divided by the dilution ratio of 4.6.

[0247] (2) Second reagent (0.001% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing" was changed to "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.005% (w / v) after mixing." This was designated "Second Reagent (0.001% FA-195)."

[0248] The "Sanix FA-195" contained in this second reagent (0.001% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.001% (w / v), which is the concentration in the second reagent (0.001% FA-195) of 0.005% (w / v) divided by the dilution ratio of 4.6.

[0249] (3) Second reagent (0.0016% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing" was changed to "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.0075% (w / v) after mixing." This was designated "Second Reagent (0.0016% FA-195)."

[0250] The "Sanix FA-195" contained in this second reagent (0.0016% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0016% (w / v), which is the concentration in the second reagent (0.0016% FA-195) of 0.00754% (w / v) divided by the dilution ratio of 4.6.

[0251] (4) Second reagent (0.0022% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix FA-195 was mixed into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing" was changed to "Sanix FA-195 was mixed into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing." This was designated "Second Reagent (0.0022% FA-195)."

[0252] The "Sanix FA-195" contained in this second reagent (0.0022% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0022% (w / v), which is the concentration in the second reagent (0.0022% FA-195) of 0.01% (w / v) divided by the dilution ratio of 4.6.

[0253] (5) Second reagent (0.0054% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing" was changed to "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.025% (w / v) after mixing." This was designated "Second Reagent (0.0054% FA-195)."

[0254] The "Sanix FA-195" contained in this second reagent (0.0054% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0054% (w / v), which is the concentration in the second reagent (0.0054% FA-195) of 0.025% (w / v) divided by the dilution ratio of 4.6.

[0255] (6) Second reagent (0.01% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing" was changed to "Sanix FA-195 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.05% (w / v) after mixing." This was designated "Second Reagent (0.01% FA-195)."

[0256] The "Sanix FA-195" contained in this second reagent (0.01% FA-195) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.01% (w / v), which is the concentration in the second reagent (0.01% FA-195) of 0.05% (w / v) divided by the dilution ratio of 4.6.

[0257] (7) Second reagent (0% FA-195) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) In (g) of (1) above, "Sanix FA-195" was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.002% (w / v) after mixing," but "Sanix FA-195 was not mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture," so a second reagent containing no "Sanix FA-195" was prepared in accordance with the procedure described in (g) of (1) above. This was designated "Second Reagent (0% FA-195)."

[0258] [2] Sample 1. Sample (HMGB1 concentration: 0 ng / mL) The buffer solution containing 1% (w / v) bovine serum albumin (BSA) was designated as "sample (HMGB1 concentration: 0 ng / mL)."

[0259] 2. Sample (HMGB1 concentration: 5 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 5 ng / mL. This was designated "sample (HMGB1 concentration: 5 ng / mL)."

[0260] 3. Sample (HMGB1 concentration: 10 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 10 ng / mL. This was designated "sample (HMGB1 concentration: 10 ng / mL)."

[0261] 4. Sample (HMGB1 concentration: 20 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 20 ng / mL. This was designated "sample (HMGB1 concentration: 20 ng / mL)."

[0262] 5. Sample (HMGB1 concentration: 40 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 40 ng / mL. This was designated "sample (HMGB1 concentration: 40 ng / mL)."

[0263] 6. Sample (HMGB1 concentration: 80 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 80 ng / mL. This was designated "sample (HMGB1 concentration: 80 ng / mL)."

[0264] [3] Shaking treatment The second reagent (0% FA-195), second reagent (0.0004% FA-195), second reagent (0.001% FA-195), second reagent (0.0016% FA-195), second reagent (0.0022% FA-195), second reagent (0.0054% FA-195), and second reagent (0.01% FA-195) prepared in 2 of [1] above were each divided into half, placed in containers, and sealed.

[0265] One half of the mixture was refrigerated (2 to 8°C) and shaken at 100 rpm for at least 3 days, and this second reagent was designated as "second reagent (with shaking)." The other half of the mixture was left standing in a refrigerator (2 to 8°C) for at least 3 days, and this second reagent was designated as "second reagent (without shaking)."

[0266] [4] Measurement of HMGB1 in samples 1. Measurement using the second reagent (with shaking) Measurement of HMGB1 in the sample in [2] was carried out using the first reagent in [1] 1 as the first reagent for measuring HMGB1 in the sample, and the second reagent in [3] (with shaking) as the second reagent.

[0267] Measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation, Japan). The first reagent in item 1 of item 1 of item 1 of item 2 of item 2 of item 2 of item 1 of item 1 of item 2 of item 2 of item 3 ... Specifically, 24 μL of the sample in [2] above was used, 120 μL of the first reagent in [1] 1 above was used as the first reagent, and 40 μL of each of the second reagents in [3] above (with shaking) was used as the second reagent, and the change in absorbance was measured using the two-point-end method. The reaction temperature was 37°C, and the reaction time was a total of 10 minutes from the time when the first reagent in [1]-1 was added to and mixed with the sample in [2]. The change in absorbance was measured at a measurement wavelength of 800 nm after the first reagent in [1]-1 was added to and mixed with the sample in [2].

[0268] 2. Measurement using the second reagent (without shaking) Measurement of HMGB1 in the sample of [2] was carried out using the first reagent of [1] 1 as the first reagent for measuring HMGB1 in the sample, and the second reagent of [3] (without shaking) as the second reagent.

[0269] Measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation, Japan). The first reagent in item 1 of item 1 of item 1 of item 2 of item 2 of item 2 of item 1 of item 1 of item 2 of item 2 of item 3 ... Specifically, 24 μL of the sample in [2] above was used, 120 μL of the first reagent in [1] 1 above was used as the first reagent, and 40 μL of the second reagent in [3] above (without shaking) was used as the second reagent, and the change in absorbance was measured using the two-point-end method. The reaction temperature was 37°C, and the reaction time was 10 minutes in total after adding and mixing the first reagent of [1]-1 to the sample of [2], and the amount of change in absorbance was measured at a measurement wavelength of 800 nm.

[0270] [5] Measurement results 1. Measurement results when using the second reagent (0% FA-195) (with shaking) or the second reagent (0% FA-195) (without shaking) as the second reagent.

[0271] The results of measurements using either the second reagent (0% FA-195) (with shaking) or the second reagent (0% FA-195) (without shaking) as the second reagent are shown in Figure 1 as a calibration curve graph when measuring HMGB1 in the sample.

[0272] In this Figure 1, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 1, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0% FA-195) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0% FA-195) (without shaking) was used.

[0273] Figure 1 shows that the calibration curve when the second reagent (0% FA-195) (with shaking) is used is significantly different from the calibration curve when the second reagent (0% FA-195) (without shaking) is used. Furthermore, it can be seen that the slope of the calibration curve when the second reagent (0% FA-195) (with shaking) was used was significantly lower.

[0274] Furthermore, Figure 1 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a large change in absorbance of nearly 0.08 when the second reagent (0% FA-195) (with shaking) is used, but the change in absorbance is close to 0 when the second reagent (0% FA-195) (without shaking) is used.

[0275] This confirmed that shaking the carrier on which the anti-HMGB1 antibody was immobilized caused nonspecific aggregation of the carrier on which the anti-HMGB1 antibody was immobilized, resulting in an increase in the reagent blank.

[0276] 2. Measurement results when using the second reagent (0.0004% FA-195) (with shaking) or the second reagent (0.0004% FA-195) (without shaking) as the second reagent

[0277] The results of measurements using either the second reagent (0.0004% FA-195) (with shaking) or the second reagent (0.0004% FA-195) (without shaking) as the second reagent are shown in Figure 2 as a calibration curve graph when measuring HMGB1 in the sample.

[0278] In FIG. 2, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 2, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0004% FA-195) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0004% FA-195) (without shaking) was used.

[0279] Figure 2 shows that the calibration curve when the second reagent (0.0004% FA-195) (with shaking) is used is different from the calibration curve when the second reagent (0.0004% FA-195) (without shaking) is used.

[0280] Furthermore, Figure 2 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a large absorbance change of nearly 0.06 when the second reagent (0.0004% FA-195) (with shaking) is used, but the absorbance change is close to 0 when the second reagent (0.0004% FA-195) (without shaking) is used.

[0281] This confirmed that when 0.0004% (w / v) glycerin-propylene oxide-ethylene oxide adduct was present during contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody was immobilized, shaking the carrier on which the anti-HMGB1 antibody was immobilized could not be prevented from causing nonspecific aggregation, and therefore the increase in the reagent blank caused by this aggregation could not be prevented.

[0282] 3. Measurement results when using the second reagent (0.001% FA-195) (with shaking) or the second reagent (0.001% FA-195) (without shaking) as the second reagent

[0283] The results of measurements using either the second reagent (0.001% FA-195) (with shaking) or the second reagent (0.001% FA-195) (without shaking) as the second reagent are shown in Figure 3 as a calibration curve graph when measuring HMGB1 in the sample.

[0284] In this Figure 3, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 3, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.001% FA-195) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.001% FA-195) (without shaking) was used.

[0285] Figure 3 shows that the calibration curves obtained when the second reagent (0.001% FA-195) (with shaking) was used and the calibration curves obtained when the second reagent (0.001% FA-195) (without shaking) was used are parallel.

[0286] Furthermore, Figure 3 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a small change in absorbance of around 0.02 when the second reagent (0.001% FA-195) (with shaking) is used, and a change in absorbance close to 0 when the second reagent (0.001% FA-195) (without shaking) is used.

[0287] This confirmed that when 0.001% (w / v) glycerin-propylene oxide-ethylene oxide adduct is present during contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0288] 4. Measurement results when using the second reagent (0.0016% FA-195) (with shaking) or the second reagent (0.0016% FA-195) (without shaking) as the second reagent

[0289] The measurement results when the second reagent (0.0016% FA-195) (with shaking) or the second reagent (0.0016% FA-195) (without shaking) was used as the second reagent are shown in Figure 4 as a calibration curve graph when measuring HMGB1 in the sample.

[0290] In this Figure 4, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 4, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0016% FA-195) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0016% FA-195) (without shaking) was used.

[0291] Figure 4 shows that the calibration curves obtained when the second reagent (0.0016% FA-195) (with shaking) and the second reagent (0.0016% FA-195) (without shaking) are nearly identical.

[0292] Furthermore, Figure 4 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 whether the second reagent (0.0016% FA-195) (with shaking) or the second reagent (0.0016% FA-195) (without shaking) is used.

[0293] This confirmed that when 0.0016% (w / v) glycerin-propylene oxide-ethylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0294] 5. Measurement results when using the second reagent (0.0022% FA-195) (with shaking) or the second reagent (0.0022% FA-195) (without shaking) as the second reagent

[0295] The measurement results when the second reagent (0.0022% FA-195) (with shaking) or the second reagent (0.0022% FA-195) (without shaking) was used as the second reagent are shown in Figure 5 as a calibration curve graph when measuring HMGB1 in the sample.

[0296] In this Figure 5, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 5, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0022% FA-195) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0022% FA-195) (without shaking) was used.

[0297] Figure 5 shows that the calibration curves obtained when the second reagent (0.0022% FA-195) (with shaking) and the second reagent (0.0022% FA-195) (without shaking) are almost overlapping.

[0298] Furthermore, Figure 5 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 whether the second reagent (0.0022% FA-195) (with shaking) or the second reagent (0.0022% FA-195) (without shaking) is used.

[0299] This confirmed that when 0.0022% (w / v) glycerin-propylene oxide-ethylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0300] 6. Measurement results when using the second reagent (0.0054% FA-195) (with shaking) or the second reagent (0.0054% FA-195) (without shaking) as the second reagent

[0301] The measurement results when the second reagent (0.0054% FA-195) (with shaking) or the second reagent (0.0054% FA-195) (without shaking) was used as the second reagent are shown in Figure 6 as a graph of the calibration curve when measuring HMGB1 in the sample.

[0302] In this Figure 6, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 6, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0054% FA-195) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0054% FA-195) (without shaking) was used.

[0303] Figure 6 shows that the calibration curves obtained when the second reagent (0.0054% FA-195) (with shaking) and the second reagent (0.0054% FA-195) (without shaking) are almost overlapping.

[0304] Furthermore, Figure 6 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows that the change in absorbance is close to 0 when the second reagent (0.0054% FA-195) (with shaking) is used, as well as when the second reagent (0.0054% FA-195) (without shaking) is used.

[0305] This confirmed that when 0.0054% (w / v) glycerin-propylene oxide-ethylene oxide adduct is present during contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from undergoing nonspecific aggregation, thereby suppressing the increase in the reagent blank caused by this aggregation.

[0306] 7. Measurement results when using the second reagent (0.01% FA-195) (with shaking) or the second reagent (0.01% FA-195) (without shaking) as the second reagent

[0307] The measurement results when the second reagent (0.01% FA-195) (with shaking) or the second reagent (0.01% FA-195) (without shaking) was used as the second reagent are shown in Figure 7 as a calibration curve graph when measuring HMGB1 in the sample.

[0308] In FIG. 7, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 7, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.01% FA-195) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.01% FA-195) (without shaking) was used.

[0309] Figure 7 shows that the calibration curves obtained when the second reagent (0.01% FA-195) (with shaking) and the second reagent (0.01% FA-195) (without shaking) are almost overlapping.

[0310] Furthermore, Figure 7 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 when using the second reagent (0.01% FA-195) (with shaking) and when using the second reagent (0.01% FA-195) (without shaking).

[0311] This confirmed that when 0.01% (w / v) glycerin-propylene oxide-ethylene oxide adduct is present during contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0312] [Example 5] (Confirmation of the effect when glycerin-propylene oxide adduct is present) When the HMGB1 in the sample was brought into contact with a carrier on which anti-HMGB1 antibody was immobilized, the HMGB1 in the sample was measured in the presence of a glycerin-propylene oxide adduct, and a calibration curve was created to confirm the effectiveness of the present invention.

[0313] [1] Reagent for measuring HMGB1 in samples 1. Reagent 1 A solution containing 200 mM buffer (pH 8.0) was used as the first reagent.

[0314] 2. Second Reagent (1) Second reagent (0.0022% GP-400) (a) An anti-HMGB1 monoclonal antibody mixture was prepared by mixing equal amounts of the antibody (2H6) prepared in Example 1 and the antibody (2D4) prepared in Example 2. This was designated the "anti-HMGB1 monoclonal antibody mixture."

[0315] (b) The anti-HMGB1 monoclonal antibody mixture from (a) above was diluted with buffer to a concentration of 1 to 2 mg / mL, and 1 mL of this antibody solution was mixed with 1 mL of latex particles (Fujikura Kasei Co., Ltd., Japan) and allowed to stand at 2 to 8°C for 16 hours to immobilize the anti-HMGB1 monoclonal antibody mixture onto the latex particles.

[0316] (c) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which the anti-HMGB1 monoclonal antibody mixture was immobilized, and a blocking treatment was performed to obtain latex particles onto which the blocked anti-HMGB1 monoclonal antibody mixture was immobilized.

[0317] (d) The anti-HMGB1,2 monoclonal antibody prepared in Example 3 was diluted with buffer to a concentration of 1 to 2 mg / mL, and 1 mL of this antibody solution was mixed with 1 mL of latex particles (Fujikura Kasei Co., Ltd., Japan) and allowed to stand at 2 to 8°C for 16 hours to immobilize the anti-HMGB1,2 monoclonal antibody onto the latex particles.

[0318] (e) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) was added to and mixed with the latex particles onto which anti-HMGB1,2 monoclonal antibodies had been immobilized, and a blocking treatment was performed to obtain blocked latex particles onto which anti-HMGB1,2 monoclonal antibodies had been immobilized.

[0319] (f) Equal amounts of latex particles immobilized with the blocked anti-HMGB1 monoclonal antibody mixture prepared in (c) above and latex particles immobilized with blocked anti-HMGB1,2 monoclonal antibodies prepared in (e) above were mixed to prepare a mixture of latex particles immobilized with anti-HMGB1 monoclonal antibodies, which was designated the "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture."

[0320] (g) A glycerin-propylene oxide adduct represented by the following general formula (2), in which a + b + c = 5.6 moles (the number of moles is the average number of moles added), "Sannyx GP-400" (molecular weight: 420, CAS No. 25791-96-2) available from Sanyo Chemical Industries, Ltd., was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture from (f) above to a concentration of 0.01% (w / v) to prepare a second reagent. This was designated "Second Reagent (0.0022% GP-400)."

[0321] [ka]

[0322] The "Sanix GP-400" contained in this second reagent (0.0022% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0022% (w / v), which is the concentration of 0.01% (w / v) in the second reagent (0.0022% GP-400) divided by the dilution ratio of 4.6.

[0323] (2) Second reagent (0.0054% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) The second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing" was changed to "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.025% (w / v) after mixing." This was designated "Second Reagent (0.0054% GP-400)."

[0324] The "Sanix GP-400" contained in this second reagent (0.0054% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0054% (w / v), which is the concentration in the second reagent (0.0054% GP-400) of 0.025% (w / v) divided by the dilution ratio of 4.6.

[0325] (3) Second reagent (0.0076% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) The second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing" was changed to "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.035% (w / v) after mixing." This was designated "Second Reagent (0.0076% GP-400)."

[0326] The "Sanix GP-400" contained in this second reagent (0.0076% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.0076% (w / v), which is the concentration in the second reagent (0.0076% GP-400) of 0.035% (w / v) divided by the dilution ratio of 4.6.

[0327] (4) Second reagent (0.01% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) A second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing" was changed to "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.05% (w / v) after mixing." This was designated "Second Reagent (0.01% GP-400)."

[0328] The "Sanix GP-400" contained in this second reagent (0.01% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.01% (w / v), which is the concentration in the second reagent (0.01% GP-400) of 0.05% (w / v) divided by the dilution ratio of 4.6.

[0329] (5) Second reagent (0.016% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) The second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing" was changed to "Sanix GP-400 was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.075% (w / v) after mixing." This was designated "Second Reagent (0.016% GP-400)."

[0330] The "Sanix GP-400" contained in this second reagent (0.016% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.016% (w / v), which is the concentration in the second reagent (0.016% GP-400) of 0.075% (w / v) divided by the dilution ratio of 4.6.

[0331] (6) Second reagent (0.022% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) The second reagent was prepared as described in (1)(g) above, except that in (1)(g), "Sanix GP-400 was mixed into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing" was changed to "Sanix GP-400 was mixed into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.10% (w / v) after mixing." This was designated "Second Reagent (0.022% GP-400)."

[0332] The "Sanix GP-400" contained in this second reagent (0.022% GP-400) is such that the concentration of HMGB1 in the sample that coexists with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture during the measurement of HMGB1 in the sample described below is 0.022% (w / v), which is the concentration in the second reagent (0.022% GP-400) of 0.10% (w / v) divided by the dilution ratio of 4.6.

[0333] (7) Second reagent (0% GP-400) (a) An anti-HMGB1 monoclonal antibody-immobilized latex particle mixture was prepared as described in (a) to (f) of (1) above. (b) In (g) of (1) above, "Sanix GP-400" was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture to a concentration of 0.01% (w / v) after mixing," but "Sanix GP-400 was not mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture," so a second reagent containing no "Sanix GP-400" was prepared in accordance with the procedure described in (g) of (1) above. This was designated "Second Reagent (0% GP-400)."

[0334] [2] Sample 1. Sample (HMGB1 concentration: 0 ng / mL) The buffer solution containing 1% (w / v) bovine serum albumin (BSA) was designated as "sample (HMGB1 concentration: 0 ng / mL)."

[0335] 2. Sample (HMGB1 concentration: 5 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 5 ng / mL. This was designated "sample (HMGB1 concentration: 5 ng / mL)."

[0336] 3. Sample (HMGB1 concentration: 10 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 10 ng / mL. This was designated "sample (HMGB1 concentration: 10 ng / mL)."

[0337] 4. Sample (HMGB1 concentration: 20 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 20 ng / mL. This was designated "sample (HMGB1 concentration: 20 ng / mL)."

[0338] 5. Sample (HMGB1 concentration: 40 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 40 ng / mL. This was designated "sample (HMGB1 concentration: 40 ng / mL)."

[0339] 6. Sample (HMGB1 concentration: 80 ng / mL) Purified porcine HMGB1 (thymus-derived) [99.1% amino acid sequence identity with human HMGB1] was diluted with a buffer containing 1% (w / v) bovine serum albumin (BSA) to prepare a sample with an HMGB1 concentration of 80 ng / mL. This was designated "sample (HMGB1 concentration: 80 ng / mL)."

[0340] [3] Shaking treatment The second reagent (0% GP-400), second reagent (0.0022% GP-400), second reagent (0.0054% GP-400), second reagent (0.0076% GP-400), second reagent (0.01% GP-400), second reagent (0.016% GP-400), and second reagent (0.022% GP-400) prepared in 2 of [1] above were each divided into half, placed in containers, and sealed.

[0341] One half of the mixture was refrigerated (2 to 8°C) and shaken at 100 rpm for at least 3 days, and this second reagent was designated as "second reagent (with shaking)." The other half of the mixture was left standing in a refrigerator (2 to 8°C) for at least 3 days, and this second reagent was designated as "second reagent (without shaking)."

[0342] [4] Measurement of HMGB1 in samples 1. Measurement using the second reagent (with shaking) Measurement of HMGB1 in the sample in [2] was carried out using the first reagent in [1] 1 as the first reagent for measuring HMGB1 in the sample, and the second reagent in [3] (with shaking) as the second reagent.

[0343] Measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation, Japan). The first reagent in item 1 of item 1 of item 1 of item 2 of item 2 of item 2 of item 1 of item 1 of item 2 of item 2 of item 3 ... Specifically, 24 μL of the sample in [2] above was used, 120 μL of the first reagent in [1] 1 above was used as the first reagent, and 40 μL of each of the second reagents in [3] above (with shaking) was used as the second reagent, and the change in absorbance was measured using the two-point-end method. The reaction temperature was 37°C, and the reaction time was 10 minutes in total after adding and mixing the first reagent of [1]-1 to the sample of [2], and the amount of change in absorbance was measured at a measurement wavelength of 800 nm.

[0344] 2. Measurement using the second reagent (without shaking) Measurement of HMGB1 in the sample of [2] was carried out using the first reagent of [1] 1 as the first reagent for measuring HMGB1 in the sample, and the second reagent of [3] (without shaking) as the second reagent.

[0345] Measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation, Japan). The first reagent in item 1 of item 1 of item 1 of item 2 of item 2 of item 2 of item 1 of item 1 of item 2 of item 2 of item 3 ... Specifically, 24 μL of the sample in [2] above was used, 120 μL of the first reagent in [1] 1 above was used as the first reagent, and 40 μL of the second reagent in [3] above (without shaking) was used as the second reagent, and the change in absorbance was measured using the two-point-end method. The reaction temperature was 37°C, and the reaction time was 10 minutes in total after adding and mixing the first reagent of [1]-1 to the sample of [2], and the amount of change in absorbance was measured at a measurement wavelength of 800 nm.

[0346] [5] Measurement results 1. Measurement results when using the second reagent (0% GP-400) (with shaking) or the second reagent (0% GP-400) (without shaking) as the second reagent

[0347] The measurement results when the second reagent (0% GP-400) (with shaking) or the second reagent (0% GP-400) (without shaking) was used as the second reagent are shown in Figure 8 as a calibration curve graph when measuring HMGB1 in the sample.

[0348] In this Figure 8, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 8, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0% GP-400) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0% GP-400) (without shaking) was used.

[0349] Figure 8 shows that there is a large difference between the calibration curve obtained when the second reagent (0% GP-400) (with shaking) was used and the calibration curve obtained when the second reagent (0% GP-400) (without shaking) was used. Furthermore, it can be seen that the slope of the calibration curve when the second reagent (0% GP-400) (with shaking) was used was significantly lower.

[0350] Furthermore, Figure 8 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a large change in absorbance of nearly 0.08 when the second reagent (0% GP-400) (with shaking) is used, but the change in absorbance is close to 0 when the second reagent (0% GP-400) (without shaking) is used.

[0351] This confirmed that shaking the carrier on which the anti-HMGB1 antibody was immobilized caused nonspecific aggregation of the carrier on which the anti-HMGB1 antibody was immobilized, resulting in an increase in the reagent blank.

[0352] 2. Measurement results when using the second reagent (0.0022% GP-400) (with shaking) or the second reagent (0.0022% GP-400) (without shaking) as the second reagent

[0353] The measurement results when the second reagent (0.0022% GP-400) (with shaking) or the second reagent (0.0022% GP-400) (without shaking) was used as the second reagent are shown in Figure 9 as a graph of the calibration curve when measuring HMGB1 in the sample.

[0354] In this Figure 9, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 9, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0022% GP-400) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0022% GP-400) (without shaking) was used.

[0355] Figure 9 shows that the calibration curve when the second reagent (0.0022% GP-400) (with shaking) is used is different from the calibration curve when the second reagent (0.0022% GP-400) (without shaking) is used.

[0356] Furthermore, Figure 9 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a large change in absorbance of nearly 0.05 when the second reagent (0.0022% GP-400) (with shaking) is used, but the change in absorbance is close to 0 when the second reagent (0.0022% GP-400) (without shaking) is used.

[0357] This confirmed that when 0.0022% (w / v) glycerin-propylene oxide adduct was present during contact between the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody was immobilized, shaking the carrier on which the anti-HMGB1 antibody was immobilized could not be prevented from causing nonspecific aggregation, and therefore the increase in the reagent blank caused by this aggregation could not be prevented.

[0358] 3. Measurement results when using the second reagent (0.0054% GP-400) (with shaking) or the second reagent (0.0054% GP-400) (without shaking) as the second reagent

[0359] The measurement results when the second reagent (0.0054% GP-400) (with shaking) or the second reagent (0.0054% GP-400) (without shaking) was used as the second reagent are shown in Figure 10 as a calibration curve graph when measuring HMGB1 in the sample.

[0360] In this FIG. 10, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 10, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0054% GP-400) (with shaking) was used, and "●" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0054% GP-400) (without shaking) was used.

[0361] Figure 10 shows that the calibration curve when the second reagent (0.0054% GP-400) (with shaking) is used and the calibration curve when the second reagent (0.0054% GP-400) (without shaking) is used are parallel.

[0362] Furthermore, Figure 10 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a small change in absorbance of around 0.015 when the second reagent (0.0054% GP-400) (with shaking) is used, and that the change in absorbance is close to 0 when the second reagent (0.0054% GP-400) (without shaking) is used.

[0363] This confirmed that when 0.0054% (w / v) glycerin-propylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0364] 4. Measurement results when using the second reagent (0.0076% GP-400) (with shaking) or the second reagent (0.0076% GP-400) (without shaking) as the second reagent

[0365] The measurement results when the second reagent was either the second reagent (0.0076% GP-400) (with shaking) or the second reagent (0.0076% GP-400) (without shaking) were used are shown in Figure 11 as a calibration curve graph when measuring HMGB1 in the sample.

[0366] In this FIG. 11, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 11, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0076% GP-400) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.0076% GP-400) (without shaking) was used.

[0367] Figure 11 shows that the calibration curve when the second reagent (0.0076% GP-400) (with shaking) and the calibration curve when the second reagent (0.0076% GP-400) (without shaking) were used almost overlapped.

[0368] Furthermore, Figure 11 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 whether the second reagent (0.0076% GP-400) (with shaking) or the second reagent (0.0076% GP-400) (without shaking) was used.

[0369] This confirmed that when 0.0076% (w / v) glycerin-propylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutinations, thereby suppressing the increase in the reagent blank caused by such agglutinations.

[0370] 5. Measurement results when using the second reagent (0.01% GP-400) (with shaking) or the second reagent (0.01% GP-400) (without shaking) as the second reagent

[0371] The measurement results when the second reagent was either the second reagent (0.01% GP-400) (with shaking) or the second reagent (0.01% GP-400) (without shaking) are shown in Figure 12 as a calibration curve graph when measuring HMGB1 in the sample.

[0372] In this Figure 12, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the measured value, that is, the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000). In Figure 12, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.01% GP-400) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.01% GP-400) (without shaking) was used.

[0373] Figure 12 shows that the calibration curve when the second reagent (0.01% GP-400) (with shaking) and the calibration curve when the second reagent (0.01% GP-400) (without shaking) were used almost overlapped.

[0374] Furthermore, Figure 12 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 when the second reagent (0.01% GP-400) (with shaking) and when the second reagent (0.01% GP-400) (without shaking) were used.

[0375] This confirmed that when 0.01% (w / v) glycerin-propylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0376] 6. Measurement results when using the second reagent (0.016% GP-400) (with shaking) or the second reagent (0.016% GP-400) (without shaking) as the second reagent

[0377] The measurement results when the second reagent was either the second reagent (0.016% GP-400) (with shaking) or the second reagent (0.016% GP-400) (without shaking) are shown in Figure 13 as a calibration curve graph when measuring HMGB1 in the sample.

[0378] In this Figure 13, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 13, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.016% GP-400) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.016% GP-400) (without shaking) was used.

[0379] Figure 13 shows that the calibration curve when the second reagent (0.016% GP-400) (with shaking) and the calibration curve when the second reagent (0.016% GP-400) (without shaking) were used almost overlapped.

[0380] Furthermore, Figure 13 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 whether the second reagent (0.016% GP-400) (with shaking) or the second reagent (0.016% GP-400) (without shaking) is used.

[0381] This confirmed that when 0.016% (w / v) glycerin-propylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

[0382] 7. Measurement results when using the second reagent (0.022% GP-400) (with shaking) or the second reagent (0.022% GP-400) (without shaking) as the second reagent

[0383] The measurement results when the second reagent was either the second reagent (0.022% GP-400) (with shaking) or the second reagent (0.022% GP-400) (without shaking) are shown in Figure 14 as a calibration curve graph when measuring HMGB1 in a sample.

[0384] In this Figure 14, the horizontal axis represents the HMGB1 concentration in the sample (unit: ng / mL), and the vertical axis represents the change in absorbance at a measurement wavelength of 800 nm (unit: change in absorbance x 10,000), which is the measured value. In Figure 14, "□" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.022% GP-400) (with shaking) was used, and "●" indicates the measurement value (change in absorbance at a measurement wavelength of 800 nm) when the second reagent (0.022% GP-400) (without shaking) was used.

[0385] Figure 14 shows that the calibration curve when the second reagent (0.022% GP-400) (with shaking) and the calibration curve when the second reagent (0.022% GP-400) (without shaking) were used almost overlapped.

[0386] Furthermore, Figure 14 shows that the reagent blank, which is the measured value (change in absorbance at a measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng / mL, shows a change in absorbance close to 0 whether the second reagent (0.022% GP-400) (with shaking) or the second reagent (0.022% GP-400) (without shaking) is used.

[0387] This confirmed that when 0.022% (w / v) glycerin-propylene oxide adduct is present when the HMGB1 in the sample comes into contact with the carrier on which the anti-HMGB1 antibody is immobilized, shaking the carrier on which the anti-HMGB1 antibody is immobilized can prevent the carrier from forming nonspecific agglutination, thereby suppressing the increase in the reagent blank caused by this agglutination.

Claims

1. A method for measuring HMGB1 in a sample, which involves contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized, and measuring the aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized, thereby measuring the concentration of HMGB1 in the sample, characterized in that 0.005% (w / v) to 0.05% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.025% (w / v) to 0.10% (w / v) of a glycerin-propylene oxide adduct is present in a reagent containing a carrier on which an anti-HMGB1 antibody has been immobilized.

2. The method for measuring HMGB1 in a sample according to claim 1, characterized in that the concentration of the glycerin-propylene oxide-ethylene oxide adduct that is coexisted with the reagent containing the carrier on which the anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.025% (w / v).

3. The method for measuring HMGB1 in a sample according to claim 1 or 2, characterized in that the concentration of glycerin propylene oxide ethylene oxide adduct that is coexistent with a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.01% (w / v).

4. The method for measuring HMGB1 in a sample according to claim 1, characterized in that the concentration of the glycerin propylene oxide adduct that is coexisted with the reagent containing the carrier on which the anti-HMGB1 antibody is immobilized is 0.035% (w / v) to 0.10% (w / v).

5. The method for measuring HMGB1 in a sample according to any one of claims 1 to 4, wherein the carrier is a particle.

6. The method for measuring HMGB1 in a sample according to any one of claims 1 to 5, wherein the carrier is a latex particle.

7. The method for measuring HMGB1 in a sample according to any one of claims 1 to 6, wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1): 【Chemistry 1】 [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.]

8. The method for measuring HMGB1 in a sample according to any one of claims 1 to 6, wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2): 【Chemistry 2】 [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

9. A reagent for measuring HMGB1 in a sample, which measures the concentration of HMGB1 in the sample by contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized and measuring aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized bound via HMGB1, is characterized in that the reagent contains a carrier on which an anti-HMGB1 antibody has been immobilized and is coexistent with 0.005% (w / v) to 0.05% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.025% (w / v) to 0.10% (w / v) of a glycerin-propylene oxide adduct.

10. 10. A reagent for measuring HMGB1 in a sample according to claim 9, characterized in that the concentration of glycerin propylene oxide ethylene oxide adduct coexisting in a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.025% (w / v).

11. A reagent for measuring HMGB1 in a sample according to claim 9 or 10, characterized in that the concentration of a glycerin-propylene oxide-ethylene oxide adduct that is coexistent with a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.01% (w / v).

12. 10. A reagent for measuring HMGB1 in a sample according to claim 9, characterized in that the concentration of a glycerin propylene oxide adduct that is coexistent with a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.035% (w / v) to 0.10% (w / v).

13. The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 12, wherein the carrier is a particle.

14. The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 13, wherein the carrier is a latex particle.

15. The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 14, wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1): 【Chemistry 1】 [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.]

16. The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 14, wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2): 【Chemistry 2】 [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

17. A method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody has been immobilized, characterized in that in measuring HMGB1 in a sample, the concentration of HMGB1 in the sample is measured by contacting HMGB1 in the sample with a carrier onto which an anti-HMGB1 antibody has been immobilized and measuring aggregates of the carrier onto which the anti-HMGB1 antibody has been immobilized bound via HMGB1, the method further comprising coexisting 0.005% (w / v) to 0.05% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.025% (w / v) to 0.10% (w / v) of a glycerin-propylene oxide adduct in a reagent containing a carrier onto which an anti-HMGB1 antibody has been immobilized.

18. A method for suppressing nonspecific aggregation of a carrier on which an anti-HMGB1 antibody has been immobilized, as described in claim 17, characterized in that the concentration of a glycerin-propylene oxide-ethylene oxide adduct that is coexistent with a reagent containing a carrier on which an anti-HMGB1 antibody has been immobilized is 0.0075% (w / v) to 0.025% (w / v).

19. A method for inhibiting nonspecific aggregation of a carrier on which an anti-HMGB1 antibody has been immobilized, as described in claim 17 or claim 18, characterized in that the concentration of a glycerin-propylene oxide-ethylene oxide adduct that is coexistent with a reagent containing a carrier on which an anti-HMGB1 antibody has been immobilized is 0.0075% (w / v) to 0.01% (w / v).

20. A method for suppressing nonspecific aggregation of a carrier on which an anti-HMGB1 antibody has been immobilized, as described in claim 17, characterized in that the concentration of the glycerin propylene oxide adduct that is coexisted with the reagent containing the carrier on which an anti-HMGB1 antibody has been immobilized is 0.035% (w / v) to 0.10% (w / v).

21. A method for inhibiting nonspecific aggregation of a carrier having an anti-HMGB1 antibody immobilized thereon according to any one of claims 17 to 20, wherein the carrier is a particle.

22. A method for inhibiting nonspecific aggregation of a carrier having an anti-HMGB1 antibody immobilized thereon according to any one of claims 17 to 21, wherein the carrier is a latex particle.

23. The method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody is immobilized according to any one of claims 17 to 22, wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1): 【Chemistry 1】 [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.]

24. The method for inhibiting nonspecific aggregation of a carrier onto which an anti-HMGB1 antibody is immobilized according to any one of claims 17 to 22, wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2): 【Chemistry 2】 [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

25. A method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample, characterized in that the measurement of HMGB1 in a sample involves contacting HMGB1 in the sample with a carrier on which an anti-HMGB1 antibody has been immobilized, and measuring aggregates of the carrier on which the anti-HMGB1 antibody has been immobilized, which are bound via HMGB1, thereby measuring the concentration of HMGB1 in the sample, and the method comprises allowing 0.005% (w / v) to 0.05% (w / v) of a glycerin-propylene oxide-ethylene oxide adduct or 0.025% (w / v) to 0.10% (w / v) of a glycerin-propylene oxide adduct to coexist in a reagent containing a carrier on which an anti-HMGB1 antibody has been immobilized.

26. A method for suppressing an increase in reagent blank when measuring HMGB1 in a sample described in claim 25, characterized in that the concentration of glycerin propylene oxide ethylene oxide adduct coexisting in a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.025% (w / v).

27. A method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample described in claim 25 or 26, characterized in that the concentration of glycerin propylene oxide ethylene oxide adduct coexisting in a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.0075% (w / v) to 0.01% (w / v).

28. A method for suppressing an increase in reagent blank when measuring HMGB1 in a sample described in claim 25, characterized in that the concentration of glycerin propylene oxide adduct coexisting in a reagent containing a carrier on which an anti-HMGB1 antibody is immobilized is 0.035% (w / v) to 0.10% (w / v).

29. The method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample according to any one of claims 25 to 28, wherein the carrier is a particle.

30. The method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample according to any one of claims 25 to 29, wherein the carrier is a latex particle.

31. The method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample according to any one of claims 25 to 30, wherein the glycerin-propylene oxide-ethylene oxide adduct is a glycerin-propylene oxide-ethylene oxide adduct represented by the following general formula (1): 【Chemistry 1】 [In general formula (1), a+b+c is 3 to 20 moles, d+e+f is 3 to 30 moles, and g+h+i is 3 to 90 moles. (The number of moles is the average number of moles added.) The numbers in {} indicate random addition.]

32. The method for suppressing an increase in reagent blank value during measurement of HMGB1 in a sample according to any one of claims 25 to 30, wherein the glycerin-propylene oxide adduct is a glycerin-propylene oxide adduct represented by the following general formula (2): 【Chemistry 2】 [In the general formula (2), a+b+c is 3 to 12 moles (the number of moles is the average number of moles added)]

Images