JP7178607B2 - Reagent for extracting amyloid protein - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law 1. Presentation at the meeting Announcement date March 29, 2018 Name of the meeting, location The XVI International Symposium on Amyloidosis KKR Hotel Kumamoto (3-31 Chibashiro-cho, Chuo-ku, Kumamoto-shi, Kumamoto)

The present invention relates to reagents and methods for extracting amyloid protein. More specifically, the present invention relates to reagents and methods for specifically extracting amyloid protein from biological samples.

Amyloid protein is a water-insoluble fibrous protein, has a structure in which β-sheet structures are laminated (cross-β structure), and is mainly deposited in the extracellular regions of organs and tissues. When amyloid protein is stained with Congo red and observed with a polarizing microscope, it exhibits green, yellow or orange birefringence (Non-Patent Document 1).
Amyloidosis is a general term for diseases in which amyloid protein deposits outside the cells of organs and causes dysfunction. It is broadly classified into focal amyloidosis in which is deposited. Representative examples of systemic amyloidosis include AL amyloidosis (immunoglobulin amyloidosis), AA amyloidosis, Aβ2M amyloidosis (dialysis amyloidosis), familial amyloid polyneuropathy (FAP), and senile systemic amyloidosis (SSA). . Among these diseases, AL amyloidosis, FAP, and SSA are designated as intractable diseases in Japan. On the other hand, localized amyloidosis includes cerebral amyloidosis such as Alzheimer's disease, cerebral amyloid angiopathy, and prion disease (Non-Patent Document 2). Up to now, about 30 kinds of amyloid proteins have been identified as causative of amyloidosis (Non-Patent Document 1).

Pathological diagnosis of amyloidosis is performed on formalin-fixed paraffin-embedded (FFPE) sections, and immunostaining identifies amyloid species. However, about 10% of cases are difficult to identify by immunostaining. As an alternative, mass spectrometry is used, which requires extraction of amyloid from FFPE sections. Conventionally, amyloid deposition sites were collected from FFPE sections by laser microdissection (microdissection method), and formalin-crosslinked amyloid was treated with a surfactant and extracted. In this method, a pathologist identifies the site of amyloid deposition and then collects the deposition site by microdissection. However, it is not an easy method to implement in terms of human resources and costs.
In order to elucidate the pathophysiology of amyloidosis, analysis of amyloid proteins is essential. Conventionally, amyloid proteins are prepared from fresh or frozen materials by water extraction (water extraction method) through a multistep process. (Non-Patent Document 3). However, this method cannot prepare amyloid protein from FFPE sections, and requires the preparation of large amounts (in grams) of fresh or frozen material, which is labor intensive and costly.

Under such circumstances, if there is a technology to extract amyloid protein from FFPE sections easily and at low cost, it will be possible to conduct rapid diagnosis of amyloidosis and research to elucidate the pathology, but currently there is no such technology. .

Sipe et al., Amyloid 23:209-213 2016 Intractable Disease Information Center HP (http://www.nanbyou.or.jp/entry/45) Schubert et al., J Clin Invest., 47:924-933 1968

In view of the above circumstances, an object of the present invention is to provide a reagent for specifically extracting amyloid protein from a biological sample (eg, FFPE section, etc.) and an amyloid protein preparation kit containing the reagent.
Another object of the present invention is to provide a method for specifically extracting an amyloid protein from a biological sample.

After deparaffinizing FFPE sections, the present inventors used dimethyl sulfoxide (DMSO, hereinafter abbreviated as "DMSO"), which is a sulfoxide, which is a reagent that denatures (destroys) β-sheets in amyloid. Acid amides N,N-dimethylformamide (DMF, hereinafter abbreviated as "DMF"), lower alcohols methanol (methanol), fluorinated alcohols 2,2,2-trifluoro Ethanol (2,2,2-Trifluoroethanol: TFE, hereinafter abbreviated as “TFE”) and hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propanol: HFIP, hereinafter “HFIP”) It was found that amyloid protein was specifically extracted from the FFPE section when treated with (abbreviated below).
In addition, it is possible to extract amyloid protein even if the above five solvents are mixed, and in particular, treatment with a mixed solution of DMSO and TFE or DMF and TFE increases the specificity of the extracted amyloid protein. I also found out.
The present invention has been completed based on the above findings.

That is, the present invention is the following (1) to (19).
(1) A reagent for extracting an amyloid protein from a biological sample, the reagent containing a compound that denatures a β-sheet structure.
(2) The above ( 1) The reagent described in 1).
(3) The reagent according to (2) above, wherein the sulfoxide is either dimethylsulfoxide (DMSO) or diethylsulfoxide.
(4) the acid amides are N,N-dimethylformamide (DMF), N,N-diethylformamide, N,N-dimethylacetamide, N,N-diethylacetamide or N,N-dimethylpropionamide; The reagent according to (2) above, characterized in that
(5) the alcohol having 1 to 4 carbon atoms is methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-butanol, 2-methyl-1-propanol, t-butanol, allyl alcohol, 3-butene; -2-ol, cyclobutanol, 1,2-ethanediol, 1,2-propanediol or 1,3-propanediol.
(6) the fluorinated alcohols are 2,2,2-trifluoroethanol (TFE), 1,1,1-trifluoro-2-propanol, 1,1,1,3,3,3-hexafluoro- 2-propanol (HFIP), 1, 1, 1, 3, 3, 3-hexafluoro-2-methyl-2-propanol, 2, 2, 3, 4, 4, 4-hexafluoro-1-butanol or 2, The reagent according to (2) above, which is 2, 3, 3, 4, 4-hexafluoro-1, 5-pentanediol diol.
(7) A reagent for extracting amyloid protein from a biological sample, characterized by containing one or more selected from the group consisting of DMSO, DMF, methanol, HFIP and TFE (4) to (6) The reagent according to any one of.
(8) The reagent according to (7) above, characterized in that DMSO and TFE are selected.
(9) The reagent according to (7) above, characterized in that DMF and TFE are selected.
(10) The reagent according to any one of (1) to (9) above, wherein the biological sample is immobilized.
(11) The reagent according to (10) above, wherein the biological sample is immobilized with a immobilizing reagent having an aldehyde group.
(12) The reagent according to (11) above, wherein the biological sample is a formalin-fixed, paraffin-embedded section.
(13) A kit for extracting an amyloid protein from a biological sample, the kit comprising at least the reagent according to any one of (1) to (12) above.
(14) The kit according to (13) above, further comprising a wax solubilizer.
(15) A method for extracting an amyloid protein from a biological sample, the method comprising the following steps.
(a) contacting and incubating a biological sample with the reagent according to any one of (1) to (12) above;
(b) removing insolubles from the mixture after incubation in step (a); and (c) removing the reagent from the fraction obtained in step (b);
(16) The method according to (15) above, wherein the biological sample is fixed.
(17) The method according to (16) above, wherein the biological sample is immobilized with a immobilizing reagent having an aldehyde group.
(18) The method according to any one of (15) to (17) above, further comprising the step of deparaffinizing the biological sample with a wax solubilizer.
(19) A method for identifying amyloid protein in a biological sample, comprising determining the amino acid sequence of the amyloid protein extracted by the method according to any one of (15) to (18) above.

INDUSTRIAL APPLICABILITY According to the present invention, amyloid protein can be specifically extracted from a biological sample simply and at low cost. As a result, the diagnosis of amyloidosis becomes easy, and even cases that cannot be identified by immunostaining can be easily diagnosed.

Furthermore, since the present invention enables extraction of amyloid protein from FFPE, pathological analysis can be performed using accumulated FFPE, and the scope of research subjects is dramatically expanded.

The results of SDSPAGE separation of extracts from FFPE sections of Alzheimer's disease model mice using DMSO, DMF and TFE are shown. Arrows indicate extracted amyloid proteins. Fig. 2 shows the results of mass spectrometric analysis of extracts from FFPE sections of Alzheimer's disease model mice using DMSO. Figure 2 shows the results of a study on the effect of temperature and the effect of a mixed solution of DMSO, DMF and TFE on Amyloid β extraction from FFPE sections. Arrows indicate the extracted amyloid protein (Amyloid β). The results of SDSPAGE separation of extracts from FFPE sections of Alzheimer's disease model mice using DMSO, DMF, methanol, TFE and HFIP are shown. Arrows indicate the extracted amyloid protein (Amyloid β). 1 shows the results of extraction of amyloid protein from human amyloidosis specimens. Fig. 2 shows the results of mass spectrometric analysis of amyloid proteins from human amyloidosis specimens. Fig. 3 shows the results of extracting amyloid protein from non-human animal amyloidosis specimens. 1 shows the results of examining the effect of TFE on the β-sheet structure in amyloid protein.

A first embodiment of the present invention is a reagent for extracting an amyloid protein from a biological sample, the reagent containing a compound that denatures the β-sheet structure (also referred to as the "reagent of the present invention").
Here, the compound that modifies the β-sheet structure is not particularly limited as long as it can modify or destroy the β-sheet structure. Examples include sulfoxides such as dimethylsulfoxide (DMSO) and diethylsulfoxide, Acid amides such as N,N-dimethylformamide (DMF), N,N-diethylformamide, N,N-dimethylacetamide, N,N-diethylacetamide and N,N-dimethylpropionamide, methanol, ethanol , n-propanol, isopropanol, n-butanol, 2-butanol, 2-methyl-1-propanol, t-butanol, allyl alcohol, 3-buten-2-ol, cyclobutanol, 1,2-ethanediol, 1, C1-4 lower alcohols such as 2-propanediol and 1,3-propanediol, 2,2,2-trifluoroethanol (TFE), 1,1,1-trifluoro-2-propanol, 1 ,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol, 2,2,3,4, Mention may be made of compounds such as fluorinated alcohols such as 4-hexafluoro-1-butanol and 2,2,3,3,4,4-hexafluoro-1,5-pentanediol. The reagent of the present invention may contain one or more selected from the above compounds (compounds that modify the β-sheet structure). More preferred reagents of the present invention are reagents containing one or more selected from the group consisting of DMSO, DMF, methanol, HFIP and TFE.

Here, the biological sample is a sample derived from the living body of an animal, such as a biological tissue. The biological sample may be immobilized so that soluble proteins do not flow out, and the immobilizing agent is preferably an agent having an aldehyde group such as formaldehyde or glutaraldehyde. Also, the biological sample may be a formalin-fixed paraffin (FFPE) section or the like. When the biological sample is an FFPE section, it is desirable to perform a wax solubilization treatment (deparaffinization treatment). The wax solubilizer is not particularly limited as long as it contains a compound that solubilizes wax (deparaffinizes). Examples include aromatic hydrocarbons having 6 to 8 carbon atoms such as benzene, toluene, and xylene. , n-hexane, 2,2-dimethylbutane, 2,3-dimethylbutane, 2-methylpentane, 3-methylpentane, n-heptane, n-octane, n-nonane, n-decane, cyclohexane, cycloheptane and Aliphatic hydrocarbons with 6 to 10 carbon atoms such as cyclooctane, petroleum mixed hydrocarbons such as mineral oil (liquid paraffin) and petroleum benzine, natural oils such as D-limonene, dichloromethane, chloroform, 1,2 -chlorinated hydrocarbons such as dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane and tetrachloroethylene, aliphatic ethers such as diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran and tetrahydropyran, etc. Carbon disulfide and the like can be mentioned, and one or more of these compounds can be used in combination. As a wax solubilizer, more preferably one or more compounds selected from the group consisting of xylene, n-hexane, mineral oil, D-limonene, chloroform, benzene, diethyl ether, carbon disulfide and petroleum benzine. things can be mentioned. In particular, wax solubilizers that phase separate when mixed with reagents that modify the beta-sheet structure (eg, reagents including DMSO, DMF, methanol, HFIP and/or TFE) are preferred. Using such a wax solubilizer makes it possible to more easily extract amyloid protein from FFPE sections.

The reagent according to the first embodiment of the present invention is a reagent containing a compound capable of denaturing, destroying, etc. the β-sheet structure (e.g., one selected from the group consisting of DMSO, DMF, methanol, HFIP and TFE, or and may contain substances other than these compounds (e.g., water, etc.), and substances other than compounds (e.g., water, etc.) capable of denaturing, destroying, etc. the β-sheet structure. The content is 10% or less, preferably 5% or less, more preferably 2% or less.
Even if the reagent of the present invention contains only one compound that denatures the β-sheet structure, the amyloid protein can be specifically extracted from the biological sample (that is, proteins other than the amyloid protein are not extracted). However, even a reagent containing a plurality of compounds can suitably achieve the purpose. More specifically, for example, a mixture of a plurality of selected from sulfoxides DMSO, acid amides DMF, lower alcohols methanol, fluorinated alcohols TFE and HFIP may be used. In this case, mixing DMSO and TFE or DMF and TFE further improves the specificity of amyloid protein extraction. When the reagent of the present invention is prepared by mixing one or more selected from the group consisting of DMSO, DMF, methanol, HFIP and TFE, the abundance ratio of each substance in the reagent is not particularly limited. Less than 10% (by weight, the same below), 10% to less than 20%, 20% to less than 30%, 30% to less than 40%, 40% to less than 50%, 50% to less than 60%, 60% to less than 70% %, 70% or more and less than 80%, 80% or more and less than 90%, or 90% or more and less than 100%.

A second embodiment of the present invention is a kit for extracting an amyloid protein from a biological sample, the kit comprising at least the reagent of the present invention.
The kit according to the second embodiment of the present invention (also referred to as "the kit of the present invention") contains at least one reagent of the present invention, and may contain a plurality of reagents of the present invention. . In addition to the reagents of the present invention, the kit of the present invention contains wax solubilizers (e.g., aromatic hydrocarbons having 6 to 8 carbon atoms such as benzene, toluene, and xylene, n-hexane, and 2,2-dimethylbutane). C6-10 fats such as , 2,3-dimethylbutane, 2-methylpentane, 3-methylpentane, n-heptane, n-octane, n-nonane, n-decane, cyclohexane, cycloheptane and cyclooctane petroleum-based mixed hydrocarbons such as mineral oil (liquid paraffin) and petroleum benzine, natural oils such as D-limonene, dichloromethane, chloroform, 1,2-dichloroethane, 1,1,1-trichloroethane , chlorinated hydrocarbons such as 1,1,2-trichloroethane and tetrachloroethylene, aliphatic ethers such as diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran and tetrahydropyran, and carbon disulfide, etc.) It may also contain a buffer for diluting the extracted amyloid. Instructions for use of the kit of the present invention may be included in addition to reagents and the like.

A third embodiment of the present invention is a method for extracting an amyloid protein from a biological sample, comprising the following steps.
(a) contacting and incubating a biological sample with a reagent of the invention;
(b) removing insolubles from the mixture after incubation in step (a); and (c) removing the reagent from the fraction obtained in step (b);
In step (a), a biological sample containing amyloid protein (preferably a sample fixed with formaldehyde or the like) is brought into contact with the reagent of the present invention, and components in the sample (e.g., DMSO, DMF, methanol, HFIP and/or TFE) is the step of incubating the biological sample so that it permeates the surface and interior of the biological sample. The incubation temperature in step (a) is not particularly limited, but is, for example, about 10°C to about 70°C, preferably about 20°C to 60°C, more preferably about 30°C to about 50°C. . Incubation time is, for example, about 0.5 hours to about 24 hours, preferably about 1 hour to about 15 hours. When FFPE sections are used as biological samples, they are preferably deparaffinized in advance.
Step (b) is a step of removing insoluble matter from the mixture of the biological sample and the reagent of the present invention. Insoluble matter not dissolved in the reagent of the present invention can be removed as a precipitate fraction by centrifugation or the like, and the amyloid protein can be recovered in the supernatant fraction.
Step (c) is a step of removing the reagent of the present invention (eg, DMSO, DMF, methanol, HFIP and/or TFE) from the fraction obtained by removing insolubles in step (b). Removal of the reagent of the present invention can be carried out by a general method for removing organic solvents that can be easily carried out by those skilled in the art. For example, a method of removing an organic solvent from a supernatant fraction obtained by removing insoluble matter by centrifugation or the like using a vacuum dryer such as an evaporator can be used. Once the reagent of the present invention is removed, the amyloid protein extracted from the biological sample can be obtained as a dry preparation.

In a fourth embodiment of the present invention, amyloid protein is extracted from a biological sample according to the third embodiment of the present invention (hereinafter also referred to as "the amyloid protein extraction method of the present invention"), and the extracted amyloid protein is A method for identifying amyloid protein in a biological sample comprising determining the amino acid sequence.
The amino acid sequence of the amyloid protein obtained by the amyloid protein extraction method of the present invention can be determined by a conventional method. Based on the determined amino acid sequence information, the extracted amyloid protein is identified, making it possible to identify amyloidosis associated with the amyloid protein.
In addition, if a trace amount of other protein is present in addition to the extracted amyloid protein, the amyloid protein is separated from the extracted amyloid protein fraction by a method such as SDSPAGE or column chromatography, and then the amyloid protein is isolated. Amino acid sequence analysis may be performed.

The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, where this specification has been translated into English and includes the words "a", "an" and "the" in the singular, unless the context clearly indicates otherwise, the singular rather than the singular It shall include plural things.
EXAMPLES The present invention will be further described below with reference to Examples, but the Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Extraction of Amyloid β from FFPE section of Alzheimer's disease model mouse 1-1. Materials and Methods A 10 μm-thick section of a formalin-fixed paraffin embedded (FFPE) block of the brain of a 5XFAD mouse, an Alzheimer's disease model mouse, was used.
The deparaffinized sections were placed in a tube one by one, DMSO, DMF and TFE, 100 μl each of 1% NH ₃ solution and 1% formic acid solution of each solution were added and allowed to stand at 37° C. for 16 hours. After centrifuging each sample, the supernatant was transferred to another tube and heated to dryness in a vacuum dryer to obtain an extract. The resulting extracts were separated by SDSPAGE and silver stained.
Based on this result, the amino acid sequence of the extracted protein was determined. 100 μl of DMSO was added to the deparaffinized section and allowed to stand at 37° C. for 16 hours. 90 μl of 20 mM Tris-HCl, pH 8.0 was added to 10 μl of the supernatant, and 0.5 μg of trypsin was added for enzymatic digestion at 37° C. for 16 hours to prepare a peptide sample. Peptide samples were measured by LC/MS/MS (Q-Exactive plus, Thermo Fischer Scientific) and proteins were identified from the acquired data using a mascot search engine.

1-2. result
Using DMSO, DMF and TFE, a protein band (arrow in FIG. 1) was confirmed at a molecular weight of 10 kDa or less. Under alkaline and acidic conditions, some extracts were observed in the high molecular weight region of TFE.
Moreover, as a result of determining the amino acid sequence of the extract using DMSO, a peptide (LVFFAEDVGSNK: SEQ ID NO: 1) derived from the Amyloid β protein was identified. FIG. 2 shows the MSMS spectrum of the extracted peptides.

2. Investigation on the effects of temperature and mixtures of DMSO, DMF and TFE on extraction of Amyloid β from FFPE sections 2-1. Materials and Methods A 10 μm slice of a formalin-fixed, paraffin-embedded brain block of a 5XFAD mouse, an Alzheimer's disease model mouse, was used.
100 μl each of DMSO, DMF and TFE were added to the deparaffinized sections and allowed to stand at 37° C. and 60° C. for 16 hours. A 50% aqueous solution of each solution, a 50% DMSO/TFE mixture, a 50% DMF/TFE mixture, or a 50% DMSO/DMF mixture was added to the deparaffinized sections, and the sections were allowed to stand at 37°C for 16 hours. Each supernatant was subjected to SDSPAGE and silver-stained.

2-2. result
At 37°C and 60°C, there was no difference in the extraction using each solution. Amyloid β was not extracted with each 50% aqueous solution. In the 50% DMSO/TFE and 50% DMF/TFE mixtures, compared to the DMSO and DMF-only extracts, smear signals in the high-molecular region disappeared, and the extraction of proteins other than Amyloid β was suppressed. (Fig. 3).

3. Investigation on the effects of DMSO, DMF, methanol, TFE and HFIP on the extraction of Amyloid β from FFPE sections 3-1. Materials and Methods A 10 μm slice of a formalin-fixed, paraffin-embedded brain block of a 5XFAD mouse, an Alzheimer's disease model mouse, was used.
100 μl each of DMSO, DMF, methanol, TFE and HFIP were added to the deparaffinized sections and allowed to stand at 37° C. for 16 hours. Each supernatant was subjected to SDSPAGE and silver-stained.

3-2. result
Using DMSO, DMF, methanol, TFE and HFIP, a protein band was detected with a molecular weight of about 10 kDa or less similar to that in FIG.

4. Extraction of amyloid protein from human amyloidosis specimen 4-1. Materials and Methods FFPE sections of the following three human cases diagnosed as having amyloidosis by Congo red staining at Niigata University University Hospital were used.
Case 1: Kidney with AA Amyloidosis,
Case 2: Heart in a case where amyloid protein was not identified Case 3: Intestinal tract in a case where amyloid protein was not identified After adding 100 μl of DMF to each deparaffinized section, it was allowed to stand at 37°C for 16 hours. . After centrifuging the sample, the supernatant was transferred to another tube and heated to dryness in a vacuum dryer to obtain an extract. The resulting extracts were separated by SDSPAGE and silver stained.

4-2. Results In each case, specific proteins with different molecular weights were extracted (Fig. 4).

5. Identification of amyloid proteins from human amyloidosis specimens by mass spectrometry 5-1. Materials and Methods 10 μl each of the extract obtained in 3 above was subjected to mass spectrometry.
Each extract was digested with trypsin in the same manner as in 1 above and FIG. 2 to prepare a peptide sample. The obtained peptide samples were measured by LCMSMS (TSQ quantive, Thermo Fischer Scientific). For mass spectrometry, selected reaction monitoring (SRM) was performed by setting a transition for the mass of fragment peptides obtained by tryptic digestion of Serum Amyloid A, Apolipoprotein A IV, and Transthyretin.

5-2. Results Serum Amyloid A (AYSDMR: SEQ ID NO: 2), Apolipoprotein IV (SLAPYAQDTQEK: SEQ ID NO: 3, LGEVNTYAGDLQK: SEQ ID NO: 4, SELTQQLNALFQDK: SEQ ID NO: 5), Transthyretin (GSPAINVAVHVFR : SEQ ID NO: 6, ALGISFHEHAEVVFTANDSGPR: SEQ ID NO: 7, AADDTWEPFASGK: SEQ ID NO: 8) were detected (Fig. 5).

6. Extraction of amyloid protein from amyloidosis specimen of non-human animal 6-1. Materials and Methods FFPE sections of the following animal tissues in which amyloid deposition was confirmed by Congo red staining were used.
Case 1 Canine medullary carcinoma Case 2 Canine intestinal plasmacytoma Treated in the same manner as in 3 above, and subjected to SDSPAGE and silver staining.

6-2. Results In each case, specific proteins with different molecular weights were extracted (Fig. 6).

7. Confirmation of the effect of the reagent of the present invention on the β-sheet structure
We confirmed the effect of TFE on the β-sheet structure in amyloid protein.
CD spectra of amyloid proteins Amyloid β1-42 (Peptide Research Institute, Inc.) and Transthyretin (Wako Pure Chemical Industries, Ltd.) in Tris-HCl buffer and in TFE were measured and compared.
As a result, the CD spectra of amyloid protein in Tris-HCl buffer and TFE showed a negative maximum near 215 nm and a positive maximum near 195 nm, indicating a β structure, for both Amyloidβ1-42 and Transthyretin in Tris-HCl buffer. observed. In contrast, in TFE, negative peaks were observed around 208 and 222, which indicate α-helical structures for both proteins.
From the above, it was confirmed that TFE, one of the reagents of the present invention, denatures the β-sheet structure in amyloid protein.

The present invention provides reagents and methods for convenient and specific extraction of amyloid protein from biological samples. Therefore, the present invention greatly contributes to the development of medical fields such as research and diagnosis of amyloidosis.

Claims (10)

A reagent for extracting amyloid protein from formalin-fixed paraffin-embedded sections ,
Said reagent comprising either N,N-dimethylformamide (DMF), methanol, 2,2,2-trifluoroethanol (TFE), a mixture of DMSO and TFE, or a mixture of DMF and TFE . The reagent of claim 1, wherein the reagent is a mixture of DMF and TFE, or a mixture of DMSO and TFE. 3. The reagent according to claim 1, wherein the DMF content in the mixture of DMF and TFE is 40% by weight or more and less than 60% by weight. 3. The reagent according to claim 1, wherein the content ratio of DMSO in the mixed solution of DMSO and TFE is 40% by weight or more and less than 60% by weight. A kit for extracting amyloid protein from formalin-fixed, paraffin-embedded sections , the kit comprising at least the reagent according to any one of claims 1 to 4 . 6. The kit of Claim 5 , further comprising a wax solubilizer. A method for extracting amyloid protein from formalin-fixed, paraffin-embedded sections , the method comprising the steps of:
(a) contacting and incubating a biological sample with the reagent according to any one of claims 1 to 4 ;
(b) removing insolubles from the mixture after incubation in step (a); and (c) removing the reagent from the fraction obtained in step (b); The method according to claim 7, wherein the temperature of incubation in step (a) is between 20°C and 60°C. 9. The method of claim 7 or 8 , further comprising the step of deparaffinizing the formalin-fixed, paraffin-embedded sections with a wax solubilizer. A method for identifying amyloid protein in a biological sample, comprising determining the amino acid sequence of the amyloid protein extracted by the method according to any one of claims 7 to 9 .

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