JP2007097508A - Method for pretreatment of tissue material of mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the pretreatment of a mammalian tissue material for the preparation of a specimen for PrPSc (scrapie-type prion protein) detection and measurement to complete the pretreatment with reduced number of steps in a short time without exerting adverse effect on the detection and measured value of PrPSc. <P>SOLUTION: The pretreatment method contains a solubilized PrPC decomposition step to add a decomposition enzyme mixture containing a surfactant and proteinase K to the collected mammalian tissue material and homogenize the product. The invention further provides a step to add a DNA decomposition enzyme and/or an RNA decomposition enzyme and/or a collagen decomposition enzyme to the decomposition enzyme mixture. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a pretreatment method for mammalian tissue material, and particularly to an abnormal prion protein (PrPSc) that accumulates in mammalian tissue such as the onset of prion disease, mainly Western blotting (WB method) and enzyme immunization. The present invention relates to a pretreatment method of mammalian tissue material for preparing a sample for detection or measurement by a measurement method (EIA method including ELISA) or the like.

  Examples of mammalian prion diseases include Creutzfeldt-Jakob disease (CJD) as representative diseases in humans, Gerstmann-Strausler-Scheinker Syndrome (GSS), which is hereditary prion disease, , Fatal familial insomnia, Kuru, etc. CJD is sporadic CJD of unknown etiology, hereditary familial CJD, mutant CJD caused by oral infection from bovine spongiform encephalopathy (BSE) (for example, see Non-Patent Document 1), human pituitary extract preparation, It is classified into iatrogenic CJD caused by infection via human dura mater, cornea, etc., but it is common in that abnormal prion protein (PrPSc) accumulates in the central nervous system. On the other hand, in mutant CJD, PrPSc is also found in lymphoid tissues such as tonsils [see, for example, Non-Patent Document 2]. BSE is considered to be caused by infection from a mixed feed contaminated by sheep or goat scrapie organs, and similar to scrapie, degeneration is observed in the central nervous system and the like, and PrPSc is deposited, but PrPSc is deposited in lymphoid tissues. It is different from scrapie in that it is not recognized [see, for example, Non-Patent Document 3].

  BSE has been detected using histopathological, clinical, and epidemiological methods. Brain stem (medullary medulla) tissue is collected, and proteinase K (PK) is used for normal prion protein (PrPC). In the aggregate of PrPSc, in the aggregate of PrPSc, a fragment called “PrP27-30” remaining after degradation with PK is reacted with a prion protein (PrPC, PrPSc) specific antibody to detect or measure PrPSc, A test kit using the ELISA method is commercially available [see, for example, Non-Patent Document 4]. The sample preparation method (pretreatment method from medullary tissue) for detecting or measuring PrPSc with such a test kit is different for each test kit, and shows the operation procedure. For example, in the pretreatment procedure of the company A test kit, 500 ± 40 mg of medullary tissue is taken into a homogenizer bag, 15% anionic surfactant (sodium lauryl sulfate) solution mixed with 16% methanol is added, and homogenized for 2 minutes. After standing for 5 to 10 minutes, 180 μL of the homogenate (brain emulsion) is transferred to a microplate for centrifugation and centrifuged at 2750 × g for 5 minutes at 18 to 30 ° C. Next, 100 μL of the supernatant was collected in an assay plate containing 20 μL of PK solution in advance, digested with PK at 34 ° C. for 60 minutes, and then 150 μL of guanidine hydrochloride solution was added and denatured at 34 ° C. for 15 minutes. .

  In the pretreatment procedure of the B company test kit, 350 ± 40 mg of medullary tissue is placed in a 2 mL microtube containing 1.4 mL of glucose solution and beads, and completely homogenized using a bead-type crusher. Take 500 μL of the resulting homogenate in another 2 mL microtube, add 500 μL of PK solution containing 2M urea, react at 37 ° C. for 10 minutes, add 250 μL of 1-butanol, stir, 20000 × g Centrifuge for 5 minutes. After the obtained precipitate is dried for 5 minutes, 50 μL of 6M urea is added and subjected to denaturation treatment at 100 ° C. for 5 minutes, and diluted 6-fold with a diluent.

In addition to the pretreatment method as described above, for example, a tissue sample obtained by finely chopping brain tissue and spleen tissue with scissors is used as a surfactant [(8% Zwittergent 3-12 (trade name), manufactured by Calbio Chemical Co., Ltd.). ), Cercosyl (Sarkosyl (trade name), manufactured by Sigma: C ₁₅ H ₂₅ NO ₃ Na)], 100 mM sodium chloride, 5 mM magnesium chloride, 50 mM, pH = 7.5 Tris-HCl buffer (Tris) -HCl) is added to homogenize brain tissue to produce a homogenate (homogenate) (first step), and then in the second step, decomposition (digestion) is performed. (For example, refer to Patent Document 1). In the second step of digestion treatment, the homogenate obtained in the first step is subjected to decomposition (digestion) treatment using 0.5 mg / 100 mg collagenase and 40 μg / 100 mg DNase at a temperature of 37 ° C. for 4 to 12 hours. Furthermore, digestion treatment is performed at a temperature of 37 ° C. for 0.5 to 2 hours using 50 μg / 100 mg of PK (Proteinase K). Next, in the third step, after stopping the reaction, the mixture was centrifuged at 15,000 rpm and room temperature for 20 minutes, and heated and dissolved in 5% sodium dodecyl sulfate for 10 minutes to obtain a PrPSc-containing concentrate.

Japanese Patent Laid-Open No. 11-32795, (Example, FIGS. 1 to 3 etc.) Will, R.A. G. et al; Lancet Vol. 347, pp. 921-925 (1996) Hill, A.M. F. et al; Lancet Vol. 349, pp. 99-100 (1997) O'Rourke, K.M. I. et al; Clin. Microbiol. Vol. 38, pp. 3254-3259 (2000) Ken Noda, Hideki Nagai, Midori Hirasawa, Veterinary and Livestock Newspaper, 56, 9, 719-725, (2003)

  According to the pretreatment procedure of the above-mentioned Company A and Company B test kits and the PrPSc concentration method described in Patent Document 1, PrPC contained in the collected mammalian tissue material is decomposed, and the WB method, ELISA method, etc. It is suitable for detecting or measuring PrPSc, and it is possible to prepare a sample containing solubilized PrPSc at a high recovery rate, but the number of pretreatment steps is large and the operation is complicated, and the treatment takes a long time. There was a problem of requiring. In particular, in the case of the BSE inspection, a large-scale storage facility is required for freezing or refrigerated storage of each processed animal that has been dismantled in about 20 minutes until the respective inspection results are known. In addition, when the processing time in each process is simply shortened or the number of processes is reduced, the degradation of PrPC and removal of non-specific substances such as contaminating proteins become insufficient, and the recovery rate of PrPSc decreases. The problem of affecting the detection and measurement values of PrPSc occurs.

  The present invention has been made in view of the above problems, and does not adversely affect the PrPSc test and measurement values, and can complete the pretreatment within a short time with a small number of steps. Another object of the present invention is to provide a pretreatment method for mammalian tissue material for preparing a sample for detection or measurement of PrPSc.

  In order to achieve the above object, a method for pretreating mammalian tissue material according to the present invention is a pretreatment method for mammalian tissue material for preparing a sample for detection or measurement of abnormal prion protein (PrPSc); The collected mammalian tissue material is solubilized with normal prion protein (PrPC) by adding a degrading enzyme mixture containing a surfactant and proteinase K (PK) and homogenizing it. It comprises the solubilized PrPC decomposition process which decomposes | disassembles.

  In such a mammalian tissue material pretreatment method, the mammalian tissue material is selected from human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, cat It is preferable that the medullary tissue or the central nervous tissue of the medullary medulla and the degrading enzyme mixed solution contain DNA degrading enzyme and / or RNA degrading enzyme and / or collagen degrading enzyme. Furthermore, it is preferable that the surfactant contains an amphoteric surfactant, and the surfactant is preferably a combination of an amphoteric surfactant and an anionic surfactant. And the amphoteric surfactant is lauric acid amidopropyl hydroxysulfobetaine (LSB), lauric acid amidopropyl betaine (LPB), cocamidopropyl betaine (CPB), caprylic acid propyl betaine (CAPB), n-dodecyl-N, N-dimethylglycine (DDGly), laurylaminopropionic acid (LAPA), n-dodecyl-N, N- (dimethylammonio) butyric acid (DDDMA), n-dodecyl-N, N- (dimethylammonio) undecanoic acid (DDMU), 3-[(3-Colamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-Colamidopropyl) dimethylammonio] -2-hydroxy-1-propane One or more selected from sulfonic acid (CHAPSO) or a salt thereof In addition, the anionic surfactant is sodium dodecyl sulfate (SDS), lauroyl sarcosine (LSarc), cocoyl sarcosine sodium, dodecanoyl sarcosine sodium, lauroylmethyl-β-alanine sodium, cole One or more selected from sodium oxalate (SChol), sodium deoxycholate (SDChol), sodium glycocholate, and sodium taurocholate, and the blending ratio of the surfactant in the decomposing enzyme mixture is 0. .1 to 30% by weight is preferable.

  In the mammalian tissue material pretreatment method as described above, the homogenization treatment in the solubilized PrPC decomposition step is preferably performed by a bead-type crusher in which beads are placed in a container. In the solubilized PrPC decomposition step, After homogenization treatment, it is preferable to heat at a temperature of 20 ° C. or more and 70 ° C. or less, more preferably 35 ° C. or more and 55 ° C. or less. Further, the homogenization treatment in the solubilized PrPC decomposition step is performed at 20 ° C. or more. It can also be carried out under a temperature condition of 70 ° C. or lower, more preferably 35 ° C. or higher and 55 ° C. or lower. The treatment solution obtained in the solubilized PrPC decomposition step preferably includes a protein denaturation treatment step (A) in which a PK inhibitor and powdered urea are added and heated, and the solubilized PrPC For the precipitate obtained by adding the protein precipitant and stirring after centrifugation to the treatment liquid obtained in the decomposition step, it can also include a protein denaturation treatment step (B) in which urea solution is added and heated, Furthermore, it is preferable to add a diluent to the protein-denatured solution obtained in such a protein-denaturing treatment step to obtain a PrPSc detection or measurement sample. The treatment solution obtained in the solubilized PrPC decomposition step can include a PK inhibitor addition step in which a PK inhibitor is added. Further, the treatment solution obtained in such a PK inhibitor addition step can be diluted. A solution may be added to prepare a sample for detection or measurement of PrPSc.

  According to the present invention, by adding a degradative enzyme mixture containing a surfactant and PK and performing a homogenization treatment, the mammalian tissue material is solubilized and normal prion protein (PrPC) is degraded. The processing steps can be reduced without adversely affecting the detection and measurement of PrPSc, and the processing time can be shortened. In addition, any mammalian tissue material can be used in any mammal that may suffer from prion diseases such as human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, cat, etc. Applicable to tissue material. Among animal tissue materials, for example, in cattle, PrPSc is said to be accumulated most, and even a medullary tissue, which is relatively difficult to solubilize, is efficiently cleared in a short time of about 15 minutes or less. Can be solubilized. In addition to surfactant and PK, a homogenization treatment is performed using a decomposing enzyme mixture containing a DNA degrading enzyme and / or an RNA degrading enzyme and / or a collagen degrading enzyme, so that mammalian tissue material can be more reliably solubilized. It is possible to decompose PrPC. Examples of the surfactant to be added to the degradation enzyme mixture include, for example, lauramide amidopropyl hydroxysulfobetaine (LSB), lauramidopropyl betaine (LPB), cocamidopropyl betaine (CPB), propyl betaine caprate (CAPB), n -Dodecyl-N, N-dimethylglycine (DDGly), laurylaminopropionic acid (LAPA), n-dodecyl-N, N- (dimethylammonio) butyric acid (DDDMAB), n-dodecyl-N, N- (dimethyl) Ammonio) undecanoic acid (DDMU), 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-colamidopropyl) dimethylammonio] -2- Hydroxy-1-propanesulfonic acid (CHAPSO) or By using one or more amphoteric surfactants selected from these salts, mammalian tissue materials can be solubilized to a clear state in a shorter time, even for tissue materials of medium to large animals. There is. Further, in some cases, the combination of an amphoteric surfactant and an anionic surfactant as the surfactant can solubilize the mammalian tissue material more efficiently.

  In the solubilized PrPC decomposition step, after homogenization, the PrPC is more reliably decomposed by heating (incubating) at a temperature of 20 ° C. or higher and 70 ° C. or lower, more preferably 35 ° C. or higher and 55 ° C. or lower. In some cases, the sensitivity and accuracy of detection and measurement of PrPSc are further increased. When the protein denaturing treatment step (A) adopts a method in which a PK inhibitor and powdered urea are added to the treatment solution obtained in the solubilized PrPC decomposition step and heated, the amount of the treatment solution increases. This is advantageous in that a concentration step such as centrifugation or re-dissolution operation can be omitted after the protein denaturation treatment step. As the protein denaturation treatment step (B), a method is employed in which butanol is added to the treatment solution obtained in the solubilized PrPC decomposition step and stirred, and then the urea solution is added to the precipitate obtained by centrifugation and heated. In this case, non-specific substances that may reduce the sensitivity and accuracy of detection and measurement may be previously reduced or removed from the sample for detection and measurement of PrPSc.

  BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the best mode for carrying out the method for pretreatment of mammalian tissue material in mammals of the present invention will be described in detail with reference to the drawings.

[First Embodiment]
As shown in the flow of the pretreatment method in FIG. 1, the pretreatment method of mammalian tissue material according to the first embodiment is applied to mammalian tissue material collected for preparing a sample for detection or measurement of PrPSc. A solubilized PrPC decomposition step is performed in which a hydrolyzing enzyme mixture is added to perform a homogenization treatment. Examples of mammalian tissue materials used in the pretreatment method of the first embodiment include cattle, goats, sheep, deer, elk, mink, pigs, monkeys, cats and the like including humans from small animals such as mice, rats, and hamsters. Any mammalian tissue material ranging from middle animals to large animals can be applied. For example, in a BSE test, a definitive diagnosis by a histopathological examination or an immunohistochemical examination is performed on the buttock tissue of bovine medulla, which is said to have the largest amount of PrPSc, and for screening by the WB method, EIA method, etc. It is considered appropriate to prepare a sample for detection or measurement of PrPSc using a half divided in the center of the medullary medulla tissue. Bovine medullary tissue is one of the tissue materials that are relatively difficult to solubilize, but by adding a mixture of a degrading enzyme containing a surfactant and PK and performing a homogenization treatment within a short period of time. The tissue material can be solubilized and PrPC decomposed to a nearly clear state.

  As the surfactant to be added to the degradation enzyme mixture in the present embodiment, any surfactant such as an amphoteric surfactant, an anionic surfactant, a cationic surfactant, and a nonionic surfactant can be used. A plurality of surfactants may be used in combination. Among these surfactants, those having relatively high hydrophilicity are suitable, for example, the HLB value (edited by Tokiyuki Yoshida et al., Engineering Book Co., Ltd., published in 1987, “New Edition Surfactant Handbook” pp. 234-245 and the Japan Oil Chemistry Association, published by Maruzen Co., Ltd., 1990, “Revised 3rd edition, Oil and Chemical Chemistry Handbook”, pp. 489-493), good surfactants up to about 40, good 10 or more By using one having an HLB value, it may be solubilized more efficiently in a short time. Of the surfactants, amphoteric surfactants are preferably used, and amphoteric surfactants and anionic surfactants may be used in combination. In such a case, the mammal to be pretreated is used. In some cases, tissue material can be solubilized more efficiently. The PK may be a PK that is usually used for pretreatment of mammalian tissue material for preparing samples for detection or measurement of various proteins by the WB method or enzyme immunoassay (EIA method including ELISA). For example, any PK can be used. For example, in addition to PK derived from fungi such as Tritillachium album (Endopeptidase K: EC 3.4.21.64), bacteria, fungi, yeasts, animals Recombinant prokaryotic cells or recombinant PKs obtained from the products of recombinant prokaryotic cells that have been genetically modified to express a protein having PrPC degrading activity equal to or higher than that of PK using cells or the like as host cells can also be used.

  The degrading enzyme mixed solution may contain a DNA degrading enzyme and / or an RNA degrading enzyme and / or a collagen degrading enzyme in addition to the surfactant and PK, and a degrading enzyme comprising a surfactant and PK as main components. Compared to the case of a mixed solution, it is more preferable in terms of enabling tissue solubilization and PrPC decomposition within a shorter time. In addition, in the case of adding a DNA-degrading enzyme and / or an RNA-degrading enzyme and / or a collagen-degrading enzyme in addition to a surfactant and PK, a detection of PrPSc obtained by the pretreatment method of this embodiment In some cases, non-specific substances that may reduce the sensitivity and accuracy of detection and measurement may be reduced or removed in advance from the sample for measurement. The DNA degrading enzyme (DNase: deoxyribonuclease) added in this case is not particularly limited, and for example, exo DNase-I (EC 3.1.1.11), exo DNase-III (EC 3.1.11. .3), exo DNase-V (EC 3.1.11.5), exo DNase-VII (EC 3.1.11.6) and the like, which are sequentially cleaved from the end of the polynucleotide chain (Exonclease) ), And also cleave internal bonds of polynucleotide chains or oligonucleotide chains such as DNase-I (EC 3.1.21.1) and endo DNase (EC 3.1.21.2) derived from bovine pancreas Any DNase such as an end type can be used. End-type DNase is preferable in that DNA contained in mammalian tissue can be decomposed into small fragment strands in a short time, and has low specificity as a degrading enzyme, and cleaves double-stranded and single-stranded DNA. DNase-I can be used more suitably because it can be decomposed. Further, for example, DNase-I and exo-type or other end-type DNase may be used in combination.

  Examples of RNAse (RNase: ribonuclease) include exonuclease that sequentially cleaves from the end of a polynucleotide chain such as RNase-II (EC. 3.1.13.1) and, for example, derived from bovine pancreas RNase-A (EC 3.1.27.5), RNase-T1 (EC 3.1.27.3) or any other RNase such as an endonuclease that cleaves the internal bond of a polynucleotide chain (Endonclease) However, endo-type RNase is preferable in that it can be decomposed into small fragment chains in a short time, and RNase-A is more preferably used in that it is stable in a relatively wide pH range. it can. Also, a plurality of RNases may be used, such as a combination of RNase-A and other RNases. In addition to or instead of the DNA-degrading enzyme and the RNA-degrading enzyme, a nuclease that cleaves the phosphodiester bond in both the nucleotide strands of DNA and RNA can be added. Examples of such nucleases include endonuclease S1 (EC 3.1.30.1) derived from Aspergillus oryzae and endonuclease (EC 3.1.30.2) derived from Seratia marcescens. ), Micrococcus endonuclease (EC 3.1.31.1) derived from Staphylococcus aureus, and the like.

  Collagen is a major protein that accounts for about 30% of the proteins contained in mammalian tissues, but it has a triple helical structure, so it is difficult to cleave and decompose it with a general protease, For example, collagen proteins having different molecular structures that are called type I, II, III,..., XIII, XIV collagen and the like and that constitute collagen family proteins are specifically distributed in each connective tissue of mammals. When a collagenolytic enzyme that cleaves or degrades the triple helical structure of such a collagen family protein is added to the degradative enzyme mixture, it is used for detection and measurement of PrPSc obtained by the pretreatment method of the first embodiment. In some cases, non-specific proteins and polypeptides that may reduce the sensitivity and accuracy of detection and measurement may be reduced or removed in advance from the sample. Collagen degrading enzymes to be added to the degrading enzyme mixed solution are not particularly limited, and any collagen degrading enzymes can be used as long as they can degrade one or more types (types) of collagen family proteins. Collagenase, Gelatinase, Streptidase (Streptidase), which is mainly classified into metalloendopeptidases (EC 3.4.24.) And also called matrix metalloproteinase (MMP). Collagen degrading enzymes such as Stromlysin) can be suitably used. It is preferable to use one or more types of collagenolytic enzymes that degrade the type of collagen that is abundant in the animal tissue material to be pretreated, and it is more preferable to use collagenolytic enzymes capable of degrading multiple types of collagen proteins. .

  Examples of such collagenases include various collagenases derived from bacteria such as Clostridium, Bacillus, Pseudomonas, Vibrio, and Streptomyces. [Microbial collagenase: EC 3.4.24.3 (also known as: collagenase A, collagenase I, MMP-1, 8, 18, etc.]), a vertebrate that degrades a plurality of collagens including each type of I to III Interstitial collagenase [Interstitial collagenase: EC 3.4.24.7 (also known as: collagenase-1, MMP-1)] and I-III, V, VII, VIII, X Neutrophil collagenase (Neurophil collagenase: EC 3.4.24.34 (also known as collagenase-2, MMP-8, etc.)), collagenase-3 [EC 3.4.24. -(Also known as MMP-13)], collagenase-4 [EC 3.4.24. -(Alias MMP-18)] and the like. Gelatinases include, for example, gelatinase A (EC 3.4.24.24 (also known as type IV collagenase, MMP-2, etc.)) that degrades each type of collagen of IV, V, VII, and X. Gelatinase B [EC 3.4.24.35 (also known as type V collagenase, MMP-9, etc.)] that decomposes collagens of type IV and V. Examples of stromelysin include III ~ Stromelysin-1 [EC 3.4.24.17 (also known as MMP-3)], stromelysin-2 [EC 3.4.24.22] which degrades each type of collagen such as V and IX. (Alternative name: MMP-10)] In addition, in this embodiment, in addition to the above-mentioned collagenolytic enzymes, animal tissue materials are soluble. It is also possible to such commonly proteolytic enzymes and lipolytic enzymes used are added to the enzyme mixture when.

  The surfactant used when the amphoteric surfactant is added to the degradation enzyme mixture in the first embodiment is not particularly limited. For example, amide alkylbetaine, sulfobetaine, carboxybetaine, imidazolinium betaine. Other than amphoteric surfactants such as amino acid type and amino fatty acid type such as alkylaminoacetic acid type, alkylaminopropionic acid type, alkyl / dimethylaminoacetic acid type, alkylaminodipropionic acid type, etc. For example, any amphoteric surfactant such as cholate amidoalkyl / dimethylammoniopropane sulfonic acid, alkylamido taurine salt, alkylamidopropylamine oxide, lecithin and the like may be used. Two or more of the same or different types can be used in combination. Specific examples of such amphoteric surfactants include, for example, lauric acid amidopropyl hydroxysulfobetaine (LSB), lauric acid amidopropyl betaine (LPB), cocamidopropyl betaine (CPB: coconut oil fatty acid amidopropyl betaine), Propyl betaine caprate (CAPB), n-dodecyl-N, N-dimethylglycine (DDGly: EMPGEN BB trade name, manufactured by Calbiochem), laurylaminopropionic acid (LAPA: trade name Levon, Sanyo Chemical Industries, etc.), n- Dodecyl-N, N- (dimethylammonio) butyric acid (DDDMA; manufactured by Calbiochem), n-dodecyl-N, N- (dimethylammonio) undecanoic acid (DDMU; manufactured by Calbiochem), 3-[( 3-Colamide Propyl) dimethyl ammonio] -1-propanesulfonic acid (CHAPS), 3 - [(3- cholamidopropyl) dimethyl ammonio] -2-hydroxy-1 and the like propanesulfonic acid (CHAPSO). The amphoteric surfactant as described above may be an alkali metal salt, an alkaline earth metal salt, an ammonium salt, an amine salt, a salt such as hydrochloride or acetate, and an aqueous solution having an appropriate concentration, It may be an amphoteric surfactant dissolved or diluted in a dilute salt solution such as physiological saline or a pH buffer solution.

  Moreover, it does not specifically limit as a surfactant used when adding an anionic surfactant to the degradation enzyme liquid mixture in this 1st Embodiment, For example, a fatty acid type, an alkenyl succinic acid type | system | group, N-acyl sarcosine type | system | group, Carboxylic acid type such as N-acyl glutamic acid type, and sulfate type such as alkyl sulfate type, alkyl polyoxyethylene sulfate type, monoacyl glycerol type sulfate and acylaminosulfate type such as olefin sulfonic acid type, alkane Sulfonic acid type such as sulfonic acid type, sulfo fatty acid type, N-acyl taurine type, N-acyl-N-methyl tauric acid type, dialkyl sulfosuccinic acid type, etc., for example, alkyl phosphate type, alkyl polyoxyethylene phosphate type In addition to phosphate ester types such as cholic acid, deoxycholic acid, etc. That may be anionic surfactants. Specific examples of such anionic surfactants include sodium dodecyl sulfate (SDS), lauroyl sarcosine (LSarc), cocoyl sarcosine sodium, dodecanoyl sarcosine sodium, lauroylmethyl-β-alanine sodium, sodium cholate ( SChol), sodium deoxycholate (SDChol), sodium glycocholate, sodium taurocholate and the like. Two or more of the same or different types can be used in combination. Although the above anionic surfactants are mainly shown as sodium salts, they are other alkali metal salts, alkaline earth metal salts, ammonium salts and amine salts, and salts such as hydrochlorides and acetates. Alternatively, a surfactant dissolved or diluted in an aqueous solution of an appropriate concentration, a diluted salt solution such as physiological saline, or a pH buffer solution may be used.

  In addition, the surfactant used in the case of adding a cationic surfactant to the degradation enzyme mixed solution is not particularly limited, and examples thereof include alkylpolyoxyethyleneamine-based, fatty acid triethanolamine ester-based, N-acyl-N ′, Any cationic surface activity such as amine / amine salt type such as N'-diethylethylenediamine type, and quaternary ammonium salt type such as alkyltrimethylammonium type, dialkyldimethylammonium type, alkyldimethylbenzylammonium type, alkylpyridinium type, etc. It may be an agent. Two or more of the same or different types can be used in combination. Specific examples of such cationic surfactants include, for example, lauric acid triethanolamine monoester, N-lauroyl-N ′, N′-diethylethylenediamine, cetyltrimethylammonium chloride, cetyldimethyleneammonium chloride, dodecyltrimethyl Ammonium chloride, dodecyl dimethylene ammonium chloride, didecyl dimethyl ammonium, cetyl pyridinium chloride, guanidine thiocyanate and the like can be mentioned. The cationic surfactants as described above are mainly shown as chloride salts, but may be other halide salts such as bromide salts, salts such as hydrochlorides, formates and acetates, Alternatively, a surfactant dissolved or diluted in an aqueous solution of an appropriate concentration, a dilute salt solution such as physiological saline, or a pH buffer solution may be used.

  The surfactant used when adding a nonionic surfactant to the degradation enzyme mixture in the present embodiment is not particularly limited, and examples thereof include fatty acid polyoxyethylene ester, fatty acid polyoxyethylene sorbitan ester, and alkyl polyoxyethylene. Ether type, alkyl polyoxyethylene polyoxypropylene ether type, alkylphenyl polyoxyethylene ether type, alkyl polyoxyethylene amine type, alkyl polyoxyethylene amide type, fatty acid alkanolamide type, amine oxide type, fatty acid sorbitan ester type, fatty acid Any nonionic surfactant such as polyglycerol ester, fatty acid sucrose ester, alkyl polyglycoside, N-alkanoyl-N-methylglucamine and the like may be used. Two or more of the same or different types can be used in combination. Specific examples of such nonionic surfactants include, for example, polyoxyethylene-p-octylphenol (trade name NP40; p-tert-octylphenol), polyoxyethylene (80) sorbitol ester (trade name Tween 20), poly Oxyethylene (20) sorbitol ester (trade name Tween 80), polyoxyethylene glycol (9-10) -p-octylphenol (trade name Triton X100), octyl-β-D-glucoside (Octyl-β-D-Glucoside), etc. Can be mentioned. The nonionic surfactant as described above may be a nonionic surfactant dissolved or diluted in an aqueous solution of an appropriate concentration, a dilute salt solution such as physiological saline, or a pH buffer solution.

  The total blending ratio of the surfactant in the degradation enzyme mixture may be appropriately increased or decreased depending on the type of tissue material such as difficulty in solubilization of the mammalian tissue material to be pretreated, and is not particularly limited. However, it is preferable to set it as 0.1 to 30 weight%. It is more preferably 1% by weight or more and 20% by weight or less, and further preferably 3% by weight or more and 15% by weight or less. If it is less than 0.1% by weight, solubilization of the tissue material may be insufficient, and if it exceeds 30% by weight, the measured value of PrPSc may be affected. Further, the blending ratio when using a plurality of surfactants may be appropriately determined according to the selected surfactant and is not particularly limited. The amount of PK added to the decomposing enzyme mixture can be appropriately increased or decreased according to the amount used for normal PrPC decomposition. In this embodiment, the blending amount in 1 mL of the decomposing enzyme mixture is 10 μg. As mentioned above, it is preferable to set it as 300 micrograms or less, and it is more preferable to set it as 30 micrograms or more and 250 micrograms or less.

  The amount of addition of DNA-degrading enzyme and / or RNA-degrading enzyme and / or collagen-degrading enzyme to such a degrading enzyme mixture is not particularly limited, and the kind of tissue material to be pretreated and the selected DNA decomposing process are not limited. It can be appropriately determined according to the type of enzyme, RNase, or collagenase. In the present embodiment, the compounding amount of the DNA degrading enzyme is preferably 1 μg or more and 1000 μg or less, and more preferably 5 μg or more and 200 μg or less in 1 mL of the decomposing enzyme mixture. The amount of RNA-degrading enzyme is preferably 2 μg or more and 2000 μg or less, and more preferably 10 μg or more and 400 μg or less in 1 mL of the decomposing enzyme mixture. The compounding amount of the collagen degrading enzyme is preferably 0.05 mg or more and 50 mg or less, more preferably 0.25 mg or more and 10 mg or less in 1 mL of the degrading enzyme mixed solution. Furthermore, an organic solvent such as methanol, chloroform, methanol / chloroform mixed solution, 1-butanol, 2-butanol and the like can be added to the above degrading enzyme mixture, and when such an organic solvent is added, The mixing ratio in the degradation enzyme mixture is preferably 0.1 wt% or more and 10 wt% or less. In addition, for example, inorganic salts such as sodium iodide, potassium iodide, sodium chloride, magnesium chloride and the like can be added in a range that can be dissolved in the enzyme mixture of degrading enzymes. In addition, it is preferable to adjust the pH of the above-mentioned degrading enzyme mixed solution to a predetermined range. In addition to the surfactant and PK to be added, the addition state of DNA degrading enzyme, RNA degrading enzyme, collagen degrading enzyme, etc. A pH buffering agent may be appropriately selected and added according to the conditions. In addition, such a degradative enzyme mixed solution is, for example, freeze-dried or separated into a solid part prepared by blending powdered components and a liquid part such as a surfactant or a pH buffer solution. Alternatively, it can be used by dissolving it at the time of use.

  In the solubilized PrPC decomposition step in the present embodiment, the above-described degradation enzyme mixed solution is added to the mammalian tissue material to be pretreated and subjected to homogenization. The amount of the degradative enzyme mixture used can be appropriately determined according to the animal species and tissue type of the tissue material, but here, it is 2 times or more and 20 times or less the weight of the tissue material to be pretreated. Preferably, the weight is 3 times or more and 15 times or less. For the homogenization treatment, for example, a potter elvegem type that rotates a fluororesin rod such as polytetrafluoroethylene (PTFE) in a cylindrical container having an inner diameter slightly larger than the outer diameter, a metal fixed blade and a rotating blade Tissue material is crushed by (for example, manufactured by Polytron), blender type (for example, manufactured by Waring) with a high-speed rotary blade attached in a closed container, and bead together with tissue material to be processed in a container that can be closed Various homogenizers such as a bead type crusher (manufactured by Wakken Yakuhin Co., Ltd., Yasui Machine Co., Ltd.) that crush tissue material by placing, shaking, rotating, etc., and a type in which these are combined with ultrasonic treatment can be used. In addition, tissue may be crushed by reciprocating the inner cylinder in two connected syringes, and using multiple types together Possible it is. In the first embodiment, there is a case where the tissue material can be efficiently solubilized within a shorter time, and the possibility that the tissue material is scattered during the homogenization treatment is low, so that the bead type crushing is performed. It is preferable to use a machine. When using such a bead-type crushing type homogenizer, the homogenization treatment is preferably performed for 1 minute to 10 minutes, more preferably 2 minutes to 5 minutes. Further, the treatment liquid after the treatment by the homogenizer can be heated in a thermostat or a heat block maintained at a predetermined temperature of 20 ° C. or higher and 70 ° C. or lower, more preferably 35 ° C. or higher and 55 ° C. or lower. The heating time is preferably 1 minute or more and 10 minutes or less, and more preferably 2 minutes or more and 5 minutes or less. In addition, the homogenization treatment can be performed under a temperature condition of 20 ° C. or higher and 70 ° C. or lower, more preferably 35 ° C. or higher and 55 ° C. or lower, without adversely affecting the detection and measurement of PrPSc. In some cases, processing time can be shortened.

  The treatment liquid (PrPC-decomposed liquid) obtained in the solubilized PrPC decomposition process in the first embodiment as described above is in a state where the mammalian tissue material is solubilized and normal prions are decomposed. Next, a protein denaturation treatment step (A) as shown in FIG. 1 is performed on this PrPC decomposed solution. In the protein denaturation treatment step (A) of this embodiment, a PK inhibitor and powdered urea are added and heated. As the PK inhibitor, any protease inhibitor such as serine protease inhibitor, cysteine protease inhibitor, metalloendopeptidase inhibitor can be used as long as it can inhibit PK activity. An agent can also be used in combination. Here, it is more preferable to use what contains a serine protease inhibitor, for example, phenylmethanesulfonyl fluoride (PMSF), benzamidine, 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF: for example, commercial product) A serine protease inhibitor such as PEFABLOC SC (Roche Diagnostics) can be preferably used. The addition amount of the PK inhibitor may be appropriately determined according to the amount of PK added to the degradation enzyme mixture in the solubilized PrPC decomposition step in the previous stage, but in this embodiment, the concentration after addition to the PrPC decomposition solution is It is preferable to add so that it may become 0.1 mM or more and 10 mM or less, and 0.5 mM or more and 5 mM or less are more preferable. Moreover, it is good to heat, after adding powdery urea so that the density | concentration after addition may be 4-8M, More preferably, it may be 5-7M. As this heating condition, as 30 degreeC or more, 110 degreeC, for example It can be heated at the following temperature for 1 minute or more and 10 minutes or less. It is more preferable to set the heating conditions at a temperature of 50 ° C. or more and 100 ° C. or less for 3 minutes or more and 7 minutes or less.

  In the first embodiment, if necessary, the urea concentration used as a sample for detection or measurement of PrPSc by adding a diluent to the protein-denatured solution obtained by the protein-denaturing treatment step (A) as described above An adjustment step can be performed (FIG. 1). It should be noted that the protein-denatured solution obtained in the preceding protein denaturation treatment step (A) has the PrPC27 completely decomposed and PrPSc is accumulated in the mammalian tissue material before the pretreatment. As a sample for detecting or measuring PrPSc by reacting with a prion protein (PrPC, PrPSc) specific antibody, the urea concentration adjustment step is omitted, and protein denaturation treatment is performed. The spent solution can be used as it is. On the other hand, the protein-denatured solution has a high urea concentration and the like, and may affect a measurement system using, for example, the WB method or the ELISA method. In this case, the urea concentration adjusting step may be performed. The diluent may be prepared so as to be suitable for measurement for detecting or measuring PrPSc, and is not particularly limited. For example, in addition to water and physiological saline, phosphate (phosphate buffered physiological saline: PBS), tris (hydroxymethyl) aminomethane salt (Tris buffer), N-tris (hydroxymethyl) methylglycine salt System (Tricine buffer), N-2-hydroxyethylpiperazine-N'-ethanesulfonate system (HEPES buffer), 3- (morpholino) propanesulfonate system (MOPS buffer), glycine-hydrochloric acid system, A pH buffer solution such as an acetate salt, or a blocking agent solution such as albumin or skim milk that prevents non-specific adsorption in an ELISA method or the like can be suitably used. Furthermore, for example, the diluted solution can be used to increase the sensitivity of the measurement system, or to a measurement system with a compositional ingredient other than urea such as a measurement interfering substance derived from tissue material remaining in the protein-denatured solution or a surfactant or PK inhibitor. It is also possible to add substances that prevent the effects of Further, the dilution factor is not particularly limited as in the case of the diluted solution. In the present embodiment, the urea concentration after dilution is preferably 0.05 M or more and 3 M or less, in that it hardly affects a general measurement system for detecting or measuring PrPSc, 0.5 M or more, More preferably, it is 2.5M or less, and it is even better to dilute to 1M or more and 2M or less.

[Second Embodiment]
The mammalian tissue material pretreatment method according to the second embodiment mainly differs from the first embodiment in the protein denaturation treatment step, as shown in the flow of the pretreatment method in FIG. That is, as in the case of the first embodiment, a solubilized PrPC decomposition step is performed in which a degradation enzyme mixture is added to a mammalian tissue material collected to prepare a PrPSc detection or measurement sample and homogenized. . Next, in the second embodiment, the protein denaturation treatment step (B) is performed in place of the protein denaturation treatment step (A) in the first embodiment. In consideration of the difference in the processing conditions, the solubilized PrPC decomposition step in the first embodiment can be performed in accordance with the processing conditions described above. As for the solubilized PrPC decomposition step according to the second embodiment, a detailed description thereof is omitted, but as in the case of the first embodiment, the treatment liquid obtained in the solubilized PrPC decomposition step (PrPC decomposed) (Liquid) is in a state in which the mammalian tissue material is solubilized and PrPC is decomposed. In the protein denaturation treatment step (B) of the second embodiment, as shown in FIG. 2, the PrPC-decomposed solution is stirred after adding a protein precipitating agent, and then centrifuged to remove the supernatant. A urea solution is added to the resulting precipitate and heated.

As the protein precipitating agent, any protein precipitating agent can be used as long as PrPSc accumulates in the pre-treatment mammalian tissue material and can agglutinate, precipitate and precipitate a protein containing the “PrP27-30” fragment. For example, an organic solvent such as acetone, methanol, ethanol, propanol, 1-butanol and 2-butanol or a mixture of a plurality of organic solvents, salts having a salting-out effect such as ammonium sulfate, and tungsten compounds such as SiW ₁₁ , Trichloroacetic acid and the like are not particularly limited. As a protein precipitating agent in the second embodiment, a component derived from a mammalian tissue material that can be a measurement interfering substance that may affect a measurement system such as an ELISA method is mixed and coprecipitated in the precipitate. In some cases, an organic solvent or a mixture of a plurality of organic solvents is preferable, an organic solvent containing butanol is more preferable, and 2-butanol is more preferable. Further, the amount of such protein precipitating agent to be added is appropriately determined depending on the type of the protein precipitating agent selected and the type of surfactant added to the degrading enzyme mixture in the solubilized PrPC decomposition step in the previous stage. can do. For example, when using an organic solvent or an organic solvent mixed solution as a protein precipitating agent, 0.1 mL or more and 2 mL or less are preferably added to 1 mL of PrPC decomposed solution, and 0.2 mL or more and 1 mL or less are more preferable. preferable.

  Centrifugation conditions after adding the protein precipitant and stirring are not particularly limited as long as the supernatant can be separated from the precipitate in a clear state. For example, at 17000 × g, separation can be performed in 5 minutes, or centrifugation may be performed using a cooling centrifuge. After removing the supernatant, urea solution is added to the precipitate. The urea concentration of the urea solution is preferably 4 to 8M, and more preferably 5 to 7M. The amount of urea solution added can be appropriately determined according to the type and amount of mammalian tissue material collected for pretreatment, and is not particularly limited. For example, about 100 mg of collected mammalian tissue material, about 25 μL to 250 μL of urea solution may be added, and 50 μL to 150 μL is more preferable. As heating conditions in the protein denaturation treatment step (B), for example, heating can be performed at a temperature of 30 ° C. or higher and 110 ° C. or lower for 1 minute or longer and 10 minutes or shorter. It is more preferable to set the heating conditions at a temperature of 50 ° C. or more and 100 ° C. or less for 3 minutes or more and 7 minutes or less.

  In the second embodiment, if necessary, the concentration of urea used as a sample for detection or measurement of PrPSc by adding a diluent to the protein denaturation-treated solution obtained by the protein denaturation treatment step (B) as described above An adjustment process can be performed (FIG. 2). The protein-denatured solution obtained in the protein denaturation treatment step (B) has a PrP27-30 fragment in the case where PrPC is completely decomposed and PrPSc is accumulated in the mammalian tissue material before pretreatment. Is included. Therefore, as a pre-treated sample for detecting or measuring PrPSc by reacting with a prion protein (PrPC, PrPSc) specific antibody, it is possible to omit the urea concentration adjustment step and use the protein-denatured solution as it is. is there. On the other hand, the protein-denatured solution has a high urea concentration, which may affect a measurement system using, for example, the WB method or the ELISA method. In such a case, the urea concentration adjustment step may be performed. . In the urea concentration adjustment step according to the second embodiment, the usable diluent, the substance that can be further added to the diluent, the dilution factor, and the like are the same as those in the first embodiment, and are detailed in order to avoid duplication. Description is omitted.

[Third Embodiment]
As shown in the flow of the pretreatment method in FIG. 3, the pretreatment method of the mammalian tissue material according to the third embodiment mainly comprises no protein denaturation treatment step and is solubilized. The difference is that a PK inhibitor is added to the treatment liquid (PrPC decomposed liquid) obtained in the PrPC decomposition step. That is, as in the case of the first embodiment, a solubilized PrPC decomposition step is performed in which a degradation enzyme mixture is added to a mammalian tissue material collected to prepare a PrPSc detection or measurement sample and homogenized. . Next, in the third embodiment, instead of the protein denaturation treatment step (A), a PK inhibitor addition step of adding a PK inhibitor without adding urea to the PrPC-decomposed solution is performed. The solubilized PrPC decomposition step in the first embodiment can be performed in accordance with the processing conditions described above in consideration of the difference in the processing conditions from the protein denaturation processing step (A) in the first embodiment. As for the solubilized PrPC decomposition step according to the third embodiment, the repeated detailed description is omitted, but as in the case of the first embodiment, the PrPC decomposed solution obtained in the solubilized PrPC decomposition step is The mammalian tissue material is solubilized and PrPC is decomposed.

  In the PK inhibitor addition step of the third embodiment, the amount of the PK inhibitor added to the PrPC decomposition solution and the amount of the PK inhibitor added are PrPC decomposition in the protein denaturation treatment step (A) of the first embodiment. The same PK inhibitor as in the case of adding to the liquid can be used, and the same applies to the amount of PK inhibitor added, and redundant detailed description is omitted. In the third embodiment, if necessary, a diluent is added to the treatment liquid (PrPC-decomposed liquid to which the PK inhibitor has been added) obtained by the PK inhibitor addition step as described above to add PrPSc. A surfactant concentration adjusting step can be carried out as a sample for detection or measurement of water (FIG. 3). In the treatment solution obtained in the preceding PK inhibitor addition step, when PrPC is completely decomposed and PrPSc is accumulated in the mammalian tissue material before the pretreatment, the unmodified “PrP27-30” "The fragment is included. Therefore, as a sample for detecting or measuring PrPSc by reacting with a native prion protein (PrPSc) specific antibody, the surfactant concentration adjustment step is omitted, and the PrPC-decomposed solution to which the PK inhibitor is added is used as it is. It is also possible. On the other hand, the PrPC-decomposed liquid to which the PK inhibitor is added has a high surfactant concentration, which may affect a measurement system using, for example, the WB method or ELISA method. In this case, the surfactant concentration What is necessary is just to implement an adjustment process. In the surfactant concentration adjusting step according to the third embodiment, usable diluents, substances that can be further added to the diluted solution, and the like are the same as those in the urea concentration adjusting step of the first embodiment, and are redundant. Description is omitted. In the surfactant concentration adjustment step according to the third embodiment, PrPSc is detected as the dilution factor when diluting the PrPC-decomposed solution to which the PK inhibitor has been added using the diluent as described above. Or what is necessary is just to set it as the dilution rate suitable for the measurement for a measurement, and it does not specifically limit. In the present embodiment, for example, it is preferably 2 times or more and 120 times or less, and preferably 2.4 times or more and 12 times or less in that it does not affect a general measurement system for detecting or measuring PrPSc. It is more preferable to dilute it so that it becomes 3 times or more and 6 times or less. According to such a pretreatment method of the third embodiment, it is necessary to use a specific antibody against the native “PrP27-30” fragment in the PrPSc assay or measurement system, but the first or second embodiment. As compared with the case, the pretreatment can be performed within a shorter time.

  Hereinafter, the pretreatment method for mammalian tissue material of the present invention will be specifically described with reference to Examples and Test Examples, but the present invention is not limited thereby.

[Example 1] (Solubilization of tissue material)
Tissue solubilization was examined in accordance with the first embodiment, using normal mouse central nervous tissue (intracerebral tissue) and bovine medullary tissue as animal tissue materials. A PBS solution containing 3% by weight of lauroyl sarcosine (LSarc) and 1% by weight of p-octylphenol (NP40) surfactant and PK (200 μg / mL) was used as the degradation enzyme mixture. A mixture of degrading enzyme having a weight five times the amount of tissue material collected was added, and the inner cylinder was reciprocated in two linked syringes to homogenize the tissue for 2 minutes. Warmed for minutes. In this state, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was turbid. In the subsequent protein denaturation treatment step, urea was added at a concentration of 6 M, and the heating conditions were 100 ° C. and 5 minutes. In the urea concentration adjustment step, PBS was diluted to a urea concentration of 1M using a diluent. Even in this state, the pretreated liquid from the mouse brain tissue material was clear, but the pretreated liquid from the bovine medullary tissue material was turbid.

[Example 2] (Solubilization of tissue material)
The same as in Example 1 except that polyoxyethylene (80) sorbitol ester (trade name Tween 20) was added as a 2-butanol and surfactant to the decomposing enzyme mixture, and the formulation was changed as shown in Table 1. The examination was conducted under conditions. Under any conditions, as in Example 1, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was found to be turbid.

[Example 3] (Solubilization of tissue material)
Examination was performed under the same conditions as in Example 1 except that sodium chloride was added to the degradation enzyme mixture and the pH was changed as shown in Table 2. Under any conditions, as in Example 1, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was found to be turbid.

[Example 4] (Solubilization of tissue material)
1-butanol, magnesium chloride, DNase-I, RNase-A, collagenase A, bovine pancreas-derived lipase (pancrelipase) and the like are added to the degradation enzyme mixture, and only lauroyl sarcosine (LSarc) is used as a surfactant. Examination was performed under the same conditions as in Example 1 except that the combination was changed as shown. Under any conditions, as in Example 1, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was found to be turbid.

[Example 5] (Solubilization of tissue material)
DNase-I, RNase-A, collagenase A, bovine pancreatic lipase and the like are added to the degradation enzyme mixture, and a surfactant is added as octyl-β-D-glucoside (Octyl-β-D-Glucoside), 3-[( 3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonic acid (CHAPSO), Except that the combination was changed as shown in FIG. 4, the examination was performed under the same conditions as in Example 1. Under any conditions, as in Example 1, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was found to be turbid.

[Example 6] (Solubilization of tissue material)
As shown in Table 5, DNase-I, RNase-A, collagenase A, bovine pancreatic lipase and the like are added to the degradation enzyme mixture, and the surfactant is lauroyl sarcosine (LSarc) and octyl-β-D-glucoside. The examination was performed under the same conditions as in Example 1 except that the combination was changed. Under any conditions, as in Example 1, the mouse brain tissue material was clearly solubilized, but the bovine medullary tissue material was found to be turbid.

[Example 7] (Solubilization of tissue material)
Tissue solubilization was examined according to the second embodiment using bovine medullary tissue as an animal tissue material. 2-Butanol, DNase-I, RNase-A, collagenase A, bovine pancreatic lipase, etc. are added to the degradation enzyme mixture, and lauroyl sarcosine (LSarc) and octyl-β-D-glucoside surfactant and PK (200 μg) are added. / ML) was added and blended as shown in Table 6. Add 10 times the weight of degrading enzyme mixed with the amount of tissue material collected, and use a bead type crusher (trade name Bead Smash 12; manufactured by Wakken Pharmaceutical Co., Ltd. After homogenization treatment, the mixture was heated (incubated) at 37 ° C. to 42 ° C. for 5 minutes using a heat block (TAITEC dry thermo unit DTU-1B or aluminum block AL-1136B). In this state, the bovine medullary tissue material was clearly solubilized, but turbidity was observed when stored at room temperature. 4- (2-Aminoethyl) -benzenesulfonyl fluoride (AEBSF: trade name; PEFABLOC SC, manufactured by Roche Diagnostics) was added as a PK inhibitor to a concentration equivalent to 2 mM, and SiW ₁₁ 0.5 wt% Was added, followed by centrifugation at 15000 × g for 5 minutes. The supernatant was removed, and a 6M urea solution was added to the obtained precipitate in a volume of 1.5 to 3 times the tissue material collected weight, followed by protein denaturation treatment at 100 ° C. for 5 minutes. In the urea concentration adjustment step, PBS was diluted to a urea concentration of 1M using a diluent. Turbidity was observed in the treatment solution after the protein denaturation treatment step and the urea concentration adjustment step, and solubilization was insufficient. Further, in the urea concentration adjusting step treatment solution in an insufficiently solubilized state, it could not be detected by the ELISA method even when bovine r-PrPC was added. When mouse brain tissue material was used as animal tissue material, it was clearly solubilized and detectable.

[Example 8] (Solubilization of tissue material)
Tissue solubilization was examined according to the first embodiment using bovine medulla tissue as an animal tissue material. DNase-I, RNase-A and collagenase A are added to the degradation enzyme mixture, and an amphoteric surfactant [n-dodecyl-N, N-dimethylglycine (DDGly: EMPGEN BB trade name, manufactured by Calbiochem) or n- Table 7 shows surfactants combined with dodecyl-N, N- (dimethylammonio) butyrate (DDDMA; manufactured by Calbiochem)] and an anionic surfactant [lauroyl sarcosine (LSarc)] and PK (200 μg / mL). A PBS solution added and blended as described above was used. A mixture of degradative enzymes having a weight 10 times the amount of tissue material collected was added, and 2500 rpm at a bead type crusher (trade name Bead Smash 12; manufactured by Wakken Pharmaceutical Co., Ltd. After homogenizing for 2 minutes, the mixture was heated (incubated) at 37 ° C. to 42 ° C. for 5 minutes using a heat block (dry thermo unit DTU-1B or aluminum block AL-1136B manufactured by TAITEC). The treatment solution that had been subjected to the solubilized PrPC decomposition process for bovine medullary tissue material was solubilized almost clearly.

[Example 9] (Solubilization of tissue material)
Example 8 except that lauric acid amidopropyl hydroxysulfobetaine (LSB), lauric acid amidopropyl betaine (LPB), cocamidopropyl betaine (CPB: palm oil fatty acid amidopropyl betaine) was used as the amphoteric surfactant. In the same manner as in the case, each was added and blended as shown in Table 8, and tissue solubilization was examined. As in the case of Example 8, the treatment solution that had undergone the solubilized PrPC decomposition step on bovine medullary tissue material was solubilized almost clearly.

[Example 10] (Solubilization of tissue material)
Except that lauric acid amidopropyl betaine (LPB) was additionally used as an amphoteric surfactant, it was added and blended as shown in Table 9 in the same manner as in Example 8 to examine tissue solubilization. As in the case of Example 8, the treatment solution that had undergone the solubilized PrPC decomposition step on bovine medullary tissue material was solubilized almost clearly.

[Example 11] (Sample preparation for ELISA measurement)
PrPSc-added bovine medullary tissue material obtained by adding 5% by weight of a brain emulsion of a PrPSc-infected mouse to 100 mg of normal bovine medullary tissue material and 100 mg of bovine medullary tissue material as mammalian tissue material was collected in a 2 mL capacity microtube, and 4% by weight N-dodecyl-N, N- (dimethylammonio) butyrate (DDDMA; manufactured by Calbiochem), 0.01 wt% DNase-I, 0.01 wt% RNase-A, 0.5 wt% Collagenase A, 0.005 wt% PK added 50 mM, pH 7.5 Tris buffer solution 1 mL and stainless steel beads were added, and 2500 rpm with a bead type crusher (trade name; Multi-Bead Shocker, Yasui Kikai Co., Ltd.). Homogenize for 2 minutes at 37 ° C and then heat for 3 minutes at 37 ° C with an aluminum block heater. (Incubation) to obtain a solubilized PrPC decomposition step treatment liquid. Next, 25 μL of 4- (2-aminoethyl) -benzenesulfonyl fluoride (AEBSF: trade name; PEFABLOC SC, manufactured by Roche Diagnostics) was added as a PK inhibitor and heated at 100 ° C. for 2 minutes with an aluminum block heater. did. Samples derived from normal bovine medullary tissue material obtained at this stage, 0, 120, 160, 180, 190, 200 μL, and samples 200, 80, 40, 20, 10, 0 μL derived from PrPSc-added bovine medullary tissue material, respectively Mix in another microtube to prepare 5%, 2%, 1%, 0.5%, 0.25% and 0% (without PrPSc) PrPSc-infected mouse brain emulsion samples. did. 72 mg of urea is added to each sample and heated at 100 ° C. for 5 minutes to carry out a protein denaturation treatment step. After thoroughly stirring, 0.4 mL each of Block Ace (Dainippon Pharmaceutical Co., Ltd.) diluted 10-fold with purified water was added to obtain a pretreated measurement sample (final urea concentration of about 2M).

[Test Example 1] (ELISA measurement)
The test was conducted using a 96-well microplate on which a mouse anti-PrP monoclonal antibody was immobilized in advance. About each measurement sample obtained in Example 11, 50 μL of each was dispensed into 2 wells (double measurement). 50 μL of HRP-labeled mouse anti-PrP monoclonal antibody solution was added to each well, allowed to stand at room temperature for 1 hour, and then washed with PBST. Add 100 μL of chromogenic substrate solution (TMBZ: 3,3 ′, 5,5′-tetramethylbenzidine, manufactured by Sigma) and leave it at room temperature for 30 minutes, then add 100 μL of 1M sulfuric acid and add microplate. Absorbance at 450 nm was measured with a reader. As a result, as shown in FIG. 4, an increase in absorbance proportional to the amount of PrPSc-infected mouse brain emulsion was observed, and even when the rapid pretreatment method according to the present invention was a large animal tissue material, a sample for measuring PrPSc was used. It was confirmed to be effective for preparation.

It is a flowchart of the mammalian tissue material pre-processing method by the 1st Embodiment of this invention. It is a flowchart of the mammalian tissue material pre-processing method by the 2nd Embodiment of this invention. It is a flowchart of the mammalian tissue material pre-processing method by the 3rd Embodiment of this invention. It is a figure which shows an example of the PrPSc measurement by ELISA method about the pre-processing sample obtained by this invention.

Claims (15)

A pretreatment method for mammalian tissue material for preparing a sample for detection or measurement of abnormal prion protein (PrPSc), comprising:
Pretreatment of mammalian tissue material comprising a solubilized PrPC degradation step of adding a degradation enzyme mixture containing a surfactant and proteinase K (PK) to the collected mammalian tissue material and homogenizing the mixture. Method.   The mammalian tissue material is a medullary or central nervous tissue of a mammal selected from human, cow, goat, sheep, deer, elk, mink, pig, monkey, mouse, rat, hamster, cat. The pretreatment method for mammalian tissue material according to claim 1.   The method for pretreating mammalian tissue material according to claim 1 or 2, wherein the decomposing enzyme mixed solution contains a DNA degrading enzyme and / or an RNA degrading enzyme and / or a collagen degrading enzyme.   The method for pretreating mammalian tissue material according to any one of claims 1 to 3, wherein the surfactant contains an amphoteric surfactant.   The method for pretreating mammalian tissue material according to any one of claims 1 to 4, wherein the surfactant comprises a combination of an amphoteric surfactant and an anionic surfactant.   The amphoteric surfactant is lauric acid amidopropyl hydroxysulfobetaine (LSB), lauric acid amidopropyl betaine (LPB), cocamidopropyl betaine (CPB), propyl betaine caprate (CAPB), n-dodecyl-N, N -Dimethylglycine (DDGly), Laurylaminopropionic acid (LAPA), n-dodecyl-N, N- (dimethylammonio) butyric acid (DDDMA), n-dodecyl-N, N- (dimethylammonio) undecanoic acid ( DDMU), 3-[(3-Colamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-Colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfone One or more selected from acids (CHAPSO) or salts thereof Pretreatment method mammalian tissue material according to claim 4 or 5, characterized in that the at it.   The anionic surfactant is sodium dodecyl sulfate (SDS), lauroyl sarcosine (LSarc), sodium cocoyl sarcosine, sodium dodecanoyl sarcosine, sodium lauroylmethyl-β-alanine, sodium cholate (SChol), sodium deoxycholate ( The mammalian tissue material pretreatment method according to claim 5, wherein one or more of SDChol), sodium glycocholate, and sodium taurocholate are selected.   The method for pretreating mammalian tissue material according to any one of claims 1 to 7, wherein the mixing ratio of the surfactant in the decomposing enzyme mixture is 0.1 to 30% by weight.   The mammalian tissue material pretreatment method according to any one of claims 1 to 8, wherein the homogenization treatment in the solubilized PrPC decomposition step is performed by a bead type crusher in which beads are placed in a container.   The mammalian tissue material according to any one of claims 1 to 9, wherein in the solubilized PrPC decomposition step, after homogenization treatment, heating (incubation) is performed at a temperature of 20 ° C or higher and 70 ° C or lower. Pre-processing method.   11. The protein denaturation treatment step (A) in which a PK inhibitor and powdered urea are added to the treatment liquid obtained in the solubilized PrPC decomposition step and heated. A pretreatment method for mammalian tissue material according to claim 1.   A protein denaturation treatment step (B) in which a urea solution is added to a precipitate obtained by adding a protein precipitating agent to the treatment solution obtained in the solubilized PrPC decomposition step and stirring and then centrifuging; The method for pretreatment of a mammalian tissue material according to any one of claims 1 to 10, wherein:   A mammalian tissue material characterized in that a diluted solution is added to the protein-denatured solution obtained in the protein denaturation treatment step according to claim 11 or 12 to obtain a sample for detection or measurement of PrPSc. Pre-processing method.   The method for pretreatment of mammalian tissue material according to any one of claims 1 to 10, further comprising a PK inhibitor addition step of adding a PK inhibitor to the treatment solution obtained in the solubilized PrPC decomposition step. .   A pretreatment method for mammalian tissue material, wherein a diluent is added to the treatment liquid obtained in the PK inhibitor addition step according to claim 14 to obtain a sample for detection or measurement of PrPSc.

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