WO2024101329A1 - Procédé de prétraitement d'échantillon - Google Patents
Procédé de prétraitement d'échantillon Download PDFInfo
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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.
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Abstract
La présente invention aborde le problème lié à la fourniture d'un procédé de prétraitement d'un échantillon pour quantifier avec précision et commodément une protéine de biomarqueur de maladie dans l'échantillon tout en empêchant l'agrégation ou l'adsorption de la protéine de biomarqueur dans un récipient. Plus particulièrement, la présente invention concerne un procédé de prétraitement d'un échantillon contenant une protéine de biomarqueur d'agrégation, le procédé comprenant le mélange de l'échantillon avec du dodécylsulfate de sodium (SDS). De plus, la présente invention concerne un procédé de quantification d'une protéine de biomarqueur d'agrégation dans un échantillon, le procédé comprenant une étape de mélange de l'échantillon avec du SDS, et une étape, suivant l'étape susmentionnée, de détection et de quantification spécifiques de la protéine de biomarqueur dans l'échantillon.
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JP2009052906A (ja) * | 2007-08-23 | 2009-03-12 | Nippon Sekijiyuujishiya | 伝達性海綿状脳症に係る異常プリオン蛋白質の検出又は測定方法 |
JP2020038192A (ja) * | 2018-09-03 | 2020-03-12 | 学校法人同志社 | リン酸化タンパク質の組織学的検出方法及びキット |
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JP2009052906A (ja) * | 2007-08-23 | 2009-03-12 | Nippon Sekijiyuujishiya | 伝達性海綿状脳症に係る異常プリオン蛋白質の検出又は測定方法 |
JP2020038192A (ja) * | 2018-09-03 | 2020-03-12 | 学校法人同志社 | リン酸化タンパク質の組織学的検出方法及びキット |
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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) * |
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