JP2023097737A - Method and Reagent for Measuring HMGB1 in Sample, Method for Suppressing Non-Specific Aggregation of Anti-HMGB1 Antibody-Immobilized Carrier, and Method for Suppressing Increase in Reagent Blank When Measuring HMGB1 in Sample - Google Patents

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

The present invention provides a method and reagent for measuring HMGB1 (high mobility group protein-1; HMG-1) in a sample that can serve as a marker for diseases such as sepsis, and suppression of non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized. The present invention relates to a method and a method for suppressing an increase in reagent blank when measuring HMGB1 in a sample.
INDUSTRIAL APPLICABILITY The present invention is useful in the field of life sciences such as clinical examination, clinical pathology and medicine, and the field of chemistry such as analytical chemistry.

High mobility group protein (High Mobility Group Protein) was discovered in 1964 as a large amount of non-histone protein contained in the chromatin structure. Sex is extremely high.
Moreover, it is known that it exists abundantly not only in the nucleus but also in the cytoplasm.
Although its physiological action is not clearly understood, HMGB1 loosens the double helix structure when it binds to DNA, and is said to change the higher-order structure of DNA to an optimal structure during the transcription reaction, thereby enhancing transcriptional activity. , is thought to function as a broad spectrum transcription promoter and nucleosome relaxant.

There are several types of high mobility group proteins. For example, 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)), high mobility group protein I-C (HMGB I-C ) etc. can be mentioned.

In 1999, Wang et al. performed quantitative measurement of HMGB1 in serum (in blood) for the first time by Western blotting using a polyclonal antibody prepared using HMGB1 itself as an immunogen.
As a result, Wang et al. showed that HMGB1 can serve as a marker for sepsis.
They also showed that it is possible to distinguish between surviving patients and dying patients in sepsis patients by precisely measuring HMGB1 in blood.

That is, the usefulness of not only confirming the presence of HMGB1 in blood but also accurately quantifying it has been clarified (see Non-Patent Document 1).

It was previously shown that the antibody used for the measurement of HMGB1, that is, the antibody that specifically binds to HMGB1 (anti-HMGB1 antibody) can be prepared by Pakinen et al. and Rep et al. , Non-Patent Document 3).
Repp et al. stated that using this antibody, Solid-phase Enzyme Immunoassay can be performed on HMGB1. (In this solid-phase enzyme immunoassay method, purified HMGB1 is immobilized on wells of a microplate (microtiter plate), and an enzyme-labeled antibody that binds to HMGB1 is brought into contact with and acted on to HMGB1. It was confirmed that the binding antibody binds to the purified HMGB1.)
In 2000, Ruhiainen et al. reported a polyclonal antibody prepared using rat HMGB1 itself prepared from recombinant DNA by genetic engineering as an immunogen, and a portion of the amino acid sequence of HMGB1 "Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg HMGB1 in human blood was measured by the ELISA sandwich method using polyclonal antibodies prepared using a peptide consisting of "Pro Pro Ser Ala" as an immunogen (see Non-Patent Document 4).

Yamada et al. also disclosed an antibody that binds to HMGB1 but not to HMGB2, and a method and reagent for measuring HMGB1 that uses this antibody to measure only HMGB1 without measuring HMGB2 (Patent Document 1).

Furthermore, Yamada et al. describe that each of cations and anions are allowed to coexist in a sample at a concentration such that the value obtained by multiplying each molar concentration by the absolute value of the valence of the ion is 150 mM or more. A method and a reagent for measuring HMGB1 and/or HMGB2 contained therein have been disclosed (Patent Document 2).
This measurement method and measurement reagent can enhance the sensitivity of measurement.

Furthermore, Yamada et al. reported that from an avian antibody that specifically binds to the amino acid sequence represented by the following formula (I) of human HMGB1: 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 is highly productive, with high probability of obtaining a high titer antibody with high binding ability to human HMGB1.

In addition, the inventors of the present application proposed that in a method for measuring HMGB1 in a sample using a carrier on which an anti-HMGB1 antibody is immobilized, two monoclonal antibodies produced by a specific hybridoma are combined and immobilized on the same carrier. Disclosed is a method for measuring HMGB1 in a sample and the like (Patent Document 4).
This method for measuring HMGB1 in the sample allows highly sensitive measurement.

JP-A-2003-96099 Japanese Patent Application Laid-Open No. 2004-144728 WO2008/075788 JP 2020-148557 A

H. Wang et al., SCIENCE, Vol. 285, No. 9, pp. 248-251, published in 1999. J. Parkkinen et al., The Journal of Biological Chemistry, Vol. 268, No. 26, pp. 19726-19738, published in 1993 W. A. Lepp et al., Journal of Immunoassay, Vol. 10, No. 4, pp. 449-465, 1989. A. Rouhiainen et al., Thromb Haemost, Vol. 84, pp. 1087-1094, published in 2000

The present inventor contacted HMGB1 in a sample with a carrier on which an anti-HMGB1 antibody was immobilized, and measured aggregates of the carrier on which the anti-HMGB1 antibody was immobilized bound via HMGB1, thereby determining HMGB1 in the sample. In the method and reagent for measuring HMGB1 in a sample, which measures the concentration of the anti-HMGB1 antibody, the anti-HMGB1 antibody is shaken during transport or for other reasons. was found to non-specifically agglutinate the carrier on which the was immobilized, thereby increasing the reagent blank (reagent blind test) caused by the agglutination.

Specifically, the inventor of the present application determined the degree of non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier by measuring the ratio of the two wavelengths of absorbance of the measurement reagent containing the anti-HMGB1 antibody-immobilized carrier. I found that it can be evaluated.
More specifically, the absorbance at 570 nm and the absorbance at 800 nm of a measurement reagent containing a carrier on which an anti-HMGB1 antibody is immobilized are measured, and the absorbance ratio obtained by dividing the absorbance at 570 nm by the absorbance at 800 nm is calculated. find the value.
When the anti-HMGB1 antibody-immobilized carrier agglutinates non-specifically, the value of the absorbance on the long wavelength side increases. becomes smaller.
Therefore, non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier is obtained by dividing the absorbance value at 570 nm of the measurement reagent containing the anti-HMGB1 antibody-immobilized carrier by the absorbance value at 800 nm. can be evaluated.

Then, non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier increases the apparent particle diameter of the anti-HMGB1 antibody-immobilized carrier, thereby increasing the reagent blank.
In addition, non-specific agglutination of the carrier on which the anti-HMGB1 antibody is immobilized causes an antigen-antibody reaction between "HMGB1 in the sample" and "anti-HMGB1 antibody on the carrier on which the anti-HMGB1 antibody is immobilized." Since the effective number of "anti-HMGB1 antibody-immobilized carriers" is reduced, the slope of the calibration curve during measurement of HMGB1 in the sample is lowered.
Due to the increase in the reagent blank and the decrease in the slope of the calibration curve, the calibration curve for the measurement reagent containing the carrier on which the shaken anti-HMGB1 antibody was immobilized was obtained by immobilizing the anti-HMGB1 antibody that was not shaken. The calibration curve for the measurement reagent containing the carrier will change.

An object of the present invention is to contact HMGB1 in a sample with a carrier on which an anti-HMGB1 antibody is immobilized, and measure aggregates of the carrier on which an anti-HMGB1 antibody is immobilized bound via HMGB1. In the method and reagent for measuring HMGB1 in a sample, which measures the concentration of HMGB1, the anti-HMGB1 antibody is detected by shaking the carrier on which the anti-HMGB1 antibody is immobilized during transportation or by shaking for other reasons. To provide a method and reagent for measuring HMGB1 in a sample that suppresses non-specific agglutination of a carrier on which HMGB1 is immobilized, thereby suppressing an increase in reagent blank caused by the agglutination.
In addition, it suppresses non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier due to shaking of the anti-HMGB1 antibody-immobilized carrier during transport or other reasons. An object of the present invention is to provide a method for suppressing non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized.
Furthermore, non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier may occur due to shaking of the anti-HMGB1 antibody-immobilized carrier during transport or other reasons. An object of the present invention is to provide a method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample, which can suppress an increase in reagent blank due to the measurement of HMGB1 in a sample.

The inventors of the present application have repeatedly studied suppression of non-specific agglutination of carriers on which anti-HMGB1 antibodies are immobilized, and suppression of increase in reagent blanks caused by the agglutination. We have found that the above problems can be solved by coexisting a glycerin/propylene oxide/ethylene oxide adduct or a glycerin/propylene oxide adduct in a specific concentration range at the time of contact with a carrier on which an antibody is immobilized, and have completed the present invention. Arrived.

That is, the present invention consists of the following inventions.
(1) The concentration of HMGB1 in the sample is determined by contacting the HMGB1 in the sample with the anti-HMGB1 antibody-immobilized carrier and measuring the aggregate of the anti-HMGB1 antibody-immobilized carrier bound via HMGB1. 0.001% (w/v) to 0.01% (w/v) at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized is measured. of HMGB1 in a sample, characterized in that a glycerin/propylene oxide/ethylene oxide adduct of 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is coexistent. Measuring method.
(2) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0054% (w/v) /v).
(3) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0022% (w/v) /v).
(4) The concentration of the glycerol/propylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact with the ), the method for measuring HMGB1 in a sample according to (1) above.
(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) The glycerin/propylene oxide/ethylene oxide adduct according to any one of (1) to (6) above, wherein the adduct is a glycerin/propylene oxide/ethylene oxide adduct represented by the following general formula (1): Method for measuring HMGB1 in samples.

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

(8) HMGB1 in the sample according to any one of (1) to (6) above, wherein the glycerin/propylene oxide adduct is a glycerin/propylene oxide adduct represented by the following general formula (2): How to measure.

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

(9) The concentration of HMGB1 in the sample is determined by contacting the HMGB1 in the sample with the anti-HMGB1 antibody-immobilized carrier and measuring the aggregate of the anti-HMGB1 antibody-immobilized carrier bound via HMGB1. 0.001% (w/v) to 0.01% (w/v) at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized in the reagent for measuring HMGB1 in the sample that measures of the glycerin/propylene oxide/ethylene oxide adduct of 0.0054% (w/v) to 0.022% (w/v) of the glycerin/propylene oxide/ A reagent for measuring HMGB1 in a sample, characterized by containing an ethylene oxide adduct or a glycerin/propylene oxide adduct.
(10) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0054% (w/v) /v), the reagent for measuring HMGB1 in the sample according to (9) above.
(11) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0022% (w/v) /v), the reagent for measuring HMGB1 in the sample according to (9) or (10).
(12) The concentration of the glycerol/propylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact with the ), the reagent for measuring HMGB1 in the sample according to (9) above.
(13) A reagent for measuring HMGB1 in a sample according to any one of (9) to (12) above, wherein the carrier is a particle.
(14) A reagent for measuring HMGB1 in a sample according to any one of (9) to (13), wherein the carrier is latex particles.
(15) The glycerin/propylene oxide/ethylene oxide adduct according to any one of (9) to (14) above, wherein the adduct is a glycerin/propylene oxide/ethylene oxide adduct represented by the following general formula (1): A reagent for measuring HMGB1 in a sample.

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

(16) HMGB1 in the sample according to any one of (9) to (14) above, wherein the glycerin/propylene oxide adduct is a glycerin/propylene oxide adduct represented by the following general formula (2): measurement reagent.

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

(17) The concentration of HMGB1 in the sample is determined by contacting the HMGB1 in the sample with the anti-HMGB1 antibody-immobilized carrier and measuring the aggregate of the anti-HMGB1 antibody-immobilized carrier bound via HMGB1. In the measurement of HMGB1 in the sample, 0.001% (w / v) to 0.01% (w / v) at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody are immobilized An anti-HMGB1 antibody is immobilized, characterized in that 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. A method for suppressing non-specific aggregation of the carrier.
(18) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0054% (w/v) /v).
(19) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0022% (w/v) /v).
(20) The concentration of the glycerol/propylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact with each other is adjusted from 0.0076% (w/v) to 0.022% (w/v ), the method for suppressing non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier according to (17) above.
(21) A method for suppressing non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier according to any one of (17) to (20) above, wherein the carrier is a particle.
(22) A method for suppressing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier according to any one of (17) to (20), wherein the carrier is latex particles.
(23) The glycerin/propylene oxide/ethylene oxide adduct according to any one of (17) to (22) above, wherein the adduct of glycerin/propylene oxide/ethylene oxide is represented by the following general formula (1): A method for suppressing non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized.

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

(24) the anti-HMGB1 antibody of any one of (17) to (22) above, wherein the glycerin/propylene oxide adduct is a glycerin/propylene oxide adduct represented by the following general formula (2); A method for suppressing non-specific aggregation of immobilized carriers.

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

(25) The concentration of HMGB1 in the sample is determined by contacting the HMGB1 in the sample with the anti-HMGB1 antibody-immobilized carrier and measuring the aggregate of the anti-HMGB1 antibody-immobilized carrier bound via HMGB1. In the measurement of HMGB1 in the sample, 0.001% (w / v) to 0.01% (w / v) at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody are immobilized When measuring HMGB1 in a sample, characterized in that 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. method for suppressing the rise of the reagent blank.
(26) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0054% (w/v) /v).
(27) The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is adjusted from 0.0016% (w/v) to 0.0022% (w/v) /v).
(28) The concentration of the glycerin-propylene oxide adduct coexisting when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact with the ).
(29) A method for suppressing an increase in reagent blank when measuring HMGB1 in a sample according to any one of (25) to (28) above, wherein the carrier is a particle.
(30) A method for suppressing an increase in reagent blank when measuring HMGB1 in a sample according to any one of (25) to (29) above, wherein the carrier is latex particles.
(31) The glycerin/propylene oxide/ethylene oxide adduct according to any one of (25) to (30) above, wherein the adduct of glycerin/propylene oxide/ethylene oxide is a glycerin/propylene oxide/ethylene oxide adduct represented by the following general formula (1): A method for suppressing an increase in reagent blank when measuring HMGB1 in a sample.

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

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

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

In the method and reagent for measuring HMGB1 in a sample, the carrier on which the anti-HMGB1 antibody is immobilized is shaken during transportation or shaken for other reasons, causing the carrier on which the anti-HMGB1 antibody is immobilized to become non-existent. It suppresses the occurrence of specific agglutination, thereby suppressing an increase in reagent blanks caused by the agglutination.

In addition, it suppresses non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier due to shaking of the anti-HMGB1 antibody-immobilized carrier during transport or other reasons. It is possible.

Furthermore, non-specific aggregation of the anti-HMGB1 antibody-immobilized carrier may occur due to shaking of the anti-HMGB1 antibody-immobilized carrier during transport or other reasons. It is possible to suppress the increase in the reagent blank that occurs.

When measuring HMGB1 in a sample using the second reagent (0% FA-195) (with shaking) and the second reagent (0% FA-195) (without shaking) as the second reagent It is a diagram showing a graph of the calibration curve of.

HMGB1 in samples when using the second reagent (0.0004% FA-195) (with shaking) and the second reagent (0.0004% FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.001% FA-195) (with shaking) and the second reagent (0.001% FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.0016% FA-195) (with shaking) and the second reagent (0.0016% FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.0022% FA-195) (with shaking) and the second reagent (0.0022% FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.0054% FA-195) (with shaking) and the second reagent (0.0054% FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.01%, FA-195) (with shaking) and the second reagent (0.01%, FA-195) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

When measuring HMGB1 in a sample using the second reagent (0% GP-400) (with shaking) and the second reagent (0% GP-400) (without shaking) as the second reagent It is a diagram showing a graph of the calibration curve of.

HMGB1 in samples when using the second reagent (0.0022% GP-400) (with shaking) and the second reagent (0.0022% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.0054% GP-400) (with shaking) and the second reagent (0.0054% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.0076% GP-400) (with shaking) and the second reagent (0.0076% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.01% GP-400) (with shaking) and the second reagent (0.01% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.016% GP-400) (with shaking) and the second reagent (0.016% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

HMGB1 in samples when using the second reagent (0.022% GP-400) (with shaking) and the second reagent (0.022% GP-400) (without shaking) as the second reagent is a diagram showing a graph of a calibration curve when measuring .

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

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

Examples of the anti-HMGB1 antibody include monoclonal antibodies, polyclonal antibodies, antisera, antibody fragments [Fab and F(ab') ₂ , etc.], or single-chain antibodies (scFv) that can bind to HMGB1. can be mentioned.

This anti-HMGB1 antibody is an antibody (chimeric antibody, humanized antibody, fully humanized antibody, etc.) in which the amino acid sequence of an animal species different from that of the animal to be immunized with the immunogen is changed by gene recombination technology, etc. good too.

The anti-HMGB1 antibody is preferably a monoclonal antibody.

Moreover, in the present invention, two or more anti-HMGB1 antibodies may be used.

(2) Immunogens Immunogens for producing anti-HMGB1 antibodies in the present invention are described below.
All or part of HMGB1 can be used as an immunogen for producing an anti-HMGB1 antibody in the present invention.
That is, all or part of HMGB1, such as HMGB1 derived from humans, mammals such as bovines, pigs, dogs, cats, mice, or rats, or birds such as chickens, or HMGB1 obtained by genetic recombination, can be used. .

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

There is also a report that an antibody can recognize an amino acid sequence consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, 147, 201-210, published in 1992).
Therefore, the minimum unit of the amino acid sequence of the immunogen of the anti-HMGB1 antibody in the present invention is the amino acid sequence of all or part of the amino acid sequence of HMGB1, or one or more amino acid sequences of all or part of these amino acid sequences. By deleting, substituting, inserting, adding or modifying one (usually 1 to 8, preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2) amino acid residues Among the obtained amino acid sequences, an amino acid sequence consisting of three consecutive amino acid residues can be considered, so a tripeptide consisting of an amino acid sequence consisting of these three consecutive amino acid residues, or other amino acids or A peptide or the like can be considered as the minimum immunogen unit of the anti-HMGB1 antibody in the present invention.

As the immunogen, peptides or proteins containing all or part of the amino acid sequence of HMGB1, or 1 to several (usually 1 to 8) in the whole or part of the amino acid sequence of HMGB1 , preferably 1 to 6, more preferably 1 to 4, particularly preferably 1 to 2) amino acid residues are deleted, substituted, inserted, added or modified. Alternatively, proteins and the like can be obtained from body fluids, cells, tissues, organs, etc. of humans, etc. by extraction, purification, etc. by known methods.

In the present invention, the method for obtaining a peptide or protein containing all or part of the amino acid sequence of HMGB1 is not particularly limited, and may be obtained by any method, for example, by a known method. can be done.

The immunogen can be synthesized by a peptide synthesis method such as a liquid phase method or a solid phase method, and an automatic peptide synthesizer may be used. Chemistry IV", Tokyo Kagaku Doujin, 1975, Izumiya et al., "Fundamentals and Experiments of Peptide Synthesis", Maruzen, 1985, Japanese Biochemical Society, "Sequel Biochemical Experiment Lecture 2 Protein Chemistry Part", Tokyo Kagaku Doujin, 1987 It can be synthesized according to the method described in 2003, etc., and it is also easy to produce mutants by subjecting the above amino acid sequences to deletion, substitution, insertion or addition.
Modifications such as introduction of non-natural amino acids, chemical modification of each amino acid residue, and introduction of cysteine residues to cyclize the molecule to stabilize the structure may also be performed.

Furthermore, the above-mentioned immunogen may be prepared from DNA or RNA having a corresponding nucleic acid base sequence using genetic engineering technology. Doujin, 1986, Japanese Biochemical Society ed. "Sequel Biochemical Experiment Lecture 1 Gene Research Method II", Tokyo Kagaku Dojin, 1986, Japanese Biochemical Society ed. "Sequel Biochemical Experiment Lecture 1 Gene Research Method III", Tokyo Kagaku Dojin, 1987 etc. may be referred and prepared.

By the way, when the immunogen is a low-molecular-weight substance, it is common to immunize an animal or the like with an immunogen-binding carrier (carrier) bound to the immunogen. There is also a report that a specific antibody against this was produced as a source (Kiyama et al., "The 112th Annual Meeting of the Pharmaceutical Society of Japan, 3", p. 122, published in 1992). It is not mandatory to use

When an immunogen-binding carrier (carrier) is used for antibody production, the carrier (carrier) includes keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken serum albumin, poly-L- Known immunogen binding carriers such as lysine, polyalanyllysine, dipalmityllysine, tetanus toxoid and polysaccharides can be used.

Methods for conjugating the immunogen and immunogen-binding carrier (carrier) include the glutaraldehyde method, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide method, the maleimidobenzoyl-N-hydroxysuccinimide ester method, and the bisdiazotization method. Known conjugation methods such as the benzidine method or the N-succimidyl-3-(2-pyridyldithio)propionic acid method can be used.
Immunogens can also be prepared by adsorbing immunogens to immunogen-binding carriers such as nitrocellulose particles, polyvinylpyrrolidone or liposomes.

(3) Method for Preparing Polyclonal Anti-HMGB1 Antibody A polyclonal antibody that can specifically bind to HMGB1, ie, an anti-HMGB1 antibody that is a polyclonal antibody, can be prepared by the following procedure.

As an immunogen for production of this polyclonal anti-HMGB1 antibody, the immunogen described above can be used.

The above immunogen or a conjugate of the above immunogen and an immunogen-binding carrier (carrier) is administered to a mammal (mouse, guinea pig, hamster, rabbit, rat, sheep, goat, cow, horse, donkey, camel, etc.). ) or birds (such as chickens, ducks, or ostriches).

By the way, the immunological dose of the immunogen or the conjugate of the immunogen and an immunogen-binding carrier (carrier) depends on the immunogen, the immunogen-binding carrier (carrier), the type of animal to be immunized, the site of immunization injection, etc. In the case of mice, 0.1 μg to 5 mg of the immunogen or a conjugate of the immunogen and an immunogen-binding carrier (carrier) is injected per mouse. is preferred.

It is preferable to add and mix the above-mentioned immunogen or the above-mentioned immunogen and immunogen-binding carrier (carrier) with an adjuvant for immunization injection.
Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, chemically synthesized adjuvant, and pertussis adjuvant can be used.
The immunization injection may be performed subcutaneously, intravenously, intraperitoneally, or at a site such as the back.

After the initial immunization, subcutaneous, intravenous, intraperitoneal or dorsal site subcutaneous, intravenous, intraperitoneal or dorsal booster injections of the above immunogen or a conjugate of the above immunogen and an immunogen-binding carrier (carrier). do.
The number of booster injections is generally 2 to 6 times.
Also in this case, it is preferable that the above-mentioned immunogen or a combination of the above-mentioned immunogen and an immunogen-binding carrier (carrier) is added and mixed with an adjuvant and injected for additional immunization.
After the initial 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 antiserum containing antibodies.

This antiserum is subjected to salting-out with ammonium sulfate, sodium sulfate, etc., ion-exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods to purify the antibody to obtain a polyclonal antibody.

A polyclonal antibody that can bind to HMGB1 (an anti-HMGB1 antibody that is a polyclonal antibody) can be obtained by the above procedure.

By the way, when an animal or the like is immunized with a conjugate of an immunogen and an immunogen-binding carrier (carrier), antibodies against this immunogen-binding carrier (carrier) are present in the polyclonal antibodies obtained. Therefore, it is preferable to remove antibodies from such immunogen-binding carriers (carriers).

As this removal treatment method, an immunogen-binding carrier (carrier) is added to the obtained polyclonal antibody solution to remove aggregates formed, or an immunogen-binding carrier (carrier) is added to an insolubilized solid phase. A method of immobilizing and removing by affinity chromatography or the like can be used.

(4) Method for preparing anti-HMGB1 antibody, which is a monoclonal antibody As a method for obtaining a monoclonal antibody capable of specifically binding to HMGB1, that is, an anti-HMGB1 antibody, which is a monoclonal antibody, the cell fusion method of Keller et al. Koehler et al., Nature, vol.256, pp.495-497, 1975), or antibody-producing cells such as tumorigenic cells caused by viruses such as Epstan-Barr virus.
Monoclonal antibodies can be prepared by the cell fusion method by the following procedures.

First, the immunogen or a conjugate of the immunogen and an immunogen-binding carrier (carrier) is fed to mammals (mouse, nude mouse, rat, etc., for example, inbred mouse BALB/c) or birds ( chickens, etc.). The immunization dose of the above-mentioned immunogen or the combination of the above-mentioned immunogen and an immunogen-binding carrier (carrier) can be appropriately determined according to the type of animal to be immunized, the site of immunization injection, and the like. In some cases, 0.1 μg to 5 mg of the above-mentioned immunogen or a combination of the above-mentioned immunogen and an immunogen-binding carrier (carrier) is preferably injected per animal.

The immunogen or the immunogen-binding carrier (carrier) is preferably added and mixed with an adjuvant for immunization injection. Known adjuvants such as complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant and pertussis adjuvant can be used.
The immunization injection may be performed subcutaneously, intravenously, intraperitoneally, or at a site such as the back.

After the initial immunization, subcutaneous, intravenous, intraperitoneal or dorsal site subcutaneous, intravenous, intraperitoneal or dorsal booster injections of the above immunogen or a conjugate of the above immunogen and an immunogen-binding carrier (carrier). do. The number of booster injections is generally 2 to 6 times. In this case as well, the immunogen or the conjugate of the immunogen and an immunogen-binding carrier (carrier) is preferably added and mixed with an adjuvant for booster injection.

After the initial 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, the immunogen, or the immunogen and the immunogen-binding carrier (carrier) is dissolved in physiological saline (0.9% aqueous sodium chloride solution) and injected intravenously or intraperitoneally for final immunization. 3 to 5 days after the final immunization, antibody-producing cells such as splenocytes, lymph node cells or peripheral lymphocytes from the immunized animal are obtained.

Antibody-producing cells obtained from this immunized animal are fused with myeloma cells (myeloma cells) of mammals (mice, nude mice, rats, etc.). Cell lines deficient in enzymes such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) are preferred, for example the HGPRT deficient cell line from BALB/c mice, strain P3-X63-Ag8 (ATCC TIB9), P3-X63-Ag8-U1 strain (Cancer Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8 653 strain (JCRB 0028) Alternatively, SP2/O-Ag-14 strain (JCRB 0029) or the like can be used.

Cell fusion can be performed using fusion promoters such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
When myeloma cells are HGPRT-deficient strains or TK-deficient strains, fusion of cells having antibody-producing ability and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine Only cells (hybridomas) can be selectively cultured and grown.

The thus-obtained hybridoma culture supernatant is subjected to immunization by ELISA, Western blotting, or the like using the immunogen, the conjugate of the immunogen and an immunogen-binding carrier (carrier), or HMGB1. A hybridoma that produces an antibody that binds to HMGB1 can be selected by measuring by a chemical assay.

By combining this hybridoma selection method with a known cloning method such as the limiting dilution method, a cell line producing an antibody that binds to HMGB1 can be isolated and obtained.

This monoclonal antibody-producing cell line is cultured in an appropriate medium, and a monoclonal antibody (anti-HMGB1 antibody, which is a monoclonal antibody) can be obtained from the culture supernatant. may be used, and in this case, it is preferable in terms of facilitating purification of the antibody, and a medium such as DMEM medium, RPMI1640 medium, or ASF medium 103 can be used.
In addition, a monoclonal antibody-producing cell line is injected into the peritoneal cavity of mammals that are compatible with this and have been pre-stimulated with pristane or the like. antibodies) can also be obtained.

The monoclonal antibody thus obtained is purified by methods such as salting-out with ammonium sulfate or sodium sulfate, ion-exchange chromatography, gel filtration, affinity chromatography, or a combination of these methods. Monoclonal antibodies (anti-HMGB1 antibodies that are monoclonal antibodies) can be obtained.

2. Carrier The "carrier" in the "anti-HMGB1 antibody-immobilized carrier" includes, for example, polystyrene, styrene-styrenesulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate 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, erythrocytes, silica, alumina, carbon black, metallic compounds, metals, ceramics, magnetic particles, and the like.

In the present invention, particles are preferred as carriers.

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

3. Immobilization In the present invention, the "anti-HMGB1 antibody-immobilized carrier" is obtained by adsorbing and binding the anti-HMGB1 antibody and the carrier by a known method such as physical adsorption, chemical binding, or a combination thereof. Thus, an anti-HMGB1 antibody can be prepared by being "immobilized" on a carrier.

In the case of the physical adsorption method, the anti-HMGB1 antibody and the carrier are mixed and brought into contact in a solution such as a buffer, or the anti-HMGB1 antibody dissolved in a buffer or the like is brought into contact with the carrier, according to known methods. , the immobilization of the anti-HMGB1 antibody to the carrier can be performed.

In addition, when the chemical binding method is used, the Japanese Society of Clinical Pathology ed. Ed. "Shinsei Kagaku Jikken Koza 1 Protein IV", published by Tokyo Kagaku Dojin, 1991, anti-HMGB1 antibody and carrier are combined with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imidoester or maleimide. The anti-HMGB1 antibody can be immobilized on the carrier by mixing and contacting with and reacting the anti-HMGB1 antibody with the respective amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, or the like of the carrier.

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

In addition, if it is necessary to further treat the surface of the carrier on which the anti-HMGB1 antibody has been immobilized, bovine serum albumin (BSA), casein, or gelatin may be added to the surface of the carrier to which the anti-HMGB1 antibody has been immobilized. The carrier may be blocked (masked) by a known method such as contacting and coating with a protein such as ovalbumin or a salt thereof, a surfactant, or skim milk powder.

4. Samples Samples in the present invention include body fluids such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, sweat, tears, ascites or amniotic fluid; feces; organs such as blood vessels or liver; Any biological sample that may contain HMGB1, such as an organ, tissue or cell extract, is eligible.

The form of the sample used for the measurement is preferably a liquid. If the sample is not liquid, it may be treated as a liquid sample by subjecting it to a known method such as extraction or solubilization.

Moreover, the sample may be subjected to a concentration treatment, if necessary.

Also, the sample may be diluted by adding a diluent before the measurement.
For example, dilution may be performed by adding a diluent to the sample prior to contacting and binding the sample with the anti-HMGB1 antibody.
Various aqueous solvents can be used as this diluent.
For example, aqueous solvents such as water, physiological saline, tris(hydroxymethyl)aminomethane buffer [Tris buffer], phosphate buffer, and various buffers such as phosphate buffered physiological saline can be used.
The pH of this buffer is preferably in the range of pH5-pH10.

5. Measurement of Aggregates of Anti-HMGB1 Antibody-Immobilized Carriers The concentration of HMGB1 in the sample is measured by contacting the "carrier" and measuring the aggregate of the "anti-HMGB1 antibody-immobilized carrier" bound via HMGB1.

That is, the "HMGB1 in the sample" and the "anti-HMGB1 antibody-immobilized carrier" are brought into contact to form an immune complex aggregate of the "anti-HMGB1 antibody-immobilized carrier" bound via HMGB1. The transmitted light or scattered light is measured by an optical method or visually. In other words, it measures the formation of aggregates of "complexes due to antigen-antibody reaction". (Agglutination method)

In the present invention, 0.001% (w/v) to 0.01% (w/v) of glycerol is added to the contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier". A propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) glycerin/propylene oxide adduct is coexistent.

Methods for measuring aggregates of the carrier on which the anti-HMGB1 antibody is immobilized include, for example, the latex turbidimetric method, the latex agglutination method, the hemagglutination method, the particle agglutination method, and the like.

Measurement of aggregates of a carrier on which an anti-HMGB1 antibody is immobilized, production of immune complex aggregates such as latex turbidimetric method, latex agglutination method, hemagglutination method or particle agglutination method, transmission light and scattering When light is measured optically or visually, phosphate buffer, glycine buffer, Tris buffer, Good's buffer, or the like can be used as a solvent.

When the latex turbidimetric method is used as the measurement principle, the particle size of the latex particles used as a carrier is not particularly limited, but the latex particles combine via the substance to be measured (HMGB1) to form aggregates. The average particle size of the latex particles is preferably 0.04 to 1 μm for reasons such as the degree of aggregation and ease of measurement of the aggregates produced.

Further, when the latex turbidimetric method is used as the measurement principle, the concentration of the latex particles immobilized with the anti-HMGB1 antibody includes the concentration of HMGB1 in the sample, the distribution density of the antibody in the present invention on the surface of the latex particles, The optimal concentration varies depending on various conditions such as the particle size of the latex particles and the mixing ratio of the sample and the measurement reagent, so it cannot be generalized.

However, usually, a sample and a measurement reagent are mixed, and an antigen-antibody reaction is performed between an "anti-HMGB1 antibody" immobilized on latex particles and "HMGB1" contained in the sample. The concentration of the HMGB1 antibody-immobilized latex particles is 0.005 to 1% (w/v) at the time of contact between the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized latex particles. In this case, the concentration of "anti-HMGB1 antibody" is such that when "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized latex particles" come into contact with each other, "immobilized latex particles" are included in the reagent for measuring HMGB1 in the sample.

In addition, when the measurement principle is an indirect agglutination method such as a latex agglutination method, a hemagglutination method, or a particle agglutination method, the particle size of the particles used as a carrier is not particularly limited, but the average particle size is It 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 1-10, more preferably in the range of 1-2.

Examples of containers used for measurement when indirect agglutination methods such as latex agglutination method, hemagglutination method, or particle agglutination method are used as the principle of measurement include glass, polystyrene, polyvinyl chloride, and polymethacrylate. Test tubes, microplates (microtiter plates) or trays, etc., can be mentioned. It is preferable that the bottom surface of the solution containing portion of these containers (wells of microplates, etc.) be U-shaped, V-shaped, UV-shaped, or the like, sloping from the center of the bottom to the periphery.

The measurement can be performed by a known method or the like. For example, when measuring by an optical method, the sample is reacted with the "anti-HMGB1 antibody-immobilized carrier", and the endpoint method or rate method is used. measures transmitted light and scattered light.
In the case of visual measurement, the sample is allowed to react with the "anti-HMGB1 antibody-immobilized carrier" in the container such as a plate or microplate, and the state of agglutination is visually determined. In addition, you may measure using apparatuses, such as a microplate reader, instead of this measuring visually.

An example of measurement operation is given below.
For example, first, when the measurement reagent containing the "anti-HMGB1 antibody-immobilized carrier" and the "HMGB1 in the sample" and the "anti-HMGB1 antibody-immobilized carrier (latex particles)" come into contact, 0.0. 001% (w/v) to 0.01% (w/v) of glycerin-propylene oxide-ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of glycerin-propylene A measurement reagent containing a buffer solution (aqueous solvent) containing the glycerin/propylene oxide/ethylene oxide adduct or the glycerin/propylene oxide adduct at a concentration that allows the coexistence of the oxide adduct is prepared and prepared.

Next, for example, when the sample is in contact with "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier (latex particles)", 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 at a concentration that allows the coexistence of the glycerin/propylene oxide adduct. A buffer solution (aqueous solvent) containing a propylene oxide/ethylene oxide adduct or a glycerin/propylene oxide adduct” is mixed with a measurement reagent, and then this mixture is mixed with a “carrier on which an anti-HMGB1 antibody is immobilized”. A reagent for measurement is mixed to bring "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier" into contact with each other.
In the present invention, 0.001% (w/v) to 0.01% (w/v) glycerol- A propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is coexistent.

By contacting "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier", "anti-HMGB1 antibody" of "anti-HMGB1 antibody-immobilized carrier" and "HMGB1" contained in the sample of the antigen-antibody reaction.
Then, an aggregate (...-[HMGB1]-[anti-HMGB1 antibody-immobilized carrier]-[HMGB1 ]-[Carrier on which anti-HMGB1 antibody is immobilized]-[HMGB1]-...) is measured.

The measurement of the produced aggregates is carried out by measuring the absorbance of the transmitted light or scattered light of the reaction mixture during the measurement reaction in which the aggregates are present, by the endpoint method or the rate method. .

Then, the measured values such as absorbance obtained by measuring the sample are compared with the measured values such as absorbance obtained by measuring a standard substance (a sample with a known concentration of HMGB1), and Calculate the concentration (quantitative value) of HMGB1.

In addition, the measurement of the absorbance of transmitted light or scattered light may be performed by measuring transmitted light or scattered light, and may be one-wavelength measurement or two-wavelength measurement (two difference or ratio by wavelength).

The measurement wavelength is generally selected from 340 nm to 1,000 nm.

In addition, the measurement of HMGB1 in the present invention may be performed manually, or may be performed using a device such as a measuring device.

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

In addition, the measurement of HMGB1 in the present invention may be performed by a one-step method (single-reagent method) or by a method including a plurality of operation steps such as a two-step method (two-reagent method).

In the following, a more specific description will be given by taking as an example the case of measuring HMGB1 in a sample by a method using the latex nephelometric method as the measurement principle.

[1] First, the following reagents for measuring HMGB1 are prepared and prepared.
First reagent:
0.001% (w/v) to 0.01% (w/v) of glycerol/propylene oxide at the time of contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier (latex particles)"・Ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of the glycerin/propylene oxide/ethylene oxide adduct or glycerin at a concentration that allows the coexistence of the glycerin/propylene oxide adduct・Buffer containing propylene oxide adduct (aqueous solvent)
Second reagent:
Buffer containing "anti-HMGB1 antibody-immobilized latex particles"

[2] A given amount of a sample such as serum and a given amount of the first reagent are mixed, and the mixture is allowed to stand at a given temperature for a given period of time.
Note that the mixing ratio (quantity ratio) of the sample and the first reagent may be appropriately selected.
Moreover, the temperature during the standing is preferably a constant temperature within the range of room temperature (1° C. to 30° C.) or slightly warm (30° C. to 40° C.). (For example, 37°C, etc.)

[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, and the reaction mixture is left to stand at a certain temperature for a certain period of time.
As a result, the "HMGB1 in the sample" and the "latex particles on which the anti-HMGB1 antibody is immobilized" are brought into contact.
In the present invention, 0.001% (w/v) to 0.01% (w/v) is added when the "HMGB1 in the sample" and the "anti-HMGB1 antibody-immobilized latex particles" contact each other. of glycerin/propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of glycerin/propylene oxide adduct.
Moreover, the amount of the second reagent to be added may be appropriately selected.
The temperature during the standing is preferably a constant temperature within the range of room temperature (1° C. to 30° C.) or slightly warm (30° C. to 40° C.). (For example, 37°C, etc.)
Furthermore, the standing time 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.

By adding and mixing the second reagent to the mixed solution of the sample and the first reagent, an antigen-antibody reaction between the "anti-HMGB1 antibody immobilized on the latex particles" and the "HMGB1 contained in the sample" is caused. .

Then, by this antigen-antibody reaction, "...-[HMGB1]-[anti-HMGB1 antibody-immobilized latex particles]-[HMGB1]-[anti-HMGB1 antibody-immobilized latex particles]-[HMGB1]-. . . ” and the like are formed, and aggregates of “anti-HMGB1 antibody-immobilized latex particles” bound via HMGB1 are generated.

[4] Then, in an analyzer, a spectrophotometer, or the like, the reaction mixture is irradiated with light, and a decrease in transmitted light intensity (increase in absorbance ) or by measuring the increase in scattered light intensity, the amount of the complex aggregate produced, that is, the amount of HMGB1 contained in the sample is determined.

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

6. Glycerin/Propylene Oxide/Ethylene Oxide Adduct (1) Method for Measuring HMGB1 in Sample, Method for Suppressing Non-Specific Aggregation of Anti-HMGB1 Antibody-Immobilized Carrier, and Suppression of Rise in Reagent Blank When Measuring HMGB1 in Sample Method In the method of the present invention for measuring HMGB1 in a sample, the method for suppressing non-specific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized, and the method for suppressing an increase in reagent blank when measuring HMGB1 in a sample, 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) of glycerin/propylene oxide adduct is coexistent.

That is, 0.001% (w/v) to 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide is added to the contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier". Coexistence of the adduct or 0.0054% (w/v) to 0.022% (w/v) glycerol/propylene oxide adduct with "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier" Let

Here, 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.

The concentration of the glycerin/propylene oxide/ethylene oxide adduct at the time of contact between “HMGB1 in the sample” and “anti-HMGB1 antibody-immobilized carrier” was determined by shaking the “anti-HMGB1 antibody-immobilized carrier” during transport. Suppresses non-specific agglutination of the "anti-HMGB1 antibody-immobilized carrier" by being shaken for some reason, and suppresses an increase in the reagent blank caused by the agglutination. In terms of the effect of being able to /v) is more preferred.

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).

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

In the present invention, non-specific aggregation of the "anti-HMGB1 antibody-immobilized carrier" is caused by shaking the "anti-HMGB1 antibody-immobilized carrier" during transportation or by shaking for other reasons. From the viewpoint of the effect of suppressing the occurrence of and suppressing the increase in the reagent blank caused by the aggregation, in the general formula (1), a + b + c is 5 mol to 18 mol, and d + e + f is 7 mol. ˜21 mol and g+h+i is preferably between 25 mol and 65 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, from the viewpoint of the above effect, in the general formula (1), it is more preferable that a + b + c is 7 mol to 16 mol, d + e + f is 10 mol to 19 mol, and g + h + i is 35 mol to 55 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, in terms of the above effect, in the general formula (1), a + b + c is 10 mol to 13 mol, d + e + f is 12 mol to 17 mol, and g + h + i is more preferably 40 mol to 50 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, from the viewpoint of the above effect, in the general formula (1), it is particularly preferable that a + b + c is 11 mol to 12 mol, d + e + f is 14 mol to 15 mol, and g + h + i is 44 mol to 45 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

From the viewpoint of the above effect, it is particularly preferable that a+b+c is 11.5 mol, d+e+f is 14.3 mol, and g+h+i is 44.1 mol in general formula (1). (The number of moles is the average number of moles added.) The numbers in { } are random additions.
In this general formula (1), a + b + c is 11.5 mol, d + e + f is 14.3 mol, and g + h + i is 44.1 mol [(The number of moles is the average number of added moles.) { } is random addition. ] Examples of the glycerin/propylene oxide/ethylene oxide adduct include “Sannics FA-195” (molecular weight: 3,370, CAS No. 9082-00-2) sold by Sanyo Chemical Industries, Ltd. .

(2) Reagent for measuring HMGB1 in a sample In the reagent for measuring HMGB1 in a sample of the present invention, 0.001% (w/v) of HMGB1 in the sample is contacted with the carrier on which the anti-HMGB1 antibody is immobilized. -0.01% (w/v) of glycerin/propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of glycerin/propylene oxide adduct in the coexistence The "measuring reagent for HMGB1 in a sample" contains the glycerin/propylene oxide/ethylene oxide adduct or the glycerin/propylene oxide adduct at a concentration such that

That is, 0.001% (w/v) to 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide is added to the contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier". The adduct or 0.0054% (w/v) to 0.022% (w/v) of the glycerol/propylene oxide adduct is allowed to coexist with "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier." The "measuring reagent for HMGB1 in a sample" contains the glycerin/propylene oxide/ethylene oxide adduct or the glycerin/propylene oxide adduct at a concentration that can be obtained.

Here, 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.

The concentration of the glycerin/propylene oxide/ethylene oxide adduct at the time of contact between “HMGB1 in the sample” and “anti-HMGB1 antibody-immobilized carrier” was determined by shaking the “anti-HMGB1 antibody-immobilized carrier” during transport. Suppresses non-specific agglutination of the "anti-HMGB1 antibody-immobilized carrier" by being shaken for some reason, and suppresses an increase in the reagent blank caused by the agglutination. In terms of the effect of being able to /v) is more preferred.
In the reagent for measuring HMGB1 in a sample of the present invention, the above concentration of glycerin/propylene oxide/ethylene oxide adduct can coexist when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. The above-mentioned glycerin/propylene oxide/ethylene oxide adduct of is contained in a reagent for measuring HMGB1 in a sample.

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).

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

In the present invention, non-specific aggregation of the "anti-HMGB1 antibody-immobilized carrier" is caused by shaking the "anti-HMGB1 antibody-immobilized carrier" during transportation or by shaking for other reasons. From the viewpoint of the effect of suppressing the occurrence of and suppressing the increase in the reagent blank caused by the aggregation, in the general formula (1), a + b + c is 5 mol to 18 mol, and d + e + f is 7 mol. ˜21 mol and g+h+i is preferably between 25 mol and 65 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, from the viewpoint of the above effect, in the general formula (1), it is more preferable that a + b + c is 7 mol to 16 mol, d + e + f is 10 mol to 19 mol, and g + h + i is 35 mol to 55 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, in terms of the above effect, in the general formula (1), a + b + c is 10 mol to 13 mol, d + e + f is 12 mol to 17 mol, and g + h + i is more preferably 40 mol to 50 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

Further, from the viewpoint of the above effect, in the general formula (1), it is particularly preferable that a + b + c is 11 mol to 12 mol, d + e + f is 14 mol to 15 mol, and g + h + i is 44 mol to 45 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions.

From the viewpoint of the above effect, it is particularly preferable that a+b+c is 11.5 mol, d+e+f is 14.3 mol, and g+h+i is 44.1 mol in general formula (1). (The number of moles is the average number of moles added.) The numbers in { } are random additions.
In this general formula (1), a + b + c is 11.5 mol, d + e + f is 14.3 mol, and g + h + i is 44.1 mol [(The number of moles is the average number of added moles.) { } is random addition. ] Examples of the glycerin/propylene oxide/ethylene oxide adduct include “Sannics FA-195” (molecular weight: 3,370, CAS No. 9082-00-2) sold by Sanyo Chemical Industries, Ltd. .

In addition, in the reagent for measuring HMGB1 in the sample of the present invention, "0.001% (w / v) to 0.01% (w / v), preferably from 0.0016% (w/v) to 0.0054% (w/v), more preferably from 0.0016% (w/v) to 0.0022% (w/v) Containing the glycerin/propylene oxide/ethylene oxide adduct at a concentration that allows the coexistence of the glycerin/propylene oxide/ethylene oxide adduct in the "measuring reagent for HMGB1 in the sample" will be described below.

When the reagent for measuring HMGB1 in the sample is a single reagent system, the concentration of the glycerin/propylene oxide/ethylene oxide adduct at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized is a% (w/v). and the weight of the glycerin/propylene oxide/ethylene oxide adduct at that time is bg (grams), the volume of the reagent for measuring HMGB1 in the sample at that time is cmL, and the volume of the sample at that time is dmL. % (w/v) = (bg (grams) x 100) ÷ (cmL + dmL).
Converting this formula, bg (grams) x 100 = a% (w/v) x (cmL + dmL).
Dividing both sides by cmL yields (bg (grams) x 100) / cmL = {a% (w/v) x (cmL + dmL)} / cmL.
Then, 0.001% (w/v) to 0.01% (w/v) of a glycerin/propylene oxide/ethylene oxide adduct coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. Assuming that the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the reagent for measuring HMGB1 in the sample is e% (w/v), e% (w/v) = (bg (grams) x 100) ÷ cmL, so from the above formula, e% (w/v) = (bg (grams) x 100) ÷ cmL = {a% (w/v) x (cmL + dmL)} ÷ cmL.

Further, when the reagent for measuring HMGB1 in the sample is a two-reagent system and the carrier on which the anti-HMGB1 antibody is immobilized is contained in the second reagent of the reagent for measuring HMGB1 in the sample, HMGB1 in the sample and Assuming that the concentration of the glycerin/propylene oxide/ethylene oxide adduct at the time of contact with the carrier on which the anti-HMGB1 antibody is immobilized is a% (w/v), the glycerin in the second reagent of the HMGB1 measurement reagent in the sample at that time・The weight of the propylene oxide/ethylene oxide adduct is b2 g (grams), the volume of the first reagent for measuring HMGB1 in the sample is c1 mL, and the second reagent for measuring HMGB1 in the sample is c1 mL. Assuming that the volume of the sample is c2 mL and the volume of the sample at that time is d mL, a% (w/v) = (b2 g (grams) x 100) ÷ (c1 mL + c2 mL + d mL).
Then, 0.001% (w/v) to 0.01% (w/v) of a glycerin/propylene oxide/ethylene oxide adduct coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. Assuming that the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the reagent for measuring HMGB1 in the sample is e2% (w/v), b2g (gram) = (e2% (w /v)×c2 mL)÷100.
By substituting b2g (grams) in this formula for b2g (grams) in the previous formula, a% (w/v) = (e2% (w/v) x c2mL)/(c1mL + c2mL + dmL).
Converting this formula yields e2% (w/v)={a% (w/v)×(c1 mL+c2 mL+d mL)}/c2 mL.

When the reagent for measuring HMGB1 in the sample is a two-reagent system and the carrier on which the anti-HMGB1 antibody is immobilized is contained in the second reagent of the reagent for measuring HMGB1 in the sample, HMGB1 in the sample and Assuming that the concentration of the glycerin/propylene oxide/ethylene oxide adduct at the time of contact with the carrier on which the anti-HMGB1 antibody is immobilized is a% (w/v), the glycerin in the first reagent for measuring HMGB1 in the sample at that time・Assuming that the weight of the propylene oxide/ethylene oxide adduct is b1 g (grams), and the weight of the glycerin/propylene oxide/ethylene oxide adduct in the second reagent of the reagent for measuring HMGB1 in the sample at that time is b2 g (grams), Let c1 mL be the volume of the first reagent for measuring HMGB1 in the sample at that time, let c2 mL be the volume of the second reagent for measuring HMGB1 in the sample at that time, and let d mL be the volume of the sample at that time, a % (w/v) = {(b1 g (gram) + b2 g (gram)) x 100} ÷ (c1 mL + c2 mL + dmL).
Converting this formula, (b1g (grams) + b2g (grams)) x 100 = a% (w/v) x (c1mL + c2mL + dmL).
Converting this formula, b1g (grams) x 100 + b2g (grams) x 100 = a% (w/v) x (c1mL + c2mL + dmL).
Converting this formula, b1g (grams)×100={a% (w/v)×(c1mL+c2mL+dmL)}−(b2g (grams)×100).
Dividing both sides by c1mL yields (b1g (grams) x 100) ÷ c1mL = [{a% (w/v) x (c1mL + c2mL + dmL)} - (b2g (grams) x 100)] ÷ c1mL.
Then, 0.001% (w/v) to 0.01% (w/v) of a glycerin/propylene oxide/ethylene oxide adduct coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. Assuming that the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the first reagent for measuring HMGB1 in the sample is e1% (w/v), e1% (w/v) = (b1 g (grams) × 100) ÷ c1 mL, so from the above formula, e1% (w / v) = (b1 g (grams) × 100) ÷ c1 mL = [{a% (w / v) × (c1 mL + c2 mL + dmL)} −(b2 g (grams)×100)]÷c1 mL.

Also, when converting the above formula b1g (gram) × 100 + b2g (gram) × 100 = a% (w/v) × (c1mL + c2mL + dmL), b2g (gram) × 100 = {a% (w/v) × (c1mL + c2mL + dmL )}-(b1g (grams)×100).
Dividing both sides by c2mL yields (b2g (grams) x 100) ÷ c2mL = [{a% (w/v) x (c1mL + c2mL + dmL)} - (b1g (grams) x 100)] ÷ c2mL.
Then, 0.001% (w/v) to 0.01% (w/v) of a glycerin/propylene oxide/ethylene oxide adduct coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. Assuming that the concentration of the glycerin-propylene oxide-ethylene oxide adduct in the second reagent of the reagent for measuring HMGB1 in the sample is e2% (w/v), e2% (w/v) = (b2 g (grams) x 100) ÷ c2mL, so from the above formula, e2% (w/v) = (b2g (grams) x 100) ÷ c2mL = [{a% (w/v) x (c1mL + c2mL + dmL)} −(b1 g (grams)×100)]÷c2 mL.

7. Glycerin/propylene oxide adduct (1) Method for measuring HMGB1 in a sample, method for suppressing non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized, and method for suppressing an increase in reagent blank when measuring HMGB1 in a sample Present In the method for measuring HMGB1 in a sample, the method for suppressing non-specific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized, and the method for suppressing an increase in the reagent blank when measuring HMGB1 in a sample, according to the invention, HMGB1 in the sample and 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) of glycerin/propylene oxide adduct is coexistent.

That is, 0.001% (w/v) to 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide is added to the contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier". Coexistence of the adduct or 0.0054% (w/v) to 0.022% (w/v) glycerol/propylene oxide adduct with "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier" Let

Here, in the present invention, the glycerin/propylene oxide adduct is not particularly limited, and any glycerin/propylene oxide adduct can be used.

The concentration of the glycerin-propylene oxide adduct at the time of contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier" is determined by shaking the "anti-HMGB1 antibody-immobilized carrier" during transport. Shaking due to this or other reasons can suppress non-specific agglutination of the "anti-HMGB1 antibody-immobilized carrier" and suppress an increase in the reagent blank caused by the agglutination. From the viewpoint of the effect that it can be made, it is preferably 0.0076% (w/v) to 0.022% (w/v).

In the present invention, the glycerin/propylene oxide adduct is preferably a glycerin/propylene oxide adduct represented by the following general formula (2).

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

In the present invention, non-specific aggregation of the "anti-HMGB1 antibody-immobilized carrier" is caused by shaking the "anti-HMGB1 antibody-immobilized carrier" during transportation or by shaking for other reasons. In general formula (2), a+b+c is preferably 4 mol to 10 mol, from the viewpoint of the effect of suppressing the occurrence of and suppressing the increase in reagent blank caused by the agglutination. (The number of moles is the average number of added moles.)

Further, from the viewpoint of the above effect, it is more preferable that a+b+c in general formula (2) is 4.5 mol to 8 mol. (The number of moles is the number of molar additions.)

Further, from the viewpoint of the above effect, it is more preferable that a+b+c in general formula (2) is 5 mol to 6 mol. (The number of moles is the average number of added moles.)

From the viewpoint of the above effect, it is particularly preferable that a+b+c in general formula (2) is 5.6 mol. (The number of moles is the average number of added moles.)
In this general formula (2), a + b + c is 5.6 mol (the number of moles is the average number of moles added). -400" (molecular weight: 420, CAS No. 25791-96-2).

(2) Reagent for measuring HMGB1 in a sample In the reagent for measuring HMGB1 in a sample of the present invention, 0.001% (w/v) of HMGB1 in the sample is contacted with the carrier on which the anti-HMGB1 antibody is immobilized. -0.01% (w/v) of glycerin/propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of glycerin/propylene oxide adduct in the coexistence The "measuring reagent for HMGB1 in a sample" contains the glycerin/propylene oxide/ethylene oxide adduct or the glycerin/propylene oxide adduct at a concentration such that

That is, 0.001% (w/v) to 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide is added to the contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier". The adduct or 0.0054% (w/v) to 0.022% (w/v) of the glycerol/propylene oxide adduct is allowed to coexist with "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier." The "measuring reagent for HMGB1 in a sample" contains the glycerin/propylene oxide/ethylene oxide adduct or the glycerin/propylene oxide adduct at a concentration that can be obtained.

Here, in the present invention, the glycerin/propylene oxide adduct is not particularly limited, and any glycerin/propylene oxide adduct can be used.

The concentration of the glycerin-propylene oxide adduct at the time of contact between "HMGB1 in the sample" and "anti-HMGB1 antibody-immobilized carrier" is determined by shaking the "anti-HMGB1 antibody-immobilized carrier" during transport. Shaking due to this or other reasons can suppress non-specific agglutination of the "anti-HMGB1 antibody-immobilized carrier" and suppress an increase in the reagent blank caused by the agglutination. From the viewpoint of the effect that it can be made, it is preferably 0.0076% (w/v) to 0.022% (w/v).
In the reagent for measuring HMGB1 in a sample of the present invention, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact with each other, the concentration of the glycerin/propylene oxide adduct can coexist. A glycerin-propylene oxide adduct is included in a reagent for measuring HMGB1 in a sample.

In the present invention, the glycerin/propylene oxide adduct is preferably a glycerin/propylene oxide adduct represented by the following general formula (2).

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

In the present invention, non-specific aggregation of the "anti-HMGB1 antibody-immobilized carrier" is caused by shaking the "anti-HMGB1 antibody-immobilized carrier" during transportation or by shaking for other reasons. In general formula (2), a+b+c is preferably 4 mol to 10 mol, from the viewpoint of the effect of suppressing the occurrence of and suppressing the increase in reagent blank caused by the agglutination. (The number of moles is the average number of added moles.)

Further, from the viewpoint of the above effect, it is more preferable that a+b+c in general formula (2) is 4.5 mol to 8 mol. (The number of moles is the number of molar additions.)

Further, from the viewpoint of the above effect, it is more preferable that a+b+c in general formula (2) is 5 mol to 6 mol. (The number of moles is the average number of added moles.)

From the viewpoint of the above effect, it is particularly preferable that a+b+c in general formula (2) is 5.6 mol. (The number of moles is the average number of added moles.)
In this general formula (2), a + b + c is 5.6 mol (the number of moles is the average number of moles added). -400" (molecular weight: 420, CAS No. 25791-96-2).

In addition, in the reagent for measuring HMGB1 in the sample of the present invention, "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 at a concentration that allows the coexistence of the glycerin/propylene oxide adduct with the " The “contained in a reagent for measuring HMGB1 in a sample” will be explained below.

When the reagent for measuring HMGB1 in a sample is a single-reagent system, the concentration of the glycerin-propylene oxide adduct at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized is defined as a% (w/v), If the weight of the glycerin/propylene oxide adduct at that time is bg (grams), the volume of the reagent for measuring HMGB1 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) x 100) / (cmL + dmL).
Converting this formula, bg (grams) x 100 = a% (w/v) x (cmL + dmL).
Dividing both sides by cmL yields (bg (grams) x 100) / cmL = {a% (w/v) x (cmL + dmL)} / cmL.
Then, 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is allowed to coexist when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. When the concentration of the glycerin-propylene oxide adduct in the reagent for measuring HMGB1 in the sample is e% (w/v), e% (w/v) = (bg (gram) x 100) ÷ cmL Therefore, from the above formula, e % (w/v) = (bg (grams) x 100) / cmL = {a % (w/v) x (cmL + dmL)} / cmL.

Further, when the reagent for measuring HMGB1 in the sample is a two-reagent system and the carrier on which the anti-HMGB1 antibody is immobilized is contained in the second reagent of the reagent for measuring HMGB1 in the sample, HMGB1 in the sample and Assuming that the concentration of the glycerin-propylene oxide adduct at the time of contact with the carrier on which the anti-HMGB1 antibody is immobilized is a% (w/v), the glycerin-propylene in the second reagent of the reagent for measuring HMGB1 in the sample at that time Let the weight of the oxide adduct be b2 g (grams), the volume of the first reagent for measuring HMGB1 in the sample at that time be c1 mL, and the volume of the second reagent for measuring HMGB1 in the sample at that time be c2 mL. Assuming that the volume of the sample at that time is dmL, a% (w/v)=(b2g (grams)×100)/(c1mL+c2mL+dmL).
Then, 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is allowed to coexist when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. If the concentration of the glycerin-propylene oxide adduct in the second reagent of the HMGB1 measurement reagent in the sample is e2% (w/v), then b2g (gram) = (e2% (w/v) x c2 mL)/100.
By substituting b2g (grams) in this formula for b2g (grams) in the previous formula, a% (w/v) = (e2% (w/v) x c2mL)/(c1mL + c2mL + dmL).
Converting this formula yields e2% (w/v)={a% (w/v)×(c1 mL+c2 mL+d mL)}/c2 mL.

When the reagent for measuring HMGB1 in the sample is a two-reagent system and the carrier on which the anti-HMGB1 antibody is immobilized is contained in the second reagent of the reagent for measuring HMGB1 in the sample, HMGB1 in the sample and The concentration of the glycerin-propylene oxide adduct at the time of contact with the carrier on which the anti-HMGB1 antibody was immobilized was set to a% (w/v), and the glycerin-propylene in the first reagent of the reagents for measuring HMGB1 in the sample at that time. Let the weight of the oxide adduct be b1 g (grams), let the weight of the glycerin-propylene oxide adduct in the second reagent of the reagent for measuring HMGB1 in the sample at that time be b2 g (grams), and let the weight of HMGB1 in the sample at that time be b2 g (grams). If the volume of the first reagent for measuring HMGB1 in the sample is c1 mL, the volume of the second reagent for measuring 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 )={(b1g (gram)+b2g (gram))×100}÷(c1mL+c2mL+dmL).
Converting this formula, (b1g (grams) + b2g (grams)) x 100 = a% (w/v) x (c1mL + c2mL + dmL).
Converting this formula, b1g (grams) x 100 + b2g (grams) x 100 = a% (w/v) x (c1mL + c2mL + dmL).
Converting this formula, b1g (grams)×100={a% (w/v)×(c1mL+c2mL+dmL)}−(b2g (grams)×100).
Dividing both sides by c1mL yields (b1g (grams) x 100) ÷ c1mL = [{a% (w/v) x (c1mL + c2mL + dmL)} - (b2g (grams) x 100)] ÷ c1mL.
Then, 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is allowed to coexist when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. When the concentration of the glycerin-propylene oxide adduct in the first reagent for measuring HMGB1 in the sample is e1% (w/v), e1% (w/v) = (b1g (gram) x 100) ÷ c1 mL, so from the above formula, e1% (w / v) = (b1 g (grams) x 100) ÷ c1 mL = [{a% (w / v) x (c1 mL + c2 mL + dmL)} - (b2 g ( gram)×100)]÷c1 mL.

Also, when converting the above formula b1g (gram) × 100 + b2g (gram) × 100 = a% (w/v) × (c1mL + c2mL + dmL), b2g (gram) × 100 = {a% (w/v) × (c1mL + c2mL + dmL )}-(b1g (grams)×100).
Dividing both sides by c2mL yields (b2g (grams) x 100) ÷ c2mL = [{a% (w/v) x (c1mL + c2mL + dmL)} - (b1g (grams) x 100)] ÷ c2mL.
Then, 0.0054% (w/v) to 0.022% (w/v) of a glycerin/propylene oxide adduct is allowed to coexist when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact. When the concentration of the glycerin-propylene oxide adduct in the second reagent of the HMGB1 measurement reagent in the sample is e2% (w/v), e2% (w/v) = (b2g (gram) x 100) ÷ c2mL, so from the above formula, e2% (w / v) = (b2g (grams) x 100) ÷ c2mL = [{a% (w / v) x (c1mL + c2mL + dmL)} - (b1g ( gram)×100)]÷c2 mL.

8. Method for Suppressing Non-Specific Aggregation of Anti-HMGB1 Antibody-Immobilized Carrier The "method for suppressing non-specific aggregation of anti-HMGB1 antibody-immobilized carrier" of the present invention comprises immobilizing HMGB1 and anti-HMGB1 antibody in a sample. The concentration of HMGB1 in the sample is measured by contacting the carrier and measuring the aggregate of the carrier on which the anti-HMGB1 antibody bound via HMGB1 is immobilized. 0.001% (w/v) to 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide adduct or 0.0054% ( w/v) to 0.022% (w/v) of glycerin/propylene oxide adduct is coexistent.

This makes it possible to suppress non-specific aggregation of the carrier on which the anti-HMGB1 antibody is immobilized.

"Sample", "anti-HMGB1 antibody-immobilized carrier", "measurement of HMGB1 concentration in sample by measuring aggregates of anti-HMGB1 antibody-immobilized carrier bound via HMGB1", "Coexistence of glycerin/propylene oxide/ethylene oxide adduct" and "coexistence of glycerin/propylene oxide adduct" are as described above.

9. Method for suppressing an increase in reagent blank when measuring HMGB1 in a sample The "method for suppressing an increase in reagent blank when measuring HMGB1 in a sample" of the present invention comprises HMGB1 in a sample and a carrier on which an anti-HMGB1 antibody is immobilized. In the measurement of HMGB1 in the sample, the concentration of HMGB1 in the sample is measured by measuring the aggregate of the carrier on which the anti-HMGB1 antibody bound via HMGB1 is immobilized. 0.001% (w/v) to 0.01% (w/v) of a glycerin/propylene oxide/ethylene oxide adduct or 0.0054% (w/v) at the time of contact with the carrier on which the HMGB1 antibody is immobilized It is characterized by the coexistence of up to 0.022% (w/v) of a glycerin/propylene oxide adduct.

As a result, it is possible to suppress an increase in the reagent blank due to non-specific agglutination in the carrier on which the anti-HMGB1 antibody is immobilized.

"Sample", "anti-HMGB1 antibody-immobilized carrier", "measurement of HMGB1 concentration in sample by measuring aggregates of anti-HMGB1 antibody-immobilized carrier bound via HMGB1", "Coexistence of glycerin/propylene oxide/ethylene oxide adduct" and "coexistence of glycerin/propylene oxide adduct" are as described above.

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

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

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 problem.

One volume of bovine HMGB1 (whole length) as the immunogen was mixed with one volume of FREUND adjuvant (DIFCO LABORATORIES) as a chemically synthesized adjuvant.

(2) Next, mice (BALB/c) were intraperitoneally injected with 300 to 500 μg/mouse/injection of a mixture of the above HMGB1 solution and FREUND complete adjuvant as an immunogen, and 2 weeks and 4 weeks later. , mice were injected intraperitoneally with the mixture of HMGB1 solution described above and incomplete FREUND adjuvant.

(3) Four weeks after the final immunization, a stock solution of bovine HMGB1 (full-length) was boosted with 300 μg/mouse, and the following day, a pair of spleen cells and myeloma cells (P3U1) from the immunized mouse was administered. The mixture was mixed at a ratio of 1 to 10:1, polyethylene glycol [PEG 1500; Roche (Switzerland)] was added in a conventional manner for cell fusion, and grown hybridoma colonies were screened.

Specifically, cell fusion was performed as follows.
The mixed spleen cells and myeloma cells (P3U1) were centrifuged to remove the supernatant, suspended in 1 mL of polyethylene glycol [PEG1500; Roche (Switzerland)] at room temperature for 1 minute, and stirred at 37°C for 1 minute. bottom.

An operation of adding 1 mL of serum-free medium over 1 minute was performed twice, and then 7 mL of serum-free medium was added over 2 minutes.

After washing the cells several times, they 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% CO ₂ .

As a method for selecting grown monoclonal antibody-producing cell lines (fused cell lines), 7 to 14 days after cell fusion, bovine HMGB1 (full length) was immobilized, and the fused cell culture supernatant was used as the primary antibody. It was performed in the system of ELISA method.

Specifically, this ELISA method 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 added to phosphate-buffered saline (0.9% aqueous sodium chloride solution). and phosphate-buffered saline (0.9% sodium chloride aqueous solution) as a control were each prepared in a 96-well microtiter plate [Thermo Fisher Scientific Inc. (USA, State of Illinois)] and allowed to stand at 5°C for 16 to 24 hours (or 37°C for 2 hours) to immobilize each of the HMGB1 and HMGB2 on the wells of the microtiter plate, Furthermore, it was blocked by standing in phosphate buffered saline containing 1% BSA at 5°C for 16 to 24 hours (or 37°C for 2 hours).

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

(iii) Next, 100 μL of each of these fused cell culture supernatant solutions was injected as a sample into the wells of the microtiter plate washed in (ii) above, allowed to stand at 37° C. for 2 hours, and Each of the HMGB1 and HMGB2 immobilized on the wells of the titer plate was allowed to react with the monoclonal antibody contained in each fusion cell culture supernatant solution.

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

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

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

(vii) Next, a coloring solution consisting of 0.045% 3,3′,5,5′-tetramethylbenzidine hydrochloride aqueous solution (pH 2.0) containing 0.2 mM EDTA·disodium, and 5 mM 100 μL of a chromogenic substrate solution prepared by mixing 1:1 with a substrate solution consisting of a 60 mM disodium phosphate aqueous solution (pH 4.3) containing hydrogen oxide, 41 mM citric acid, and 0.2 mM EDTA.disodium. , was injected into each well of the microtiter plate washed in (vi) above, allowed to stand at room temperature for 30 minutes, allowed to react, and developed color.

After that, 100 μL of a reaction stopping solution consisting of 0.7N sulfuric acid was dispensed into each well to stop the coloring reaction.

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

Based on the above measurements, a fused cell line producing an antibody that binds to HMGB1 was selected, and one clone was established from among the grown fused cell lines and named 181208 2H6 strain.

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

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

After culturing, IgG in the supernatant was allowed to bind to a protein A column [GE Healthcare Bio-Sciences (Sweden)].

Bound IgG was eluted with 100 mM aqueous citric acid (pH 3.0).

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 a monoclonal antibody-producing cell line (181208 2H6). .

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

The 181208 2H6 strain, which is a monoclonal antibody-producing cell line of the antibody (2H6), is a patent microorganism depositary center [NPMD] of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). has been deposited on December 20, 2018 as "Accession number: NITE P-02843".

[Example 2] (Preparation of antibody (2D4))
At a different time from Example 1, the procedures described in (1) to (4) of Example 1 were performed to prepare anti-HMGB1 monoclonal antibodies again.

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

An anti-HMGB1 monoclonal antibody could be obtained from the 181212 2D4 monoclonal antibody-producing cell line.

This monoclonal antibody was named "antibody (2D4)".

The 181212 2D4 strain, which is a monoclonal antibody-producing cell line of the antibody (2D4), is a patent microorganism depositary center [NPMD] of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). has been deposited on December 20, 2018 as "Accession number: NITE P-02842".

[Example 3] (Preparation of anti-HMGB1,2 monoclonal antibody)
Separately from Examples 1 and 2, the procedures described in (1) to (4) of Example 1 were performed to prepare monoclonal antibodies that bind to HMGB1 and HMGB2.

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

A monoclonal antibody that binds to HMGB1 and HMGB2 was obtained from the 181212 5D1 monoclonal antibody-producing cell line.

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

The 181212 5D1 strain, which is a monoclonal antibody-producing cell line for anti-HMGB1,2 monoclonal antibodies, was obtained from the Patent Microorganism Depositary Center [NPMD] of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). No.) on December 20, 2018 as "Accession number: NITE P-02844".

[Example 4] (Confirmation of effect when coexistence of glycerin/propylene oxide/ethylene oxide adduct)
When HMGB1 in a sample and a carrier on which an anti-HMGB1 antibody is immobilized are contacted, HMGB1 in the sample is measured in the presence of a glycerin/propylene oxide/ethylene oxide adduct, a calibration curve is created, and the effect of the present invention is obtained. It was confirmed.

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

2. Second reagent (1) Second reagent (0.0004% FA-195)
(a) The antibody (2H6) prepared in Example 1 above and the antibody (2D4) prepared in Example 2 above were mixed in equal amounts to prepare a mixture of anti-HMGB1 monoclonal antibodies. This was termed "anti-HMGB1 monoclonal antibody mixture".

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

(c) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) is added to the latex particles on which the anti-HMGB1 monoclonal antibody mixture has been immobilized, and the mixture is blocked. to obtain latex particles on which the anti-HMGB1 monoclonal antibody mixture was immobilized.

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

(e) Anti-HMGB1,2 monoclonal antibody-immobilized latex particles are mixed with 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) for blocking. Latex particles on which the treated anti-HMGB1,2 monoclonal antibody was immobilized were obtained.

(f) The latex particles on which the blocked anti-HMGB1 monoclonal antibody mixture prepared in (c) above was immobilized, and the blocked anti-HMGB1,2 monoclonal antibody prepared in (e) above were immobilized. and latex particles were mixed in equal amounts to prepare a mixture of latex particles on which the anti-HMGB1 monoclonal antibody was immobilized. This was named "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture".

(g) In the glycerin/propylene oxide/ethylene oxide adduct represented by the following general formula (1), a+b+c is 11.5 mol, d+e+f is 14.3 mol, and g+h+i is 44.1 mol [(The number of moles is the average number of moles added.) The numbers in { } are random additions. ] After mixing "Sunnix FA-195" (molecular weight: 3,370, CAS No. 9082-00-2) sold by Sanyo Chemical Industries, Ltd., which is an adduct of glycerin, propylene oxide, and ethylene oxide, 0. 002% (w/v) was mixed with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture of (f) to prepare a second reagent. This was named "second reagent (0.0004% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.0004% FA-195) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0004, which is the value obtained by dividing the concentration 0.002% (w/v) in the second reagent (0.0004% FA-195) by the dilution ratio 4.6 % (w/v).

(2) Second reagent (0.001% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. Change "mix" to "mix 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.005% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.001% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.001% FA-195) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.001 of the value obtained by dividing the concentration 0.005% (w / v) in the second reagent (0.001% FA-195) by the dilution ratio 4.6 % (w/v).

(3) Second reagent (0.0016% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. Change "mix" to "mix 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.0075% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.0016% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.0016% FA-195) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0016, the value obtained by dividing the concentration 0.00754% (w / v) in the second reagent (0.0016% FA-195) by the dilution ratio 4.6 % (w/v).

(4) Second reagent (0.0022% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. Change "mixing" to "mix 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.01% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.0022% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.0022% FA-195) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0022, the value obtained by dividing the concentration 0.01% (w / v) in the second reagent (0.0022% FA-195) by the dilution ratio 4.6 % (w/v).

(5) Second reagent (0.0054% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. Change "mix" to "mix 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.025% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.0054% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.0054% FA-195) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0054, the value obtained by dividing the concentration 0.025% (w / v) in the second reagent (0.0054% FA-195) by the dilution ratio 4.6 % (w/v).

(6) Second reagent (0.01% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. Change "mixing" to "mix 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.05% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.01% FA-195)".

The "Sannix FA-195" contained in this second reagent (0.01% FA-195) is HMGB1 in the sample and anti-HMGB1 monoclonal antibody-immobilized latex in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.01 of the value obtained by dividing the concentration 0.05% (w / v) in the second reagent (0.01% FA-195) by the dilution ratio 4.6 % (w/v).

(7) Second reagent (0% FA-195)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix FA-195" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.002% (w/v) after mixing. The description of (1) (g) above except that the place of "mixing" is changed to "without mixing 'Sannix FA-195' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture." A second reagent containing no "Sannix FA-195" was prepared. This was named "second reagent (0%/FA-195)".

[2] Sample 1. Sample (HMGB1 concentration: 0 ng/mL)
A buffer containing 1% (w/v) bovine serum albumin (BSA) was named "sample (HMGB1 concentration: 0 ng/mL)".

2. Sample (HMGB1 concentration: 5 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 5 ng/mL. A sample with a HMGB1 concentration of 5 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 5 ng/mL)".

3. Sample (HMGB1 concentration: 10 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 10 ng/mL. A sample with a HMGB1 concentration of 10 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 10 ng/mL)".

4. Sample (HMGB1 concentration: 20 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 20 ng/mL. A sample with a HMGB1 concentration of 20 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 20 ng/mL)".

5. Sample (HMGB1 concentration: 40 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 40 ng/mL. A sample with a HMGB1 concentration of 40 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 40 ng/mL)".

6. Sample (HMGB1 concentration: 80 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 80 ng/mL. A sample with a HMGB1 concentration of 80 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 80 ng/mL)".

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

One of the halves was refrigerated (2 to 8 ° C.) and shaken at 100 times / minute for 3 days or more, and these second reagents were designated as "second reagent (with shaking)". .
The other half was refrigerated (2 to 8° C.) and allowed to stand for 3 days or longer.

[4] Measurement of HMGB1 in sample 1. Measurement using second reagent (with shaking) Using the first reagent of 1 of the above [1] as the first reagent of the HMGB1 measurement reagent in the sample, and the second reagent of the above [3] as the second reagent Using the second reagent of each of the reagents (with shaking), HMGB1 in the sample of [2] was measured.

The measurement was performed using a Hitachi 7180 automatic analyzer (Hitachi High Tech [Japan]).
The first reagent of 1 of the above [1] was added and mixed with the sample of the above [2], and after 5 minutes, each of the second reagents of the second reagent of the above [3] (with shaking) was added and mixed.
Specifically, 24 μL of the sample of [2] is used, 120 μL of the first reagent of 1 of [1] is used as the first reagent, and the second reagent of [3] is used as the second reagent ( Using 40 μL of each of the second reagents (with shaking), the amount of change in absorbance was measured by the two-point-end method.
The reaction temperature was 37 ° C., the reaction time was 10 minutes after adding and mixing the first reagent of 1 of 1 to the sample of [2], and the sample of [2] was measured at a measurement wavelength of 800 nm. The amount of change in absorbance was measured after adding and mixing the first reagent in 1 of [1] above.

2. Measurement using the second reagent (without shaking) Using the first reagent in 1 of the above [1] as the first reagent of the HMGB1 measurement reagent in the sample, and the second reagent in the above [3] as the second reagent HMGB1 in the sample of [2] was measured using the second reagent of each of the reagents (without shaking).

The measurement was performed using a Hitachi 7180 automatic analyzer (Hitachi High Tech [Japan]).
The first reagent of 1 of the above [1] was added and mixed with the sample of the above [2], and after 5 minutes, each of the second reagents of the second reagent of the above [3] (without shaking) was added and mixed.
Specifically, 24 μL of the sample of [2] is used, 120 μL of the first reagent of 1 of [1] is used as the first reagent, and the second reagent of [3] is used as the second reagent ( Using 40 μL of each of the second reagents (without shaking), the amount of change in absorbance was measured by the two-point-end method.
The reaction temperature was 37° C., the reaction time was 10 minutes after adding and mixing the first reagent of 1 of 1 above to the sample of 2 above, and the amount of change in absorbance was measured at a measurement wavelength of 800 nm. rice field.

[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

The 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, HMGB1 in the sample is shown in FIG. 1 as a graph of the calibration curve when measuring .

In FIG. 1, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 1, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0% FA-195) (with shaking), and "●" indicates Measured values (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0%, FA-195) (without shaking) are shown.

From this FIG. 1, the calibration curve when using the second reagent (0% FA-195) (with shaking) and the second reagent (0% FA-195) (without shaking) It can be seen that there is a large deviation between the calibration curves.
In addition, it can be seen that the slope of the calibration curve when using the second reagent (0%, FA-195) (with shaking) is greatly reduced.

Further, from FIG. 1, the reagent blank, which is the measured value (change in absorbance at the measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng/mL, is the second reagent (0% FA-195) (vibration With shaking), the amount of absorbance change has increased to around 0.08, but when the second reagent (0%, FA-195) (without shaking) is used, the amount of absorbance change is It can be seen that it is close to 0.

From this, it was confirmed that shaking the anti-HMGB1 antibody-immobilized carrier causes non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier, which causes an increase in the reagent blank. was taken.

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

The measurement result 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, FIG. 2 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 2, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 2, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0004% FA-195) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0004%, FA-195) (without shaking).

From this FIG. 2, the calibration curve when using the second reagent (0.0004% FA-195) (with shaking) and the second reagent (0.0004% FA-195) (without shaking) It can be seen that the calibration curves when used are far apart.

Further, from FIG. 2, 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, is the second reagent (0.0004% FA-195) (with shaking), the absorbance change has increased to around 0.06, but when using the second reagent (0.0004% FA-195) (without shaking) It can be seen that the amount of change in absorbance is close to zero.

From this, when 0.0004% (w/v) of glycerin/propylene oxide/ethylene oxide adduct was present at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody were immobilized, anti-HMGB1 antibody When the carrier on which the anti-HMGB1 antibody is immobilized is shaken, non-specific agglutination of the carrier on which the anti-HMGB1 antibody is immobilized cannot be suppressed, thereby preventing an increase in the reagent blank caused by the agglutination. It was confirmed that it could not be suppressed.

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

The measurement result 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, FIG. 3 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 3, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 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) is used, and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.001% FA-195) (without shaking).

From this FIG. 3, the calibration curve when using the second reagent (0.001% FA-195) (with shaking) and the second reagent (0.001% FA-195) (without shaking) It can be seen that the calibration curves when used are parallel.

Further, from FIG. 3, 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, is the second reagent (0.001% FA-195) (with shaking), it was found that the absorbance change was small at around 0.02, and when the second reagent (0.001% FA-195) (without shaking) was used, It can be seen that the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.001% (w/v) glycerin/propylene oxide/ethylene oxide adduct is present, the anti-HMGB1 antibody can be suppressed from causing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier by shaking the carrier on which the . confirmed that it can be done.

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

The measurement result 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, FIG. 4 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 4, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000). .
In FIG. 4, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0016% FA-195) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0016%, FA-195) (without shaking).

From this FIG. 4, the calibration curve when using the second reagent (0.0016% FA-195) (with shaking) and the second reagent (0.0016% FA-195) (without shaking) It can be seen that the calibration curves when used are almost close to each other.

Further, from FIG. 4, 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, is the second reagent (0.0016% FA-195) (with shaking) and the second reagent (0.0016%, FA-195) (without shaking), the amount of change in absorbance is close to zero.

From this, when 0.0016% (w/v) of glycerin/propylene oxide/ethylene oxide adduct was present at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody were immobilized, anti-HMGB1 antibody can be suppressed from causing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier by shaking the carrier on which the . confirmed that it can be done.

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

The measurement result 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, FIG. 5 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 5, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 5, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0022% FA-195) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0022%, FA-195) (without shaking).

From this FIG. 5, the calibration curve when using the second reagent (0.0022% FA-195) (with shaking) and the second reagent (0.0022% FA-195) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 5, 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, is the second reagent (0.0022%, FA-195) (with shaking) and the second reagent (0.0022%, FA-195) (without shaking), the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.0022% (w/v) glycerin/propylene oxide/ethylene oxide adduct is present, the anti-HMGB1 antibody can be suppressed from causing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier by shaking the carrier on which the . confirmed that it can be done.

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

The 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, FIG. 6 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 6, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 6, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0054% FA-195) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0054%, FA-195) (without shaking).

From this FIG. 6, the calibration curve when using the second reagent (0.0054% FA-195) (with shaking) and the second reagent (0.0054% FA-195) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 6, 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, is the second reagent (0.0054% FA-195) (with shaking) and the second reagent (0.0054%, FA-195) (without shaking), the change in absorbance is close to zero.

From this, when 0.0054% (w/v) of glycerin/propylene oxide/ethylene oxide adduct was present at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody were immobilized, anti-HMGB1 antibody can be suppressed from causing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier by shaking the carrier on which the . confirmed that it can be done.

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

The measurement result 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, FIG. 7 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 7, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 7, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.01% FA-195) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.01%, FA-195) (without shaking).

From this FIG. 7, the calibration curve when using the second reagent (0.01% FA-195) (with shaking) and the second reagent (0.01% FA-195) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 7, 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, is the second reagent (0.01% FA-195) (with shaking) and the second reagent (0.01%, FA-195) (without shaking), the change in absorbance is close to zero.

From this, when 0.01% (w/v) of glycerin/propylene oxide/ethylene oxide adduct was present at the time of contact with the carrier on which HMGB1 in the sample and anti-HMGB1 antibody were immobilized, anti-HMGB1 antibody can be suppressed from causing non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier by shaking the carrier on which the . confirmed that it can be done.

[Example 5] (Confirmation of effect when coexisting glycerin/propylene oxide adduct)
When the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are brought into contact with each other, the HMGB1 in the sample is measured in the presence of the glycerin-propylene oxide adduct, a calibration curve is prepared, and the effect of the present invention is confirmed. bottom.

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

2. Second reagent (1) Second reagent (0.0022% GP-400)
(a) The antibody (2H6) prepared in Example 1 above and the antibody (2D4) prepared in Example 2 above were mixed in equal amounts to prepare a mixture of anti-HMGB1 monoclonal antibodies. This was termed "anti-HMGB1 monoclonal antibody mixture".

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

(c) 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) is added to the latex particles on which the anti-HMGB1 monoclonal antibody mixture has been immobilized, and the mixture is blocked. to obtain latex particles on which the anti-HMGB1 monoclonal antibody mixture was immobilized.

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

(e) Anti-HMGB1,2 monoclonal antibody-immobilized latex particles are mixed with 6.0 mL of Tris buffer (pH 7.4) containing 5% bovine serum albumin (BSA) for blocking. Latex particles on which the treated anti-HMGB1,2 monoclonal antibody was immobilized were obtained.

(f) The latex particles on which the blocked anti-HMGB1 monoclonal antibody mixture prepared in (c) above was immobilized, and the blocked anti-HMGB1,2 monoclonal antibody prepared in (e) above were immobilized. and latex particles were mixed in equal amounts to prepare a mixture of latex particles on which the anti-HMGB1 monoclonal antibody was immobilized. This was named "anti-HMGB1 monoclonal antibody-immobilized latex particle mixture".

(g) Glycerin/propylene oxide adduct represented by the following general formula (2), in which a+b+c is 5.6 mol (the number of moles is the average number of moles added). After mixing "Sunnix GP-400" (molecular weight: 420, CAS No. 25791-96-2) sold by Sanyo Chemical Industries, Ltd., which is a product, to a concentration of 0.01% (w / v) A second reagent was prepared by mixing with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture of (f) above. This was named "second reagent (0.0022% GP-400)".

It should be noted that "Sannix GP-400" contained in this second reagent (0.0022% GP-400) is the HMGB1 in the sample and the anti-HMGB1 monoclonal antibody-immobilized latex in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0022, the value obtained by dividing the concentration 0.01% (w / v) in the second reagent (0.0022% GP-400) by the dilution ratio 4.6 % (w/v).

(2) Second reagent (0.0054% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. Change "mixing" to read "mix 'Sunnix GP-400' into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.025% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.0054% GP-400)".

The "Sannix GP-400" contained in this second reagent (0.0054% GP-400) is the HMGB1 in the sample and the anti-HMGB1 monoclonal antibody-immobilized latex in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.0054, which is the value obtained by dividing the concentration 0.025% (w/v) in the second reagent (0.0054% GP-400) by the dilution ratio 4.6 % (w/v).

(3) Second reagent (0.0076% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. Change "mixing" to read "mix 'Sannix GP-400' into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.035% (w/v)." Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.0076% GP-400)".

The "Sannix GP-400" contained in this second reagent (0.0076% GP-400) is used in the measurement of HMGB1 in the sample described later, and the HMGB1 and anti-HMGB1 monoclonal antibody-immobilized latex in the sample. The concentration coexisting when in contact with the particle mixture is 0.0076, which is the value obtained by dividing the concentration 0.035% (w/v) in the second reagent (0.0076% GP-400) by the dilution ratio 4.6. % (w/v).

(4) Second reagent (0.01% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. Change “Mixing” to “Mix “Sunnix GP-400” into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.05% (w/v)”. Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.01% GP-400)".

The "Sannix GP-400" contained in this second reagent (0.01% GP-400) is the latex immobilized with HMGB1 and anti-HMGB1 monoclonal antibody in the sample in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.01 of the value obtained by dividing the concentration 0.05% (w / v) in the second reagent (0.01% GP-400) by the dilution ratio 4.6 % (w/v).

(5) Second reagent (0.016% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. Change "mixing" to "mix 'Sannix GP-400' into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.075% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.016% GP-400)".

It should be noted that "Sannix GP-400" contained in this second reagent (0.016% GP-400) is HMGB1 in the sample and anti-HMGB1 monoclonal antibody-immobilized latex in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.016, the value obtained by dividing the concentration 0.075% (w / v) in the second reagent (0.016% GP-400) by the dilution ratio 4.6 % (w/v).

(6) Second reagent (0.022% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. Change "Mixing" to "Mix "Sunnix GP-400" into the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so that the concentration after mixing is 0.10% (w/v)". Otherwise, the second reagent was prepared as described in (1)(g) above. This was named "second reagent (0.022% GP-400)".

The "Sannix GP-400" contained in this second reagent (0.022% GP-400) is the HMGB1 in the sample and the anti-HMGB1 monoclonal antibody-immobilized latex in the measurement of HMGB1 in the sample described later. The concentration coexisting when in contact with the particle mixture is 0.022, the value obtained by dividing the concentration 0.10% (w / v) in the second reagent (0.022% GP-400) by the dilution ratio 4.6 % (w/v).

(7) Second reagent (0% GP-400)
(a) A mixture of anti-HMGB1 monoclonal antibody-immobilized latex particles was prepared as described in (a) to (f) of (1) above.
(b) In (1) (g) above, "Sannix GP-400" is added to the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture so as to have a concentration of 0.01% (w/v) after mixing. The description of (1) (g) above except that the place of "mixing" is changed to "without mixing 'Sunnix GP-400' with the anti-HMGB1 monoclonal antibody-immobilized latex particle mixture." to prepare a second reagent that does not contain "Sannix GP-400". This was named "second reagent (0% GP-400)".

[2] Sample 1. Sample (HMGB1 concentration: 0 ng/mL)
A buffer containing 1% (w/v) bovine serum albumin (BSA) was named "sample (HMGB1 concentration: 0 ng/mL)".

2. Sample (HMGB1 concentration: 5 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 5 ng/mL. A sample with a HMGB1 concentration of 5 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 5 ng/mL)".

3. Sample (HMGB1 concentration: 10 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 10 ng/mL. A sample with a HMGB1 concentration of 10 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 10 ng/mL)".

4. Sample (HMGB1 concentration: 20 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 20 ng/mL. A sample with a HMGB1 concentration of 20 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 20 ng/mL)".

5. Sample (HMGB1 concentration: 40 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 40 ng/mL. A sample with a HMGB1 concentration of 40 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 40 ng/mL)".

6. Sample (HMGB1 concentration: 80 ng/mL)
Purified porcine HMGB1 (from the thymus) [99.1% amino acid sequence homology to human HMGB1] containing 1% (w/v) bovine serum albumin (BSA) such that the HMGB1 concentration was 80 ng/mL. A sample with a HMGB1 concentration of 80 ng/mL was prepared by diluting with buffer.
This was named "sample (HMGB1 concentration: 80 ng/mL)".

[3] Shaking treatment Second reagent (0% GP-400), second reagent (0.0022% GP-400), second reagent (0.0054%) prepared in 2 of [1]・ GP-400), second reagent (0.0076% GP-400), second reagent (0.01% GP-400), second reagent (0.016% GP-400) and second The reagent (0.022% GP-400) was divided into halves, placed in containers, and sealed.

One of the halves was refrigerated (2 to 8 ° C.) and shaken at 100 times / minute for 3 days or more, and these second reagents were designated as "second reagent (with shaking)". .
The other half was refrigerated (2 to 8° C.) and allowed to stand for 3 days or longer.

[4] Measurement of HMGB1 in sample 1. Measurement using second reagent (with shaking) Using the first reagent of 1 of the above [1] as the first reagent of the HMGB1 measurement reagent in the sample, and the second reagent of the above [3] as the second reagent Using the second reagent of each of the reagents (with shaking), HMGB1 in the sample of [2] was measured.

The measurement was performed using a Hitachi 7180 automatic analyzer (Hitachi High Tech [Japan]).
The first reagent of 1 of the above [1] was added and mixed with the sample of the above [2], and after 5 minutes, each of the second reagents of the second reagent of the above [3] (with shaking) was added and mixed.
Specifically, 24 μL of the sample of [2] is used, 120 μL of the first reagent of 1 of [1] is used as the first reagent, and the second reagent of [3] is used as the second reagent ( Using 40 μL of each of the second reagents (with shaking), the amount of change in absorbance was measured by the two-point-end method.
The reaction temperature was 37° C., the reaction time was 10 minutes after adding and mixing the first reagent of 1 of 1 above to the sample of 2 above, and the amount of change in absorbance was measured at a measurement wavelength of 800 nm. rice field.

2. Measurement using the second reagent (without shaking) Using the first reagent in 1 of the above [1] as the first reagent of the HMGB1 measurement reagent in the sample, and the second reagent in the above [3] as the second reagent HMGB1 in the sample of [2] was measured using the second reagent of each of the reagents (without shaking).

The measurement was performed using a Hitachi 7180 automatic analyzer (Hitachi High Tech [Japan]).
The first reagent of 1 of the above [1] was added and mixed with the sample of the above [2], and after 5 minutes, each of the second reagents of the second reagent of the above [3] (without shaking) was added and mixed.
Specifically, 24 μL of the sample of [2] is used, 120 μL of the first reagent of 1 of [1] is used as the first reagent, and the second reagent of [3] is used as the second reagent ( Using 40 μL of each of the second reagents (without shaking), the amount of change in absorbance was measured by the two-point-end method.
The reaction temperature was 37° C., the reaction time was 10 minutes after adding and mixing the first reagent of 1 of 1 above to the sample of 2 above, and the amount of change in absorbance was measured at a measurement wavelength of 800 nm. rice field.

[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

The 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 are HMGB1 in the sample. is shown in FIG. 8 as a graph of the calibration curve when measuring .

In FIG. 8, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 8, "□" indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0% GP-400) (with shaking), and "●" indicates Measured values (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0% GP-400) (without shaking) are shown.

From this FIG. 8, the calibration curve when using the second reagent (0% GP-400) (with shaking) and the calibration curve when using the second reagent (0% GP-400) (without shaking) It can be seen that there is a large deviation between the calibration curves.
Also, it can be seen that the slope of the calibration curve when using the second reagent (0% GP-400) (with shaking) is greatly reduced.

Further, from FIG. 8, the reagent blank, which is the measured value (change in absorbance at the measurement wavelength of 800 nm) when the HMGB1 concentration in the sample is 0 ng/mL, is the second reagent (0% GP-400) (vibration When the second reagent (0% GP-400) (without shaking) is used, the amount of absorbance change increases to around 0.08. It can be seen that it is close to 0.

From this, it was confirmed that shaking the anti-HMGB1 antibody-immobilized carrier causes non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier, which causes an increase in the reagent blank. was taken.

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

The 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, FIG. 9 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 9, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 9, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0022% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0022% GP-400) (without shaking).

From this FIG. 9, the calibration curve when using the second reagent (0.0022% GP-400) (with shaking) and the second reagent (0.0022% GP-400) (without shaking) It can be seen that the calibration curves when used are far apart.

Further, from FIG. 9, 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, is the second reagent (0.0022% GP-400) (with shaking), the absorbance change has increased to around 0.05, but when using the second reagent (0.0022% GP-400) (without shaking) It can be seen that the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.0022% (w/v) glycerin-propylene oxide adduct is present, the anti-HMGB1 antibody is immobilized. It is not possible to suppress the occurrence of non-specific agglutination of the anti-HMGB1 antibody-immobilized carrier due to shaking of the solidified carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that it was not possible.

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

The measurement result 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, FIG. 10 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 10, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 10, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0054% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0054% GP-400) (without shaking).

From this FIG. 10, the calibration curve when using the second reagent (0.0054% GP-400) (with shaking) and the second reagent (0.0054% GP-400) (without shaking) It can be seen that the calibration curves when used are parallel.

Further, from FIG. 10, 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, is the second reagent (0.0054% GP-400) (with shaking), it was found that the absorbance change was small at around 0.015, and when the second reagent (0.0054% GP-400) (without shaking) was used, It can be seen that the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.0054% (w/v) glycerin-propylene oxide adduct is present, the anti-HMGB1 antibody is immobilized. Shaking the anti-HMGB1 antibody-immobilized carrier can suppress non-specific agglutination of the carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that

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

The measurement result 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, FIG. 11 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 11, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 11, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0076% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.0076% GP-400) (without shaking).

From this FIG. 11, the calibration curve when using the second reagent (0.0076% GP-400) (with shaking) and the second reagent (0.0076% GP-400) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 11, 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, is the second reagent (0.0076% GP-400) (with shaking) and the second reagent (0.0076% GP-400) (without shaking), the change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.0076% (w/v) glycerin-propylene oxide adduct is coexistent, the anti-HMGB1 antibody is immobilized. Shaking the anti-HMGB1 antibody-immobilized carrier can suppress non-specific agglutination of the carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that

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

The measurement result 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, FIG. 12 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 12, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 12, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.01% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.01% GP-400) (without shaking).

From this FIG. 12, the calibration curve when using the second reagent (0.01% GP-400) (with shaking) and the second reagent (0.01% GP-400) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 12, 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, is the second reagent (0.01% GP-400) (with shaking) and the second reagent (0.01% GP-400) (without shaking), the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.01% (w/v) glycerin-propylene oxide adduct is present, the anti-HMGB1 antibody is immobilized. shaking the carrier to which the anti-HMGB1 antibody is immobilized can suppress non-specific agglutination of the carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that

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

The measurement result 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, FIG. 13 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 13, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000), which is the measured value. .
In FIG. 13, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.016% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.016% GP-400) (without shaking).

From this FIG. 13, the calibration curve when using the second reagent (0.016% GP-400) (with shaking) and the second reagent (0.016% GP-400) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 13, 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, is the second reagent (0.016% GP-400) (with shaking) and the second reagent (0.016% GP-400) (without shaking), the amount of change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.016% (w/v) glycerin-propylene oxide adduct is present, the anti-HMGB1 antibody is immobilized. Shaking the anti-HMGB1 antibody-immobilized carrier can suppress non-specific agglutination of the carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that

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

The measurement result 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, FIG. 14 shows a graph of the calibration curve when measuring HMGB1 in the sample.

In FIG. 14, the horizontal axis indicates the concentration of HMGB1 in the sample (unit: ng/mL), and the vertical axis indicates the amount of change in absorbance at a measurement wavelength of 800 nm (unit: amount of change in absorbance×10,000). .
In FIG. 14, “□” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.022% GP-400) (with shaking), and “● ” indicates the measured value (change in absorbance at a measurement wavelength of 800 nm) when using the second reagent (0.022% GP-400) (without shaking).

From this FIG. 14, the calibration curve when using the second reagent (0.022% GP-400) (with shaking) and the second reagent (0.022% GP-400) (without shaking) It can be seen that the calibration curves when used almost overlap.

Further, from FIG. 14, 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, is the second reagent (0.022% GP-400) (with shaking) and the second reagent (0.022% GP-400) (without shaking), the change in absorbance is close to zero.

From this, when HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized are contacted, when 0.022% (w/v) glycerin-propylene oxide adduct is coexistent, the anti-HMGB1 antibody is immobilized. Shaking the anti-HMGB1 antibody-immobilized carrier can suppress non-specific agglutination of the carrier, thereby suppressing an increase in the reagent blank caused by the agglutination. It was confirmed that

Claims (32)

The concentration of HMGB1 in the sample is measured by contacting the HMGB1 in the sample with the carrier on which the anti-HMGB1 antibody is immobilized, and measuring the aggregate of the carrier on which the anti-HMGB1 antibody is immobilized bound via HMGB1. , in a method for measuring HMGB1 in a sample, 0.001% (w/v) to 0.01% (w/v) of glycerin- A method for measuring HMGB1 in a sample, characterized in that a 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 concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0054% (w/v). 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 coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0022% (w/v). The method for measuring HMGB1 in a sample according to claim 1 or 2, characterized in that: The concentration of the glycerin-propylene oxide adduct that coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is 0.0076% (w/v) to 0.022% (w/v). The method for measuring HMGB1 in a sample according to claim 1, characterized by: The method for measuring HMGB1 in a sample according to any one of claims 1 to 4, wherein the carrier is a particle. The method for measuring HMGB1 in a sample according to any one of claims 1 to 5, wherein the carrier is latex particles. HMGB1 in the 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): Measuring method.

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

[In general formula (2), a+b+c is 3 mol to 12 mol. (The number of moles is the average number of added moles.)] The concentration of HMGB1 in the sample is measured by contacting the HMGB1 in the sample with the carrier on which the anti-HMGB1 antibody is immobilized, and measuring the aggregate of the carrier on which the anti-HMGB1 antibody is immobilized bound via HMGB1. , in a reagent for measuring HMGB1 in a sample, 0.001% (w/v) to 0.01% (w/v) of glycerin- Propylene oxide/ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of the glycerin/propylene oxide/ethylene oxide adduct at a concentration that allows the coexistence of the glycerin/propylene oxide/ethylene oxide adduct or a reagent for measuring HMGB1 in a sample, characterized by containing a glycerin/propylene oxide adduct. The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0054% (w/v). The reagent for measuring HMGB1 in the sample according to claim 9, characterized by: The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0022% (w/v). The reagent for measuring HMGB1 in a sample according to claim 9 or 10, characterized by: The concentration of the glycerin-propylene oxide adduct that coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is 0.0076% (w/v) to 0.022% (w/v). 10. The reagent for measuring HMGB1 in a sample according to claim 9, characterized by: The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 12, wherein the carrier is a particle. The reagent for measuring HMGB1 in a sample according to any one of claims 9 to 13, wherein the carrier is latex particles. HMGB1 in the 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): measuring reagent.

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

[In general formula (2), a+b+c is 3 mol to 12 mol. (The number of moles is the average number of added moles.)] The concentration of HMGB1 in the sample is measured by contacting the HMGB1 in the sample with the carrier on which the anti-HMGB1 antibody is immobilized, and measuring the aggregate of the carrier on which the anti-HMGB1 antibody is immobilized bound via HMGB1. , 0.001% (w/v) to 0.01% (w/v) of glycerol/propylene at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized, in the measurement of HMGB1 in the sample A carrier on which an anti-HMGB1 antibody is immobilized, characterized in that an oxide-ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of a glycerin-propylene oxide adduct is coexistent. A method for suppressing non-specific aggregation. The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0054% (w/v). 18. A method for suppressing non-specific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized according to claim 17, characterized by: The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0022% (w/v). 19. A method for suppressing non-specific aggregation of a carrier on which an anti-HMGB1 antibody is immobilized according to claim 17 or 18, characterized by: The concentration of the glycerin-propylene oxide adduct that coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is 0.0076% (w/v) to 0.022% (w/v). 18. A method for suppressing non-specific aggregation of the carrier on which the anti-HMGB1 antibody is immobilized according to claim 17, characterized by: 21. The method for suppressing non-specific agglutination of an anti-HMGB1 antibody-immobilized carrier according to any one of claims 17 to 20, wherein the carrier is a particle. The method for suppressing nonspecific agglutination of a carrier on which an anti-HMGB1 antibody is immobilized according to any one of claims 17 to 21, wherein the carrier is latex particles. The anti-HMGB1 antibody 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): A method for suppressing non-specific aggregation of a carrier that has been transformed.

[In general formula (1), a+b+c is 3 to 20 mol, d+e+f is 3 to 30 mol, and g+h+i is 3 to 90 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions. ] The anti-HMGB1 antibody-immobilized carrier 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): A method for suppressing non-specific aggregation.

[In general formula (2), a+b+c is 3 mol to 12 mol. (The number of moles is the average number of added moles.)] The concentration of HMGB1 in the sample is measured by contacting the HMGB1 in the sample with the carrier on which the anti-HMGB1 antibody is immobilized, and measuring the aggregate of the carrier on which the anti-HMGB1 antibody is immobilized bound via HMGB1. , 0.001% (w/v) to 0.01% (w/v) of glycerol/propylene at the time of contact with the carrier on which HMGB1 in the sample and the anti-HMGB1 antibody are immobilized, in the measurement of HMGB1 in the sample A reagent blank for measuring HMGB1 in a sample, characterized in that an oxide-ethylene oxide adduct or 0.0054% (w/v) to 0.022% (w/v) of a glycerin-propylene oxide adduct is coexistent. method for suppressing the rise of The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0054% (w/v). 26. The method for suppressing an increase in reagent blank during measurement of HMGB1 in a sample according to claim 25, characterized by: The concentration of the glycerin/propylene oxide/ethylene oxide adduct coexisting when the HMGB1 in the sample and the anti-HMGB1 antibody-immobilized carrier are brought into contact is 0.0016% (w/v) to 0.0022% (w/v). 27. The method according to claim 25 or 26 for suppressing an increase in reagent blank during measurement of HMGB1 in a sample, characterized by: The concentration of the glycerin-propylene oxide adduct that coexists when the HMGB1 in the sample and the carrier on which the anti-HMGB1 antibody is immobilized is brought into contact is 0.0076% (w/v) to 0.022% (w/v). 26. The method according to claim 25 for suppressing an increase in reagent blank when measuring HMGB1 in a sample. 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. 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 latex particles. HMGB1 measurement in the 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): method for suppressing the rise of the reagent blank at time.

[In general formula (1), a+b+c is 3 to 20 mol, d+e+f is 3 to 30 mol, and g+h+i is 3 to 90 mol. (The number of moles is the average number of moles added.) The numbers in { } are random additions. ] A reagent blank for measuring 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): method for suppressing the rise of

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

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