Pretreatment method of specimen

Abstract

To provide a pretreatment method of a specimen for accurately and simply performing quantification of a biomarker protein by preventing aggregation of a disease biomarker protein in the specimen or adsorption thereof to a container.SOLUTION: A pretreatment method of a specimen containing an aggregation biomarker protein, includes a step of mixing the specimen with sodium dodecyl sulfate (SDS). A quantification method of an aggregation biomarker protein in a specimen, includes the steps of: mixing the specimen with sodium dodecyl sulfate (SDS); and after the mixing step, specifically detecting and quantifying the biomarker protein in the specimen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for pre-treating a specimen derived from a subject. [Background technology]

[0002] Antigen-antibody reactions are commonly used to quantify disease biomarker proteins. Measuring the concentrations of Alzheimer's disease biomarkers, such as amyloid beta (Aβ) and tau, in patient samples such as blood (serum, plasma), urine, or cerebrospinal fluid can be used to diagnose the possibility of Alzheimer's disease and evaluate the disease progression. However, disease biomarker proteins, particularly aggregating proteins such as Aβ and tau, tend to aggregate easily. These proteins can adsorb to glass or resin containers used for cryopreservation of patient samples, or bind to other molecules in the patient sample, causing them to lose their antigenicity. Furthermore, when a sample is frozen in a container and stored for a long period of time, or when the sample is repeatedly frozen and thawed, there is a known problem in the quantification method for Alzheimer's disease biomarkers that uses antigen-antibody reactions, in that the measured values ​​decrease over time (Non-Patent Document 1).

[0003] To avoid such problems in quantifying aggregating proteins as disease biomarkers due to long-term storage of patient-derived samples or repeated freezing and thawing, conventional approaches to quality control of measurement results have been adopted by creating and adhering to a standardized protocol for the time from sample collection to centrifugation and the number of freezing cycles. However, such a protocol does not fundamentally eliminate the time-dependent changes in measurement values. Meanwhile, methods have been proposed in which formic acid, urea, guanidine hydrochloride, or the like is added to samples to eliminate Aβ aggregation or its binding or adsorption to other substances (Patent Document 1). These reagents are commonly used to solubilize insoluble aggregates, but they have a strong protein-denaturing effect. Therefore, when quantifying aggregating disease biomarker proteins using an antigen-antibody reaction, using these reagents to dissolve protein aggregation can result in changes in antigenicity due to denaturation of the biomarker protein or changes in antigen-binding ability due to denaturation of the antibody itself, potentially preventing accurate quantification. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-232172 [Non-patent literature]

[0005] [Non-Patent Document 1] Doblhammer et al., Alzheimers Dement. 11:291-300 2019. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above circumstances, the present invention provides a method for pretreating a sample, which prevents aggregation of a disease biomarker protein in the sample, binding to other molecules in the sample, or adsorption to a container, and enables accurate and simple quantification of the biomarker protein using antibodies or the like. [Means for solving the problem]

[0007] The inventors investigated the quantification results of amyloid beta (Aβ), a biomarker protein for aggregating diseases, when dissolved in various solvents. The amount of Aβ in a solvent was measured using a polymer photonic crystal sensor (see, for example, WO2010 / 044274 ), an example of a protein quantification method using an antigen-antibody reaction. It was found that dissolving Aβ in serum reduced the apparent measured concentration compared to when the solvent was phosphate-buffered saline (PBS), and that this change increased with freezing. The difference in the measured Aβ concentration between PBS and serum is thought to be due to binding to other molecules in serum and loss of antigenicity in serum. Furthermore, the decrease in apparent measured concentration after long-term freezing and storage is thought to be due to Aβ adsorption to the inner surface of the container. Therefore, the amount of Aβ was measured by adding PBS, formic acid, Triton, SDS, and urea to serum. The decrease in apparent measured concentration was eliminated only when formic acid and SDS were added. As mentioned above, formic acid has the potential to denature antibody molecules, which are essential for antigen-antibody reactions. Therefore, it was suggested that SDS would be a suitable substance to add to the specimen sample during pretreatment for measuring Aβ levels in serum.

[0008] The present invention has been completed based on the above findings. That is, the present invention includes the following (1) to (14). (1) A method for pretreating 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-prone 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 10 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 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. (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 10 w / v %. (9) The method according to (7) or (8) above, wherein in 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. (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, 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, the kit comprising SDS or the container described in (13) above. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0009] According to the present invention, it becomes possible to quantify with high accuracy the amount of aggregating biomarker protein in an easily collected biological tissue sample, particularly in a liquid sample such as blood, using an antibody or the like. [Brief explanation of the drawings]

[0010] [Figure 1] The results of examining the effects of various substances on the aggregation properties of amyloid β42 (Aβ42) are shown. [Figure 2] 1 shows the results of examining the effect of SDS on the aggregation properties of Aβ42. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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, comprising: This method involves mixing a sample with sodium dodecyl sulfate (SDS). The sample pretreatment method according to this 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 this embodiment is carried out before the step of quantifying the amount of the aggregating biomarker protein in the sample, particularly before the step of quantifying using molecules such as antibodies, aptamers (peptide aptamers, nucleic acid (DNA and RNA) aptamers, etc.) (herein also referred to as "molecular-specific recognition factors").

[0012] In this embodiment, the "aggregating biomarker protein" refers to a protein that serves as an indicator for determining whether or not a given disease has developed, or is likely to develop, or for determining the activity and progression of an already diagnosed disease, 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, and transthyretin, serum amyloid A, or the like as biomarker proteins for amyloidosis. Immunoglobulin light chains and prions are known as biomarker proteins for Creutzfeldt-Jakob disease.

[0013] Aβ aggregates are a major component of senile plaques, a hallmark of Alzheimer's disease. Neurofibrillary tangles, also characteristic of Alzheimer's disease, are induced by the aggregation of highly phosphorylated tau protein. Aβ is cleaved from its precursor protein, amyloid-β precursor protein (APP), by β-secretase and γ-secretase and secreted extracellularly. The two main molecular species of Aβ are Aβ40 (40 amino acids) and Aβ42 (42 amino acids). It has been reported that the amount of Aβ42 in cerebrospinal fluid (CSF) decreases, and the Aβ42 / Aβ40 ratio in plasma decreases as Alzheimer's disease progresses. Therefore, quantifying the amount of Aβ (especially Aβ42) in blood and CSF can be used to assess the likelihood of Alzheimer's disease onset and the progression of the disease after onset. Furthermore, the term "subject" includes not only humans but also non-human animals.

[0014] The "specimen" in this embodiment refers to a specimen collected from a subject, and is not particularly limited, but is preferably a liquid specimen, such as blood (plasma and serum), cerebrospinal fluid, nasal mucus, urine, and a suspension of biological tissue (biological tissue suspended or solubilized in an appropriate solvent (such as physiological saline)).

[0015] Those skilled in the art can determine the optimal concentration of SDS to be mixed with a sample (final concentration in the sample) for each type of sample through preliminary experiments. For example, the concentration may be about 1 w / v% (weight / volume percent) to about 15 w / v%, preferably about 3 w / v% to about 10 w / v%, and more preferably about 4 w / v% to about 7 w / v%. SDS is added to and mixed with the sample so that the SDS is uniformly distributed throughout the sample. It is desirable to avoid excessive stirring, which may result in denaturation of proteins contained in the sample. The step of "mixing the sample with SDS" may involve adding SDS to the collected sample and mixing the mixture. Alternatively, SDS may be added to a container in advance, and the sample may be added thereto and mixed. Alternatively, SDS may be added to a sample after a certain period of time has elapsed after storage at low temperatures (e.g., about −80°C or −20°C) and then mixed. The form of "SDS" is not particularly limited; it may be a powder or may be dissolved in an appropriate solvent.

[0016] The second embodiment is a method for quantifying an aggregation biomarker protein in a sample, which comprises 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.

[0017] Detection and quantification of aggregation biomarker proteins, including Aβ, can be performed using molecules (also referred to herein as "molecular-specific recognition factors") such as antibodies and aptamers (peptide aptamers, nucleic acid (DNA and RNA) aptamers, etc.). When antibodies are used, aggregation biomarker proteins can be detected and quantified using immunoassay-based methods, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), immunochromatography, immunomagnetic reduction (IMR), surface plasmon resonance (SPR), and quartz crystal microbalance (QCM). Other methods for detecting antigen-antibody reactions and quantifying the reaction amount include methods using optical sensors, such as polymer photonic crystal sensors.

[0018] Furthermore, when aptamers are used to detect and quantify aggregating biomarker proteins, aptamers, like antibodies, have the property of specifically recognizing molecules, and therefore many of the methods applied to detect antigen-antibody reactions (described above) can be used.

[0019] Steps (a) and (b) of the second embodiment may be performed consecutively, or step (b) may be performed after a certain period of time has elapsed since step (a) was performed. 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. Furthermore, 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 a low temperature (for example, at about -80°C or -20°C) and then SDS may be added and mixed after a certain period of time has elapsed.

[0020] 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 aggregation tend to aggregate immediately after a sample is collected in a container and adhere to the wall of the container. This aggregation can be induced during frozen storage of the sample or by freezing and thawing the sample, making it difficult to accurately measure the amount of the biomarker protein. Therefore, when storing a sample containing an aggregating biomarker protein, aggregation of the biomarker protein can be prevented by adding SDS to the sample, mixing the sample, and then storing the sample 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 sample, see the description in the first embodiment.

[0021] 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 tendencies aggregate immediately after a sample is collected in a container such as a blood collection tube, adhering to the wall of the container. Therefore, to prevent aggregation-prone biomarker proteins from aggregating in the container and adhering to the wall of the container, SDS can be placed in the container beforehand, allowing for easy and accurate quantification of the biomarker protein. The material of the "container for collecting a sample" according to this embodiment is not particularly limited, and may be made of resin or glass. The form is also not particularly limited. The SDS contained in the "container for collecting a sample" may be in any form (e.g., powder, solution, etc.). Those skilled in the art can appropriately select the amount of SDS to be placed depending on the type of sample and the type of biomarker protein contained in the sample. Assuming that the aggregating 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 15 w / v%, preferably about 3 w / v% to about 10 w / v%, and more preferably about 4 w / v% to about 7 w / v%.

[0022] 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 according to this embodiment may include, in addition to SDS or a container containing SDS, reagents and diluents used to quantify the biomarker protein, as well as antibodies or aptamers for quantifying the biomarker protein.

[0023] 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" means a numerical range of ±10%. The present invention will be further explained below by showing examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Example]

[0024] 1. Experimental Method 1-1. Sample collection To measure Aβ in blood, blood is collected using standard blood collection techniques. The blood is collected in a polypropylene container, which is resistant to adsorption of coagulating proteins. After mixing by inversion, the blood is quickly centrifuged to remove blood cell components. Typically, the clotting reaction begins immediately after blood collection, and a clot is formed by the action of platelets and the high-molecular-weight fibrin formed from fibrinogen in the plasma. The clot is separated into the lower layer by centrifugation, and serum is obtained as the supernatant. When collecting serum, a coagulation promoter to promote clot formation may be added to the blood collection container beforehand. Alternatively, if blood is collected in a blood collection container containing an anticoagulant such as EDTA, sodium citrate, or heparin, and then centrifuged, blood cell components are separated into the lower layer, and plasma is obtained as the supernatant. Either serum or plasma, obtained as the supernatant after centrifugation of collected blood, can be used to measure Aβ. In this example, serum was used.

[0025] 1-2. Sample pretreatment method As a pretreatment, an SDS solution prepared using PBS as a solvent was added to the collected blood sample (serum or plasma) so that the final SDS concentration 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 were the final concentrations after addition) was also prepared for comparison and used in the measurements.

[0026] 1-3. Aβ quantification method A blood sample containing SDS was dropped onto the surface of a polymer photonic crystal sensor pre-adsorbed with anti-Aβ antibodies and incubated at 37°C for 2 hours. The sample was then washed three times with PBS. The reflectance spectrum of the washed photonic crystal sensor surface was measured using a spectrometer. The measured spectrum was then numerically subtracted from the reflectance spectrum of the photonic crystal sensor surface measured before the blood sample was dropped. The change in peak intensity of the interference light from the photonic crystal sensor was calculated as a ratio. The decrease in peak intensity of the interference light before and after the sample drop, i.e., the smaller the peak intensity ratio, the greater the amount of Aβ bound to the anti-Aβ antibodies. In practice, a calibration curve can be drawn using an appropriate dilution series of Aβ standard solutions, and the Aβ concentration in the sample can be measured by comparing it with the measured values ​​of the blood sample.

[0027] 2.Results 2-1. Examination of the effects of various substances on Aβ aggregation Equal volumes of Aβ (200 μM) solutions were mixed with various solvents, and the amount of Aβ in each mixture was quantified (Figure 11). The Aβ solutions used here are as follows (the concentrations shown are final concentrations): Sample 1: 6 mM 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)

[0028] As an example of a biomarker protein quantification method using an antigen-antibody reaction, we measured the amount of Aβ42 in a sample using a polymer photonic crystal sensor. The vertical axis of the graph in Figure 1 represents the ratio of the reflected light intensity of the sensor before and after applying the sample to the sensor substrate. A smaller ratio indicates a greater decrease in reflected light intensity upon sample application, indicating a higher amount of Aβ in the sample. When Aβ was dissolved in serum, the apparent measured concentration decreased (increased sensor reflected light intensity ratio, Sample 4) compared to when the solvent was PBS (Sample 2). Furthermore, this change increased upon freezing (Sample 3). This result may be due to Aβ adsorption to the inner surface of the container or binding to other molecules in the serum, resulting in loss of antigenicity. When PBS, formic acid, Triton, SDS, or urea was added to serum (Samples 5–9), the decrease in 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 due to high concentrations of urea being precipitated on the sensor substrate. Formic acid may denature antibody molecules, which are essential for antigen-antibody reactions, so SDS is thought to be a more suitable substance to add to samples during measurement pretreatment.

[0029] 2-2. Examination of the effect of SDS on Aβ aggregation Figure 2 shows that the apparent decrease in measured concentration of Aβ42 dissolved in serum can be eliminated by adding 5% SDS. When a dilution series of Aβ42 solutions prepared in PBS was measured using a polymer photonic crystal sensor, changes in sensor reflectance intensity correlated with concentration were observed (circles; ●). Here, the plot remained almost flat at concentrations below 1 μM, and no significant changes in reflected light intensity were observed below 1 μM. The sensitivity of the sensor can be adjusted by the antibody concentration adsorbed to the sensor substrate, but under the experimental conditions shown in this plot, concentrations below 1 μM are considered to be the limit of the sensor's sensitivity. When measuring a dilution series of Aβ42 solutions prepared in serum, changes in sensor reflected light intensity were small (triangles; ▲). However, adding 5% SDS to serum restored the sensor reflectance intensity to the same level as in PBS (circles; ●), demonstrating that the true Aβ42 concentration could be measured (circles; ■). [Industrial Applicability]

[0030] The present invention enables accurate measurement of the amount of aggregating disease biomarker proteins in a sample, and is therefore expected to be useful in the medical field and other fields.

Claims

1. A method for pretreatment of a sample containing an aggregation biomarker protein, comprising: The method further comprises mixing sodium dodecyl sulfate (SDS) with the sample.

2. A method for preserving a specimen containing an aggregation biomarker protein, comprising: The method further comprises adding SDS to the sample and mixing.

3. 3. The method according to claim 1, wherein the concentration of SDS in the sample is about 3 w / v% to about 10 w / v%.

4. The method of 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. The method of claim 1 or 2, wherein the sample is a liquid sample.

6. 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. 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.

8. 8. The method according to claim 7, wherein in step (a), the concentration of SDS in the sample is about 3 w / v% to about 10 w / v%.

9. The method according to claim 7 or 8, wherein in step (b), the biomarker protein is detected using an antibody or an aptamer.

10. The method of 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.

11. The method of claim 7 or 8, wherein the sample is a liquid sample.

12. 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, 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, the kit comprising SDS or the container described in claim 13.

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