JP7425459B2 - Stabilized HMGB1-containing solution - Google Patents

Description

The present invention relates to a HMGB1-containing solution used when measuring HMGB1 (high mobility group protein-1; HMG-1) in a sample, which can be a marker for diseases such as sepsis.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

High Mobility Group Proteins were discovered in 1964 as a large number of non-histone proteins contained in the chromatin structure, and are universally contained in all higher animals and plants, and their primary structure is conserved among species. The quality is extremely high.
It is also known that it is abundant not only in the nucleus but also in the cytoplasm.
Although its physiological effects are not clearly understood, HMGB1 relaxes the double helix structure when binding to DNA, which changes the higher-order structure of DNA to an optimal structure during the transcription reaction, increasing transcriptional activity. , is thought to function as a very broad transcriptional promoter and nucleosome relaxer.

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.

In 1999, Wang et al. performed quantitative measurement of HMGB1 in serum (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 be a marker for sepsis.
They also showed that it is possible to distinguish between patients who will survive and those who will die from sepsis by precisely measuring HMGB1 in the blood.
That is, the usefulness of not only simply confirming the presence of HMGB1 in blood but also precisely quantifying it has been revealed (see Non-Patent Document 1).

By the way, in order to quantitatively measure the substance to be measured contained in a sample, it is common to perform calibration using a standard solution of known concentration, and also to ensure that the quantitative measurement is performed accurately and precisely. In order to confirm that the concentration of the sample is known, it is common practice to perform accuracy control using a measurement sample with a known concentration.
When measuring HMGB1, an HMGB1-containing solution is often used as a standard solution with a known concentration used for calibration and a measurement sample with a known concentration used for quality control.

However, proteins in aqueous solutions are often denatured and degraded depending on storage conditions such as storage temperature, resulting in changes in the protein's higher-order structure and decreased reactivity with antibodies. Therefore, if such an aqueous protein solution is used as a standard solution for calibration or a measurement sample for quality control, it may cause erroneous measurement values.

In order to prevent a decrease in reactivity with antibodies, there are two methods: lyophilize and store an aqueous protein solution, and then add an appropriate amount of water to dissolve the protein before using it as an aqueous solution, or store it frozen. etc. are commonly taken.
However, in the freeze-drying method, water must be added at the time of use, which makes the operation complicated, and there is a risk that errors may occur due to the dissolution operation.
In cryopreservation methods, measurement values may change depending on repeated freezing and thawing, thawing method, and thawing state.
Furthermore, in either method, the stability after the liquid state is obtained is often poor.

Methods other than freeze-drying and freezing include methods for stabilizing heme proteins characterized by the coexistence of transition metals in a sample containing heme proteins, and methods for stabilizing heme proteins characterized by coexistence of divalent metal ions with pulmonary surfactant proteins. A method of stabilizing the antigenic activity of pulmonary surfactant protein by stabilizing the antigenic activity of lung surfactant protein is known (see Patent Document 1 and Patent Document 2).
However, there is no example in which the stabilization of HMGB1-containing solutions has been sufficiently studied.

As mentioned above, conventional HMGB1-containing solutions have insufficient storage stability, and lyophilized or frozen HMGB1-containing solutions must be used, which poses problems in terms of usability and accuracy. This caused a problem in the measurement of HMGB1. Therefore, it has been desired to improve the storage stability of HMGB1-containing solutions.

H. Wang et al., SCIENCE, Vol. 285, No. 9, pp. 248-251, published in 1999.

Japanese Patent Application Publication No. 2001-249132 Japanese Patent Application Publication No. 2016-183112

Therefore, an object of the present invention is to provide an HMGB1-containing solution that is stable in liquid form for a long period of time by improving the storage stability of the HMGB1-containing solution.

As a result of intensive studies aimed at solving the above problems, the present inventors have discovered that by adding a substance having a cationic moiety to an HMGB1-containing solution, the denaturation or decomposition of HMGB1 or its effectiveness can be improved even in a liquid state. The present inventors have discovered that it is possible to stabilize the temperature for a long period of time without causing a decrease in the temperature, and have completed the present invention.

That is, the present invention provides the following inventions.
(1) An HMGB1-containing solution containing HMGB1 and a substance having a cationic site.
(2) The HMGB1-containing solution according to (1) above, wherein the cation moiety is an ion of a Group 1 element, an ion of a Group 2 element, or an ammonium ion.
(3) A method for stabilizing HMGB1 in a solution, which comprises adding a substance having a cationic moiety to a HMGB1-containing solution.
(4) The method for stabilizing HMGB1 according to claim 3, wherein the cation moiety is an ion of a Group 1 element, an ion of a Group 2 element, or an ammonium ion.
(5) A standard solution for measuring HMGB1, which is a standard solution for use in measuring HMGB1 in a sample, and is characterized by containing a substance having a cationic moiety.
(6) The standard solution for measuring HMGB1 according to claim 5, wherein the cation moiety is an ion of a Group 1 element, an ion of a Group 2 element, or an ammonium ion.

The HMGB1-containing solution of the present invention is an HMGB1-containing solution that has improved storage stability by containing HMGB1 and a substance having a cationic moiety.
Further, the method for stabilizing HMGB1 of the present invention is a method that can stabilize HMGB1 in a liquid state for a long period of time.
By using the stabilized HMGB1-containing solution as a standard solution, it is possible to provide error-free and accurate HMGB1 measurement values in disease diagnosis and the like.

The present invention will be described in detail below, but the following embodiments are illustrative for explaining the present invention, and the present invention is not limited to these embodiments. Moreover, the present invention can be implemented in various forms without departing from the gist thereof.

[1] HMGB1-containing solution 1. Overview In the present invention, HMGB1 in a solution state can be stabilized by containing a substance having a cationic site in an HMGB1-containing solution.

2. HMGB1
In the present invention, examples of HMGB1 include those derived from humans or other animals.
Further, HMGB1 can be obtained, for example, by extraction, purification, etc. from body fluids, cells, tissues, organs, etc. of humans or other animals by known methods. Furthermore, HMGB1 can also be prepared and obtained by genetic recombination of humans or other animals.

The methods for obtaining HMGB1 from human thymus, pig thymus, bovine thymus, human placenta, neutrophils, HL-60 cell line, etc. are described in the following documents (H. Goodwin et al., Biochemica Biophysica Acta, Vol. 405, pp. 280-291, published in 1975; M. Yoshida et al., J. Biochem., Vol. 95, pp. 117-124, published in 1980; Y. Adachi et al., J. Chromatogr, Vol. 530, pp. 39- Volume 46, published in 1992).

Furthermore, a method for preparing HMGB1 by genetic recombination is described in the following document (A. Mistry et al., Bio Techniques, Vol. 22, pp. 718-729, published in 1997).

3. Substance Having a Cation Site The HMGB1-containing solution of the present invention contains a substance having a cation site. In the present invention, the substance having a cationic site is not particularly limited, and any substance having a cationic site may be used.

(1) Cation Site In the present invention, examples of the cation site include metal ions, ammonium ions, and the like.

Examples of the metal ion include ions of Group 1 elements, Group 2 elements, and other metals.

Examples of Group 1 elements include lithium, sodium, potassium, and rubidium.

Examples of Group 2 elements include beryllium, magnesium, strontium, and barium.

Examples of other metals include gallium, indium, and germanium.

In the present invention, the cation moiety is preferably, for example, an ion of a Group 1 element, an ion of a Group 2 element, or an ammonium ion.

(2) Substance having a cationic site In the present invention, the substance having a cationic site is not particularly limited, and any substance having a cationic site may be used.
Examples of the substance having a cationic site include a hydroxide of a cationic site, a salt of a cationic site, and the like.

Examples of the salt of this cation site include salts with halogen ions, salts with acid groups, and the like.

Examples of the halogen ions include fluorine ions, chloride ions, bromide ions, and iodine ions.

Examples of the acid group include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, and citric acid.

Specific examples of substances having a cationic moiety include potassium chloride, sodium chloride, potassium nitrate, sodium sulfate, sodium acetate, sodium phosphate, sodium citrate, sodium bromide, lithium chloride, magnesium chloride, magnesium sulfate, Examples include ammonium chloride and ammonium sulfate.

In the present invention, preferred examples of the substance having a cation site include potassium chloride, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium phosphate, lithium chloride, magnesium sulfate, and ammonium sulfate.

In the present invention, the concentration of the substance having a cationic moiety is not particularly limited as long as it can sufficiently stabilize HMGB1 in the solution, and the optimum concentration varies depending on the concentration of HMGB1 in the solution. Among these, it may be at least 150 mM, preferably at least 200 mM, more preferably at least 500 mM, particularly preferably at least 1,000 mM.

In addition, the upper limit of the concentration of the substance having a cationic moiety in the solution is not particularly limited, but considering costs etc., it is preferably 3,000 mM or less, more preferably 2,000 mM or less, and 1,500 mM or less. is particularly preferred.

In addition, the substance having a cation site used in the present invention may be used alone or in combination of two or more types.

4. pH of solution
The pH of the HMGB1-containing solution of the present invention is preferably 9.0 or less, particularly preferably in the range of 4.4 to 8.0.

Furthermore, as the buffer used to bring the HMGB1-containing solution of the present invention to the above-mentioned pH range, conventionally known buffers having a buffering capacity within the above-mentioned pH range can be used as appropriate.
Examples of buffers that can be used include phosphoric acid, citric acid, tris(hydroxymethyl)aminomethane, imidazole, glycylglycine, MES, Bis-Tris, ADA, ACES, Bis-Tris propane, and PIPES. , MOPSO, MOPS, BES, HEPES, TES, DIPSO, TAPSO, POPSO, HEPPS, HEPPSO, Tricine, Bicine, TAPS, CHES, CAPSO, or CAPS, or salts thereof.

5. Other Constituent Components In addition to the substance having a cationic moiety, the HMGB1-containing solution of the present invention may appropriately contain a known preservative or the like as necessary.

In the present invention, in order to stabilize HMGB1 in a solution, it is sufficient to simply make a substance having a cationic site exist in the HMGB1-containing solution, and no other operations are particularly necessary.

[2] Standard solution for HMGB1 measurement 1. Outline The present invention also provides a standard solution for measuring HMGB1, which is characterized by containing a substance having a cationic moiety in the standard solution used for measuring HMGB1 in a sample.

2. HMGB1
Details of HMGB1 in the standard solution for measuring HMGB1 of the present invention are as described in "[1] HMGB1-containing solution" above.

Further, the concentration of HMGB1 contained in the standard solution for measuring HMGB1 of the present invention is not particularly limited, and may be appropriately selected from the concentration range normally used as a standard solution for measuring HMGB1.

3. Substance Having a Cation Site The details of the substance having a cation site in the standard solution for measuring HMGB1 of the present invention are as described in "[1] HMGB1-containing solution" above.

Furthermore, in the present invention, the concentration of the substance having a cationic moiety in the standard solution for measuring HMGB1 is not particularly limited as long as it can sufficiently stabilize HMGB1 in the standard solution. Although the optimum concentration varies depending on the concentration of HMGB1 in the standard solution, it is sufficient that it is 150 mM or more in the standard solution, preferably 200 mM or more, more preferably 500 mM or more, and particularly preferably 1,000 mM or more. It is.

In addition, the upper limit of the concentration of the substance having a cationic moiety in the standard solution is not particularly limited, but considering costs etc., it is preferably 3,000mM or less, more preferably 2,000mM or less, and 1,500mM or less. The following are particularly preferred.

4. pH of standard solution
The pH of the standard solution for measuring HMGB1 of the present invention is preferably 9.0 or less, particularly preferably in the range of 4.4 to 8.0.

Further, as the buffer used to bring the standard solution for measuring HMGB1 of the present invention to the above-mentioned pH range, conventionally known buffers having a buffering capacity within the above-mentioned pH range can be used as appropriate.

The standard solution for measuring HMGB1 of the present invention can be prepared by adding the substance having a cationic moiety described above to a solution containing HMGB1. For example, the substance having a cationic moiety and HMGB1 are added to pure water. It can be prepared by dissolving it and adjusting the pH using a conventionally known buffer agent or the like as described above.

Further, the standard solution for measuring HMGB1 of the present invention can be used as a measurement kit for measuring HMGB1 in a sample in combination with a measurement reagent for measuring HMGB1 in a sample.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] (Confirmation of stabilizing effect of HMGB1 in solution according to the present invention-1)
The stabilizing effect of sodium chloride on HMGB1 in solution was confirmed.

1. Preparation of HMGB1-containing solution A solution containing porcine HMGB1 prepared in Reference Example 1 described later was thoroughly dialyzed with potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4).
After this dialysis, the protein concentration of the solution containing HMGB1 was determined using a protein assay (manufactured by Bio-Rad).
Then, this solution containing HMGB1 was diluted with 10mM Good's buffer (pH 7.4) containing sodium chloride at the following concentration and 1% BSA, and five types of HMGB1 concentration of 80ng/mL with different sodium chloride concentrations were prepared. HMGB1-containing solutions were prepared respectively.
Sodium chloride: 0mM, 150mM, 300mM, 500mM or 1,000mM

2. Storage of HMGB1-containing solutions The five types of HMGB1-containing solutions prepared in 1 above were stored under refrigeration (2 to 8°C).

3. Measurement of HMGB1 The HMGB1 concentration of each of the five types of HMGB1-containing solutions was measured at the start of storage (0 days) and after 3, 61, 131, 193, 327, 469, and 594 days of storage.

Note that HMGB1 was measured by enzyme immunoassay (sandwich method) using the measurement reagent described in (1) below.

(1) Measurement reagent (a) Peroxidase-labeled antibody A peroxidase-labeled antibody prepared in Reference Example 2 described later, in which peroxidase is conjugated to an antibody that binds to HMGB1 and HMGB2, is used as an enzyme in the sandwich method of enzyme immunoassay. It was used as a labeled antibody.
(b) Antibody immobilized on a microplate The antibody immobilized on a microplate, prepared in Reference Example 3 described later, which binds to HMGB1 but does not bind to HMGB2, was immobilized on each well of the microplate. It was used as an immobilized antibody in the sandwich method.
(c) Dilution reagent 100mM Good's buffer (pH 9.0) containing 0.1% BSA was prepared and used as a dilution reagent.
(d) Washing solution Phosphate buffered saline (pH 7.2) containing 0.05% Tween 20 was prepared and used as a washing solution.
(e) Peroxidase substrate solution An aqueous hydrogen peroxide solution containing 3,3',5,5'-tetramethylbenzidine (TMBZ) was used as a peroxidase substrate solution.
(f) Reaction stop solution A 6N sulfuric acid aqueous solution was prepared and used as a reaction stop solution.
(g) Standard solution for measuring HMGB1 A solution containing HMGB1 prepared in the same manner as in Example 1, 1 was diluted with 10 mM Good's buffer (pH 7.4) containing 1% BSA, and the HMGB1 concentration was 320 ng/mL. A standard solution for HMGB1 measurement was prepared and stored frozen at -80°C.
Note that this standard solution for HMGB1 measurement was diluted with the diluent reagent in (c) of (1) above so that the HMGB1 concentration was 80 ng/mL before use.

(2) Measurement by enzyme immunoassay (sandwich method) (a) Each of the five types of HMGB1-containing solutions prepared in 1 above was diluted 41 times with the diluent reagent in (c) of (1) above.
(b) Add 100 μL of the above five types of HMGB1-containing solutions to each well of the microplate immobilized antibody in (b) of (1) above, let stand at 37°C for 2 hours, and then plate the microplate. An antigen-antibody reaction was performed between the immobilized antibody and HMGB1 contained in the HMGB1-containing solution.
(c) Next, each well of the immobilized antibody on the microplate was washed with the washing solution of (d) in (1) above.
(d) The peroxidase-labeled antibody in (a) of (1) above was diluted 6,000 times with Good's buffer (pH 7.0) containing 0.1% BSA. Next, 100 μL of this was added to each well of the immobilized antibody on the microplate that had been washed as described above, and then left at 25° C. for 2 hours.
This caused a reaction in which the peroxidase-labeled antibody was bound to HMGB1 bound to the antibody immobilized on the microplate.
(e) Thereafter, each well of the immobilized antibody on the microplate was washed with the washing solution of (d) of (1) above.
(f) Next, 100 μL of the peroxidase substrate solution of (e) in (1) above was added to each well of the immobilized antibody on the microplate. Then, the reaction was carried out at room temperature.
(g) 20 minutes after the addition of the peroxidase substrate solution, 100 μL of the reaction stop solution from 1 was added to each well of the immobilized antibody on the microplate to stop the reaction of the labeled peroxidase. .
(h) Next, the absorbance (450 nm) of the solution of the microplate-immobilized antibody in each well was measured using a microplate reader (Model 680; manufactured by Bio-Rad).
(i) Table 1 shows the measurement results of HMGB1 concentration obtained by the above operations.
In addition, each measurement value (HMGB1 concentration) shown in Table 1 is obtained by subtracting the absorbance when physiological saline (150mM sodium chloride aqueous solution) is used as a sample, and subtracting the absorbance from the standard solution for HMGB1 measurement in (g) of (1) above. It was converted from

(3) Measurement results Table 1 shows the measurement results of HMGB1 concentration. The values shown in Table 1 are the HMGB1 concentrations (ng/mL) obtained in the measurements, and the numbers in parentheses represent the residual rate of HMGB1 when the start of storage (day 0) is taken as 100%. This is what I did.

As is clear from Table 1, in the HMGB1-containing solution without sodium chloride (0 mM), the HMGB1 concentration decreased as the storage period progressed, and the HMGB1 concentration at the start of storage was taken as 100%. It can be seen that the percentage decreases to 75.3% after 61 days of storage, and to 54.6% after 131 days of storage. Even after that, it can be seen that the HMGB1 concentration continued to decrease and decreased to 4.3% on the 594th day.
On the other hand, it can be seen that in all the HMGB1-containing solutions in which sodium chloride is present, the residual rate of HMGB1 is significantly improved compared to the case where sodium chloride is not present.
In this manner, it was confirmed that HMGB1 in a solution state could be stabilized by the presence of sodium chloride in the HMGB1-containing solution.

[Example 2] (Confirmation of stabilizing effect of HMGB1 in solution according to the present invention-2)
The stabilizing effect of HMGB1 in solution by a substance having a cationic moiety was confirmed.

1. Preparation of HMGB1-containing solution Nine types of HMGB1-containing solutions were prepared respectively.

2. Storage of HMGB1-containing solutions The nine types of HMGB1-containing solutions prepared in 1 above were stored under refrigeration (2 to 8°C).

3. Measurement of HMGB1 At the start of storage (0 days) and after 6 days, 10 days, 64 days, 90 days, and 276 days, the HMGB1 concentration of each of the nine types of HMGB1-containing solutions was measured in the same manner as in Example 1-3. was measured.

(3) Measurement results Table 2 shows the measurement results of HMGB1 concentration. The values shown in Table 2 are the HMGB1 concentrations (ng/mL) obtained in the measurements, and the numbers in parentheses represent the residual rate of HMGB1 when the start of storage (day 0) is taken as 100%. This is what I did.

As is clear from Table 2, in the HMGB1-containing solution without the presence of a substance having a cationic moiety (0 mM), the HMGB1 concentration decreased as the storage period progressed, and the HMGB1 concentration at the start of storage was reduced to 100%. It can be seen that the percentage decreases to 77.1% after 64 days of storage, to 48.5% after 90 days of storage, and to 40.6% after 276 days of storage.
On the other hand, it can be seen that in all cases where a substance having a cationic site is present in the HMGB1-containing solution, the residual rate of HMGB1 is significantly improved compared to the case where a substance having a cationic site is not present.
In this way, it was confirmed that HMGB1 in a solution state could be stabilized by the presence of a substance having a cationic moiety in the HMGB1-containing solution.

[Example 3] (Confirmation of stabilizing effect of HMGB1 in solution according to the present invention-3)
The stabilizing effect of HMGB1 in solution by a substance having a cationic moiety was confirmed.

1. Preparation of HMGB1-containing solution Three types of HMGB1-containing solution were prepared in the same manner as in Example 1-1, except that the substances having cationic moieties listed in Table 3 were contained (or not) at the respective concentrations listed in Table 3. HMGB1-containing solutions were prepared respectively.

2. Storage of HMGB1-containing solutions The three types of HMGB1-containing solutions prepared in 1 above were stored under refrigeration (2 to 8°C).

3. Measurement of HMGB1 At the start of storage (0 days), 102 days after storage, and 169 days after storage, the HMGB1 concentration of each of the two types of HMGB1-containing solutions was measured in the same manner as in Example 1-3.

4. Measurement Results Table 3 shows the measurement results of HMGB1 concentration. The values shown in Table 3 are the HMGB1 concentrations (ng/mL) obtained in the measurements, and the numbers in parentheses represent the residual rate of HMGB1 when the start of storage (day 0) is taken as 100%. This is what I did.

As is clear from Table 3, in the HMGB1-containing solution without the presence of a substance having a cationic moiety (0 mM), the HMGB1 concentration decreased as the storage period progressed, and the HMGB1 concentration at the start of storage was reduced to 100%. %, it can be seen that it decreases to 57.5% after 102 days of storage and to 37.8% after 169 days of storage.
On the other hand, it can be seen that in all cases where a substance having a cationic site is present in the HMGB1-containing solution, the residual rate of HMGB1 is significantly improved compared to the case where a substance having a cationic site is not present.
In this way, it was confirmed that HMGB1 in a solution state could be stabilized by the presence of a substance having a cationic moiety in the HMGB1-containing solution.

[Reference Example 1] (Preparation of pig HMGB1)
(1) Pig thymus was mixed with physiological saline in a blender. This was repeated about three times to thoroughly wash.
(2) This was centrifuged using a centrifuge (7,000×G, 10 minutes), and the supernatant was discarded.
(3) Thymocytes were transferred to another container, 750 mM perchloric acid was added, and the mixture was placed in a mixer.
(4) Centrifuge this in a centrifuge (7,000xG, 10 minutes), separate the supernatant, filter it through a glass filter with a pore size of 0.45 μm, and filter the glass filter with 750mM perchlorine. Washed with acid.
(5) The filtrate was added to acetone with strong stirring.
(6) This filtrate was centrifuged using a centrifuge (7,000×G, 10 minutes), the supernatant was separated, and acetone was further added.
(7) The precipitate was collected, dried in a fume hood overnight, dissolved in 7.5 mM sodium borate buffer (pH 9.0), and dialyzed.
(8) This was passed through a CM-Sephadex column to purify HMGB1 derived from pig thymus.

[Reference Example 2] (Preparation of peroxidase-labeled antibody)
A peroxidase-labeled antibody was prepared by labeling an antibody (monoclonal antibody) that binds to HMGB1 and HMGB2 with peroxidase.

(1) Introduction of maleimide group into peroxidase 4 mg of peroxidase (derived from horseradish) was dissolved in 0.3 mL of 0.1 M phosphate buffer (pH 7.0). To this, 1.0 mg of N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid dissolved in 60 μL of N,N'-dimethylformamide was added, and the mixture was reacted at 30°C for 60 minutes. I let it happen.
Thereafter, this was dialyzed overnight against 0.1M phosphate buffer (pH 6.0).
Through the above operations, a maleimide group was introduced into the peroxidase.

(2) Introduction of thiol groups into antibodies Antibodies that bind to HMGB1 and HMGB2 (monoclonal antibodies) [in-house preparation] were added to a 0.1M phosphate buffer solution (pH 6.5) containing a concentration of 10 mg/mL. A solution of 0.6 mg of S-acetylmercaptosuccinic anhydride dissolved in 10 μL of N,N'-dimethylformamide was added to 0.5 mL, and the mixture was reacted at room temperature for 30 minutes.

Then, to this were added 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Tris-HCl buffer (pH 7.0), and 0.1 mL of 1 M hydroxylamine hydrochloride (pH 7.0), respectively. and left at 30°C for 5 minutes.

Next, this was passed through a Sephadex G-25 column equilibrated with 0.1M phosphate buffer (pH 6.0) containing 5mM EDTA, and simple gel filtration chromatography was performed to remove excess S- Acetylmercaptosuccinic anhydride was removed and the antibody fraction was collected.
Through the above operations, a thiol group was introduced into the antibody (monoclonal antibody) that binds to HMGB1 and HMGB2.

(3) Preparation of labeled antibody The maleimide group-introduced peroxidase prepared in (1) above and the thiol group-introduced antibody prepared in (2) above were mixed one-on-one and reacted at 30°C for 20 hours. , peroxidase was introduced (labeled) into the antibody.

Thereafter, this was passed through an Ultragel AcA34 column equilibrated with 0.1M phosphate buffer (pH 6.5) to perform gel filtration chromatography.
Each fraction of this gel filtration chromatography was subjected to 10% polyacrylamide electrophoresis for confirmation, and only the antibody fraction bound to peroxidase was collected to avoid contamination with unbound peroxidase.

This peroxidase-bound antibody fraction was concentrated to obtain a peroxidase-bound antibody, that is, a peroxidase-labeled antibody.
Then, the protein concentration of the solution containing this peroxidase-labeled antibody was measured.

[Reference Example 3] (Microplate immobilized antibody)
An antibody (monoclonal antibody) that binds to HMGB1 but does not bind to HMGB2 was immobilized on a microplate to prepare an antibody immobilized on a microplate.

(1) An antibody that binds to HMGB1 but does not bind to HMGB2 (monoclonal antibody) [in-house preparation] was added to phosphate buffered saline (5.59mM disodium hydrogen phosphate, 1.47mM potassium dihydrogen phosphate, 137mM After adjusting the concentration to 10 μg/mL with sodium chloride, 2.68 mM potassium chloride (pH 7.2), add 100 μL per well to a 96-well microplate (manufactured by Nunc), and leave it at 37°C for at least 4 days. The antibody was adsorbed to each well of the microplate and immobilized.

(2) Add 200 μL per well of 10 mM phosphate buffer (pH 8.0) containing 1% BSA to the microplate on which this antibody has been immobilized, and perform blocking by standing in the refrigerator overnight or more. Ta.

By the above operations, a microplate-immobilized antibody was prepared in which an antibody (monoclonal antibody) that binds to HMGB1 but does not bind to HMGB2 was immobilized on a microplate.

Claims (1)

In a solution, characterized in that a substance selected from the group consisting of potassium chloride, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium phosphate, lithium chloride, magnesium sulfate, or ammonium sulfate is added to the HMGB1-containing solution. HMGB1 stabilization method.