WO2024087429A1 - Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia - Google Patents

Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia Download PDF

Info

Publication number
WO2024087429A1
WO2024087429A1 PCT/CN2023/078564 CN2023078564W WO2024087429A1 WO 2024087429 A1 WO2024087429 A1 WO 2024087429A1 CN 2023078564 W CN2023078564 W CN 2023078564W WO 2024087429 A1 WO2024087429 A1 WO 2024087429A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
amyloid precursor
fragments
antibody
urine
Prior art date
Application number
PCT/CN2023/078564
Other languages
French (fr)
Chinese (zh)
Inventor
戴正乾
顾柏俊
Original Assignee
湖南乾康科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 湖南乾康科技有限公司 filed Critical 湖南乾康科技有限公司
Priority to CN202380009304.1A priority Critical patent/CN116829956A/en
Priority to PCT/CN2023/078564 priority patent/WO2024087429A1/en
Publication of WO2024087429A1 publication Critical patent/WO2024087429A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • 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

Definitions

  • the present invention belongs to the field of biomedical technology, and in particular relates to the use of antibodies for detecting a protein biomarker group in preparing a kit for diagnosing AD, MCI and other types of Alzheimer's disease.
  • a ⁇ aggregation a hallmark feature of AD, occurs many years before the clinical manifestation of the disease. Although it is mainly synthesized in the brain, researchers have found that the peripheral system plays a vital role in clearing brain-derived A ⁇ . It is estimated that about 60% of brain A ⁇ is transported to the periphery for clearance.
  • Brain-derived A ⁇ is transported to the periphery by one or more of the following ways: crossing the blood-brain barrier and the blood-CSF barrier, interstitial fluid flow, and CSF exit pathways, including arachnoid villi and glial lymphatic pathways.
  • a ⁇ is cleared by monocytes, macrophages, neutrophils, lymphocytes, and hepatocytes through phagocytosis or endocytosis, and then excreted in urine or bile, and degraded by A ⁇ degrading enzymes and A ⁇ binding proteins in the blood.
  • Amyloid precursor protein is a single transmembrane protein expressed at high levels in the brain and can be rapidly metabolized by a variety of proteases, including ⁇ -, ⁇ -, ⁇ -, and ⁇ -secretases. Decomposition produces a large number of APP fragment products, including A ⁇ 40 and A ⁇ 42. Continuous proteolysis of APP to produce neurotoxic A ⁇ peptides, such as A ⁇ 42, is a key step in the development of AD. In patients with sporadic AD, especially in APOE ⁇ 4-negative individuals, we found that the degradation of APP is increased. However, except for A ⁇ 40 and A ⁇ 42, other APP fragments in urine have not been reported.
  • the object of the present invention is to
  • the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  • the diagnosis is performed by detecting the protein biomarker panel from a biological sample derived from a human being, and wherein the step of detecting the protein biomarker panel comprises performing an in vitro assay.
  • the biological sample is urine.
  • the antibody is an antibody that uses a sequence of an amyloid precursor protein fragment as an antigen.
  • the antibodies include A ⁇ antibodies and anti-Cystatin C antibodies.
  • the A ⁇ antibodies include mouse anti-human A ⁇ monoclonal antibodies, N-terminal monoclonal antibodies against amyloid precursor protein, and polyclonal antibodies against the C-terminus of amyloid precursor protein; specifically, the anti-A ⁇ antibodies include W0-2 clone antibody, aducanumab clone antibody, and N-terminal monoclonal antibodies against amyloid precursor protein (APP) (22C11 clone), and polyclonal antibodies against the C-terminus of amyloid precursor protein (APP) (Ab369).
  • the protein biomarker panel is detected by western blotting.
  • the present invention also aims to:
  • a protein biomarker group for diagnosing AD, MCI and other types of Alzheimer's disease is provided, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  • the diagnosis is performed by detecting the protein biomarker panel from a biological sample derived from a human being, and wherein the step of detecting the protein biomarker panel comprises performing an in vitro assay.
  • the biological sample is urine.
  • the present invention also aims to:
  • a method for diagnosing AD, MCI and other types of Alzheimer's disease comprises detecting a protein biomarker group in a biological sample, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  • the biological sample is human urine.
  • the method is to detect the protein biomarker group in the biological sample by using different antibodies through protein immunoblotting.
  • the present invention detects fragments of amyloid precursor protein in urine for early screening and auxiliary diagnosis of AD, MIC and other types of Alzheimer's disease; the fragments contain multiple amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  • the present invention investigated the presence of A ⁇ and APP fragments in urine and determined that APP fragments have high potential to serve as novel biomarkers for the early diagnosis of AD or other types of dementia.
  • the present invention uses Western blotting to detect amyloid precursor protein (APP) and A ⁇ in the urine of cognitively normal people and dementia patients (Alzheimer's disease (AD) or frontotemporal dementia (FTD)) using different antibodies.
  • Multiple APP fragments were identified, including ⁇ 14KD, ⁇ 28KD, ⁇ 56KD and ⁇ 68KD protein bands.
  • these APP fragments were found to be significantly increased in the urine of AD or FTD patients.
  • Subsequent immunoprecipitation and mass spectrometry confirmed the presence of APP fragments. Therefore, quantitative or qualitative detection of urine APP fragments can be used for early diagnosis of AD or other types of dementia patients.
  • Figure 1 Western blotting for A ⁇ .
  • Synthetic A ⁇ 42 was dissolved in a minimal amount of DMSO and then added to fresh urine collected from healthy young donors. The sample was concentrated using the TCA/Acton method.
  • Figure 2 Western blotting for A ⁇ .
  • a ⁇ monomers were solubilized in F-12 medium and allowed to aggregate at 4°C for at least 24 hours. Oligomers were diluted with PBS or urine collected from healthy young donors. Samples were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes. The immunoblots were boiled for 5 min and probed with anti-A ⁇ 42 mAb (clone W0-2, 0.2 ⁇ g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL).
  • Figure 3 Western blotting for A ⁇ and APP.
  • Synthetic A ⁇ 40 (8-250 ng) and three APP/PS1 mouse brain tissue extracts (50 ⁇ g protein each, animal number #L53, L29 and L03) were separated using 4-20% Tricine gel and transferred to nitrocellulose membrane.
  • the immunoblot was boiled for 5 min and probed with anti-A ⁇ 42 monoclonal antibody (clone W0-2, 1 ⁇ g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL).
  • Figure 4 Western blotting to detect APP fragments in urine.
  • the following donors were concentrated by freeze drying (upper panel) or TAC/Acton method (lower panel): urine from A ⁇ PET-negative healthy controls (HC), A ⁇ PET-positive Alzheimer's disease (AD) or frontotemporal dementia (FTD) patients was frozen and dried.
  • the samples (2 ⁇ g protein each) were separated by 4-20% Tricine gel and transferred to nitrocellulose membrane after separation.
  • the immunoblot was boiled for 5 min and then stained with Detection was performed with anti-A ⁇ 42 monoclonal antibody (clone W0-2, 0.2 ⁇ g/mL) and anti-mouse IgG secondary antibody (0.02 ⁇ g/mL).
  • FIG. 5 Western blotting to detect APP fragments in urine.
  • Urine from A ⁇ PET-negative healthy controls (HC), A ⁇ PET-positive Alzheimer's disease (AD), frontotemporal dementia (FTD) patients and other donors was collected and frozen and concentrated using the freeze-drying concentration method (upper figure, 10-fold concentration, 30 ⁇ L) or the TAC/Acton method (lower figure, 2 ⁇ g protein each).
  • the samples were separated with 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation.
  • the immunoblot was boiled for 5 minutes and detected with anti-A ⁇ 42 monoclonal antibody (Aducanumab, 0.1 ⁇ g/mL) and enzyme-labeled anti-human IgG secondary antibody (0.02 ⁇ g/mL).
  • FIG. 6 Western blotting to detect APP fragments in urine.
  • TAC/Acton method was used to freeze-concentrate urine from A ⁇ PET-negative healthy controls (HC), A ⁇ PET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients.
  • Samples (2 ⁇ g protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation.
  • the immunoblot was boiled for 5 min, reacted with anti-A ⁇ 42 monoclonal antibody (produced by Bio-east, 0.2 ⁇ g/mL), and then detected with enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL) primers.
  • FIG. 7 Western blotting to detect APP fragments in urine.
  • TAC/Acton method was used to freeze-concentrate urine from A ⁇ PET-negative healthy controls (HC), A ⁇ PET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients.
  • Samples (2 ⁇ g protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation.
  • the immunoblot was boiled for 5 min, reacted with anti-APP-C-terminal polyclonal antibody (Ab369, Sigma-Aldrich, 0.1 ⁇ g/mL), and then detected with enzyme-labeled anti-rabbit secondary antibody (0.02 ⁇ g/mL) primers.
  • FIG. 8 Western blotting for detecting APP fragments in urine.
  • TAC/Acton method was used to freeze-concentrate urine from A ⁇ PET-negative healthy controls (HC), A ⁇ PET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients.
  • Samples (2 ⁇ g protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation.
  • the immunoblots were boiled for 5 min, reacted with anti-APP-N-terminal antibody (clone 22C11, Sigma-Aldrich, 0.1 ⁇ g/mL), and then detected with enzyme-labeled anti-mouse IgG secondary Ab (0.02 ⁇ g/mL) primers.
  • FIG. 9 Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 ⁇ g of protein was separated using 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation. The immunoblot was boiled for 5 minutes and initiated with anti-A ⁇ 42 monoclonal antibody (clone W0-2, 0.2 ⁇ g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL). Aggregated A ⁇ 42 oligomers (5.7 pg), APP/PS1 mouse brain extract (2 ⁇ g) and synthetic A ⁇ 42 (0.5 and 1.0 ng) were used as controls. The right side shows the Western blotting image of the control group with a shorter exposure time. Symbols represent: ⁇ : CN; ⁇ : AD; ⁇ : MCI or memory impairment. Open: A ⁇ PET negative; Solid: A ⁇ PET positive.
  • FIG. 10 Western blotting for APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 ⁇ g of protein was separated using 4-20% Tricine gel and transferred to nitrocellulose membrane. The immunoblot was boiled for 5 min and detected with anti-A ⁇ 42 monoclonal antibody (Aducanumab, 40 ng/mL) and enzyme-labeled anti-human IgG secondary antibody (0.02 ⁇ g/mL). Synthetic A ⁇ 42 (0.1 and 0.2 ⁇ g), APP/PS1 mouse brain extract (2 ⁇ g) and aggregated A ⁇ 42 oligomers 5.7 pg were used as controls. Symbols represent: ⁇ : CN; ⁇ : AD; ⁇ : MCI or memory impairment. Open: A ⁇ PET negative; Solid: A ⁇ PET positive.
  • Figure 11 Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 ⁇ g of protein was separated using 4-20% Tricine gel and transferred to nitrocellulose membrane after separation. The immunoblot was boiled for 5min, reacted with anti-APP N-terminal monoclonal antibody (22C11, 0.1 ⁇ g/mL), and then detected with enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL). Synthetic A ⁇ 42 (0.1 and 0.2 ⁇ g), APP/PS1 mouse brain extract (2 ⁇ g) and aggregated A ⁇ 42 oligomers (5.7pg) were used as controls. Symbols represent: ⁇ : CN; ⁇ : AD; ⁇ : MCI or memory impairment. Open: A ⁇ PET negative; solid: A ⁇ PET positive.
  • Figure 12 Western blotting for APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 ⁇ g of protein was separated with 4-20% Tricine gel and transferred to nitrocellulose membrane. The immunoblot was boiled for 5 min, reacted with rabbit anti-APP c-terminal antibody (Ab369, 0.1 ⁇ g/mL), and then detected with enzyme-labeled anti-rabbit IgG secondary antibody (0.02 ⁇ g/mL). Aggregated A ⁇ 42 oligomers (5.7 pg), APP/PS1 mouse brain extract (2 ⁇ g), and synthetic A ⁇ 42 (0.5 and 1.0 ng) were used as controls. The right side shows the Western blotting image of the control group with a shorter exposure time. Symbols represent: ⁇ : CN; ⁇ : AD; ⁇ : MCI or memory impairment. Open: A ⁇ PET negative; Real: A ⁇ PET positive.
  • Figure 13 Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 ⁇ g of protein was separated using 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation. The immunoblot was boiled for 5 minutes and detected with anti-cystatin C monoclonal antibody (0.1 ⁇ g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 ⁇ g/mL). Synthetic A ⁇ 42 (0.1 and 0.2 ⁇ g), APP/PS1 mouse brain extract (50 ⁇ g) and aggregated A ⁇ 42 oligomers (0.1 ⁇ g) were used as controls. Symbols represent: ⁇ : CN; ⁇ : AD; ⁇ : MCI or memory impairment. Open: A ⁇ PET negative; Real: A ⁇ PET positive.
  • FIG. 14 SDS-PAGE gel results of urine from HC healthy subjects and AD patients after immunoprecipitation with W0-2 antibody.
  • the 6 lanes in W0-2 are duplicate lanes.
  • the gel was stained with Coomassie Blue R250 and photographed after destaining in destaining solution. Arrows indicate the approximate location of the cut gel lane.
  • the gel was cut horizontally on the lane. 20 ⁇ g of protein was loaded into each lane from each 20 ⁇ L sample.
  • MlgG mouse IgG control, 50 ⁇ g
  • W0-2 W0-2 antibody 20 ⁇ g
  • a ⁇ 42 synthetic A ⁇ 42, 20 ⁇ g
  • IP immunoprecipitation reaction.
  • Tris-Tricine gradient gel was prepared and stacked, and the samples were mixed with buffer and heated at 96°C for 5 min. Approximately 40 ⁇ L of the sample mixture and SeeBlue Plus (Thermo Fisher) prestained protein standards were added to the gel. The proteins were separated by electrophoresis and then transferred to a nitrocellulose membrane. The membrane was boiled in 10 mm phosphate buffered saline (PBS) for 5 min and blocked overnight with 5% skim milk powder in Tris buffered saline plus Tween-20 (TBST), pH (7.2).
  • PBS phosphate buffered saline
  • Tween-20 Tween-20
  • the blot was washed several times with TBST, and then TBST containing a primary antibody diluted 1:1000-1:25000 (pre-confirmed) and 5% skim milk powder was added, overnight at 4°C. After washing, the blot was incubated with a TBST containing a HRP-conjugated secondary antibody diluted 1:10 000-1:30 000 and 5% skim milk powder for 1-2 hours. After washing, the blot image was captured with a Gel Doc device using an ultrasensitive ECL reagent. First, Western blotting was used to detect synthetic A ⁇ with or without TCA/acetone treatment. The results showed that the treatment (TCA precipitation) and control (urine) did not affect the detection of A ⁇ ( Figure 1).
  • urine was collected from 5 cognitively normal healthy controls with negative A ⁇ PET scans (centiloid score ⁇ 15), 5 samples of clinically diagnosed AD with A ⁇ PET scan centiloid score >25, and 2 patients with frontotemporal dementia.
  • the urine was concentrated 10 times by freeze-drying or TCA precipitation.
  • Western blotting analysis was performed with A ⁇ antibody (clone W0-2). All samples had a ⁇ 28KD band, but AD and FTD patients showed stronger staining, while healthy controls had weaker staining. Another 14-16KD band was only visible in some AD and FTD patients. Different processes, such as freeze-drying or TAC/acetone precipitation, did not show significant differences (Figure 4).
  • the present invention used a different A ⁇ antibody, named aducanumab.
  • aducanumab In addition to the ⁇ 28KD and 14-16KD bands found with the W0-2 antibody, aducanumab also found two other high molecular weight bands, one at ⁇ 56KD and the other at ⁇ 68KD (Figure 5). These two bands were very obvious in AD and FTD patients, but were very weak in HC samples ( Figure 5). Similarly, there was no significant difference in the results of freeze-dried urine or TCA precipitated urine protein (Figure 5).
  • APP with a full length of about ⁇ 100KD was found in one HC, one AD and one FTD.
  • the ⁇ 68KD band was also visible in some AD patients ( Figure 8).
  • the above results confirmed the presence of APP in urine, and the APP content in AD and FTD patients was significantly higher than that in the HC group.
  • the protein concentration in each urine sample was determined by a BCA assay kit (Pierce TM , Thermo Fisher), and 250 ng of protein was separated with a 4-20% Tricine gel and transferred to a nitrocellulose membrane.
  • Urine samples were centrifuged at 14000G for 40 min at 4°C. The supernatant was taken and the pH was adjusted to 7.6 with 500 mM potassium borate buffer. Urine samples (1 mL) were mixed with 5 mL of colloidal gold coated with W0-2 antibody. The mixture was placed on a stirrer at 4°C overnight. Then centrifuged at 14000G, 4°C for 30min. The supernatant was discarded and washed three times with 1mL PBS (14000G, 4°C for 30min). After discarding the supernatant, 120 ⁇ L 1 ⁇ Tricine buffer was added and the precipitant was boiled at 95°C for 5min.
  • the protein concentration was determined by the BCA protein method, and 20 ⁇ g of protein was added to a 16.5% pre-prepared Tricine gel. The gel was run at 70mA for 45min. W-02 antibody (20 ⁇ g) and A ⁇ 42 monomer were added as controls in different channels. After electrophoresis, the gel was stained with Coomassie Blue R250 solution (0.1% Coomassie Blue R-250, 40% ethanol and 10% acetic acid) for 3 hours and then destained with destaining solution (10% ethanol and 7.5% acetic acid).
  • the unique bands ( ⁇ 4, 7, 14, 28, 56 and 68KD) of these AD sample urine were segmented and sequenced by mass spectrometry (MS).
  • MS mass spectrometry
  • the sequence was used to perform Protein Blast detection on UniProtKB and Swiss-Prot databases, with a 100% APP positive rate and an E value of 1 ⁇ 10 -6 .
  • the sequence "LVFFAEDVGSNK” identified by mass spectrometry (MS) is located at residues 688 to 699 of APP, and after processing, it is part of A ⁇ 40 and A ⁇ 42.
  • a sequence different from APP in the WYFDVTEGK test was found in the ⁇ 68kD protein band.
  • Protein Blast detection was performed on UniProtKB and Swiss-Prot databases using WYFDVTEGK, and the positive rate of APP was 100% and the E value was 1 ⁇ 10 -5 .
  • the sequence identified by mass spectrometry was located at residues 307 to 315 of APP, and these positions were the soluble part of APP after processing.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Electrochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Use of an antibody for detecting a protein biomarker group in the preparation of a kit for diagnosing Alzheimer's disease (AD), mild cognitive impairment (MCI) and other types of senile dementia. The protein biomarker group comprises a plurality of amyloid precursor protein (APP) fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are as shown in SEQ ID NO.1 to SEQ ID NO.7.

Description

用于检测蛋白生物标志物组的抗体在制备诊断AD、MCI和其它类型老年痴呆的试剂盒中的用途Use of antibodies for detecting protein biomarker groups in the preparation of kits for diagnosing AD, MCI and other types of Alzheimer's disease 技术领域Technical Field
本发明属于生物医学技术领域，具体涉及用于检测蛋白生物标志物组的抗体在制备诊断AD、MCI和其它类型老年痴呆的试剂盒中的用途。The present invention belongs to the field of biomedical technology, and in particular relates to the use of antibodies for detecting a protein biomarker group in preparing a kit for diagnosing AD, MCI and other types of Alzheimer's disease.
背景技术Background technique
由于全球范围内阿尔茨海默病病例数量的迅速增加，如今迫切需要找到能够在阿尔茨海默病早期阶段充分检测到的新型生物标志物来进行疾病的早期干预。Aβ聚集，是AD的标志特征，早于疾病临床表现之前多年出现。尽管其主要在大脑中合成，但研究人员发现外周系统在清除脑源性Aβ方面起着至关重要的作用。据估计，约60％的脑Aβ转运到外周被清除。脑源性Aβ通过以下一种或多种方式运输到外周:穿过血脑屏障和血-CSF屏障、间质液流动和CSF出口通路，包括蛛网膜绒毛和胶质淋巴通路。而在外周，Aβ被单核细胞、巨噬细胞、中性粒细胞、淋巴细胞和肝细胞等通过吞噬或内吞作用清除，之后经尿液或胆汁排出，通过Aβ降解酶以及血液中的Aβ结合蛋白降解。由于尿液排泄被认为是人体清除废物和不需要的代谢物的最重要的方式，其可溶性Aβ1-40(Aβ40)和Aβ1-42(Aβ42)是尿液的正常成分并在阿尔茨海默病患者中发生改变也就不足为奇了。然而，关于尿液Aβ的研究仍处于长期被忽视的状态。Due to the rapid increase in the number of cases of Alzheimer's disease worldwide, there is an urgent need to find new biomarkers that can be fully detected in the early stages of Alzheimer's disease for early intervention of the disease. Aβ aggregation, a hallmark feature of AD, occurs many years before the clinical manifestation of the disease. Although it is mainly synthesized in the brain, researchers have found that the peripheral system plays a vital role in clearing brain-derived Aβ. It is estimated that about 60% of brain Aβ is transported to the periphery for clearance. Brain-derived Aβ is transported to the periphery by one or more of the following ways: crossing the blood-brain barrier and the blood-CSF barrier, interstitial fluid flow, and CSF exit pathways, including arachnoid villi and glial lymphatic pathways. In the periphery, Aβ is cleared by monocytes, macrophages, neutrophils, lymphocytes, and hepatocytes through phagocytosis or endocytosis, and then excreted in urine or bile, and degraded by Aβ degrading enzymes and Aβ binding proteins in the blood. Since urinary excretion is considered the most important way for the body to eliminate waste and unwanted metabolites, it is not surprising that soluble Aβ 1-40 (Aβ40) and Aβ 1-42 (Aβ42) are normal components of urine and are altered in patients with Alzheimer's disease. However, the study of urinary Aβ has been neglected for a long time.
淀粉样前体蛋白(APP)是一种在脑内高水平表达的单一跨膜蛋白，可被多种蛋白酶快速代谢分解，包括α-、β-、γ-和η-分泌酶。分解产生大量的APP碎片产物，包括Aβ40和Aβ42。APP连续蛋白水解产生神经毒性Aβ肽，如Aβ42，是AD发生的关键步骤。在散发性AD患者尤其是在APOEε4阴性个体中我们发现APP的分解处理在增加。但除Aβ40、Aβ42外，尿中其他APP片段未见报道。Amyloid precursor protein (APP) is a single transmembrane protein expressed at high levels in the brain and can be rapidly metabolized by a variety of proteases, including α-, β-, γ-, and η-secretases. Decomposition produces a large number of APP fragment products, including Aβ40 and Aβ42. Continuous proteolysis of APP to produce neurotoxic Aβ peptides, such as Aβ42, is a key step in the development of AD. In patients with sporadic AD, especially in APOEε4-negative individuals, we found that the degradation of APP is increased. However, except for Aβ40 and Aβ42, other APP fragments in urine have not been reported.
发明内容Summary of the invention
本发明的目的在于，The object of the present invention is to
提供用于检测蛋白生物标志物组的抗体在制备诊断AD、MCI和其它类型老年痴呆的试剂盒中的用途，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。 Provided is the use of antibodies for detecting a protein biomarker group in preparing a kit for diagnosing AD, MCI and other types of Alzheimer's disease, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
优选地，所述诊断是从来源于人类的生物样本中检测所述蛋白生物标志物组，并且其中检测所述蛋白生物标志物组的步骤包括进行生物体外检测。Preferably, the diagnosis is performed by detecting the protein biomarker panel from a biological sample derived from a human being, and wherein the step of detecting the protein biomarker panel comprises performing an in vitro assay.
优选地，所述生物样本为尿液。Preferably, the biological sample is urine.
优选地，所述抗体是以淀粉样前体蛋白片段的其中一段序列为抗原的抗体。Preferably, the antibody is an antibody that uses a sequence of an amyloid precursor protein fragment as an antigen.
更优选地，所述抗体包括Aβ抗体和抗Cystatin C抗体体。More preferably, the antibodies include Aβ antibodies and anti-Cystatin C antibodies.
更优选地，所述Aβ抗体包括针对鼠抗人Aβ单克隆抗体、针对淀粉样前体蛋白的N端单抗和针对淀粉样前体蛋白的C端的多克隆抗体；具体而言，所述抗Aβ抗体包括W0-2克隆抗体、aducanumab克隆抗体以及针对淀粉样前体蛋白(APP)N端单抗抗体(22C11克隆)、针对淀粉样前体蛋白(APP)C端的多克隆抗体(Ab369)。More preferably, the Aβ antibodies include mouse anti-human Aβ monoclonal antibodies, N-terminal monoclonal antibodies against amyloid precursor protein, and polyclonal antibodies against the C-terminus of amyloid precursor protein; specifically, the anti-Aβ antibodies include W0-2 clone antibody, aducanumab clone antibody, and N-terminal monoclonal antibodies against amyloid precursor protein (APP) (22C11 clone), and polyclonal antibodies against the C-terminus of amyloid precursor protein (APP) (Ab369).
优选地，所述蛋白生物标志物组通过蛋白免疫印迹法检测。Preferably, the protein biomarker panel is detected by western blotting.
本发明的目的还在于，The present invention also aims to:
提供一种用于诊断AD、MCI和其它类型老年痴呆的蛋白生物标志物组，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。A protein biomarker group for diagnosing AD, MCI and other types of Alzheimer's disease is provided, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
优选地，所述诊断是从来源于人类的生物样本中检测所述蛋白生物标志物组，并且其中检测所述蛋白生物标志物组的步骤包括进行生物体外检测。Preferably, the diagnosis is performed by detecting the protein biomarker panel from a biological sample derived from a human being, and wherein the step of detecting the protein biomarker panel comprises performing an in vitro assay.
优选地，所述生物样本为尿液。Preferably, the biological sample is urine.
本发明的目的还在于，The present invention also aims to:
提供一种诊断AD、MCI和其它类型老年痴呆的方法，所述方法是检测生物样本中的蛋白生物标志物组，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。A method for diagnosing AD, MCI and other types of Alzheimer's disease is provided, wherein the method comprises detecting a protein biomarker group in a biological sample, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
优选地，所述生物样本是人类尿液。Preferably, the biological sample is human urine.
优选地，所述方法是通过蛋白免疫印迹法，用不同抗体检测生物样本中的蛋白生物标志物组。Preferably, the method is to detect the protein biomarker group in the biological sample by using different antibodies through protein immunoblotting.
本发明通过检测尿液中的淀粉样前体蛋白的片段来早期筛查和辅助诊断AD、MIC和其它类型老年痴呆；所述片段包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。The present invention detects fragments of amyloid precursor protein in urine for early screening and auxiliary diagnosis of AD, MIC and other types of Alzheimer's disease; the fragments contain multiple amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
在寻找基于尿液的AD生物标志物的过程中，本发明研究了尿液中Aβ和APP片段的存在，并确定APP片段具有很高的潜力使其可以作为早期诊断AD或其他类型痴呆的新型生物标志物。 In the search for urine-based AD biomarkers, the present invention investigated the presence of Aβ and APP fragments in urine and determined that APP fragments have high potential to serve as novel biomarkers for the early diagnosis of AD or other types of dementia.
总之，本发明运用蛋白免疫印迹法(Western blotting)，针对认知正常人群和痴呆患者(阿尔茨海默病(AD)或额颞叶痴呆患者(FTD))尿液中的β淀粉样蛋白，用不同抗体对其中淀粉样前体蛋白(APP)和Aβ进行检测。由此鉴定出多个APP片段，包括～14KD、～28KD、～56KD和～68KD蛋白条带。与年龄匹配的认知正常对照组相比，AD或FTD患者的尿液中发现明显升高的这些APP片段。后续的免疫沉淀和质谱法进一步分析证实了APP片段的存在。因此，定量或定性检测尿液APP片段可用于进行对AD或其他类型的痴呆患者的早期诊断。In summary, the present invention uses Western blotting to detect amyloid precursor protein (APP) and Aβ in the urine of cognitively normal people and dementia patients (Alzheimer's disease (AD) or frontotemporal dementia (FTD)) using different antibodies. Multiple APP fragments were identified, including ~14KD, ~28KD, ~56KD and ~68KD protein bands. Compared with the age-matched cognitively normal control group, these APP fragments were found to be significantly increased in the urine of AD or FTD patients. Subsequent immunoprecipitation and mass spectrometry further analysis confirmed the presence of APP fragments. Therefore, quantitative or qualitative detection of urine APP fragments can be used for early diagnosis of AD or other types of dementia patients.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1:蛋白免疫印迹(Western blotting)检测Aβ。图的上部分：将合成Aβ42溶解在最小量的DMSO中，然后添加到从健康年轻供体收集的新鲜尿液中。样品采用TCA/Acton法进行浓缩。图的下部分：Aβ42溶于DMSO中，并用PBS稀释。用4-20％的Tricine凝胶分离Aβ42，转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗Aβ42单抗(克隆W0-2,1μg/mL)和酶标抗小鼠IgG次级抗体(0.05μg/mL)检测。由此可发现Aβ42的单体、二聚体和四聚体。Figure 1: Western blotting for Aβ. Upper part of the figure: Synthetic Aβ42 was dissolved in a minimal amount of DMSO and then added to fresh urine collected from healthy young donors. The sample was concentrated using the TCA/Acton method. Lower part of the figure: Aβ42 was dissolved in DMSO and diluted with PBS. Aβ42 was separated using 4-20% Tricine gel and transferred to a nitrocellulose membrane. The immunoblot was boiled for 5 min and probed with anti-Aβ42 mAb (clone W0-2, 1 μg/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.05 μg/mL). Monomers, dimers, and tetramers of Aβ42 were detected.
图2:蛋白免疫印迹(Western blotting)检测Aβ。将Aβ单体在F-12介质中超声溶解，在4℃中放置至少24小时形成聚集。用PBS或从健康年轻供体收集的尿液来稀释低聚物。样品用4-20％Tricine凝胶来进行分离，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗Aβ42单抗(克隆W0-2,0.2μg/mL)和酶标抗小鼠IgG次级抗体(0.02μg/mL)检测。Figure 2: Western blotting for Aβ. Aβ monomers were solubilized in F-12 medium and allowed to aggregate at 4°C for at least 24 hours. Oligomers were diluted with PBS or urine collected from healthy young donors. Samples were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes. The immunoblots were boiled for 5 min and probed with anti-Aβ42 mAb (clone W0-2, 0.2 μg/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 μg/mL).
图3:蛋白免疫印迹(Western blotting)检测Aβ和APP。用4-20％Tricine凝胶分离合成Aβ40(8-250ng)和3个APP/PS1小鼠脑组织提取物(各50μg蛋白量，动物编号#L53,L29和L03)，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗Aβ42单抗(克隆W0-2,1μg/mL)和酶标抗小鼠IgG次级抗体(0.02μg/mL)检测。Figure 3: Western blotting for Aβ and APP. Synthetic Aβ40 (8-250 ng) and three APP/PS1 mouse brain tissue extracts (50 μg protein each, animal number #L53, L29 and L03) were separated using 4-20% Tricine gel and transferred to nitrocellulose membrane. The immunoblot was boiled for 5 min and probed with anti-Aβ42 monoclonal antibody (clone W0-2, 1 μg/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 μg/mL).
图4:蛋白免疫印迹(Western blotting)检测尿液中APP片段。采用冷冻干燥法(上图)或TAC/Acton法(下图)浓缩以下供体：AβPET阴性的健康对照(HC)、AβPET阳性的阿尔茨海默病(AD)或额颞叶痴呆(FTD)患者人群的尿液进行冷冻和干燥。用4-20％Tricine凝胶分离样品(每个2μg蛋白量)，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用 抗Aβ42单抗(克隆W0-2,0.2μg/mL)和抗小鼠IgG次级抗体(0.02μg/mL)检测。Figure 4: Western blotting to detect APP fragments in urine. The following donors were concentrated by freeze drying (upper panel) or TAC/Acton method (lower panel): urine from AβPET-negative healthy controls (HC), AβPET-positive Alzheimer's disease (AD) or frontotemporal dementia (FTD) patients was frozen and dried. The samples (2 μg protein each) were separated by 4-20% Tricine gel and transferred to nitrocellulose membrane after separation. The immunoblot was boiled for 5 min and then stained with Detection was performed with anti-Aβ42 monoclonal antibody (clone W0-2, 0.2 μg/mL) and anti-mouse IgG secondary antibody (0.02 μg/mL).
图5:蛋白免疫印迹(Western blotting)检测尿液中APP片段。收集AβPET阴性的健康对照(HC)、AβPET阳性的老年痴呆症(AD)、额颞叶痴呆(FTD)患者等供体的尿液，采用冻干浓缩法(上图、10倍浓缩、30μL)或TAC/Acton法(下图、各2μg蛋白量)对尿液进行冷冻浓缩。样品用4-20％Tricine凝胶分离，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗Aβ42单抗(Aducanumab,0.1μg/mL)和酶标抗人IgG次级抗体(0.02μg/mL)检测。Figure 5: Western blotting to detect APP fragments in urine. Urine from AβPET-negative healthy controls (HC), AβPET-positive Alzheimer's disease (AD), frontotemporal dementia (FTD) patients and other donors was collected and frozen and concentrated using the freeze-drying concentration method (upper figure, 10-fold concentration, 30 μL) or the TAC/Acton method (lower figure, 2 μg protein each). The samples were separated with 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation. The immunoblot was boiled for 5 minutes and detected with anti-Aβ42 monoclonal antibody (Aducanumab, 0.1 μg/mL) and enzyme-labeled anti-human IgG secondary antibody (0.02 μg/mL).
图6:蛋白免疫印迹(Western blotting)检测尿液中APP片段。采用TAC/Acton法对AβPET阴性的健康对照(HC)、AβPET阳性的阿尔茨海默病(AD)和额颞叶痴呆(FTD)患者等供体的尿液进行冷冻浓缩。用4-20％Tricine凝胶分离样品(每个2μg蛋白量)，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗Aβ42单抗(产自Bio-east,0.2μg/mL)反应，再用酶标抗小鼠IgG次级抗体(0.02μg/mL)引物检测。Figure 6: Western blotting to detect APP fragments in urine. TAC/Acton method was used to freeze-concentrate urine from AβPET-negative healthy controls (HC), AβPET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients. Samples (2 μg protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation. The immunoblot was boiled for 5 min, reacted with anti-Aβ42 monoclonal antibody (produced by Bio-east, 0.2 μg/mL), and then detected with enzyme-labeled anti-mouse IgG secondary antibody (0.02 μg/mL) primers.
图7:蛋白免疫印迹(Western blotting)检测尿液中APP片段。采用TAC/Acton法对AβPET阴性的健康对照(HC)、AβPET阳性的阿尔茨海默病(AD)和额颞叶痴呆(FTD)患者等供体的尿液进行冷冻浓缩。用4-20％Tricine凝胶分离样品(每个2μg蛋白量)，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗APP-C端多克隆抗体(Ab369,Sigma-Aldrich,0.1μg/mL)反应，再用酶标抗兔二级抗体(0.02μg/mL)引物检测。Figure 7: Western blotting to detect APP fragments in urine. TAC/Acton method was used to freeze-concentrate urine from AβPET-negative healthy controls (HC), AβPET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients. Samples (2 μg protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation. The immunoblot was boiled for 5 min, reacted with anti-APP-C-terminal polyclonal antibody (Ab369, Sigma-Aldrich, 0.1 μg/mL), and then detected with enzyme-labeled anti-rabbit secondary antibody (0.02 μg/mL) primers.
图8:蛋白免疫印迹(Western blotting)检测尿液中APP片段。采用TAC/Acton法对AβPET阴性的健康对照(HC)、AβPET阳性的阿尔茨海默病(AD)和额颞叶痴呆(FTD)患者等供体的尿液进行冷冻浓缩。用4-20％Tricine凝胶分离样品(每个2μg蛋白量)，分离完成后转移到硝化纤维膜上。将免疫印迹煮沸5min，用抗APP-N-端抗体(克隆22C11,Sigma-Aldrich,0.1μg/mL)反应，再用酶标抗小鼠IgG次级Ab(0.02μg/mL)引物检测。Figure 8: Western blotting for detecting APP fragments in urine. TAC/Acton method was used to freeze-concentrate urine from AβPET-negative healthy controls (HC), AβPET-positive Alzheimer's disease (AD) and frontotemporal dementia (FTD) patients. Samples (2 μg protein each) were separated using 4-20% Tricine gel and transferred to nitrocellulose membranes after separation. The immunoblots were boiled for 5 min, reacted with anti-APP-N-terminal antibody (clone 22C11, Sigma-Aldrich, 0.1 μg/mL), and then detected with enzyme-labeled anti-mouse IgG secondary Ab (0.02 μg/mL) primers.
图9:蛋白免疫印迹(Western blotting)检测尿液中APP片段。将从患有或没有AD的老年人收集的冷冻人尿进行解冻，用4-20％的Tricine凝胶分离每个含有0.25μg蛋白质的样品，分离完成后转移到硝化纤维膜上。免疫印迹煮沸5min，用抗Aβ42单抗(克隆W0-2,0.2μg/mL)和酶标抗小鼠IgG次级抗体(0.02μg/mL)启动。以聚合的Aβ42寡聚物(5.7pg)、 APP/PS1小鼠脑提取物(2μg)和合成Aβ42(0.5和1.0ng)互为对照。右侧显示了曝光时间较短的对照组的免疫蛋白印迹(Western blotting)图像。符号代表：○:CN；□:AD；△:MCI或记忆障碍者。开放:AβPET阴性；实心:AβPET阳性。Figure 9: Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 μg of protein was separated using 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation. The immunoblot was boiled for 5 minutes and initiated with anti-Aβ42 monoclonal antibody (clone W0-2, 0.2 μg/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 μg/mL). Aggregated Aβ42 oligomers (5.7 pg), APP/PS1 mouse brain extract (2 μg) and synthetic Aβ42 (0.5 and 1.0 ng) were used as controls. The right side shows the Western blotting image of the control group with a shorter exposure time. Symbols represent: ○: CN; □: AD; △: MCI or memory impairment. Open: AβPET negative; Solid: AβPET positive.
图10:蛋白免疫印迹(Western blotting)检测尿液中APP片段。将从患有或没有AD的老年人收集的冷冻人尿进行解冻，用4-20％的Tricine凝胶分离每个含有0.25μg蛋白质的样品，分离完成后转移到硝化纤维膜上。免疫印迹煮沸5min，用抗Aβ42单抗(Aducanumab,40ng/mL)，再用酶标抗人IgG次级抗体(0.02μg/mL)检测。以合成的Aβ42(0.1和0.2μg)、APP/PS1小鼠脑提取物(2μg)和聚集的Aβ42低聚物5.7pg为对照。符号代表：○:CN；□:AD；△:MCI或记忆障碍者。开放:AβPET阴性；实心:AβPET阳性。Figure 10: Western blotting for APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 μg of protein was separated using 4-20% Tricine gel and transferred to nitrocellulose membrane. The immunoblot was boiled for 5 min and detected with anti-Aβ42 monoclonal antibody (Aducanumab, 40 ng/mL) and enzyme-labeled anti-human IgG secondary antibody (0.02 μg/mL). Synthetic Aβ42 (0.1 and 0.2 μg), APP/PS1 mouse brain extract (2 μg) and aggregated Aβ42 oligomers 5.7 pg were used as controls. Symbols represent: ○: CN; □: AD; △: MCI or memory impairment. Open: AβPET negative; Solid: AβPET positive.
图11:蛋白免疫印迹(Western blotting)检测尿液中APP片段。将从患有或没有AD的老年人收集的冷冻人尿进行解冻，用4-20％的Tricine凝胶分离每个含有0.25μg蛋白质的样品，分离完成后转移到硝化纤维膜上。免疫印迹煮沸5min，用抗APP N端单抗(22C11,0.1μg/mL)反应，再用酶标抗小鼠IgG次级抗体(0.02μg/mL)检测。以合成的Aβ42(0.1和0.2μg)、APP/PS1小鼠脑提取物(2μg)和聚集的Aβ42低聚物(5.7pg)为对照。符号代表：○:CN；□:AD；△:MCI或记忆障碍者。开放:AβPET阴性；实心:AβPET阳性。Figure 11: Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25μg of protein was separated using 4-20% Tricine gel and transferred to nitrocellulose membrane after separation. The immunoblot was boiled for 5min, reacted with anti-APP N-terminal monoclonal antibody (22C11, 0.1μg/mL), and then detected with enzyme-labeled anti-mouse IgG secondary antibody (0.02μg/mL). Synthetic Aβ42 (0.1 and 0.2μg), APP/PS1 mouse brain extract (2μg) and aggregated Aβ42 oligomers (5.7pg) were used as controls. Symbols represent: ○: CN; □: AD; △: MCI or memory impairment. Open: AβPET negative; solid: AβPET positive.
图12:蛋白免疫印迹(Western blotting)检测尿液中APP片段。将从患有或没有AD的老年人收集的冷冻人尿进行解冻，用4-20％的Tricine凝胶分离每个含有0.25μg蛋白质的样品，分离完成后转移到硝化纤维膜上。免疫印迹煮沸5min，用兔抗APP c端抗体(Ab369,0.1μg/mL)反应，再用酶标抗兔IgG次级抗体(0.02μg/mL)检测。以聚合的Aβ42寡聚物(5.7pg)、APP/PS1小鼠脑提取物(2μg)和合成的Aβ42(0.5和1.0ng)为对照。右侧显示了曝光时间较短的对照组的Western blotting图像。符号代表：○:CN；□:AD；△:MCI或记忆障碍者。开放:AβPET阴性；实相:AβPET阳性。Figure 12: Western blotting for APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 μg of protein was separated with 4-20% Tricine gel and transferred to nitrocellulose membrane. The immunoblot was boiled for 5 min, reacted with rabbit anti-APP c-terminal antibody (Ab369, 0.1 μg/mL), and then detected with enzyme-labeled anti-rabbit IgG secondary antibody (0.02 μg/mL). Aggregated Aβ42 oligomers (5.7 pg), APP/PS1 mouse brain extract (2 μg), and synthetic Aβ42 (0.5 and 1.0 ng) were used as controls. The right side shows the Western blotting image of the control group with a shorter exposure time. Symbols represent: ○: CN; □: AD; △: MCI or memory impairment. Open: AβPET negative; Real: AβPET positive.
图13:蛋白免疫印迹(Western blotting)检测尿液中APP片段。将从患有或没有AD的老年人收集的冷冻人尿进行解冻，用4-20％的Tricine凝胶分离每个含有0.25μg蛋白质的样品，分离完成后转移到硝化纤维膜上。免疫印迹煮沸5min，用抗胱氨酸抑素C单抗(0.1μg/mL)和酶标抗小鼠IgG次级抗体(0.02μg/mL)检测。以合成的Aβ42(0.1和0.2μg)、 APP/PS1小鼠脑提取物(50μg)和聚集的Aβ42低聚物(0.1μg)为对照。符号代表：○:CN；□:AD；△:MCI或记忆障碍者。开放:AβPET阴性；实相:AβPET阳性。Figure 13: Western blotting to detect APP fragments in urine. Frozen human urine collected from elderly people with or without AD was thawed, and each sample containing 0.25 μg of protein was separated using 4-20% Tricine gel and transferred to a nitrocellulose membrane after separation. The immunoblot was boiled for 5 minutes and detected with anti-cystatin C monoclonal antibody (0.1 μg/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02 μg/mL). Synthetic Aβ42 (0.1 and 0.2 μg), APP/PS1 mouse brain extract (50 μg) and aggregated Aβ42 oligomers (0.1 μg) were used as controls. Symbols represent: ○: CN; □: AD; △: MCI or memory impairment. Open: Aβ PET negative; Real: Aβ PET positive.
图14。HC健康人群和AD患者尿液经W0-2抗体免疫沉淀后的SDS-PAGE凝胶结果。W0-2内的6个跑带为重复跑带。凝胶用考马斯蓝R250染色，在脱色液中脱色后拍照。箭头表示切割凝胶跑带的大致位置。凝胶在跑带上水平切割。20μL样品中各取20μg蛋白装入每个通道。MlgG＝小鼠lgG对照，50μg；W0-2＝W0-2抗体20μg；Aβ42:合成Aβ42,20μg；IP＝免疫沉淀反应。Figure 14. SDS-PAGE gel results of urine from HC healthy subjects and AD patients after immunoprecipitation with W0-2 antibody. The 6 lanes in W0-2 are duplicate lanes. The gel was stained with Coomassie Blue R250 and photographed after destaining in destaining solution. Arrows indicate the approximate location of the cut gel lane. The gel was cut horizontally on the lane. 20μg of protein was loaded into each lane from each 20μL sample. MlgG = mouse IgG control, 50μg; W0-2 = W0-2 antibody 20μg; Aβ42: synthetic Aβ42, 20μg; IP = immunoprecipitation reaction.
具体实施方式Detailed ways
在免疫印迹法Western blotting实验中，尿液样本采用冻干法浓缩至1/10体积处理，或采用三氯乙酸(TCA)沉淀法处理。即，在1mL尿液中加入100μL 0.15％脱氧胆酸盐，在室温(RT)中放置10min。加入TCA(100μL)，将混合物在4℃下放置1小时，然后在4℃下以16000g离心力离心40min。丢弃上清液，加入500μL冷藏丙酮。样品在16000g离心力下离心40min，将干燥物溶解于50μL 2×SDS缓冲液中，用SDS-page进行分析。In Western blotting experiments, urine samples were concentrated to 1/10 volume by freeze drying or by trichloroacetic acid (TCA) precipitation. That is, 100 μL 0.15% deoxycholate was added to 1 mL of urine and placed at room temperature (RT) for 10 min. TCA (100 μL) was added, the mixture was placed at 4°C for 1 hour, and then centrifuged at 16000g for 40 min at 4°C. The supernatant was discarded and 500 μL refrigerated acetone was added. The sample was centrifuged at 16000g for 40 min, and the dried material was dissolved in 50 μL 2×SDS buffer and analyzed by SDS-page.
制备6-20％的Tris-Tricine梯度凝胶且堆叠，将样品与缓冲液混合，在96℃下加热5min。将约40μL的样品混合物以及SeeBlue Plus(赛默飞)预染色蛋白标准品加到凝胶中。蛋白质通过电泳分离，然后转移到硝化纤维膜上。在10mm磷酸盐缓冲盐水(PBS)中煮沸5min，用5％脱脂奶粉在Tris缓冲盐水加吐温-20(TBST)中封闭过夜，pH值(7.2)。用TBST清洗印迹几次，然后加含1:1000-1:25000(事先确认)稀释的一抗和5％脱脂奶粉的TBST，4℃过夜。清洗后，用含有1:10 000～1:30 000稀释HRP偶联二抗和5％脱脂奶粉的TBST培养1-2小时。清洗后，使用超灵敏ECL试剂，用Gel Doc设备捕获印迹图像。首先用免疫印迹Western blotting检测有或没有进行TCA/丙酮处理的合成Aβ。结果表明，结果处理(TCA沉淀)的和对照的(尿液)，均不影响Aβ的检测(图1)。而且，随着Aβ承载量增加(>30ng)，Aβ二聚体(～8KD)逐渐可见。进一步增加Aβ承载超过250ng，可观察到四聚体存在(～16KD)(图1)。随后检测了溶解在PBS或尿液中的聚集Aβ，在低蛋白量(20ng或更少)下，PBS或尿液中均未观察到聚集Aβ的明显差异，表明尿液不太可能影响Aβ聚集自然形成(图2)。为了确保系统能够检测APP，制备了3只APP/PS1转基因小鼠的脑提取物，并使用免疫印迹Western blotting进行检测。观察到多个含APP片段或低聚物的蛋白条带，其中APP总长(～100KD)最为突出，其次是可能的C端片段(CTF)99条带(～14 KD)(图3)。值得注意的是，尚未发现比四聚物更大的Aβ低聚物(图1-3)。A 6-20% Tris-Tricine gradient gel was prepared and stacked, and the samples were mixed with buffer and heated at 96°C for 5 min. Approximately 40 μL of the sample mixture and SeeBlue Plus (Thermo Fisher) prestained protein standards were added to the gel. The proteins were separated by electrophoresis and then transferred to a nitrocellulose membrane. The membrane was boiled in 10 mm phosphate buffered saline (PBS) for 5 min and blocked overnight with 5% skim milk powder in Tris buffered saline plus Tween-20 (TBST), pH (7.2). The blot was washed several times with TBST, and then TBST containing a primary antibody diluted 1:1000-1:25000 (pre-confirmed) and 5% skim milk powder was added, overnight at 4°C. After washing, the blot was incubated with a TBST containing a HRP-conjugated secondary antibody diluted 1:10 000-1:30 000 and 5% skim milk powder for 1-2 hours. After washing, the blot image was captured with a Gel Doc device using an ultrasensitive ECL reagent. First, Western blotting was used to detect synthetic Aβ with or without TCA/acetone treatment. The results showed that the treatment (TCA precipitation) and control (urine) did not affect the detection of Aβ (Figure 1). Moreover, as the Aβ loading increased (>30ng), Aβ dimers (~8KD) gradually became visible. Further increasing the Aβ loading to more than 250ng, tetramers were observed (~16KD) (Figure 1). Aggregated Aβ dissolved in PBS or urine was then detected. At low protein levels (20ng or less), no significant differences in aggregated Aβ were observed in either PBS or urine, indicating that urine is unlikely to affect the natural formation of Aβ aggregation (Figure 2). To ensure that the system could detect APP, brain extracts from three APP/PS1 transgenic mice were prepared and detected using Western blotting. Multiple protein bands containing APP fragments or oligomers were observed, of which the total length of APP (~100KD) was the most prominent, followed by the possible C-terminal fragment (CTF) 99 band (~14 KD) (Figure 3). It is noteworthy that Aβ oligomers larger than tetramers have not been found (Figures 1-3).
接下来，收集5例AβPET扫描阴性(centiloid评分<15)的认知正常健康对照，5例临床诊断为AD的样本，AβPET扫描centiloid评分>25,和2例额颞叶痴呆患者的尿液。尿液经冻干或TCA沉淀浓缩10次。用Aβ抗体进行Western blotting分析(克隆W0-2)。所有样本均有～28KD条带，但AD和FTD患者表现染色较强，而健康对照组染色较弱。另一条14-16KD带仅在部分AD和FTD患者中可见。不同的工艺，如冷冻干燥或TAC/丙酮沉淀，没有显著差异(图4)。Next, urine was collected from 5 cognitively normal healthy controls with negative Aβ PET scans (centiloid score <15), 5 samples of clinically diagnosed AD with Aβ PET scan centiloid score >25, and 2 patients with frontotemporal dementia. The urine was concentrated 10 times by freeze-drying or TCA precipitation. Western blotting analysis was performed with Aβ antibody (clone W0-2). All samples had a ~28KD band, but AD and FTD patients showed stronger staining, while healthy controls had weaker staining. Another 14-16KD band was only visible in some AD and FTD patients. Different processes, such as freeze-drying or TAC/acetone precipitation, did not show significant differences (Figure 4).
为了验证结果，本发明使用了一种不同的Aβ抗体，名为aducanumab。除了用W0-2抗体可发现的～28KD和14-16KD条带外，aducanumab还发现了另外两个高分子条带，一个在～56KD，另一个在～68KD(图5)。这两个条带在AD和FTD患者中很明显，但在HC样本中表现非常弱(图5)。同样，冻干尿或TCA沉淀尿蛋白的结果没有显著差异(图5)。To verify the results, the present invention used a different Aβ antibody, named aducanumab. In addition to the ~28KD and 14-16KD bands found with the W0-2 antibody, aducanumab also found two other high molecular weight bands, one at ~56KD and the other at ~68KD (Figure 5). These two bands were very obvious in AD and FTD patients, but were very weak in HC samples (Figure 5). Similarly, there was no significant difference in the results of freeze-dried urine or TCA precipitated urine protein (Figure 5).
使用另一种Aβ抗体来进一步验证这一发现。对相同的样品进行免疫印迹Western blotting检测，并使用从Bioeast Biotech(博岳生物，杭州，中国)购买的Aβ抗体以及我公司自行研发的QK-02进行检测。检测结果与W0-2结果相似(图6)。Another Aβ antibody was used to further validate this finding. The same samples were subjected to Western blotting and tested with Aβ antibodies purchased from Bioeast Biotech (Boyue Biotech, Hangzhou, China) and QK-02 developed by our company. The results were similar to those of W0-2 (Figure 6).
使用兔抗APP蛋白C端多克隆抗体，我们在AD和FTD患者中发现了APP-C端片段(CTF)99，在两名FTD患者中发现了CTF83，其中一名患者显示了极强的染色(图7)。结果表明，之前由W0-2、aducanumab和Bioeast抗体识别的14-16KD条带更有可能是CTF99和CTF83条带，而不是Aβ低聚体。运用鼠抗APP蛋白N端单克隆抗体(克隆22C11)，在FTD、AD中发现～28KD和～56KD条带，HC样本中表现显色非常弱(图8)。在一个HC、一个AD和一个FTD中发现全长约～100KD的APP。部分AD患者也可见～68KD带(图8)。以上结果证实了APP在尿中存在，且AD和FTD患者的APP含量明显高于HC组结果。本发明进一步检测了42例认知正常对照组(CN,n＝19)、MCI患者(n＝15)和AD患者(n＝8)在未浓缩冷冻尿液样本中APP的存在。通过BCA检测试剂盒(PierceTM，赛默飞)测定每个尿样中的蛋白质浓度，用4-20％Tricine凝胶分离250ng蛋白，转移到硝化纤维膜上。用Aβ抗体W0-2(图9)和Aducanumab(图10)、APP N端抗22C11抗体(图11)、APP C端抗Ab369抗体(图12)检测印迹。同时以聚合Aβ、APP/PS1小鼠脑提取物和合成Aβ42作为对照。这些结果证实尿中存在可测量的APP及其碎片。为了平衡加样量，半胱氨酸蛋白酶抑制剂Cystatin C的抗体也被用于检测这些尿液样本(图13)。Using rabbit anti-APP protein C-terminal polyclonal antibody, we found APP-C-terminal fragment (CTF) 99 in AD and FTD patients, and CTF83 in two FTD patients, one of which showed extremely strong staining (Figure 7). The results showed that the 14-16KD bands previously recognized by W0-2, aducanumab and Bioeast antibodies were more likely to be CTF99 and CTF83 bands rather than Aβ oligomers. Using mouse anti-APP protein N-terminal monoclonal antibody (clone 22C11), ~28KD and ~56KD bands were found in FTD and AD, and the color development was very weak in HC samples (Figure 8). APP with a full length of about ~100KD was found in one HC, one AD and one FTD. The ~68KD band was also visible in some AD patients (Figure 8). The above results confirmed the presence of APP in urine, and the APP content in AD and FTD patients was significantly higher than that in the HC group. The present invention further detected the presence of APP in unconcentrated frozen urine samples from 42 cognitively normal controls (CN, n = 19), MCI patients (n = 15) and AD patients (n = 8). The protein concentration in each urine sample was determined by a BCA assay kit (Pierce TM , Thermo Fisher), and 250 ng of protein was separated with a 4-20% Tricine gel and transferred to a nitrocellulose membrane. The blot was detected with Aβ antibodies W0-2 (Figure 9) and Aducanumab (Figure 10), APP N-terminal anti-22C11 antibody (Figure 11), and APP C-terminal anti-Ab369 antibody (Figure 12). At the same time, polymerized Aβ, APP/PS1 mouse brain extracts and synthetic Aβ42 were used as controls. These results confirm the presence of measurable APP and its fragments in urine. In order to balance the loading amount, antibodies to the cysteine protease inhibitor Cystatin C were also used to detect these urine samples (Figure 13).
为了进一步验证免疫印迹Western blotting的结果，使用了质谱分析仪鉴定尿液中的APP片段。简易步骤，将210mL双蒸馏水加热至沸腾，加入2mL 1％氯金酸。让混合物再煮 沸2min，然后加入1.5mL 1％柠檬酸三钠。不断加热搅拌溶液，直到它变成清澈的红色。然后，继续搅拌5min。待溶液冷却至室温后，加入更多的水，使最终体积达到200毫升。通过400-700nm光谱法检测金溶液的质量。峰值波长为520nm，估计粒径为20nm。峰宽约1～2nm，峰外径大于1。To further verify the results of Western blotting, a mass spectrometer was used to identify APP fragments in urine. Simple steps: Heat 210 mL of double distilled water to boiling and add 2 mL of 1% chloroauric acid. Let the mixture boil for another 2 minutes. Boil for 2 minutes, then add 1.5mL of 1% trisodium citrate. Keep heating and stirring the solution until it turns a clear red color. Then, continue stirring for 5 minutes. After the solution cools to room temperature, add more water to bring the final volume to 200 mL. The quality of the gold solution is tested by 400-700nm spectroscopy. The peak wavelength is 520nm and the estimated particle size is 20nm. The peak width is about 1-2nm, and the peak outer diameter is greater than 1.
用0.1M K2CO3调节溶液pH至pH＝8.5。缓慢加入抗Aβ抗体(W0-2克隆)至终浓度为10μg/mL，慢速搅拌10min，加入聚乙烯吡咯烷酮(PVP，平均MW 10,000,Sigma)使最终浓度为0.02％。最后，加入0.1％的聚乙二醇(PEG，平均MW 20,000,Sigma)溶液。混合物在14000G离心力下4℃离心40min。离心后，除去上清，重新悬浮于15mL水中。将5名认知正常对照组和5名AD患者的尿液样本混合，因此每组的最终体积为10mL。尿样在14000G离心力下4℃离心40min。取用上清液，用500mM硼酸钾缓冲液将pH调至7.6。尿样(1mL)与5mL包被W0-2抗体的胶体金混合。将混合液放在4℃的搅拌器上过夜。然后在14000G下，4℃下离心30min。丢弃上清液，用1mL PBS冲洗3次(14000G,4℃30min)。废弃上清液后，加入120μL 1×Tricine缓冲液，沉淀剂在95℃下煮沸5min。采用BCA蛋白法测定蛋白质浓度，将20μg蛋白质加入16.5％的预制备Tricine凝胶中。凝胶在70mA下运行45min。在不同的通道添加W-02抗体(20μg)和Aβ42单体作为对照。电泳完成后，凝胶用考马斯蓝R250溶液(0.1％考马斯蓝R-250,40％乙醇和10％乙酸)染色3小时，然后用去染色液(10％乙醇和7.5％乙酸)脱色。The pH of the solution was adjusted to pH = 8.5 with 0.1M K 2 CO 3. Anti-Aβ antibody (W0-2 clone) was slowly added to a final concentration of 10 μg/mL, stirred slowly for 10 min, and polyvinyl pyrrolidone (PVP, average MW 10,000, Sigma) was added to a final concentration of 0.02%. Finally, 0.1% polyethylene glycol (PEG, average MW 20,000, Sigma) solution was added. The mixture was centrifuged at 14000G for 40 min at 4°C. After centrifugation, the supernatant was removed and resuspended in 15 mL of water. Urine samples from 5 cognitively normal controls and 5 AD patients were mixed so that the final volume of each group was 10 mL. Urine samples were centrifuged at 14000G for 40 min at 4°C. The supernatant was taken and the pH was adjusted to 7.6 with 500 mM potassium borate buffer. Urine samples (1 mL) were mixed with 5 mL of colloidal gold coated with W0-2 antibody. The mixture was placed on a stirrer at 4°C overnight. Then centrifuged at 14000G, 4°C for 30min. The supernatant was discarded and washed three times with 1mL PBS (14000G, 4°C for 30min). After discarding the supernatant, 120μL 1× Tricine buffer was added and the precipitant was boiled at 95°C for 5min. The protein concentration was determined by the BCA protein method, and 20μg of protein was added to a 16.5% pre-prepared Tricine gel. The gel was run at 70mA for 45min. W-02 antibody (20μg) and Aβ42 monomer were added as controls in different channels. After electrophoresis, the gel was stained with Coomassie Blue R250 solution (0.1% Coomassie Blue R-250, 40% ethanol and 10% acetic acid) for 3 hours and then destained with destaining solution (10% ethanol and 7.5% acetic acid).
结果显示，AD尿样中存在独特的条带(图14)。Aβ42单体的阳性对照为预期的4.5KD大小。然而，在HC样本和AD样本的凝胶上，于～7KD也观察到微弱的条带，这表明单体形成了少量的Aβ42二聚体。小鼠lgG道如预期显示出明确的非特异性结合，尤其是在AD尿样本凝胶中，证实了本实验中使用的IP是有效的，结果是有效的。The results showed that there were unique bands in AD urine samples (Figure 14). The positive control of Aβ42 monomer was the expected size of 4.5KD. However, a faint band was also observed at ~7KD on the gels of HC samples and AD samples, indicating that the monomer formed a small amount of Aβ42 dimers. The mouse IgG tract showed clear nonspecific binding as expected, especially in the AD urine sample gel, confirming that the IP used in this experiment was effective and the results were valid.
对这些AD样本尿液的独特条带(～4,7,14,28,56和68KD)进行切分，并通过质谱(MS)进行蛋白测序。使用该序列对UniProtKB和Swiss-Prot数据库进行Protein Blast检测，APP阳性率100％，E值为1×10-6。质谱(MS)鉴定的序列“LVFFAEDVGSNK”位于APP的残基号688～699，经处理后为Aβ40和Aβ42的一部分。The unique bands (~4, 7, 14, 28, 56 and 68KD) of these AD sample urine were segmented and sequenced by mass spectrometry (MS). The sequence was used to perform Protein Blast detection on UniProtKB and Swiss-Prot databases, with a 100% APP positive rate and an E value of 1×10 -6 . The sequence "LVFFAEDVGSNK" identified by mass spectrometry (MS) is located at residues 688 to 699 of APP, and after processing, it is part of Aβ40 and Aβ42.
在～68kD蛋白条带中发现了与APP在WYFDVTEGK测试中不同的序列。使用WYFDVTEGK对UniProtKB和Swiss-Prot数据库进行Protein Blast检测，APP的阳性率为100％，E值为1×10-5。质谱鉴定的序列位于APP的残基号307～315，这些位置经过处理后是APP的可溶性部分。A sequence different from APP in the WYFDVTEGK test was found in the ~68kD protein band. Protein Blast detection was performed on UniProtKB and Swiss-Prot databases using WYFDVTEGK, and the positive rate of APP was 100% and the E value was 1×10 -5 . The sequence identified by mass spectrometry was located at residues 307 to 315 of APP, and these positions were the soluble part of APP after processing.
包被抗Aβ抗体的金纳米颗粒免疫沉淀仅在AD患者尿液中可见蛋白条带，而在健康对照 组尿液中未见蛋白条带，提示AD患者APP代谢物谱与健康人不同。这一观察结果与免疫印迹Western blotting结果一致，患者中APP片段数量较高。由此可得，APP片段具有很高的潜力使其可以作为AD或其他类型痴呆的早期诊断的新型生物标志物。 Immunoprecipitation of gold nanoparticles coated with anti-Aβ antibodies revealed protein bands only in the urine of AD patients, but not in healthy controls. No protein bands were found in the urine of the AD group, suggesting that the APP metabolite profile of AD patients is different from that of healthy people. This observation is consistent with the results of Western blotting, which showed that the number of APP fragments in patients was higher. Therefore, APP fragments have a high potential to serve as a new biomarker for the early diagnosis of AD or other types of dementia.

Claims (10)

  1. 用于检测蛋白生物标志物组的抗体在制备诊断AD、MCI和其它类型老年痴呆的试剂盒中的用途，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。The use of antibodies for detecting a protein biomarker group in the preparation of a kit for diagnosing AD, MCI and other types of Alzheimer's disease, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  2. 如权利要求1所述的用途，其特征在于，所述诊断是从来源于人类的生物样本中检测所述蛋白生物标志物组，并且其中检测所述蛋白生物标志物组的步骤包括进行生物体外检测；所述生物样本为尿液。The use according to claim 1 is characterized in that the diagnosis is to detect the protein biomarker group from a biological sample derived from a human, and wherein the step of detecting the protein biomarker group comprises performing an in vitro detection; the biological sample is urine.
  3. 如权利要求1所述的用途，其特征在于，所述抗体是以淀粉样前体蛋白的其中一段序列为抗原的抗体。The use according to claim 1, characterized in that the antibody is an antibody with a sequence of amyloid precursor protein as an antigen.
  4. 如权利要求3所述的用途，其特征在于，所述抗体包括抗Aβ抗体，抗淀粉样前体蛋白抗体和抗Cystatin C抗体。The use as described in claim 3 is characterized in that the antibodies include anti-Aβ antibodies, anti-amyloid precursor protein antibodies and anti-Cystatin C antibodies.
  5. 如权利要求4所述的用途，其特征在于，所述Aβ抗体包括鼠抗人Aβ单克隆抗体、抗人Aβ自身免疫性抗体、针对淀粉样前体蛋白N端抗体和针对淀粉样前体蛋白C端的抗体。The use according to claim 4 is characterized in that the Aβ antibody includes mouse anti-human Aβ monoclonal antibody, anti-human Aβ autoimmune antibody, antibody against the N-terminus of amyloid precursor protein and antibody against the C-terminus of amyloid precursor protein.
  6. 如权利要求1所述的用途，其特征在于，所述蛋白生物标志物组通过蛋白免疫印迹法检测。The use according to claim 1, characterized in that the protein biomarker panel is detected by protein immunoblotting.
  7. 一种用于诊断AD、MCI和其它类型老年痴呆的蛋白生物标志物组，其特征在于，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。A protein biomarker group for diagnosing AD, MCI and other types of Alzheimer's disease, characterized in that the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  8. 如权利要求7所述的蛋白生物标志物组，其特征在于，所述诊断是从来源于人类的生物样本中检测所述蛋白生物标志物组，并且其中检测所述蛋白生物标志物组的步骤包括进行生物体外检测；所述生物样本为尿液。The protein biomarker panel as described in claim 7 is characterized in that the diagnosis is to detect the protein biomarker panel from a biological sample derived from a human, and wherein the step of detecting the protein biomarker panel includes performing an in vitro detection; the biological sample is urine.
  9. 一种诊断AD、MIC和其它类型老年痴呆的方法，其特征在于，所述方法是检测生物样本中的蛋白生物标志物组，所述蛋白生物标志物组包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。 A method for diagnosing AD, MIC and other types of Alzheimer's disease, characterized in that the method is to detect a protein biomarker group in a biological sample, wherein the protein biomarker group comprises a plurality of amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
  10. 通过检测尿液中的淀粉样前体蛋白的片段来早期筛查和辅助诊断AD、MIC和其它类型老年痴呆的应用；所述片段包含多个作为蛋白生物标志物的淀粉样前体蛋白片段，所述淀粉样前体蛋白片段的序列如SEQ ID NO.1至SEQ ID NO.7所示。 The invention relates to an application of early screening and auxiliary diagnosis of AD, MIC and other types of Alzheimer's disease by detecting fragments of amyloid precursor protein in urine; the fragments contain multiple amyloid precursor protein fragments as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
PCT/CN2023/078564 2023-02-28 2023-02-28 Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia WO2024087429A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202380009304.1A CN116829956A (en) 2023-02-28 2023-02-28 Use of antibodies for detecting protein biomarker sets in the preparation of kits for diagnosis of AD, MCI and other types of senile dementia
PCT/CN2023/078564 WO2024087429A1 (en) 2023-02-28 2023-02-28 Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/078564 WO2024087429A1 (en) 2023-02-28 2023-02-28 Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia

Publications (1)

Publication Number Publication Date
WO2024087429A1 true WO2024087429A1 (en) 2024-05-02

Family

ID=88114978

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/078564 WO2024087429A1 (en) 2023-02-28 2023-02-28 Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia

Country Status (2)

Country Link
CN (1) CN116829956A (en)
WO (1) WO2024087429A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1339066A (en) * 1998-09-24 2002-03-06 法玛西雅厄普约翰美国公司 Alzheimer's disease secretase
CN101724057A (en) * 2008-10-24 2010-06-09 中国科学院上海生命科学研究院 Application of amyloid precursor protein N-end structure in transport and metabolism process
CN102326083A (en) * 2009-02-18 2012-01-18 弗·哈夫曼-拉罗切有限公司 Method for inhibiting neurodegeneration
CN102604959A (en) * 2012-02-21 2012-07-25 北京交通大学 Alzheimer disease pathogenic gene and application thereof
CN112740044A (en) * 2018-05-01 2021-04-30 阿尔斯特大学 Method for diagnosis or prognosis of neurological disorders

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1339066A (en) * 1998-09-24 2002-03-06 法玛西雅厄普约翰美国公司 Alzheimer's disease secretase
CN101724057A (en) * 2008-10-24 2010-06-09 中国科学院上海生命科学研究院 Application of amyloid precursor protein N-end structure in transport and metabolism process
CN102326083A (en) * 2009-02-18 2012-01-18 弗·哈夫曼-拉罗切有限公司 Method for inhibiting neurodegeneration
CN102604959A (en) * 2012-02-21 2012-07-25 北京交通大学 Alzheimer disease pathogenic gene and application thereof
CN112740044A (en) * 2018-05-01 2021-04-30 阿尔斯特大学 Method for diagnosis or prognosis of neurological disorders

Also Published As

Publication number Publication date
CN116829956A (en) 2023-09-29

Similar Documents

Publication Publication Date Title
AU2003250102B2 (en) Fragments of beta-amyloid as targets for vaccination against alzheimer disease
US8318171B2 (en) Neurotoxic oligomers
JP5657382B2 (en) Highly sensitive immunoassay and biological target peptide and protein measurement kits
EP2174142A2 (en) Use of a camelid single-domain antibody for detecting an oligomeric form of an amyloid beta peptide and its applications
KR101804624B1 (en) Composition for diagnosing neurological disease using nasal mucus
Grundke‐Iqbal et al. Alzheimer neurofibrillary tangles: antiserum and immunohistological staining
JP3032290B2 (en) Diagnostic tools for demyelinating neuropathy, especially multiple sclerosis
AU2001268828A1 (en) Neurotoxic oligomers
WO2024087429A1 (en) Use of antibody for detecting protein biomarker group in preparation of kit for diagnosing ad, mci and other types of senile dementia
KR102033776B1 (en) Diagnostic composition of alzheimer&#39;s disease severity comprising agents measuring expression level of tau protein and diagnostics method of alzheimer&#39;s disease severity using the same
TW202035438A (en) Diagnostic drug and diagnostic method for alzheimer&#39;s disease
KR20100132964A (en) Method for detection of pneumococcus
US20120135542A1 (en) Methods and materials for diagnosing light chain amyloidosis
WO2019095403A1 (en) Aβ KIT, DETECTION METHOD AND USE
KR20120129023A (en) Proteomic biomarkers for the diagnosis and therapy of age-realted macular deteneration
LU503936B1 (en) The application of antibodies for detecting protein biomarker group in the preparation of kits for diagnosing Alzheimer&#39;s disease, mild cognitive impairment and other senile dementia
US20190049464A1 (en) Blood biomarker for detecting arteriosclerosis
JP7383043B2 (en) Cross-linked product of amyloid β protein (Aβ) that can be a substitute for amylospheroid (ASPD) and analysis of ASPD
WO2023002967A1 (en) Aβ BIOMARKER IN ALZHEIMER&#39;S DISEASE MODEL MOUSE AND METHOD FOR ANALYZING SAME
JP2009052933A (en) Detecting method and diagnosing kit of prion disease
KR101908610B1 (en) Method for screening of major urinary protein and diagnosis of Prion disease
JP2005506551A5 (en)
KR100956764B1 (en) Method for diagnosis of stroke using hemoglobin-alpha subunit as bio-marker
CN117589996A (en) Diagnostic use of highly toxic amyloid oligomers
Buonora et al. Autoimmune profiling reveals PRDX6 as a candidate TBI biomarker

Legal Events

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

Ref document number: 23881032

Country of ref document: EP

Kind code of ref document: A1