WO2024101329A1 - Method for pretreating specimen - Google Patents

Method for pretreating specimen Download PDF

Info

Publication number
WO2024101329A1
WO2024101329A1 PCT/JP2023/039965 JP2023039965W WO2024101329A1 WO 2024101329 A1 WO2024101329 A1 WO 2024101329A1 JP 2023039965 W JP2023039965 W JP 2023039965W WO 2024101329 A1 WO2024101329 A1 WO 2024101329A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
sds
biomarker protein
specimen
container
Prior art date
Application number
PCT/JP2023/039965
Other languages
French (fr)
Japanese (ja)
Inventor
優 長島
純 竹澤
達郎 遠藤
Original Assignee
国立大学法人 東京大学
公立大学法人大阪
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人 東京大学, 公立大学法人大阪 filed Critical 国立大学法人 東京大学
Publication of WO2024101329A1 publication Critical patent/WO2024101329A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for pretreatment of a specimen derived from a subject.
  • antigen-antibody reactions are used to quantify disease biomarker proteins.
  • concentration of proteins such as amyloid beta (A ⁇ ) and tau, which are Alzheimer's disease biomarkers
  • patient samples such as blood (serum, plasma), urine, or cerebrospinal fluid to diagnose the possibility of Alzheimer's disease and evaluate the disease progression.
  • coagulable proteins such as A ⁇ and tau are prone to coagulation themselves, and are adsorbed to glass or resin containers used for cryopreservation of patient samples, or bind to other molecules in the patient sample, losing their antigenicity.
  • Non-Patent Document 1 when a sample is placed in a container and frozen for a long period of time, or when the sample is repeatedly frozen and thawed, there is a known problem in that the measurement value decreases over time in the Alzheimer's disease biomarker quantification method that uses an antigen-antibody reaction.
  • the present invention provides a method for pretreating a sample that prevents disease biomarker proteins in the sample from agglutinating, binding to other molecules in the sample, or adsorption to a container, and enables accurate and easy quantification of the biomarker proteins using antibodies, etc.
  • the inventors have investigated the quantitative results of amyloid beta (A ⁇ ), an aggregating disease biomarker protein, when it is dissolved in various solvents.
  • a ⁇ amyloid beta
  • the amount of A ⁇ in the solvent was measured using a polymer photonic crystal sensor (see, for example, WO2010/044274, etc.) as an example of a protein quantitative method using an antigen-antibody reaction. It was found that when A ⁇ was dissolved in serum, the apparent measured concentration decreased compared to when the solvent was phosphate-buffered saline (PBS), and the amount of change increased by freezing.
  • PBS phosphate-buffered saline
  • the present invention has been completed based on the above findings. That is, the present invention relates to the following (1) to (14).
  • the method according to (1) or (2) above, wherein the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
  • biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain and prion.
  • sample is a liquid sample.
  • liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine and a suspension of biological tissue.
  • a method for quantifying an aggregation biomarker protein in a sample comprising the steps of: (a) mixing a sample with sodium dodecyl sulfate (SDS); (b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
  • SDS sodium dodecyl sulfate
  • the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
  • the biomarker protein is detected using an antibody or an aptamer.
  • biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion, as well as post-translationally modified versions of these proteins.
  • sample is a liquid sample.
  • liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine, tears, sweat, saliva, skin exudate, brain interstitial fluid, and a suspension of biological tissue.
  • a container for storing a sample containing an aggregation biomarker protein the container containing SDS.
  • a kit for quantifying an aggregation biomarker protein in a sample comprising SDS or the container described in (13) above.
  • the symbol "to” indicates a numerical range including both values on either side of it.
  • the present invention makes it possible to quantify with high accuracy the amount of aggregating biomarker proteins in easily collected biological tissue samples, particularly liquid samples such as blood, using antibodies or the like.
  • FIG. 1 shows the results of an investigation into the effects of various substances on the aggregation properties of amyloid ⁇ 42 (A ⁇ 42).
  • FIG. 2 shows the results of investigating the effect of SDS on the aggregation property of A ⁇ 42.
  • FIG. 3 shows the results of investigating the effect of the concentration of added SDS on the adsorption of proteins in serum to a container.
  • the first embodiment is a method for pretreatment of a specimen containing an aggregation biomarker protein, the method comprising mixing the specimen with sodium dodecyl sulfate (SDS).
  • SDS sodium dodecyl sulfate
  • the sample pretreatment method according to the present embodiment is a method for preventing accurate quantification from being impossible due to aggregation of the biomarker protein in the sample before measuring the amount of the aggregating biomarker protein in the sample.
  • the pretreatment method in the present embodiment is carried out before a step of quantifying the amount of the aggregating biomarker protein in the sample, particularly using a molecule (herein also referred to as a "molecule-specific recognition factor") such as an antibody, an aptamer (peptide aptamer, nucleic acid (DNA and RNA) aptamer, etc.).
  • a molecule herein also referred to as a "molecule-specific recognition factor”
  • an antibody an aptamer (peptide aptamer, nucleic acid (DNA and RNA) aptamer, etc.).
  • the "aggregating biomarker protein” refers to a protein that serves as an indicator for determining whether or not a certain disease has developed, or is likely to develop, or for judging the activity and progression of a disease that has already been diagnosed, and refers to a protein that has the property of easily aggregating with other proteins or with other molecules, or that has the property of easily adhering to the wall of a container for collecting a sample as a result of aggregation.
  • Immunoglobulin light chains and prions are known as biomarker proteins for Creutzfeldt-Jakob disease.
  • examples of the above-mentioned "aggregation biomarker protein” include proteins such as amyloid beta, tau protein, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion, which are post-translationally modified (e.g., phosphorylated, glycosylated, ubiquitinated, nitrosylated, methylated, acetylated, lipidated, etc.).
  • a ⁇ aggregates are the main component of senile plaques, which are characteristic of Alzheimer's disease.
  • neurofibrillary tangles which are also characteristic of Alzheimer's disease, are induced by the aggregation of highly phosphorylated tau protein.
  • a ⁇ is excised from its precursor protein, amyloid- ⁇ precursor protein (APP), by ⁇ -secretase and ⁇ -secretase, and secreted outside the cell.
  • APP amyloid- ⁇ precursor protein
  • a ⁇ 42 which consists of 42 amino acids, but these are not the only molecular species of A ⁇ .
  • a ⁇ 42 in cerebrospinal fluid decreases, and the ratio of A ⁇ 42/A ⁇ 40 in plasma decreases. Therefore, by quantifying the amount of A ⁇ (especially A ⁇ 42) in blood and cerebrospinal fluid, it is possible to understand the possibility of developing Alzheimer's disease and the progression of the disease after onset. Furthermore, “subjects” include not only humans but also non-human animals.
  • sample in this embodiment refers to a sample collected from a subject, and although there is no particular limitation, a liquid sample is preferable, such as blood (including plasma and serum), cerebrospinal fluid, nasal mucus, urine, tears, sweat, saliva, skin exudate, cerebral interstitial fluid, and biological tissue suspensions (biological tissue suspended or solubilized in an appropriate solvent (such as physiological saline)).
  • blood including plasma and serum
  • cerebrospinal fluid such as blood (including plasma and serum)
  • nasal mucus such as urine, tears, sweat, saliva, skin exudate, cerebral interstitial fluid
  • biological tissue suspensions biological tissue suspended or solubilized in an appropriate solvent (such as physiological saline)
  • a person skilled in the art can determine the optimal concentration of SDS to be mixed with the specimen (final concentration in the specimen) for each type of specimen through preliminary experiments, but to give an example, it may be about 1 w/v% (weight/volume percent) to about 25 w/v%, preferably about 3 w/v% to about 20 w/v%, more preferably about 10 w/v% to about 17 w/v%, and most preferably about 15 w/v%.
  • the addition and mixing of SDS to the specimen is performed so that SDS is uniformly distributed in the specimen, but it is desirable to avoid denaturation of proteins contained in the specimen due to excessive stirring as much as possible.
  • the step of "mixing the specimen with SDS” may involve adding SDS to the collected specimen and mixing, or adding SDS to the container in which the specimen is collected in advance, adding the specimen to it, and mixing.
  • SDS may be added to a specimen that has been stored at low temperature (for example, at about -80°C or -20°C) for a certain period of time, and then mixing.
  • the form of "SDS” is not particularly limited, and it may be in the form of a powder or dissolved in an appropriate solvent.
  • a second embodiment is a method for quantifying an aggregation biomarker protein in a sample, comprising the following steps (a) and (b): (a) mixing a sample with sodium dodecyl sulfate (SDS); (b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
  • steps (a) and (b) comprising the following steps (a) and (b): (a) mixing a sample with sodium dodecyl sulfate (SDS); (b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
  • SDS sodium dodecyl sulfate
  • Detection and quantification of aggregating biomarker proteins, including A ⁇ can be carried out by methods using molecules such as antibodies and aptamers (peptide aptamers, nucleic acid (DNA and RNA) aptamers, etc.) (herein also referred to as "molecular-specific recognition factors").
  • molecules such as antibodies and aptamers (peptide aptamers, nucleic acid (DNA and RNA) aptamers, etc.) (herein also referred to as "molecular-specific recognition factors").
  • aggregating biomarker proteins can be detected and quantified by immunoassay-based methods, such as Enzyme-Linked Immunosorbent Assay (ELISA) (including digital ELISA), Enzyme Immuno Assay (EIA), Immuno-Chromatography, Immunomagnetic Reduction (IMR), Surface Plasmon Resonance (SPR), Chemiluminescent Immunoassays (CLIA) such as Electrochemiluminescence Immunoassay (ECLIA) and Chemiluminescent Enzyme Immunoassay (CLEIA), and Quartz Crystal Microbalance (QCM).
  • ELISA Enzyme-Linked Immunosorbent Assay
  • EIA Enzyme Immuno Assay
  • IMR Immunomagnetic Reduction
  • SPR Surface Plasmon Resonance
  • Chemiluminescent Immunoassays CLIA
  • ELIA Electrochemiluminescence Immunoassay
  • CLIA Chemiluminescent Enzyme Immunoas
  • aptamers when aptamers are used to detect and quantify aggregating biomarker proteins, many of the methods applied to detect antigen-antibody reactions (mentioned above) can be used because aptamers, like antibodies, have the property of specifically recognizing molecules.
  • Steps (a) and (b) in the second embodiment may be performed consecutively, or step (b) may be performed a certain period of time after step (a).
  • step (b) may be performed immediately after SDS is added to and mixed with the sample, or step (b) may be performed several days, months, or years after SDS is added to and mixed with the sample.
  • step (a) SDS may be added to and mixed with the sample immediately after collection from the subject, or the sample may be stored at low temperature (e.g., at approximately -80°C or -20°C) and SDS may be added and mixed after a certain period of time has passed.
  • the third embodiment is a method for preserving a sample containing an aggregation biomarker protein, which comprises adding SDS to the sample and mixing it.
  • Many biomarker proteins characterized by aggregating properties aggregate immediately after a specimen is collected in a container and adhere to the wall of the container. Aggregation may be induced during the frozen storage period of the specimen or by freezing and thawing the specimen, making it difficult to accurately measure the amount of the biomarker protein. Therefore, when storing a specimen containing an aggregating biomarker protein, aggregation of the biomarker protein can be prevented by adding SDS to the specimen, mixing the specimen, and then storing the specimen at an appropriate temperature, for example, a low temperature (e.g., ⁇ 80° C. or ⁇ 20° C.). For the amount (concentration) of SDS to be added to and mixed with the specimen, see the description in the first embodiment.
  • a low temperature e.g., ⁇ 80° C. or ⁇ 20° C.
  • the fourth embodiment is a container for collecting a specimen containing an aggregation biomarker protein, which contains SDS.
  • SDS is stored in the container for collecting the specimen in advance, so that the quantification of the biomarker protein can be performed easily and accurately.
  • the material of the "container for collecting the specimen" according to this embodiment is not particularly limited, and may be made of resin or glass. The form is also not particularly limited.
  • the form of the SDS stored in the "container for collecting the specimen” may be any form (powder, solution, etc.).
  • a person skilled in the art can appropriately select the amount of SDS stored depending on the type of specimen and the type of biomarker protein contained in the specimen.
  • the SDS concentration in the sample after collection may be about 1 w/v% (weight/volume percent) to about 25 w/v%, preferably about 3 w/v% to about 20 w/v%, more preferably about 10 w/v% to about 17 w/v%, and most preferably about 15 w/v%.
  • the fifth embodiment is a kit for quantifying an aggregating biomarker protein in a sample, which includes at least SDS or a container according to the fourth embodiment (i.e., a container for collecting a sample containing an aggregating biomarker protein, in which SDS is stored).
  • the kit of this embodiment may include, in addition to SDS or a container in which SDS is stored, a reagent used for quantifying a biomarker protein, a diluent, and an antibody or aptamer for quantifying the biomarker protein.
  • Experimental method 1-1 Collection of specimens
  • blood collected using standard blood collection techniques is used. Blood is collected in a polypropylene container that is less susceptible to adsorption of coagulant proteins, and after mixing by inversion, it is quickly centrifuged to remove blood cell components.
  • the coagulation reaction begins immediately after blood is collected, and a blood clot is formed by the action of high molecular weight fibrin formed from fibrinogen in plasma and platelets.
  • the blood clot is separated into the lower layer by centrifugation, and serum is obtained as the supernatant.
  • a coagulation promoter to promote the formation of a blood clot may be added to the blood collection container in advance.
  • blood is collected in a blood collection container that contains an anticoagulant such as EDTA, sodium citrate, or heparin, and centrifuged after collection, blood cell components are separated into the lower layer, and plasma is obtained as the supernatant.
  • an anticoagulant such as EDTA, sodium citrate, or heparin
  • plasma is obtained as the supernatant.
  • serum was used.
  • Sample pretreatment method As a pretreatment, SDS solution prepared using PBS as a solvent was added to the collected blood sample (serum or plasma) so that the final concentration of SDS was 5%, and the sample was mixed well.
  • serum to which 50% formic acid, 50% Triton, and 9M urea had been added (solvent: PBS, concentrations: final concentrations after addition) was also prepared for comparison and used in the measurements.
  • a ⁇ quantification method The blood sample to which SDS has been added is dropped onto the surface of a polymer photonic crystal sensor to which anti-A ⁇ antibodies have been adsorbed, and incubated at 37°C for 2 hours. It is then washed three times with PBS. The reflection spectrum of the surface of the photonic crystal sensor after washing was measured using a spectrometer. The measured spectrum was numerically subtracted from the reflection spectrum data of the surface of the photonic crystal sensor before the blood sample was dropped, and the change in the peak intensity of the interference light from the photonic crystal sensor was calculated as a ratio value.
  • the A ⁇ concentration in the sample can be measured by preparing an appropriate dilution series of A ⁇ standard solutions, drawing a calibration curve, and comparing it with the measured value of the blood sample.
  • Sample 1 6mM A ⁇ 42 in PBS (not frozen)
  • Sample 2 100 ⁇ M A ⁇ 42 in PBS (not frozen)
  • Sample 3 100 ⁇ M A ⁇ 42 in serum (frozen)
  • Sample 4 100 ⁇ M A ⁇ 42 in serum (not frozen)
  • Sample 5 100 ⁇ M A ⁇ 42 in serum + 0.5 ⁇ PBS (not frozen)
  • Sample 6 100 ⁇ M A ⁇ 42 in serum + 50% formic acid (not frozen)
  • Sample 7 100 ⁇ M A ⁇ 42 in serum + 50 % Triton (not frozen)
  • Sample 8 100 ⁇ M A ⁇ 42 in serum + 5% SDS (not frozen)
  • Sample 9 100 ⁇ M A ⁇ 42 in serum + 9M urea (not frozen).
  • the amount of A ⁇ 42 in a sample was measured using a polymer photonic crystal sensor.
  • the vertical axis of the graph in Figure 1 is the ratio of the reflected light intensity of the sensor before and after the sample was applied to the sensor substrate. The smaller the ratio value, the greater the decrease in reflected light intensity due to the application of the sample, indicating a larger amount of A ⁇ in the sample.
  • the apparent measured concentration decreased (the ratio of the reflected light intensity of the sensor increased, Sample 4) compared to when the solvent was PBS (Sample 2), and the amount of change increased by freezing (Sample 3).
  • the present invention makes it possible to accurately measure the amount of aggregating disease biomarker protein in a sample, and is therefore expected to be useful in the medical field and the like.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The present invention addresses the problem of providing a method for pretreating a specimen to accurately and conveniently quantify a disease biomarker protein in the specimen while preventing aggregation or adsorption of the biomarker protein to a container. Specifically, the present invention relates to a method for pretreating a specimen containing an aggregating biomarker protein, the method comprising mixing the specimen with sodium dodecyl sulfate (SDS). Moreover, the present invention relates to a method for quantifying an aggregating biomarker protein in a specimen, the method including a step of mixing the specimen with SDS, and a step of, after the aforesaid step, specifically detecting and quantifying the biomarker protein in the specimen.

Description

検体の前処理方法Sample pretreatment method
　本発明は、被験者由来の検体の前処理方法に関する。 The present invention relates to a method for pretreatment of a specimen derived from a subject.
　一般に、疾患バイオマーカータンパク質の定量には、抗原抗体反応が用いられる。なかでも、アルツハイマー病バイオマーカーであるアミロイドβ（Amyloidβ；Aβ）やタウ（tau）といったタンパク質の濃度を、血液（血清、血漿）、尿または髄液などの患者由来の検体中で測定することで、アルツハイマー病の罹患の可能性の診断や病勢評価を行うことができる。しかし、疾患バイオマーカータンパク質の中でも、特にAβやtauをはじめとした凝集性のタンパク質は、それ自身が凝集しやすい性質を持ち、患者由来検体の凍結保存に用いられるガラスや樹脂製の容器に吸着したり、患者検体中のほかの分子に結合して抗原性を失ったりする。
　また、検体を容器に入れて凍結し長期間保存したり、頻回の凍結・融解を繰り返したりすると、抗原抗体反応を用いたアルツハイマー病バイオマーカー定量法において、経時的に測定値が低下していく問題点が知られている（非特許文献１)。 In general, antigen-antibody reactions are used to quantify disease biomarker proteins. In particular, the concentration of proteins such as amyloid beta (Aβ) and tau, which are Alzheimer's disease biomarkers, can be measured in patient samples such as blood (serum, plasma), urine, or cerebrospinal fluid to diagnose the possibility of Alzheimer's disease and evaluate the disease progression. However, among disease biomarker proteins, coagulable proteins such as Aβ and tau are prone to coagulation themselves, and are adsorbed to glass or resin containers used for cryopreservation of patient samples, or bind to other molecules in the patient sample, losing their antigenicity.
Furthermore, when a sample is placed in a container and frozen for a long period of time, or when the sample is repeatedly frozen and thawed, there is a known problem in that the measurement value decreases over time in the Alzheimer's disease biomarker quantification method that uses an antigen-antibody reaction (Non-Patent Document 1).
　このような、患者由来検体の長期保存、または凍結・融解の繰り返しによる、疾患バイオマーカーとしての凝集性タンパク質の定量における問題を回避するために、従来は、検体採取から遠心までの時間および凍結の回数などを標準化したプロトコルを作成し、これを遵守することにより、測定結果の品質管理を行う方策がとられていた。しかしながら、このような方策は、測定値の経時的変化を本質的に解消する解決方法ではなかった。一方、Aβの凝集性または他の物質との結合もしくは吸着性を解消するために、検体に、ギ酸、尿素または塩酸グアニジンなどを添加する方法が提案されている（特許文献１）。これらの試薬は、不溶性凝集体の可溶化に汎用されているが、タンパク質の変性作用が強い。そのため、抗原抗体反応を用いて凝集性の疾患バイオマーカータンパク質の定量を行う場合に、これらの試薬を用いてタンパク質の凝集を解消すると、バイオマーカータンパク質の変性による抗原性の変化または抗体自体の変性による抗原結合能の変化などが生じ、正確な定量ができない可能性がある。 In order to avoid problems in the quantification of aggregating proteins as disease biomarkers due to long-term storage of patient-derived samples or repeated freezing and thawing, a standard protocol was created for the time from sample collection to centrifugation and the number of times the sample was frozen, and measures were taken to control the quality of the measurement results by following this protocol. However, such measures were not a solution that essentially eliminated the change in measurement value over time. On the other hand, a method has been proposed in which formic acid, urea, guanidine hydrochloride, or the like is added to the sample to eliminate the aggregating property of Aβ or its binding or adsorption to other substances (Patent Document 1). These reagents are widely used to solubilize insoluble aggregates, but have a strong denaturing effect on proteins. Therefore, when quantifying aggregating disease biomarker proteins using an antigen-antibody reaction, if these reagents are used to eliminate protein aggregation, changes in antigenicity due to denaturation of the biomarker protein or changes in antigen binding ability due to denaturation of the antibody itself may occur, making it impossible to accurately quantify the protein.
特開2011-232172号公報JP 2011-232172 A
　上記事情に鑑み、本発明は、検体中における疾患バイオマーカータンパク質の凝集や検体中の他の分子への結合、または容器への吸着を防ぎ、抗体等による当該バイオマーカータンパク質の定量を正確かつ簡便に実施するための検体の前処理方法を提供する。 In view of the above circumstances, the present invention provides a method for pretreating a sample that prevents disease biomarker proteins in the sample from agglutinating, binding to other molecules in the sample, or adsorption to a container, and enables accurate and easy quantification of the biomarker proteins using antibodies, etc.
　発明者らは、凝集性の疾患バイオマーカータンパク質であるアミロイドβ（Amyloidβ；Aβ）を、様々な溶媒に溶解した際に、その定量結果がどのようになるかを検討した。溶媒中のAβの量は、抗原抗体反応を用いたタンパク質の定量法の一例として、ポリマー性フォトニック結晶センサー（例えば、WO2010/044274などを参照のこと）を用いて実施した。血清にAβを溶解すると、溶媒がリン酸緩衝生理食塩水（PBS）であった場合と比較して、見かけ上の測定濃度が下がり、さらにその変化量は凍結操作によって増大することが分かった。溶媒がPBSの場合と血清の場合におけるAβの測定濃度の違いは、溶媒が血清の場合に、血清中の他の分子と結合して抗原性を失ったりするためと考えられる。また凍結して長期保存した場合に見かけ上の測定濃度が下がる理由は、Aβが容器の内面に吸着するためと考えられる。そこで、血清にPBS、ギ酸、Triton、SDSおよび尿素を添加して、Aβ量の測定を行ったところ、ギ酸とSDSを添加したときのみ、見かけ上の測定濃度の低下が解消された。上述のように、ギ酸は抗原抗体反応に必須の抗体分子を変性させてしまう可能性があるため、血清中のAβ量の測定の前処理の際に検体試料に添加する物質としては、SDSが適していることが示唆された。 The inventors have investigated the quantitative results of amyloid beta (Aβ), an aggregating disease biomarker protein, when it is dissolved in various solvents. The amount of Aβ in the solvent was measured using a polymer photonic crystal sensor (see, for example, WO2010/044274, etc.) as an example of a protein quantitative method using an antigen-antibody reaction. It was found that when Aβ was dissolved in serum, the apparent measured concentration decreased compared to when the solvent was phosphate-buffered saline (PBS), and the amount of change increased by freezing. The difference in the measured concentration of Aβ when the solvent was PBS and when the solvent was serum is thought to be due to the fact that when the solvent was serum, Aβ binds to other molecules in the serum and loses its antigenicity. In addition, the reason why the apparent measured concentration decreases when frozen and stored for a long period of time is thought to be because Aβ is adsorbed to the inner surface of the container. Therefore, PBS, formic acid, Triton, SDS, and urea were added to serum and the amount of Aβ was measured, and the decrease in the apparent measured concentration was eliminated only when formic acid and SDS were added. As mentioned above, formic acid can denature antibody molecules that are essential for antigen-antibody reactions, so it was suggested that SDS would be a suitable substance to add to specimen samples during pretreatment for measuring Aβ levels in serum.
　本発明は以上の知見に基づいて、完成されたものである。
　すなわち、本発明は以下の（１）～（１４）である。
（１）凝集性バイオマーカータンパク質を含む検体の前処理方法であって、検体にドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合することを含む、前記方法。
（２）凝集性バイオマーカータンパク質を含む検体の保存方法であって、検体にSDSを添加し、混合することを含む、前記方法。
（３）検体中のSDSの濃度が、約3w/v%～約20w/v%である、上記（１）または（２）に記載の方法。
（４）前記バイオマーカータンパク質が、アミロイドβ、タウ、TDP43、α-シヌクレイン、ポリグルタミン、トランスサイレチン、血清アミロイドA、免疫グロブリン軽鎖およびプリオンからなるグループより選択されるいずれかである、上記（１）または（２）に記載の方法。
（５）前記検体が、液性検体である、上記（１）または（２）に記載の方法。
（６）前記液性検体が、血液、脳脊髄液、鼻粘液、尿および生体組織の懸濁液からなるグループより選択されるいずれかである、上記（１）または（２）に記載の方法。
（７）検体中の凝集性バイオマーカータンパク質の定量方法であって、以下の（ａ）および（ｂ）の工程を含む、前記方法である。
（ａ）検体とドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合する工程、
（ｂ）工程（ａ）後、検体中のバイオマーカータンパク質を特異的に検出し、定量する工程。
（８）前記工程（ａ）において、前記検体中のSDSの濃度が、約3w/v%～約20w/v%である、上記（７）に記載の方法。
（９）前記工程（ｂ）において、バイオマーカータンパク質を抗体またはアプタマーにより検出する、上記（７）または（８）に記載の方法。
（１０）前記バイオマーカータンパク質が、アミロイドβ、タウ、TDP43、α-シヌクレイン、ポリグルタミン、トランスサイレチン、血清アミロイドA、免疫グロブリン軽鎖およびプリオン、ならびにこれらのタンパク質の翻訳後修飾されたものからなるグループより選択されるいずれかである、上記（７）または（８）に記載の方法。
（１１）前記検体が、液性検体である、上記（７）または（８）に記載の方法。
（１２）前記液性検体が、血液、脳脊髄液、鼻粘液、尿、涙、汗、唾液、皮膚浸出液、脳の間質液および生体組織の懸濁液からなるグループより選択されるいずれかである、上記（７）または（８）に記載の方法。
（１３）凝集性バイオマーカータンパク質を含む検体を収納するための容器であって、SDSが収納されている、前記容器。
（１４）検体中の凝集性バイオマーカータンパク質を定量するためのキットであって、SDSまたは上記（１３）に記載の容器を含む、前記キット。
　なお、本明細書において「～」の符号は、その左右の値を含む数値範囲を示す。 The present invention has been completed based on the above findings.
That is, the present invention relates to the following (1) to (14).
(1) A method for pretreatment of a sample containing an aggregation biomarker protein, the method comprising mixing sodium dodecyl sulfate (SDS) with the sample.
(2) A method for preserving a sample containing an aggregation biomarker protein, the method comprising adding SDS to the sample and mixing it.
(3) The method according to (1) or (2) above, wherein the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
(4) The method according to (1) or (2) above, wherein the biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain and prion.
(5) The method according to (1) or (2) above, wherein the sample is a liquid sample.
(6) The method according to (1) or (2) above, wherein the liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine and a suspension of biological tissue.
(7) A method for quantifying an aggregation biomarker protein in a sample, comprising the steps of:
(a) mixing a sample with sodium dodecyl sulfate (SDS);
(b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
(8) The method according to (7) above, wherein in the step (a), the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
(9) The method according to (7) or (8) above, wherein in the step (b), the biomarker protein is detected using an antibody or an aptamer.
(10) The method according to (7) or (8) above, wherein the biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion, as well as post-translationally modified versions of these proteins.
(11) The method according to (7) or (8) above, wherein the sample is a liquid sample.
(12) The method according to (7) or (8) above, wherein the liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine, tears, sweat, saliva, skin exudate, brain interstitial fluid, and a suspension of biological tissue.
(13) A container for storing a sample containing an aggregation biomarker protein, the container containing SDS.
(14) A kit for quantifying an aggregation biomarker protein in a sample, comprising SDS or the container described in (13) above.
In this specification, the symbol "to" indicates a numerical range including both values on either side of it.
　本発明により、採取が容易な生体組織検体、特に血液などの液性検体中の凝集性バイオマーカータンパク質の量を、抗体などを用いて高い精度で定量することが可能となる。 The present invention makes it possible to quantify with high accuracy the amount of aggregating biomarker proteins in easily collected biological tissue samples, particularly liquid samples such as blood, using antibodies or the like.
図１は、Amyloid β42（Aβ42）の凝集性に対する種々の物質が及ぼす影響を検討した結果を示す。Figure 1 shows the results of an investigation into the effects of various substances on the aggregation properties of amyloid β42 (Aβ42). 図２は、Aβ42の凝集性に対するSDSの効果を検討した結果を示す。FIG. 2 shows the results of investigating the effect of SDS on the aggregation property of Aβ42. 図３は、血清中のタンパク質の容器への吸着に対する、添加するSDS濃度の影響を検討した結果を示す。FIG. 3 shows the results of investigating the effect of the concentration of added SDS on the adsorption of proteins in serum to a container.
　以下、本発明を実施するための形態について説明する。
　第１の実施形態は、凝集性バイオマーカータンパク質を含む検体の前処理方法であって、検体とドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合することを含む方法である。
　本実施形態にかかる検体の前処理方法は、検体中の凝集性バイオマーカータンパク質の量を測定する前に、検体中において当該バイオマーカータンパク質が凝集することにより、正確な定量ができなくなることを防ぐための方法である。本実施形態における前処理方法は、凝集性バイオマーカータンパク質の検体中の量を定量する工程であって、特に、抗体、アプタマー（ペプチドアプタマー、核酸（DNAおよびRNA）アプタマーなど）などの分子（ここでは、「分子特異的認識因子」とも記載する）を用いて定量する工程の前に実施する。 Hereinafter, an embodiment of the present invention will be described.
The first embodiment is a method for pretreatment of a specimen containing an aggregation biomarker protein, the method comprising mixing the specimen with sodium dodecyl sulfate (SDS).
The sample pretreatment method according to the present embodiment is a method for preventing accurate quantification from being impossible due to aggregation of the biomarker protein in the sample before measuring the amount of the aggregating biomarker protein in the sample. The pretreatment method in the present embodiment is carried out before a step of quantifying the amount of the aggregating biomarker protein in the sample, particularly using a molecule (herein also referred to as a "molecule-specific recognition factor") such as an antibody, an aptamer (peptide aptamer, nucleic acid (DNA and RNA) aptamer, etc.).
　本実施形態において、「凝集性バイオマーカータンパク質」とは、任意の疾患を発症しているかどうか、または、発症する可能性があるかどうか、あるいは、すでに診断された疾患の活動性や進行度を判断するための指標となるタンパク質のことであり、当該タンパク質同士、または他の分子と凝集し易い性質を有し、あるいは、凝集の結果検体を採取する容器の壁面などに付着し易くなる性質を有するタンパク質のことである。凝集性バイオマーカータンパク質として、特に限定はしないが、アルツハイマー病のバイオマーカータンパク質としてアミロイドβ（Amyloidβa）（以下「Aβ」とも記載する）、アルツハイマー病、進行性核上麻痺、または皮質基底核変性症のバイオマーカータンパク質としてタウタンパク質（Tau protein）、筋萎縮性側索硬化症のバイオマーカータンパク質としてTDP-43、パーキンソン病または多系統萎縮症のバイオマーカータンパク質としてα-シヌクレイン（α-synuclein）、ハンチントン病、脊髄小脳失調症1型、脊髄小脳失調症2型、脊髄小脳失調症3型（=マシャド・ジョセフ病)、脊髄小脳失調症6型、脊髄小脳失調症7型、歯状核赤核淡蒼球ルイ体萎縮症または球脊髄性筋萎縮症のなどのポリグルタミン病バイオマーカータンパク質としてポリグルタミン、アミロイドーシスのバイオマーカータンパク質として、トランスサイレチン、血清アミロイドAまたは 免疫グロブリン軽鎖、クロイツフェルト・ヤコブ病のバイオマーカータンパク質としてプリオンなどが知られている。
　さらに、上記「凝集性バイオマーカータンパク質」の例として、アミロイドβ、タウタンパク質、TDP43、α-シヌクレイン、ポリグルタミン、トランスサイレチン、血清アミロイドA、免疫グロブリン軽鎖およびプリオンなどのタンパク質が翻訳後修飾（例えば、リン酸化、グリコシル化、ユビキチン化、ニトロシル化、メチル化、アセチル化または脂質化など）されたタンパク質なども挙げることができる。 In this embodiment, the "aggregating biomarker protein" refers to a protein that serves as an indicator for determining whether or not a certain disease has developed, or is likely to develop, or for judging the activity and progression of a disease that has already been diagnosed, and refers to a protein that has the property of easily aggregating with other proteins or with other molecules, or that has the property of easily adhering to the wall of a container for collecting a sample as a result of aggregation. Examples of aggregation biomarker proteins include, but are not limited to, amyloid βa (hereinafter also referred to as "Aβ") as a biomarker protein for Alzheimer's disease, tau protein as a biomarker protein for Alzheimer's disease, progressive supranuclear palsy, or corticobasal degeneration, TDP-43 as a biomarker protein for amyotrophic lateral sclerosis, α-synuclein as a biomarker protein for Parkinson's disease or multiple system atrophy, polyglutamine as a biomarker protein for polyglutamine diseases such as Huntington's disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (= Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, dentatorubral-pallidoluysian atrophy, or spinal-bulbar muscular atrophy, transthyretin, serum amyloid A, or amyloidosis as a biomarker protein for amyloidosis. Immunoglobulin light chains and prions are known as biomarker proteins for Creutzfeldt-Jakob disease.
Further, examples of the above-mentioned "aggregation biomarker protein" include proteins such as amyloid beta, tau protein, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion, which are post-translationally modified (e.g., phosphorylated, glycosylated, ubiquitinated, nitrosylated, methylated, acetylated, lipidated, etc.).
　なかでも、Aβの凝集体はアルツハイマー病に特徴的な老人斑の主要な構成成分である。また、同じくアルツハイマー病に特徴的な神経原繊維変化は、高度にリン酸化されたタウタンパク質が凝集することで誘導される。Aβは、前駆タンパク質であるアミロイドβ前駆体タンパク質（Amyloid-β precursor protein；APP）から、βセクレターゼおよびγセクレターゼによって切りだされ、細胞外へと分泌される。Aβの主な分子種として、40個のアミノ酸からなるAβ40と42個のアミノ酸からなるAβ42が存在することが知られているが、これらのアミノ酸長の分子種に限られるものではない。アルツハイマー病が進行すると、脳脊髄液中のAβ42の量が減少することや、血漿中のAβ42／Aβ40の比が減少することなどが報告されている。従って、血液や脳脊髄液中のAβの量（特に、Aβ42）、を定量することで、アルツハイマー病の発症の可能性や発症後の病態の進行状態などを把握することが可能である。また、「被験者」には、ヒトのみならず、非ヒト動物も含まれる。 In particular, Aβ aggregates are the main component of senile plaques, which are characteristic of Alzheimer's disease. Furthermore, neurofibrillary tangles, which are also characteristic of Alzheimer's disease, are induced by the aggregation of highly phosphorylated tau protein. Aβ is excised from its precursor protein, amyloid-β precursor protein (APP), by β-secretase and γ-secretase, and secreted outside the cell. It is known that the main molecular species of Aβ are Aβ40, which consists of 40 amino acids, and Aβ42, which consists of 42 amino acids, but these are not the only molecular species of Aβ. It has been reported that as Alzheimer's disease progresses, the amount of Aβ42 in cerebrospinal fluid decreases, and the ratio of Aβ42/Aβ40 in plasma decreases. Therefore, by quantifying the amount of Aβ (especially Aβ42) in blood and cerebrospinal fluid, it is possible to understand the possibility of developing Alzheimer's disease and the progression of the disease after onset. Furthermore, "subjects" include not only humans but also non-human animals.
　本実施形態における「検体」は、被験者から採取された検体であって、特に限定はしないが、液性検体が好ましく、例えば、血液（血漿および血清を含む）、脳脊髄液、鼻粘液、尿、涙、汗、唾液、皮膚浸出液、脳の間質液および生体組織の懸濁液（生体組織を適切な溶媒（生理食塩水など）に懸濁または可溶化したもの）などが挙げられる。 The "sample" in this embodiment refers to a sample collected from a subject, and although there is no particular limitation, a liquid sample is preferable, such as blood (including plasma and serum), cerebrospinal fluid, nasal mucus, urine, tears, sweat, saliva, skin exudate, cerebral interstitial fluid, and biological tissue suspensions (biological tissue suspended or solubilized in an appropriate solvent (such as physiological saline)).
　検体と混合するSDSの濃度（検体中の最終濃度）は、当業者であれば、検体の種類毎に最適な濃度を予備的な実験によって決定することができるが、敢えて例示するならば、約1w/v%（重量体積パーセント）～約25w/v%、好ましくは、約3w/v%～約20 w/v%、より好ましくは、約10w/v%～約17w/v%であってもよく、最もこのましくは約15w/v%である。検体へのSDSの添加および混合は、SDSが検体中に均一になるように行うが、過度な撹拌による検体中に含まれるタンパク質の変性は極力避ける方が望ましい。ここで、「検体とSDSを混合する」工程は、採取した検体にSDSを添加し、混合してもよく、あるいは、予め検体を採取する容器にSDSを添加しておき、そこへ検体を加え、混合してもよい。また、低温保存（例えば、-80℃程度または-20℃程度にて保存）して、一定期間経過した検体にSDSを添加し、混合してもよい。「SDS」の形態は、特に限定されず、粉末であっても、適当な溶媒に溶解させた形態であってもよい。 A person skilled in the art can determine the optimal concentration of SDS to be mixed with the specimen (final concentration in the specimen) for each type of specimen through preliminary experiments, but to give an example, it may be about 1 w/v% (weight/volume percent) to about 25 w/v%, preferably about 3 w/v% to about 20 w/v%, more preferably about 10 w/v% to about 17 w/v%, and most preferably about 15 w/v%. The addition and mixing of SDS to the specimen is performed so that SDS is uniformly distributed in the specimen, but it is desirable to avoid denaturation of proteins contained in the specimen due to excessive stirring as much as possible. Here, the step of "mixing the specimen with SDS" may involve adding SDS to the collected specimen and mixing, or adding SDS to the container in which the specimen is collected in advance, adding the specimen to it, and mixing. In addition, SDS may be added to a specimen that has been stored at low temperature (for example, at about -80°C or -20°C) for a certain period of time, and then mixing. The form of "SDS" is not particularly limited, and it may be in the form of a powder or dissolved in an appropriate solvent.
　第２の実施形態は、検体中の凝集性バイオマーカータンパク質の定量方法であって、以下の（ａ）および（ｂ）の工程を含む方法である。
（ａ）検体とドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合する工程、
（ｂ）工程（ａ）後、検体中のバイオマーカータンパク質を特異的に検出し、定量する工程。 A second embodiment is a method for quantifying an aggregation biomarker protein in a sample, comprising the following steps (a) and (b):
(a) mixing a sample with sodium dodecyl sulfate (SDS);
(b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
　Aβをはじめとした凝集性バイオマーカータンパク質の検出およびその定量は、抗体、アプタマー（ペプチドアプタマー、核酸（DNAおよびRNA）アプタマーなど）などの分子（ここでは、「分子特異的認識因子」とも記載する）を用いた方法によって実施することが可能である。抗体を用いた場合、免疫学的測定法に基づいた方法、例えば、酵素結合免疫吸着法（Enzyme-Linked Immuno Sorbent Assay；ELISA）（デジタルELISAを含む）、酵素免疫測定法（Enzyme Immuno Assay；EIA）、イムノクロマトグラフィー（Immuno-Chromatography）法、免疫磁気還元（Immunomagnetic Reduction；IMR）法、表面プラズモン共鳴（Surface Plasmon Resonance；SPR）法、電気化学発光免疫測定法（Electrochemiluminescence Immunoassay；ECLIA）や化学発光酵素免疫測定法（Chemiluminescent Enzyme Immunoassay；CLEIA）などの化学発光免疫測定法（Chemiluminescent Immunoassay；CLIA）および水晶振動子（Quartz crystal microbalance；QCM）法などにより、凝集性バイオマーカータンパク質の検出および定量が可能である。抗原抗体反応を検出し、反応量を定量するその他の方法として、光学式センサーを使用した方法、例えば、ポリマー製フォトニック結晶センサーを用いた方法を挙げることができる。 Detection and quantification of aggregating biomarker proteins, including Aβ, can be carried out by methods using molecules such as antibodies and aptamers (peptide aptamers, nucleic acid (DNA and RNA) aptamers, etc.) (herein also referred to as "molecular-specific recognition factors"). When antibodies are used, aggregating biomarker proteins can be detected and quantified by immunoassay-based methods, such as Enzyme-Linked Immunosorbent Assay (ELISA) (including digital ELISA), Enzyme Immuno Assay (EIA), Immuno-Chromatography, Immunomagnetic Reduction (IMR), Surface Plasmon Resonance (SPR), Chemiluminescent Immunoassays (CLIA) such as Electrochemiluminescence Immunoassay (ECLIA) and Chemiluminescent Enzyme Immunoassay (CLEIA), and Quartz Crystal Microbalance (QCM). Other methods for detecting antigen-antibody reactions and quantifying the amount of reaction include methods using optical sensors, such as a polymer photonic crystal sensor.
　また、凝集性バイオマーカータンパク質の検出およびその定量にアプタマーを用いる場合、アプタマーは、抗体と同様に、特異的に分子を認識する性質を有しているため、抗原抗体反応を検出するために適用される方法（上述）の多くの方法を利用することができる。 In addition, when aptamers are used to detect and quantify aggregating biomarker proteins, many of the methods applied to detect antigen-antibody reactions (mentioned above) can be used because aptamers, like antibodies, have the property of specifically recognizing molecules.
　第２の実施形態の工程（ａ）と工程（ｂ）は連続的に実施しても、工程（ａ）の実施後一定期間を置いて工程（ｂ）を実施してもよい。例えば、検体にSDSを添加混合した後、すぐに工程（ｂ）を実施してもよく、あるいは、検体にSDSを添加混合した後、数日、数ヶ月または数年経過後に工程（ｂ）を実施してもよい。また、工程（ａ）において、被験者から採取した検体にすぐにSDSを添加混合してもよく、あるいは、当該検体を低温保存（例えば、-80℃程度または-20℃程度にて保存）して、一定期間経過後にSDSを添加混合してもよい。 Steps (a) and (b) in the second embodiment may be performed consecutively, or step (b) may be performed a certain period of time after step (a). For example, step (b) may be performed immediately after SDS is added to and mixed with the sample, or step (b) may be performed several days, months, or years after SDS is added to and mixed with the sample. Also, in step (a), SDS may be added to and mixed with the sample immediately after collection from the subject, or the sample may be stored at low temperature (e.g., at approximately -80°C or -20°C) and SDS may be added and mixed after a certain period of time has passed.
　第３の実施形態は、凝集性バイオマーカータンパク質を含む検体の保存方法であって、当該検体にSDSを添加し、混合することを含む方法である。
　凝集性を特徴とするバイオマーカータンパク質の中には、容器に検体を採取した直後から凝集し、容器の壁面などに付着するものも多く、検体の冷凍保存期間中に、または検体を凍結融解することによって凝集が誘導され、当該バイオマーカータンパク質の量を正確に測定することが困難になることがある。そこで、凝集性バイオマーカータンパク質を含む検体を保存する場合、当該検体にSDSを添加し、混合したのち適温、例えば、低温（例えば、-80℃または-20℃）で保存することで、当該バイオマーカータンパク質の凝集を防ぐことができる。検体に添加し、混合するSDSの量（濃度）に関しては、第１の実施形態に記載箇所を参照のこと。 The third embodiment is a method for preserving a sample containing an aggregation biomarker protein, which comprises adding SDS to the sample and mixing it.
Many biomarker proteins characterized by aggregating properties aggregate immediately after a specimen is collected in a container and adhere to the wall of the container. Aggregation may be induced during the frozen storage period of the specimen or by freezing and thawing the specimen, making it difficult to accurately measure the amount of the biomarker protein. Therefore, when storing a specimen containing an aggregating biomarker protein, aggregation of the biomarker protein can be prevented by adding SDS to the specimen, mixing the specimen, and then storing the specimen at an appropriate temperature, for example, a low temperature (e.g., −80° C. or −20° C.). For the amount (concentration) of SDS to be added to and mixed with the specimen, see the description in the first embodiment.
　第４の実施形態は、凝集性バイオマーカータンパク質を含む検体を採取するための容器であって、SDSが収納されている容器である。
　上述のように、凝集性を特徴とするバイオマーカータンパク質の中には、例えば、採血管などの容器に検体を採取した直後から凝集し、容器の壁面などに付着するものも多い。そこで、凝集性バイオマーカータンパク質が、検体採取用の容器中で凝集すること、および容器壁面への付着することを防ぐために、検体採取用の容器に予めSDSを収納しておくことで、バイオマーカータンパク質の定量を簡便かつ正確に行うことができる。本実施形態にかかる「検体を採取するための容器」の材質は、特に限定されず、樹脂性であってもガラス製であってもよい。また、形態も特に限定されない。「検体を採取するための容器」に収納されているSDSの形状は、いかなる形状（粉末状、溶液状など）であってもよい。また、収納されているSDSの量については、当業者であれば、検体の種類や当該検体に含まれるバイオマーカータンパク質の種類によって、適宜選択することができる。凝集性バイオマーカータンパク質がAβであると仮定した場合、例えば、検体を採取後の検体中SDS濃度が、約1w/v%（重量体積パーセント）～約25w/v%、好ましくは、約3w/v%～約20w/v%、より好ましくは、約10w/v%～約17w/v%であってもよく、最もこのましくは約15w/v%であってもよい。 The fourth embodiment is a container for collecting a specimen containing an aggregation biomarker protein, which contains SDS.
As described above, many biomarker proteins characterized by aggregation tend to aggregate immediately after a specimen is collected in a container such as a blood collection tube, and adhere to the wall of the container. In order to prevent the aggregation-prone biomarker protein from aggregating in the container for collecting the specimen and from adhering to the wall of the container, SDS is stored in the container for collecting the specimen in advance, so that the quantification of the biomarker protein can be performed easily and accurately. The material of the "container for collecting the specimen" according to this embodiment is not particularly limited, and may be made of resin or glass. The form is also not particularly limited. The form of the SDS stored in the "container for collecting the specimen" may be any form (powder, solution, etc.). In addition, a person skilled in the art can appropriately select the amount of SDS stored depending on the type of specimen and the type of biomarker protein contained in the specimen. Assuming that the aggregation biomarker protein is Aβ, for example, the SDS concentration in the sample after collection may be about 1 w/v% (weight/volume percent) to about 25 w/v%, preferably about 3 w/v% to about 20 w/v%, more preferably about 10 w/v% to about 17 w/v%, and most preferably about 15 w/v%.
　第５の実施形態は、検体中の凝集性バイオマーカータンパク質を定量するためのキットであって、少なくともSDS、または第４の実施形態にかかる容器（すなわち、凝集性バイオマーカータンパク質を含む検体を採取するための容器であって、SDSが収納されている容器）を含む、キットである。
　本実施形態にかかるキットには、SDSまたはSDSが収納されている容器の他、バイオマーカータンパク質の定量に使用する試薬、希釈液の他、当該バイオマーカータンパク質の定量のための抗体もしくはアプタマーなどが含まれていてもよい。 The fifth embodiment is a kit for quantifying an aggregating biomarker protein in a sample, which includes at least SDS or a container according to the fourth embodiment (i.e., a container for collecting a sample containing an aggregating biomarker protein, in which SDS is stored).
The kit of this embodiment may include, in addition to SDS or a container in which SDS is stored, a reagent used for quantifying a biomarker protein, a diluent, and an antibody or aptamer for quantifying the biomarker protein.
　本明細書が英語に翻訳されて、単数形の「a」、「an」および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものも含むものとする。また、本明細書において、「約」とは±10%の数値範囲を意味する。
　以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 When this specification is translated into English and includes the singular words "a,""an," and "the," it is intended to include the plural as well as the singular, unless the context clearly indicates otherwise. Also, in this specification, "about" refers to a numerical range of ±10%.
The present invention will be further described below with reference to examples. However, these examples are merely illustrative of embodiments of the present invention and are not intended to limit the scope of the present invention.
１．実験方法
１－１．検体の採取
　血液中のAβの測定を行うためには、標準的な採血手技を用いて採取した血液を用いる。血液は、凝集性タンパク質の吸着を起こしにくいポリプロピレン容器に採取し、転倒混和後、速やかに遠心して血球成分を取り除く。通常、血液は採取するとすぐに凝固反応が始まり、血漿中のフィブリノーゲンから形成された高分子のフィブリンと血小板の作用によって血餅が形成される。血餅は遠心分離によって下層に分離され、その上清として血清が得られる。血清を採取する際には、あらかじめ採血容器に血餅の形成を促進するための凝固促進剤を入れておいてもよい。一方、あらかじめEDTAやクエン酸ナトリウム、ヘパリンなどの抗凝固剤を入れた採血容器で採血後に遠心すると、血球成分が下層に分離され、上清として血漿が得られる。採血した血液の遠心後の上清として得られる、血清および血漿のいずれの血液検体であってもAβの測定に用いることができる。本実施例においては、血清を用いた。 1. Experimental method 1-1. Collection of specimens In order to measure Aβ in blood, blood collected using standard blood collection techniques is used. Blood is collected in a polypropylene container that is less susceptible to adsorption of coagulant proteins, and after mixing by inversion, it is quickly centrifuged to remove blood cell components. Usually, the coagulation reaction begins immediately after blood is collected, and a blood clot is formed by the action of high molecular weight fibrin formed from fibrinogen in plasma and platelets. The blood clot is separated into the lower layer by centrifugation, and serum is obtained as the supernatant. When collecting serum, a coagulation promoter to promote the formation of a blood clot may be added to the blood collection container in advance. On the other hand, if blood is collected in a blood collection container that contains an anticoagulant such as EDTA, sodium citrate, or heparin, and centrifuged after collection, blood cell components are separated into the lower layer, and plasma is obtained as the supernatant. Either blood sample, serum or plasma, obtained as the supernatant after centrifugation of collected blood can be used to measure Aβ. In this embodiment, serum was used.
１－２．検体の前処理方法
　採取した血液検体(血清あるいは血漿)に、前処置として、PBSを溶媒として調整したSDS溶液を、SDSの終濃度が5%となるように添加し、よく混和した。
なお、本実施例においては、50%ギ酸、50% Triton、9M尿素を添加(溶媒はPBS, 濃度はそれぞれ添加後の終濃度)した血清も比較のために準備し、測定に用いた。 1-2. Sample pretreatment method As a pretreatment, SDS solution prepared using PBS as a solvent was added to the collected blood sample (serum or plasma) so that the final concentration of SDS was 5%, and the sample was mixed well.
In this example, serum to which 50% formic acid, 50% Triton, and 9M urea had been added (solvent: PBS, concentrations: final concentrations after addition) was also prepared for comparison and used in the measurements.
１－３．Aβの定量方法
　SDSを添加した血液検体は、抗Aβ抗体を吸着させたポリマー性フォトニック結晶センサーの表面に滴下し、37℃で2時間incubateする。その後、PBSで3回洗浄した。洗浄後のフォトニック結晶センサーの表面の反射スペクトルを分光器を用いて測定した。測定スペクトルは、あらかじめ測定しておいた血液検体滴下前のフォトニック結晶センサーの表面の反射スペクトルのデータと数値的に差分をとり、フォトニック結晶センサーからの干渉光のピーク強度の変化を、比の値として計算した。検体滴下前後で干渉光のピーク強度が低下するほど、すなわち、ピーク強度の比の値が小さいほど、抗Aβ抗体に結合したAβの量が多いことを意味する。実際には、適当な希釈系列のAβ標準溶液を準備して検量線を描き、血液検体の測定値と比較することにより、検体中のAβ濃度が測定できる。 1-3. Aβ quantification method The blood sample to which SDS has been added is dropped onto the surface of a polymer photonic crystal sensor to which anti-Aβ antibodies have been adsorbed, and incubated at 37°C for 2 hours. It is then washed three times with PBS. The reflection spectrum of the surface of the photonic crystal sensor after washing was measured using a spectrometer. The measured spectrum was numerically subtracted from the reflection spectrum data of the surface of the photonic crystal sensor before the blood sample was dropped, and the change in the peak intensity of the interference light from the photonic crystal sensor was calculated as a ratio value. The lower the peak intensity of the interference light before and after the sample is dropped, that is, the smaller the ratio of the peak intensities, the greater the amount of Aβ bound to the anti-Aβ antibody. In practice, the Aβ concentration in the sample can be measured by preparing an appropriate dilution series of Aβ standard solutions, drawing a calibration curve, and comparing it with the measured value of the blood sample.
１－４．血清中のタンパク質の容器への吸着に対するSDS濃度の影響の検討
　まず、終濃度35 w/v%のSDS溶液を調製（溶媒： PBS）した。次に、凍結コントロール血清（L スイトロール（登録商標）EX）中へ、35 w/v%に調製 した SDS 溶液を添加し、 SDS 終濃度が0～30 w/v%となるように調整した。SDS溶液を添加した各種血清溶液をポリスチレン製96 ウェルプレートのウェルへ滴下（滴下量： 50μL）した。96ウェルプレートを室温下で1 時間静置した後、PBS を用いて各ウェルを3 回洗浄した。各ウェルに吸着したタンパク質をQuant iTTM Protein Assay Kitを用いて蛍光染色し、蛍光顕微鏡を用いて蛍光画像を取得した。 1-4. Study of the effect of SDS concentration on the adsorption of serum proteins to the container First, an SDS solution with a final concentration of 35 w/v% was prepared (solvent: PBS). Next, the SDS solution adjusted to 35 w/v% was added to the frozen control serum (L-Suitrol (registered trademark) EX) to adjust the final SDS concentration to 0-30 w/v%. Various serum solutions to which SDS solution was added were dropped (drop amount: 50 μL) into the wells of a polystyrene 96-well plate. After leaving the 96-well plate at room temperature for 1 hour, each well was washed three times with PBS. The proteins adsorbed to each well were fluorescently stained using the Quant iT TM Protein Assay Kit, and fluorescent images were obtained using a fluorescent microscope.
２．結果
２－１．Aβの凝集性に対する種々の物質が及ぼす影響の検討
　種々の溶媒に同体積のAβ（200μM）溶液を混合し、各混合液中のAβの量を定量した（図１）。ここで用いたAβ溶液は、以下の通りである（表示の濃度は終濃度）。
サンプル１；6mM Aβ42 in PBS（凍結なし）、
サンプル２；100μM Aβ42 in PBS（凍結なし）、
サンプル３；100μM Aβ42 in 血清（凍結あり）、
サンプル４；100μM Aβ42 in 血清（凍結なし）、
サンプル５；100μM Aβ42 in 血清＋0.5×PBS（凍結なし）、
サンプル６；100μM Aβ42 in 血清＋50 % ギ酸（凍結なし）、
サンプル７；100μM Aβ42 in 血清＋50 % Triton（凍結なし）、
サンプル８；100μM Aβ42 in 血清＋5 % SDS（凍結なし）、および
サンプル９；100μM Aβ42 in 血清＋9M 尿素（凍結なし） 2. Results 2-1. Examination of the effects of various substances on the aggregation of Aβ The same volume of Aβ (200 μM) solution was mixed with various solvents, and the amount of Aβ in each mixture was quantified (Figure 1). The Aβ solutions used here are as follows (the concentrations shown are the final concentrations).
Sample 1: 6mM Aβ42 in PBS (not frozen),
Sample 2: 100 μM Aβ42 in PBS (not frozen),
Sample 3: 100 μM Aβ42 in serum (frozen),
Sample 4: 100 μM Aβ42 in serum (not frozen),
Sample 5: 100 μM Aβ42 in serum + 0.5×PBS (not frozen),
Sample 6: 100 μM Aβ42 in serum + 50% formic acid (not frozen),
Sample 7: 100 μM Aβ42 in serum + 50 % Triton (not frozen),
Sample 8: 100 μM Aβ42 in serum + 5% SDS (not frozen), and Sample 9: 100 μM Aβ42 in serum + 9M urea (not frozen).
　抗原抗体反応を用いたバイオマーカータンパク質の定量法の一例として、ポリマー性フォトニック結晶センサーを用いて試料中のAβ42の量を測定した。図１のグラフの縦軸は、試料をセンサー基板にアプライした前後のセンサーの反射光強度の比の値である。この比の値が小さいほど、試料のアプライによって反射光強度が大きく低下したことを意味し、試料中のAβの量が多いことを示している。血清にAβを溶解すると、溶媒がPBSであった場合（サンプル２）と比較して、見かけ上の測定濃度が下がり（センサー反射光強度の比の値が上昇、サンプル４）、さらに、その変化量は凍結操作によって増大した（サンプル３）。このような結果が生じた原因として、Aβが容器の内面に吸着したり、血清中の他の分子と結合して抗原性を失ったりすることが考えられる。血清にPBS、ギ酸、Triton、SDSまたは尿素を添加する（サンプル５～サンプル９）と、ギ酸とSDSを添加したときのみ、見かけ上の測定濃度の低下が解消された（サンプル６およびサンプル８)。一方、PBS、Tritonを加えても、見かけ上の測定濃度は再現性をもって正しい値にはならなかった（サンプル５およびサンプル７）。また尿素を加えた場合には、見かけ上の測定濃度は正しい値と比較して上昇した（サンプル９）。この理由は、高濃度の尿素がセンサー基板上に析出したためと考えられた。ギ酸は、抗原抗体反応に必須の抗体分子を変性させてしまう可能性があり、測定の前処理の際に試料に添加する物質としては、SDSが適していると考えられる。 As an example of a method for quantifying a biomarker protein using an antigen-antibody reaction, the amount of Aβ42 in a sample was measured using a polymer photonic crystal sensor. The vertical axis of the graph in Figure 1 is the ratio of the reflected light intensity of the sensor before and after the sample was applied to the sensor substrate. The smaller the ratio value, the greater the decrease in reflected light intensity due to the application of the sample, indicating a larger amount of Aβ in the sample. When Aβ was dissolved in serum, the apparent measured concentration decreased (the ratio of the reflected light intensity of the sensor increased, Sample 4) compared to when the solvent was PBS (Sample 2), and the amount of change increased by freezing (Sample 3). The cause of this result is thought to be that Aβ was adsorbed to the inner surface of the container or bound to other molecules in the serum, causing it to lose its antigenicity. When PBS, formic acid, Triton, SDS, or urea was added to the serum (Samples 5 to 9), the decrease in the apparent measured concentration was eliminated only when formic acid and SDS were added (Samples 6 and 8). On the other hand, even when PBS or Triton was added, the apparent measured concentration did not reproducibly become the correct value (samples 5 and 7). Furthermore, when urea was added, the apparent measured concentration increased compared to the correct value (sample 9). This was thought to be because high concentrations of urea were precipitated on the sensor substrate. Formic acid may denature the antibody molecules that are essential for the antigen-antibody reaction, so SDS is thought to be a more suitable substance to add to samples during pretreatment for measurement.
２－２．Aβの凝集に対するSDSの効果の検討
　図２は、Aβ42を血清に溶解した際の見かけ上の測定濃度の低下が、5% SDSの添加によって解消できることを示した結果である。PBSを溶媒として作成したAβ42希釈系列の溶液を、ポリマー性フォトニック結晶センサーを用いて測定すると、濃度に相関のあるセンサー反射率強度の変化がみられた（丸；●）。ここで1μM以下の濃度ではプロットは、ほぼ横ばいであり、1μMの濃度以下での有意な反射光強度変化はみられなかった。センサーの感度は、センサー基板に吸着させる抗体濃度によって調節できるが、本プロットの実験条件においては、1μMの濃度以下はセンサーの感度限界であると判断できる。血清を溶媒として作成したAβ42希釈系列溶液の測定においては、センサー反射光強度の変化は小さかった（三角；▲）が、5%SDSを血清に添加することで、センサー反射率強度はPBS溶媒の結果（丸；●）と同程度に復元し、真のAβ42濃度が測定できることがわかった（丸；■）。 2-2. Study on the effect of SDS on Aβ aggregation Figure 2 shows that the apparent decrease in the measured concentration when Aβ42 is dissolved in serum can be eliminated by adding 5% SDS. When a series of dilutions of Aβ42 prepared in PBS were measured using a polymer photonic crystal sensor, a change in the sensor reflectance intensity correlated with the concentration was observed (circle; ●). Here, the plot was almost flat at concentrations below 1 μM, and no significant change in the reflected light intensity was observed below 1 μM. The sensitivity of the sensor can be adjusted by the concentration of the antibody adsorbed on the sensor substrate, but under the experimental conditions of this plot, it can be determined that the sensitivity limit of the sensor is below 1 μM. In the measurement of the series of dilutions of Aβ42 prepared in serum, the change in the sensor reflected light intensity was small (triangle; ▲), but by adding 5% SDS to serum, the sensor reflectance intensity was restored to the same level as the result of PBS solvent (circle; ●), and it was found that the true Aβ42 concentration could be measured (circle; ■).
２－３．血清中のタンパク質の容器への吸着に対するSDS濃度の影響の検討
　蛍光顕微鏡を用いて取得した蛍光画像からImage Jを用いて蛍光強度のヒストグラムを取得し、蛍光強度の最頻値を濃度に対してプロットした。結果を図３に示す。
　SDSの終濃度15 w/v%を極値として、SDS濃度の増加に伴い、ポリスチレンへのタンパク質吸着による蛍光強度増加が観察された。SDS濃度が25 w/v%程度となると 無添加の状態と同等の蛍光強度となり、 SDS添加によるタンパク質の吸着軽減の効果は無くなることが分かった。 2-3. Study of the effect of SDS concentration on the adsorption of serum proteins to the container A histogram of fluorescence intensity was obtained using Image J from the fluorescence images acquired using a fluorescence microscope, and the most frequent fluorescence intensity was plotted against the concentration. The results are shown in Figure 3.
With a final SDS concentration of 15 w/v%, an increase in fluorescence intensity due to protein adsorption to polystyrene was observed as the SDS concentration increased. When the SDS concentration reached about 25 w/v%, the fluorescence intensity was equivalent to that without SDS, and it was found that the effect of adding SDS in reducing protein adsorption was lost.
　本発明により、検体中の凝集性の疾患バイオマーカータンパク質の量を正確に測定することができる。従って、医療分野等における利用が期待される。
  INDUSTRIAL APPLICABILITY The present invention makes it possible to accurately measure the amount of aggregating disease biomarker protein in a sample, and is therefore expected to be useful in the medical field and the like.

Claims (14)

  1. 　凝集性バイオマーカータンパク質を含む検体の前処理方法であって、検体にドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合することを含む、前記方法。 A method for pretreating a sample containing an aggregating biomarker protein, the method comprising mixing sodium dodecyl sulfate (SDS) with the sample.
  2. 　凝集性バイオマーカータンパク質を含む検体の保存方法であって、検体にSDSを添加し、混合することを含む、前記方法。 A method for preserving a sample containing an aggregating biomarker protein, the method comprising adding SDS to the sample and mixing it.
  3. 　検体中のSDSの濃度が、約3w/v%～約20w/v%である、請求項１または請求項２に記載の方法。 The method according to claim 1 or 2, wherein the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
  4. 　前記バイオマーカータンパク質が、アミロイドβ、タウ、TDP43、α-シヌクレイン、ポリグルタミン、トランスサイレチン、血清アミロイドA、免疫グロブリン軽鎖およびプリオンからなるグループより選択されるいずれかである、請求項１または請求項２に記載の方法。 The method according to claim 1 or 2, wherein the biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion.
  5. 　前記検体が、液性検体である、請求項１または２に記載の方法。 The method of claim 1 or 2, wherein the sample is a liquid sample.
  6. 　前記液性検体が、血液、脳脊髄液、鼻粘液、尿および生体組織の懸濁液からなるグループより選択されるいずれかである、請求項１または請求項２に記載の方法。 The method according to claim 1 or 2, wherein the liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine, and a suspension of biological tissue.
  7. 　検体中の凝集性バイオマーカータンパク質の定量方法であって、以下の（ａ）および（ｂ）の工程を含む、前記方法である。
    （ａ）検体とドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate ；SDS）を混合する工程、
    （ｂ）工程（ａ）後、検体中のバイオマーカータンパク質を特異的に検出し、定量する工程。     A method for quantifying an aggregation biomarker protein in a sample, comprising the steps of: (a) detecting an aggregation biomarker protein in a sample;
    (a) mixing a sample with sodium dodecyl sulfate (SDS);
    (b) after step (a), specifically detecting and quantifying the biomarker protein in the sample.
  8. 　前記工程（ａ）において、前記検体中のSDSの濃度が、約3w/v%～約20w/v%である、請求項７に記載の方法。 The method according to claim 7, wherein in step (a), the concentration of SDS in the sample is about 3 w/v% to about 20 w/v%.
  9. 　前記工程（ｂ）において、バイオマーカータンパク質を抗体またはアプタマーにより検出する、請求項７または請求項８に記載の方法。 The method according to claim 7 or 8, wherein in step (b), the biomarker protein is detected using an antibody or an aptamer.
  10. 　前記バイオマーカータンパク質が、アミロイドβ、タウ、TDP43、α-シヌクレイン、ポリグルタミン、トランスサイレチン、血清アミロイドA、免疫グロブリン軽鎖およびプリオン、ならびにこれらのタンパク質の翻訳後修飾されたものからなるグループより選択されるいずれかである、請求項７または請求項８に記載の方法。 The method according to claim 7 or 8, wherein the biomarker protein is any one selected from the group consisting of amyloid beta, tau, TDP43, alpha-synuclein, polyglutamine, transthyretin, serum amyloid A, immunoglobulin light chain, and prion, as well as post-translationally modified versions of these proteins.
  11. 　前記検体が、液性検体である、請求項７または請求項８に記載の方法。 The method of claim 7 or 8, wherein the sample is a liquid sample.
  12. 　前記液性検体が、血液、脳脊髄液、鼻粘液、尿、涙、汗、唾液、皮膚浸出液、脳の間質液および生体組織の懸濁液からなるグループより選択されるいずれかである、請求項７または請求項８に記載の方法。 The method according to claim 7 or 8, wherein the liquid sample is any one selected from the group consisting of blood, cerebrospinal fluid, nasal mucus, urine, tears, sweat, saliva, skin exudate, brain interstitial fluid, and a suspension of biological tissue.
  13. 　凝集性バイオマーカータンパク質を含む検体を収納するための容器であって、SDSが収納されている、前記容器。 A container for storing a sample containing an aggregating biomarker protein, the container containing SDS.
  14. 　検体中の凝集性バイオマーカータンパク質を定量するためのキットであって、SDSまたは請求項１３に記載の容器を含む、前記キット。

          A kit for quantifying an aggregation biomarker protein in a sample, comprising SDS or the container described in claim 13.
PCT/JP2023/039965 2022-11-11 2023-11-07 Method for pretreating specimen WO2024101329A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2022-180733 2022-11-11
JP2022180733 2022-11-11

Publications (1)

Publication Number Publication Date
WO2024101329A1 true WO2024101329A1 (en) 2024-05-16

Family

ID=91032401

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/039965 WO2024101329A1 (en) 2022-11-11 2023-11-07 Method for pretreating specimen

Country Status (1)

Country Link
WO (1) WO2024101329A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009052906A (en) * 2007-08-23 2009-03-12 Nippon Sekijiyuujishiya Method for detecting or measuring abnormal prion protein related to transmissible spongiform encephalopathy
JP2020038192A (en) * 2018-09-03 2020-03-12 学校法人同志社 Method and kit for histologically detecting phosphorylated protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009052906A (en) * 2007-08-23 2009-03-12 Nippon Sekijiyuujishiya Method for detecting or measuring abnormal prion protein related to transmissible spongiform encephalopathy
JP2020038192A (en) * 2018-09-03 2020-03-12 学校法人同志社 Method and kit for histologically detecting phosphorylated protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TSUKUI, KAZUO ET AL.: "PK-resistant PrP in serum of scrapie-infected and uninfected hamsters", PROGRAMS AND ABSTRACTS OF THE 52ND ACADEMIC CONFERENCE OF THE JAPANESE SOCIETY FOR VIROLOGY, 1 November 2004 (2004-11-01) *

Similar Documents

Publication Publication Date Title
Kulenkampff et al. Quantifying misfolded protein oligomers as drug targets and biomarkers in Alzheimer and Parkinson diseases
Schoonenboom et al. Effects of processing and storage conditions on amyloid β (1–42) and tau concentrations in cerebrospinal fluid: implications for use in clinical practice
Nu et al. Blood-based immunoassay of tau proteins for early diagnosis of Alzheimer's disease using surface plasmon resonance fiber sensors
JP5032372B2 (en) Use of BNP-type peptides to predict the need for dialysis
Koel-Simmelink et al. The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA
JP6478987B2 (en) Method for measuring protein aggregates using surface FIDA
JP7448831B2 (en) Methods, biomarkers, reagent kits and devices to assist in the diagnosis of Alzheimer's dementia or mild cognitive impairment
JP2011514517A (en) Method for assaying antibodies against cyclic citrullinated peptides
Schmitz et al. Cerebrospinal fluid total and phosphorylated α-synuclein in patients with Creutzfeldt–Jakob disease and synucleinopathy
JP2019525184A (en) Histone and / or proADM as markers for organ damage
EP3570029B1 (en) Immunoassay for whole blood samples
Gargan et al. Identification of marker proteins of muscular dystrophy in the urine proteome from the mdx-4cv model of dystrophinopathy
Vascellari et al. Real-time quaking-induced conversion assays for prion diseases, synucleinopathies, and tauopathies
Singh et al. In vitro conversion assays diagnostic for neurodegenerative proteinopathies
WO2024101329A1 (en) Method for pretreating specimen
Natarajan et al. A novel time-resolved fluorescent lateral flow immunoassay for quantitative detection of the trauma brain injury biomarker-glial fibrillary acidic protein
WO2022250009A1 (en) Method for assisting differentiation of lewy body disease and alzheimer's disease, biomarker, and reagent kit
JP7348604B2 (en) Methods, biomarkers, reagent kits and devices to aid in the diagnosis of Parkinson's disease
Chang et al. Assessing plasma levels of α-synuclein and neurofilament light chain by different blood preparation methods
CN108020673A (en) A β kits, detection method and application
KR102254053B1 (en) Biomarker for detecting amyloid beta accumulation in brain of subject with normal cognitive function or mild cognitive impairment using blood sample
Vieira et al. Lab-on-a-chip technologies for minimally invasive molecular sensing of diabetic retinopathy
JP6405549B2 (en) Acute coronary syndrome marker and its use
KR20210035825A (en) Methods and related uses for identifying individuals with or at risk of developing amyloid-positive dementia based on marker molecules
JP7483109B2 (en) Ferritin measurement reagent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23888676

Country of ref document: EP

Kind code of ref document: A1