CN116908465A - Composition for protecting beta-amyloid and application thereof - Google Patents
Composition for protecting beta-amyloid and application thereof Download PDFInfo
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- CN116908465A CN116908465A CN202310692944.9A CN202310692944A CN116908465A CN 116908465 A CN116908465 A CN 116908465A CN 202310692944 A CN202310692944 A CN 202310692944A CN 116908465 A CN116908465 A CN 116908465A
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Classifications
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
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- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a composition and application thereof. The composition comprises: buffer, protease inhibitor, and at least two of NTM, DDM, DM, OTG, OG, tween-20 and Span-20. The composition can effectively reduce the loss of beta-amyloid in biological samples, particularly the loss of Abeta 40and Abeta 42in the preservation and detection period of the biological samples, can also maintain the stable ratio of Abeta 40 to Abeta 42 content, is further applied to Abeta quantitative detection in the biological samples, can reduce Abeta detection difficulty, improve the accuracy of detection results, and expands the clinical application of Abeta 42 and Abeta 40 as blood biomarkers of Alzheimer's disease or other related diseases caused by Abeta protein expression disorder.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a composition for protecting beta-amyloid, a kit and application thereof.
Background
Alzheimer's Disease (AD), a neurodegenerative disease that contributes to the progressive progression of the underlying disease, affects about 3300 thousands worldwide. The clinical manifestations are as follows: memory impairment, aphasia, disuse, disrecognition, impairment of vision space skills, executive dysfunction, personality and behavioral changes, and the like. The histological changes of patients with alzheimer's disease after death were studied and found that extracellular deposits and neurofibrillary tangles were present in the brain, consisting of aggregated beta-amyloid beta-protein (aβ) and abnormally phosphorylated Tau protein, respectively. This is thought to be one of the pathogenesis of Alzheimer's disease.
Among them, aβ is produced by cleavage of amyloid precursor protein (amyloid precursor protein, APP) which is widely present in various tissues and is intensively expressed in neuronal synapses by β -secretase and γ -secretase, and is a long peptide comprising 40 or 42 amino acids (hereinafter, abbreviated as "aβ40", "aβ42", respectively). Aβ42 has a molecular weight of about 4,514Da and Aβ40 has a molecular weight of about 4,330Da. The two peptides self-aggregate in aqueous solution to form oligomers and fibrils that are toxic to neuronal cells. Studies have shown that the ratio of aβ42 to aβ40 is promising as a clinical blood biomarker for effective diagnosis of alzheimer's disease.
Currently, aβ can be quantified by performing mass spectrometry (MALDI TOF MS) or multiplex electrochemiluminescence immunoassay after Immunoprecipitation (IP), or by ELISA, but clinical sample detection has problems of large difficulty in detecting aβ, low accuracy of detection results, inability to reflect the actual content and ratio of aβ42 and aβ40 in patient plasma levels, and the like.
There is a need to improve the stability of clinical samples, reduce the difficulty in detecting aβ in clinical samples, improve the accuracy of detection results, and help clinicians to perform comprehensive and accurate diagnosis on alzheimer's patients.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a composition for protecting beta-amyloid, a kit and application thereof, and the composition can reduce the loss of beta-amyloid in a biological sample, particularly the loss of beta-amyloid in the preservation and detection processes of the biological sample, reduce the difficulty of Abeta detection, improve the accuracy of detection results and have high clinical value.
It is to be stated that the present invention has been completed based on the following work of the inventors:
aiming at the technical problems that the detection difficulty of clinical serum or plasma samples Abeta is high and the deviation between the detection result obtained by the existing detection means and the content of Abeta 42 and Abeta 40 of human blood plasma level Abeta 40 is large, the inventor analyzes the clinical detection results of a large number of Alzheimer's patients and refers to the literature to find that: aβ42 is more hydrophobic than aβ40and self-aggregation occurs more readily, specifically with aβ40 forming dimers and tetramers, while aβ42 forms dimers, tetramers, hexamers and dodecamers. Accordingly, the inventors speculate in a large scale that: (1) The strong hydrophobicity of the Abeta causes serious loss in the biological sample preservation and detection process, so the detection rate of Abeta 40and Abeta 42in human serum is very low; (2) When the biological sample leaves the organism, the loss rate of aβ42 may be significantly greater than aβ40, such that aβ42in human serum is hardly detected or abnormally low, and thus the ratio of the obtained aβ42 to aβ40 content has no clinical reference significance. The reasons for the two aspects greatly limit the clinical application of Abeta 42 and Abeta 40 as blood biomarkers of Alzheimer's disease or other related diseases caused by Abeta protein expression disturbance.
Further, the inventors have conducted a number of screening experiments and unexpectedly found that the inclusion of a buffer, a protease inhibitor, and a nonionic surfactant of at least two of NTM, DDM, DM, OTG, OG, tween-20 and Span-20 is effective in reducing the loss of β -amyloid in a biological sample, particularly the loss of aβ40and aβ42 during low temperature storage of the biological sample, and maintaining a stable ratio of aβ40 to aβ42 content. Further test results show that after the clinical serum or plasma samples added with the composition of the invention are stored at 4 ℃ for 20 hours, the A beta 40and the A beta 42 can still be effectively detected, and the content ratio of the A beta 40and the A beta 42 is basically consistent with that of the samples which are not stored at low temperature. Therefore, the composition can be applied to the existing Abeta quantitative method or the improvement thereof, can realize the accurate Abeta quantification, and provides reference for diagnosis and treatment of clinical Alzheimer's disease or other related diseases caused by Abeta protein expression disorder.
In a first aspect of the invention, the invention provides a composition for protecting beta-amyloid. The composition comprises: a buffer, a nonionic surfactant, a protease inhibitor, the nonionic surfactant selected from at least two of NTM, DDM, DM, OTG, OG, tween-20 and Span-20. The composition of the invention can effectively reduce the loss of beta-amyloid in biological samples, especially the loss of Abeta 40and Abeta 42 during the preservation and detection of biological samples, and can also maintain the stable ratio of Abeta 40 to Abeta 42. The composition can be applied to quantitative detection of the Abeta in biological samples, can reduce the Abeta detection difficulty, improve the accuracy of detection results, and expand the clinical application of Abeta 42 and Abeta 40 as blood biomarkers of Alzheimer's disease or other related diseases caused by Abeta protein expression disorder.
According to an embodiment of the invention, the nonionic surfactant is selected from NTM and DDM.
According to an embodiment of the present invention, the mass volume percentage (g/mL) of the NTM is 0.1 to 0.3% based on the total volume of the buffer.
According to an embodiment of the present invention, the mass volume percentage (g/mL) of the NTM is 0.15-0.3% based on the total volume of the buffer.
According to an embodiment of the invention, the mass volume percentage (g/mL) of DDM is 0.1-0.3% based on the total volume of the buffer.
According to an embodiment of the invention, the mass volume percentage (g/mL) of DDM is 0.15-0.3% based on the total volume of the buffer.
The buffer is selected from one of Tris-HCl buffer, MES buffer, PBS buffer and HEPES buffer.
According to an embodiment of the invention, the pH of the buffer is between 6.9 and 7.9.
According to an embodiment of the invention, the buffer pH is 7.2-7.6.
According to an embodiment of the invention, the buffer is Tris-HCl buffer.
According to an embodiment of the invention, the concentration of Tris in the Tris-HCl buffer is 0.05-0.2M.
The protease inhibitor is at least one selected from serine protease inhibitor, trypsin inhibitor, aminopeptidase inhibitor, carboxypeptidase inhibitor, cysteine protease inhibitor, metalloprotease inhibitor and aspartic protease inhibitor.
In some specific embodiments, the trypsin inhibitor source is, including, but not limited to: bovine pancreas, ovomucoid, soybean, and lima bean.
According to an embodiment of the invention, the protease inhibitor is Cocktail.
In some embodiments, the concentration of the protease inhibitor Cocktail in the composition may be a classical working concentration known to those skilled in the art, and specifically described with reference to dilution of the Cocktail working concentration by the manufacturer, and the concentration of the Cocktail in the composition may be increased or decreased depending on the particular biological sample, including but not limited to the biological source of the sample, the mode of sample acquisition, the amount of sample protein, and the like. According to an embodiment of the invention, the composition is free of sodium chloride and potassium chloride.
The aβ protein in serum or plasma samples is unstable and often requires immediate detection of the sample or storage in a low temperature environment, such as a refrigerator; but even under low temperature storage conditions, the stability of aβ protein is obviously insufficient to support its storage for a sufficient time until detection is completed. While the storage method of packaging serum or plasma samples in dry ice can solve this problem to some extent, it is quite inconvenient. The composition of the invention can be added into biological samples, especially liquid biological samples such as serum or plasma of mammals, so that the stability of the Abeta protein can be obviously improved, and the loss of beta-amyloid can be effectively reduced. Therefore, the composition of the invention can improve the detection accuracy of the existing Abeta quantification method and reduce the Abeta detection difficulty.
In a second aspect of the invention, the invention provides a kit. The kit comprises a composition of the first aspect of the invention. The kit containing the composition can be further used for preserving and detecting biological samples, thereby achieving the purpose of controlling the loss of beta-amyloid in the biological samples.
In a third aspect of the invention, the invention proposes a use. According to an embodiment of the invention, the use is the use of a composition according to the first aspect of the invention or a kit according to the second aspect of the invention for reducing the loss of β -amyloid in a biological sample. The composition or the kit of the invention or the detection method developed by the composition or the kit of the invention can be further used for quantifying Abeta 40 and/or Abeta 42in biological samples, and clinically provides reference for diagnosis and treatment of Alzheimer's disease or other related diseases caused by Abeta protein expression disorder.
According to an embodiment of the invention, the β -amyloid is aβ40 and/or aβ42. The composition or the kit is favorable for maintaining the stability of the Abeta 40and the Abeta 42in the biological sample, and further realizes the accurate quantification of the Abeta 40and the Abeta 42.
According to an embodiment of the invention, the β -amyloid is aβ42. The composition or kit of the invention is particularly advantageous for maintaining the stability of aβ42in a biological sample. Therefore, the Aβ42 target signal with obviously improved signal-to-noise ratio can be obtained in the sample detection process.
According to an embodiment of the invention, the biological sample according to the invention is a liquid sample.
According to an embodiment of the present invention, the liquid sample includes, but is not limited to: serum or plasma or other body fluids or biological tissue disruption solutions of mammals, or serum or plasma or other body fluids or biological tissue disruption solutions of mammals further processed by clinical or biochemical laboratory or detection laboratory acceptable means such as centrifugation, separation and purification.
According to an embodiment of the invention, the biological sample is a mammalian serum or plasma sample. Clinically, doctors often diagnose Alzheimer's disease or other related diseases caused by the imbalance of the expression of Abeta proteins according to the content and the ratio of Abeta 42 and Abeta 40 of the plasma level of patients, and take the Abeta as one of the evaluation standards of the treatment effect. Thus, the compositions or kits of the invention, or the detection methods developed in accordance with the compositions or kits of the invention, are more commonly applied to control of beta-amyloid loss in human serum or plasma samples.
According to an embodiment of the invention, the biological sample is in a cryopreserved state. To maintain aβ protein stability in serum or plasma samples, it is a preferred option to maintain the biological sample in a low temperature environment.
According to an embodiment of the present invention, the preservation temperature of the low-temperature preservation is 0 to 6 ℃.
According to an embodiment of the present invention, the preservation time of the low-temperature preservation is 0.5 to 20 hours.
In order to maintain the stability of Abeta protein in serum or plasma samples, biological samples are stored in a refrigerator at 0-6 ℃, and the biological samples are convenient and easy to realize in a biochemical laboratory or a detection department. The composition or kit of the invention, or the detection method developed according to the composition or kit of the invention, allows the biological sample to be preserved for 20 hours at 0-6 ℃ and still effectively detect the Aβ40and the Aβ42, and the content ratio of the Aβ40and the Aβ42 is maintained unchanged.
In a fourth aspect of the invention, the invention provides a method of reducing the loss of β -amyloid in a biological sample. According to an embodiment of the invention, the method is to contact the biological sample with a composition according to the first aspect of the invention or to treat the biological sample with a kit according to the second aspect of the invention. The method of the invention can reduce the loss of beta-amyloid in biological samples and is used for in vitro research. In particular, the method can be used for scientific research and accurate detection of beta-amyloid in biological samples.
According to an embodiment of the present invention, the volume ratio of the composition to the biological sample is (1-2): (1-2). Thus, the loss of beta-amyloid in biological samples can be further reduced.
According to an embodiment of the invention, the β -amyloid is aβ40 and/or aβ42. The method of the invention is beneficial to maintaining the stability of the Abeta 40and Abeta 42in the biological sample and is beneficial to accurately quantifying the amounts of the Abeta 40and Abeta 42in the biological sample.
According to an embodiment of the invention, the β -amyloid is aβ42. The inventive method is particularly advantageous for maintaining the stability of aβ42in biological samples.
According to an embodiment of the invention, the biological sample is a liquid sample containing β -amyloid protein.
In some specific embodiments, the liquid sample includes, but is not limited to: derived from mammalian serum or plasma or other bodily fluids or biological tissue disruption fluids.
According to an embodiment of the present invention, further comprising: and (5) preserving the contact treatment product at a low temperature. To maintain aβ protein stability in serum or plasma samples, it is a preferred option to maintain the biological sample in a low temperature environment.
According to an embodiment of the present invention, the preservation temperature of the low-temperature preservation is 0 to 6 ℃.
According to an embodiment of the present invention, the preservation time of the low-temperature preservation is 0.5 to 20 hours.
In order to maintain the stability of Abeta protein in serum or plasma samples, biological samples are stored in a refrigerator at 0-6 ℃, and the biological samples are convenient and easy to realize in a biochemical laboratory or a detection department. The method can realize the beneficial effects that the biological sample can be stored for 20 hours at the temperature of 0-6 ℃ and can still effectively detect the Abeta 40and the Abeta 42 and the content ratio of the Abeta 40 to the Abeta 42 is kept unchanged.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a MALDI-TOF mass spectrum of a serum sample 1 of example 2 of the present invention, wherein the serum sample 1 was added with an Abeta protein protectant before being stored at low temperature and stored at 4℃for 18 hours;
FIG. 2 is a MALDI-TOF mass spectrum of a serum control sample 1 of example 2 of the present invention, wherein the serum sample was stored at 4℃for 18 hours after 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto for detection;
FIG. 3 is a MALDI-TOF mass spectrum of a serum control sample 2 of example 2 of the present invention, wherein the serum sample was stored at 4℃for 18 hours after 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added, and Abeta protein protectant was added before detection;
FIG. 4 is a MALDI-TOF mass spectrum of the serum control sample 3 of example 2 of the present invention, wherein the serum sample was directly detected after 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added;
FIG. 5 is a MALDI-TOF mass spectrum of a serum control sample 4 of example 3 of the present invention, wherein the serum sample was stored at 4℃for 1 hour after 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto, and then detected;
FIG. 6 is a MALDI-TOF mass spectrum of serum sample 2 of example 3 of the present invention, wherein the serum sample was stored at 4℃for 1 hour after 500pg/mL of Abeta 40and 50pg/mL of Abeta 42, and an Abeta protein protecting agent was added before detection.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
As used herein, the term "beta-amyloid," equivalent to "amyloid beta-protein," equivalent to "Abeta protein," is an amyloid precursor protein (amyloid p recursor protein, APP) that is widely present in various tissues and is centrally expressed at neuronal synapses, and is produced by cleavage by beta-secretase and gamma-secretase, and is a class of polypeptides based on long peptides containing 40and 42 amino acids. Wherein the long peptide comprising 40 amino acids is also referred to as "A.beta.40" or "A.beta.1-40," the amino acid sequence of which is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 1); a long peptide comprising 42 amino acids, also known as "Abeta 42" or "Abeta 1-42", the amino acid sequence of which is DAEFRHDSGYEVHHQKLVF FAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 2); quantitative studies of aβ40and aβ42in human plasma showed that: the ratio of Aβ40 to Aβ42 levels in healthy individuals is 10:1, and the ratio of both in Alzheimer's disease patients can be up to 15:1 (Shahpasand-Kroner H, klafki HW, bauer C, et al, atwo-step immunoassay for the simultaneous assessment of A. Beta. 38, Aβ40and Aβ42in human blood plasma supports the A. Beta. 42/Aβ 40ratio as a promising biomarker candidate of Alzheimer's disease.Alzheim ers Res Ther.2018Dec 8;10 (1): 121).
As used herein, the term "Abeta 38" is produced by cleavage of an amyloid precursor protein by beta-secretase and gamma-secretase, and is a long peptide comprising 38 amino acids, the amino acid sequence of which is DAEFRHDSGYEVHHQKLVFFAEDVGSNK GAIIGLMVGG (SEQ ID NO: 3).
Herein, the term "DDM" refers to n-dodecyl- β -D-maltoside.
Herein, the term "NTM" refers to n-nonyl- β -D-thiomaltoside.
Herein, the term "OTG" refers to n-octyl- β -D-thioglucoside.
Herein, the term "OG" refers to n-octyl- β -D-glucoside.
As used herein, the term "Tris-HCl" refers to Tris-hydroxymethyl aminomethane-HCl, tris (hydroxymethyl) aminomethane-hydrochloric acid.
As used herein, the term "MES" refers to 2-morpholinoethanesulfonic acid, 2-Morpholinoethanesulphonic acid.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
In this example, the components and amounts of the aβ protein protectant composition of the invention are as follows:
in this example, the above aβ protein protective agent was prepared according to the following method:
(1) 12.114g of Tris was weighed accurately, dissolved in ultrapure water, and the volume was adjusted to 1L to obtain a 0.1M Tris solution.
(2) The pH of the Tris solution of step (1) was adjusted to 7.4 using 1M HCl.
(3) Adding 2g of DDM and 2g of DDM into the solution obtained in the step (2), fully and uniformly mixing, adding a protease inhibitor Cocktail (manufacturer Roche, product number 05892791001) with a final concentration of 1X working concentration, adding the Cocktai tablet into the liquid according to the amount of 1 tablet/10 mL for dissolution by referring to the use instruction of the product, and obtaining the A beta protein protective agent.
Example 2
Before the serum sample is preserved at low temperature, the Abeta protein protective agent of the embodiment 1 is added, and then the serum sample is stored at 4 ℃ for 18 hours, and the protective effect of the Abeta protein protective agent on Abeta protein in the serum sample low temperature preservation process is further evaluated.
The blank serum in the examples of the present invention is a pooled serum of a plurality of healthy individuals, unless otherwise specified. The contents of Abeta 40and Abeta 42in the serum of the healthy human obtained clinically are very low, meanwhile, the Abeta 42in the serum of the human is hardly detected under the influence of factors such as sample preservation, transfer and the like, and the inventor skillfully adds the preset quantity of Abeta 40/Abeta 42in the serum of the healthy human so as to realize the investigation of the protection effect of the composition of the invention on Abeta protein in biological samples. Furthermore, the inventor mixes the serum of a plurality of healthy people and then adds the preset quantity of Abeta 40/Abeta 42, and evenly mixes the serum into a plurality of serum samples for sample processing and mass spectrum detection, thereby ensuring the comparability of experimental results.
In this example, serum samples were prepared and tested for aβ40and aβ42 as follows.
1.1 sample and preparation of beta-amyloid magnetic bead antibody
Serum sample 1 was prepared: the blank serum stored at-80℃was removed and thawed, 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added, the Abeta protein protectant of example 1 was added in equal volumes, and the mixture was thoroughly mixed. After the sample (hereinafter referred to as "serum sample 1") was prepared, it was stored at 4℃for 18 hours.
Serum control sample 1 was prepared: the blank serum stored at-80℃was removed and thawed, and 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto and mixed thoroughly. After the sample (hereinafter referred to as "serum control sample 1") was prepared, it was stored at 4℃for 18 hours. Before detection, the same volume of Tris-HCl buffer solution with pH of 7.4 is added and fully mixed.
Serum control sample 2 was prepared: the blank serum stored at-80℃was removed and thawed, and 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto and mixed thoroughly. After the sample (hereinafter referred to as "serum control sample 2") was prepared, it was stored at 4℃for 18 hours. The aβ protein protectant of example 1 was added in equal volumes prior to detection and thoroughly mixed.
Serum control sample 3 was prepared: the blank serum stored at-80℃was removed and thawed, and 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto and mixed thoroughly. After the sample (hereinafter referred to as "serum control sample 3") was prepared, mass spectrometry was immediately performed without storing it at a low temperature.
Beta-amyloid bead antibody preparation: the beta-amyloid antibody was added to a 30kDa ultrafiltration tube and washed 3 times with PBS buffer. PBS buffer Solution and Biotin Solution (Biotin Solution) were added and mixed by vortexing, and then reacted at room temperature for 30 minutes. After the reaction, PBS buffer solution was added to wash 3 times. All the biotinylated beta-amyloid antibodies are transferred into a magnetic bead suspension, and after vortex mixing, the mixture is turned over for 3 hours at room temperature to enable the biotinylated beta-amyloid antibodies to be fully bonded with the magnetic beads. After washing the beads 2 times with PBS buffer solution, the beads were resuspended for use.
1.2 sample processing
And after the serum sample and the serum control sample are restored to room temperature, 250 mu L of the serum sample and the serum control sample are taken into a 0.6mL centrifuge tube, 100pg of N15-Abeta 38 internal standard and 2 mu g of beta-amyloid magnetic bead antibody are added, the mixture is fully mixed, the mixture is turned over at room temperature and incubated for 30min, and after incubation is finished, the centrifuge tube is placed on a magnetic rack for magnetic separation for 1min.
The liquid in the 0.6mL centrifuge tube was discarded, 400. Mu.L PBS solution (containing 0.1% Tween 20) was added, vortexed for 30s, and then placed on a magnetic rack for magnetic separation for 1min. This operation was repeated 2 times.
After discarding the supernatant, 400. Mu.L of deionized water was added to a 0.6mL centrifuge tube and vortexed for 30s, and then placed on a magnetic rack for separation by magnetic force for 1min. After discarding the supernatant, 200. Mu.L of deionized water was added to the 0.6mL centrifuge tube, and the procedure was repeated 1 time.
The supernatant-discarded centrifuge tube was centrifuged for 30s, and then placed on a magnetic rack for magnetic separation for 30s, and a 10. Mu.L gun head was used to carefully suck out a trace amount of liquid at the bottom of the centrifuge tube. And accurately adding 10 mu L of eluent into the magnetic beads for eluting, immediately swirling for 30s to ensure that all the magnetic beads are soaked in the solution, then placing a 0.6mL centrifuge tube on a magnetic rack for magnetic separation for 30s, and obtaining supernatant, namely the treated sample to be tested.
The sample to be tested is taken as a mass spectrum detection sample in the embodiment 1.3, and is detected by a point target.
The eluent is prepared by the following steps: and dissolving trifluoroacetic acid in a 70% acetonitrile water solution according to the mass-volume ratio of 0.1%, thus obtaining the aqueous solution.
1.3 Mass Spectrometry detection
And (3) placing a hydrophobic target plate with the aperture of 2.4mm on an electric heating plate at 39 ℃ for preheating for 5min, pre-spotting a layer of matrix liquid, taking 2 mu L of sample spot targets to be detected after the matrix is crystallized, and carrying out data acquisition by using MALDI-TOF MS after all samples to be detected on the target plate are crystallized.
The matrix liquid is prepared by the following steps: alpha-Cyano-4-hydroxycinnamic acid (alpha-Cyano-4-hydroxycinnamic acid) was dissolved in 0.1% methyl diphosphonic acid (methyiphosphoranic) at a concentration of 9 mg/mL.
MALDI-TOF MS acquisition conditions were:
a laser: a semiconductor laser; laser frequency: 2000Hz; two-dimensional platform movement speed: 2mm/sec; focus Mass:4,000 Da; the acquisition mass range is as follows: 3kDa to 10kDa.
The spectrogram processing method comprises the following steps:
baseline correction: default mode; smoothing mass spectrogram: setting 9 windows by adopting a Moving-Average mode; SNR is 10; % Height to use 1; mass-to-charge ratio calibration: calibration was performed with m/z 4 330 (mass spectrum peak of aβ40) mass spectrum peak; the calibration mode is as follows: single point calibration; automatic internal calibration mass to charge ratio: m/z 4 182 (mass spectral peak of N15-A.beta.38 internal standard); automatic internal calibration limits: 1000in PPM.
MALDI-TOF mass spectrum of serum sample 1 effectively detects Abeta 40and Abeta 42 target peaks, the signal-to-noise ratio of Abeta 42 target peaks is clear, and specific mass spectrum detection results are shown in figure 1.
MALDI-TOF mass spectrum of serum control sample 1 does not detect Abeta 42 mesh peak, and specific mass spectrum detection results are shown in FIG. 2); MALDI-TOF mass spectrum of serum control sample 2 detects Abeta 42 mesh peak, but signal-to-noise ratio is low, mass spectrum detection result has no analysis meaning, and specific mass spectrum detection result is shown in figure 3.
MALDI-TOF mass spectrum of serum control sample 3 effectively detects Abeta 40and Abeta 42 target peaks, and Abeta 42 target peak has clear signal-to-noise ratio, and specific mass spectrum detection results are shown in figure 4.
The inventors further combed the test results of the above test samples, as shown in table 1.
TABLE 1 investigation of the Effect of the Aβ protein protectant of the invention on reducing Aβ protein loss in human serum samples
The above results show that:
1. the Abeta protein protective agent is added into the serum sample 1 before low-temperature preservation, abeta 40and Abeta 42 target peaks can be still effectively identified after preservation for 18 hours at 4 ℃, and the detection result can be further used for Abeta 40and Abeta 42 quantitative analysis. This is consistent with the detection of the aβ40and aβ42 target peaks for serum control sample 3 (positive control); in sharp contrast to the results of no detection of the Aβ40and Aβ42 mesh peaks of the serum control sample 1, to which the Aβprotein protectant of the invention was not added, both before low-temperature storage and before mass spectrometry.
2. The A beta protein protective agent can reduce the loss of A beta 40and A beta 42in the serum sample detection process to a certain extent. Before mass spectrum detection, serum control sample 2 of the Abeta protein protective agent is added, after the serum control sample is preserved for 18 hours at 4 ℃, abeta 40and Abeta 42 target peaks are detected, and the protective effect of the Abeta protein protective agent can be continuously shown until the sample detection is finished.
Therefore, the composition (namely the Abeta protein protective agent) can obviously reduce the loss of Abeta 40and Abeta 42in the preservation and detection process of serum or plasma samples, and can be further used for the low-temperature preservation and detection of clinical serum or plasma samples.
Protection effect evaluation test
Further, the inventors added the same amounts of aβ40and aβ42 (500 pg/mL of aβ40, 50pg/mL of aβ42) as in example 2 to a PBS solution, and immediately performed sample treatment and mass spectrometry detection, and compared the detection results with the detection results of example 2, to further evaluate the protective effect of the aβ protein protective agent of the present invention on maintaining the ratio of aβ40and aβ42 content in serum or plasma samples unchanged.
In this test, samples were prepared and aβ40and aβ42 thereof were detected as follows.
1.1 sample preparation
Aβ40/Aβ42 ratio control: the A.beta.40 and A.beta.42 protein mother solution was diluted with PBS solution to obtain 500pg/mL A.beta.40 and 50pg/mL A.beta.42, 250. Mu.L was taken as a control sample of the A.beta.40/A.beta.42 ratio in a 0.6mL centrifuge tube, and the sample was directly subjected to 1.2 steps without storage at 4 ℃.
1.2 sample processing
Adding an equal volume of Abeta protein protective agent into a 250 mu L Abeta 40/Abeta 42 ratio control sample, uniformly mixing, adding 100pg of N15-Abeta 38 internal standard and 2 mu g A beta 40 avidin magnetic bead antibody, fully mixing, turning at room temperature, incubating for 30min, and placing a centrifuge tube on a magnetic rack for magnetic separation for 1min after the incubation is finished.
Other sample processing steps are the same as in section 1.2 of example 2.
1.3 Mass Spectrometry detection
As in section 1.3 of this example.
The detection results of the ratio of Abeta 40 to Abeta 42in the serum sample of the example to the Abeta 40/Abeta 42 ratio control sample of the protection effect evaluation test are shown in Table 2.
TABLE 2 ratio of Abeta 40 to Abeta 42in control and serum samples (Abeta protein protectant added at 4 ℃ C. For 18 h)
The above test results show that the serum samples (detected after 18h storage at 4 ℃) to which the aβ protein protective agent of the present invention was added and the aβ40/aβ42 ratio control samples (detected immediately after the addition of the preset amounts of aβ40, aβ42) to which the aβ40 protective agent of the present invention was not added were substantially identical to the ratio of aβ42 to aβ40 or the ratio of aβ40 to aβ42.
The results show that the Abeta protein protective agent has good protective effect on Abeta protein in serum samples, and can effectively maintain the content ratio of Abeta 40 to Abeta 42 unchanged.
Furthermore, the Aβ protein protectant is added into biological samples, especially liquid biological samples such as serum or plasma of mammals, so that the stability of Aβ protein during sample storage can be obviously improved, the loss of β -amyloid can be effectively reduced, the detection difficulty of Aβ can be further reduced, the detection accuracy of the existing Aβ quantitative method can be improved, and references are provided for clinical diagnosis and treatment of Alzheimer's disease or other related diseases caused by the imbalance of Aβ protein expression.
Example 3
In this example, the inventors shortened the low temperature storage time of serum samples and added the aβ protein protectant of the invention prior to mass spectrometry of the samples to further investigate the effect of the aβ protein protectant of the invention on aβ40, aβ42 loss in serum or plasma samples during the detection process.
In this example, serum samples were prepared and tested for aβ40and aβ42 as follows.
1.1 sample preparation
Serum sample 2 preparation: the blank serum stored at-80℃was removed and thawed, and 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto and mixed thoroughly. After the sample (hereinafter referred to as "serum sample 2") was prepared, it was stored at 4℃for 1 hour. The aβ protein protectant of example 1 was added in equal volumes prior to detection and thoroughly mixed.
Serum control sample 4 was prepared: the blank serum stored at-80℃was removed and thawed, and 500pg/mL of Abeta 40and 50pg/mL of Abeta 42 were added thereto and mixed thoroughly. After the sample (hereinafter referred to as "serum control sample 4") was prepared, it was stored at 4℃for 1 hour. Before detection, the same volume of Tris-HCl buffer solution with pH of 7.4 is added and fully mixed.
1.2 sample processing
Reference is made to example 2.
1.3 Mass Spectrometry detection
Reference is made to example 2.
The MALDI-TOF mass spectrum detection results of the serum control sample 4 and the serum sample 2 are shown in FIG. 5 and FIG. 6.
The detection result shows that the Abeta protein protective agent can obviously improve the signal to noise ratio of Abeta 42in the detection process.
The results further demonstrate that the compositions of the invention (i.e., aβ protein protectants) are also beneficial for reducing the loss of aβ40, aβ42 during serum or plasma sample testing.
Thus, the composition of the invention can be further applied to the detection methods of Abeta 40and Abeta 42 to improve the accuracy, stability and operability of the detection methods.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A composition comprising:
buffer solution, nonionic surfactant, protease inhibitor;
the nonionic surfactant is selected from at least two of NTM, DDM, DM, OTG, OG, tween-20 and Span-20.
2. The composition of claim 1, wherein the nonionic surfactant is selected from the group consisting of NTM and DDM;
optionally, the mass volume percent (g/mL) of the NTM is 0.1-0.3% based on the total volume of the buffer;
optionally, the mass volume percent (g/mL) of the NTM is 0.15-0.3% based on the total volume of the buffer;
optionally, the mass volume percent (g/mL) of DDM is 0.1 to 0.3% based on the total volume of the buffer;
optionally, the mass volume percent (g/mL) of DDM is 0.15 to 0.3% based on the total volume of the buffer.
3. The composition of claim 1, wherein the buffer is selected from one of Tris-HCl buffer, MES buffer, PBS buffer, and HEPES buffer;
optionally, the pH of the buffer is 6.9-7.9;
optionally, the buffer has a pH of 7.2 to 7.6;
optionally, the buffer is Tris-HCl buffer;
optionally, the concentration of Tris in the Tris-HCl buffer is 0.05-0.2M.
4. The composition of claim 1, wherein the protease inhibitor is selected from at least one of serine protease inhibitors, trypsin inhibitors, aminopeptidase inhibitors, carboxypeptidase inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, and aspartic protease inhibitors;
optionally, the protease inhibitor is Cocktail.
5. The composition according to any one of claims 1 to 4, wherein the composition is free of sodium chloride and potassium chloride.
6. A kit comprising a composition according to any one of claims 1 to 5.
7. Use of the composition of any one of claims 1 to 5 or the kit of claim 6 for reducing β -amyloid loss in a biological sample;
optionally, the β -amyloid is aβ40 and/or aβ42;
optionally, the β -amyloid is aβ42;
optionally, the biological sample is a liquid sample;
optionally, the biological sample is a mammalian serum or plasma sample.
8. The use according to claim 7, wherein the biological sample is in a cryopreserved state;
optionally, the preservation temperature of the low-temperature preservation is 0-6 ℃;
optionally, the preservation time of the low-temperature preservation is 0.5-20 hours.
9. A method of reducing beta-amyloid loss in a biological sample comprising:
contacting said biological sample with the composition of any one of claims 1 to 5, or
Treating the biological sample with the kit of claim 6.
10. The method of claim 9, wherein the volume ratio of the composition to biological sample is (1-2): (1-2);
optionally, the β -amyloid is aβ40 and/or aβ42;
optionally, the β -amyloid is aβ42;
optionally, the biological sample is a liquid sample;
optionally, further comprising:
preserving the contact treatment product at low temperature;
optionally, the preservation temperature of the low-temperature preservation is 0-6 ℃;
optionally, the preservation time of the low-temperature preservation is 0.5-20 hours.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310692944.9A CN116908465A (en) | 2023-06-09 | 2023-06-09 | Composition for protecting beta-amyloid and application thereof |
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| CN202310692944.9A CN116908465A (en) | 2023-06-09 | 2023-06-09 | Composition for protecting beta-amyloid and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117700525A (en) * | 2024-02-05 | 2024-03-15 | 上海良润生物医药科技有限公司 | Polypeptide modified body and application thereof |
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- 2023-06-09 CN CN202310692944.9A patent/CN116908465A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117700525A (en) * | 2024-02-05 | 2024-03-15 | 上海良润生物医药科技有限公司 | Polypeptide modified body and application thereof |
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