CN116829956A

CN116829956A - Use of antibodies for detecting protein biomarker sets in the preparation of kits for diagnosis of AD, MCI and other types of senile dementia

Info

Publication number: CN116829956A
Application number: CN202380009304.1A
Authority: CN
Inventors: 戴正乾; 顾柏俊
Original assignee: Hunan Qiankang Technology Co ltd
Current assignee: Hunan Qiankang Technology Co ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-09-29
Also published as: WO2024087429A1

Abstract

The invention discloses application of an antibody for detecting a protein biomarker group in preparing a kit for diagnosing Alzheimer's Disease (AD), mild Cognitive Impairment (MCI) and other senile dementia, wherein the protein biomarker group comprises a plurality of amyloid precursor protein (Amyloid Precursor Protein, APP) fragments serving as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO. 1-SEQ ID NO. 7.

Description

Use of antibodies for detecting protein biomarker sets in the preparation of kits for diagnosis of AD, MCI and other types of senile dementia

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of an antibody for detecting a protein biomarker group in preparation of a kit for diagnosing AD, MCI and other types of senile dementia.

Background

Due to Alzheimer's disease worldwideThe rapid increase in the number of examples now necessitates the finding of novel biomarkers that can be adequately detected at the early stages of alzheimer's disease for early intervention of the disease. Aβ aggregation is a hallmark feature of AD, occurring years before the clinical manifestation of the disease. Although it is synthesized mainly in the brain, researchers have found that the peripheral system plays a critical role in the clearance of brain-derived aβ. It is estimated that about 60% of brain aβ transport to the periphery is cleared. Brain-derived aβ is transported to the periphery by one or more of the following means, which cross the blood brain barrier and the blood-CSF barrier, interstitial fluid flow and CSF outlet pathways, including the arachnoid villi and glial lymphatic pathways. While in the periphery, aβ is cleared by monocytes, macrophages, neutrophils, lymphocytes, hepatocytes, etc. through phagocytosis or endocytosis, and then is excreted through urine or bile, and degraded by aβ -degrading enzymes and aβ -binding proteins in blood. Since urine excretion is considered to be the most important way for the human body to remove waste and unwanted metabolites, its soluble aβ _1-40 (Aβ40) and Aβ _1-42 It is not surprising that (A.beta.42) is a normal component of urine and is altered in Alzheimer's disease patients. However, studies on urine aβ remain largely overlooked.

Amyloid Precursor Protein (APP) is a single transmembrane protein expressed at high levels in the brain and is rapidly metabolically degraded by a variety of proteases, including α -, β -, γ -and η -secretase. The decomposition produces a large amount 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. We found that there was an increase in the breakdown of APP in sporadic AD patients, especially in APOE epsilon 4 negative individuals. However, other APP fragments in urine, except for Abeta 40 and Abeta 42, are not reported.

Disclosure of Invention

The object of the present invention is to provide a device,

use of an antibody for detecting a protein biomarker panel comprising a plurality of amyloid precursor protein fragments as protein biomarkers, the sequences of the amyloid precursor protein fragments being shown in SEQ ID No.1 to SEQ ID No.7, for the preparation of a kit for diagnosing AD, MCI and other types of senile dementia is provided.

Preferably, the diagnosis is detection of the set of protein biomarkers from a biological sample derived from a human, and wherein the step of detecting the set of protein biomarkers comprises performing an in vitro detection.

Preferably, the biological sample is urine.

Preferably, the antibody is an antibody having one of the sequences of the amyloid precursor protein fragment as an antigen.

More preferably, the antibodies include aβ antibodies and anti-Cystatin C antibodies.

More preferably, the aβ antibodies include polyclonal antibodies directed against murine anti-human aβ monoclonal antibodies, N-terminal monoclonal antibodies directed against amyloid precursor protein, and C-terminal monoclonal antibodies directed against amyloid precursor protein; specifically, the anti-Abeta antibodies include W0-2 clone antibody, aducanaumab clone antibody, N-terminal monoclonal antibody (22C 11 clone) aiming at Amyloid Precursor Protein (APP), and polyclonal antibody (Ab 369) aiming at C terminal of Amyloid Precursor Protein (APP).

Preferably, the set of protein biomarkers is detected by western blotting.

It is also an object of the present invention to provide,

a protein biomarker panel for diagnosing AD, MCI and other types of senile dementia is provided, comprising a plurality of amyloid precursor protein fragments as protein biomarkers, the sequences of the amyloid precursor protein fragments being shown in SEQ ID No.1 to SEQ ID No. 7.

Preferably, the biological sample is urine.

It is also an object of the present invention to provide,

a method for diagnosing AD, MCI and other senile dementia is provided, wherein the method is to detect a protein biomarker group in a biological sample, the protein biomarker group comprises a plurality of amyloid precursor protein fragments serving as protein biomarkers, and the sequences of the amyloid precursor protein fragments are shown as SEQ ID NO. 1-SEQ ID NO. 7.

Preferably, the biological sample is human urine.

Preferably, the method is detection of a panel of protein biomarkers in a biological sample by western blotting with different antibodies.

The invention can be used for early screening and auxiliary diagnosis of AD, MIC and other senile dementia by detecting the fragment of amyloid precursor protein in urine; the fragment 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.

In the process of searching for urine-based AD biomarkers, the invention researches the existence of Abeta and APP fragments in urine and determines that the APP fragments have high potential so as to be used as novel biomarkers for early diagnosis of AD or other types of dementia.

In summary, the present invention uses Western blotting (Western blotting) to detect Amyloid Precursor Protein (APP) and aβ in urine from cognitive normal populations and dementia patients (alzheimer's disease (AD) or frontotemporal dementia patients (FTD)) using different antibodies. Thereby identifying a plurality of APP fragments, including

About 14KD, about 28KD, about 56KD and about 68KD protein bands. In comparison to the age-matched cognitive normal control group,

these APP fragments were found to be significantly elevated in urine of AD or FTD patients. Subsequent immunoprecipitation and further analysis by mass spectrometry confirm the presence of the APP fragment. Thus, the quantitative or qualitative detection of the urine APP fragment can be used to make an early diagnosis of AD or other types of dementia patients.

Drawings

FIG. 1 Western blotting (Western blotting) to detect Aβ. The upper part of the figure: synthetic aβ42 was dissolved in a minimum amount of DMSO and then added to fresh urine collected from healthy young donors. The samples were concentrated using the TCA/Acton method. The lower part of the figure: aβ42 was dissolved in DMSO and diluted with PBS. Aβ42 was separated with 4-20% Tricine gel and transferred to nitrocellulose membrane. Immunoblots were boiled for 5min and detected with anti-Abeta 42 mab (clone W0-2, 1. Mu.g/mL) and an enzyme-labeled anti-mouse IgG secondary antibody (0.05. Mu.g/mL). Monomers, dimers, and tetramers of aβ42 can be found.

FIG. 2 Western blotting (Western blotting) to detect Aβ. The aβ monomers were sonicated in F-12 medium and allowed to stand at 4 ℃ for at least 24 hours to form aggregates. The oligomers were diluted with PBS or urine collected from healthy young donors. The samples were separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min and detected with anti-Abeta 42 mab (clone W0-2, 0.2. Mu.g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL).

FIG. 3 Western blotting (Western blotting) to detect Aβ and APP. Abeta 40 (8-250 ng) and 3 APP/PS1 mouse brain tissue extracts (50. Mu.g protein amounts each, animal Nos. #L53, L29 and L03) were synthesized by separation with 4-20% tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min and detected with anti-Abeta 42 mab (clone W0-2, 1. Mu.g/mL) and an enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL).

FIG. 4 Western blotting (Western blotting) to detect APP fragments in urine. The following donors were concentrated using either the freeze-drying method (upper panel) or the TAC/Acton method (lower panel): urine from a population of patients with aβ PET negative Healthy Controls (HC), aβ PET positive Alzheimer's Disease (AD) or frontotemporal dementia (FTD) is frozen and dried. Samples (2. Mu.g protein amount each) were separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min and detected with anti-Abeta 42 mab (clone W0-2, 0.2. Mu.g/mL) and anti-mouse IgG secondary antibody (0.02. Mu.g/mL).

FIG. 5 Western blotting (Western blotting) to detect APP fragments in urine. Urine from donors such as Abeta PET negative Healthy Control (HC), abeta PET positive senile dementia (AD) and frontotemporal dementia (FTD) patients is collected, and the urine is subjected to freeze concentration by freeze-drying concentration method (upper graph, 10-fold concentration, 30. Mu.L) or TAC/Acton method (lower graph, each 2. Mu.g protein amount). The sample was separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min and detected with anti-Abeta 42 mab (Aducanaumab, 0.1. Mu.g/mL) and enzyme-labeled anti-human IgG secondary antibody (0.02. Mu.g/mL).

FIG. 6 Western blotting (Western blotting) to detect APP fragments in urine. Urine from donors such as healthy control negative for Abeta PET (HC), abeta PET positive Alzheimer's Disease (AD) and frontotemporal dementia (FTD) patients was cryo-concentrated by TAC/Acton method. Samples (2. Mu.g protein amount each) were separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min, reacted with anti-Abeta 42 mab (from Bio-east, 0.2. Mu.g/mL) and detected with an enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL) primer.

FIG. 7 Western blotting (Western blotting) to detect APP fragments in urine. Urine from donors such as healthy control negative for Abeta PET (HC), abeta PET positive Alzheimer's Disease (AD) and frontotemporal dementia (FTD) patients was cryo-concentrated by TAC/Acton method. Samples (2. Mu.g protein amount each) were separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min, reacted with anti-APP-C-terminal polyclonal antibody (Ab 369, sigma-Aldrich, 0.1. Mu.g/mL), and detected with an enzyme-labeled anti-rabbit secondary antibody (0.02. Mu.g/mL) primer.

FIG. 8 Western blotting (Western blotting) to detect APP fragments in urine. Urine from donors such as healthy control negative for Abeta PET (HC), abeta PET positive Alzheimer's Disease (AD) and frontotemporal dementia (FTD) patients was cryo-concentrated by TAC/Acton method. Samples (2. Mu.g protein amount each) were separated with 4-20% tricine gel and transferred to nitrocellulose membrane after separation. Immunoblots were boiled for 5min, reacted with anti-APP-N-terminal antibody (clone 22C11, sigma-Aldrich, 0.1. Mu.g/mL) and detected with an enzyme-labeled anti-mouse IgG secondary Ab (0.02. Mu.g/mL) primer.

FIG. 9 Western blotting (Western blotting) to detect APP fragments in urine. Frozen human urine collected from elderly persons with or without AD was thawed, samples each containing 0.25. Mu.g protein were separated with 4-20% Tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min and activated with anti-Abeta 42 mab (clone W0-2, 0.2. Mu.g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL). The polymerized Abeta 42 oligomer (5.7 pg), the APP/PS1 mouse brain extract (2 mug) and the synthetic Abeta 42 (0.5 and 1.0 ng) are used as a control. The immunoWestern blotting (Western blotting) image of the control group with a shorter exposure time is shown on the right side. The symbols represent: CN; AD; delta: MCI or dysmnesia. Opening Aβ PET negative; solid Aβ PET positive.

FIG. 10 Western blotting (Western blotting) to detect APP fragments in urine. Frozen human urine collected from elderly persons with or without AD was thawed, samples each containing 0.25. Mu.g protein were separated with 4-20% Tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min, detected with anti-Abeta 42 mab (Aducanaumab, 40 ng/mL) and then with enzyme-labeled anti-human IgG secondary antibody (0.02. Mu.g/mL). Synthetic Abeta 42 (0.1 and 0.2. Mu.g), APP/PS1 mouse brain extract (2. Mu.g) and aggregated Abeta 42 oligomer 5.7pg were used as controls. The symbols represent: CN; AD; delta: MCI or dysmnesia. Opening Aβ PET negative; solid Aβ PET positive.

FIG. 11 Western blotting (Western blotting) to detect APP fragments in urine. Frozen human urine collected from elderly persons with or without AD was thawed, samples each containing 0.25. Mu.g protein were separated with 4-20% Tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min, reacted with anti-APP N-terminal mab (22C11,0.1. Mu.g/mL) and detected with an enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL). Synthetic Abeta 42 (0.1 and 0.2. Mu.g), APP/PS1 mouse brain extract (2. Mu.g) and aggregated Abeta 42 oligomer (5.7 pg) were used as controls. The symbols represent: CN; AD; delta MCI or dysmnesia. Opening Aβ PET negative; solid Aβ PET positive.

FIG. 12 Western blotting (Western blotting) to detect APP fragments in urine. Frozen human urine collected from elderly persons with or without AD was thawed, samples each containing 0.25. Mu.g protein were separated with 4-20% Tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min, reacted with rabbit anti-APP c-terminal antibody (Ab 369, 0.1. Mu.g/mL) and then detected with an enzyme-labeled anti-rabbit IgG secondary antibody (0.02. Mu.g/mL). As controls, polymerized Abeta 42 oligomer (5.7 pg), APP/PS1 mouse brain extract (2. Mu.g) and synthetic Abeta 42 (0.5 and 1.0 ng) were used. Western blotting images of the control group with shorter exposure time are shown on the right. The symbols represent: CN; AD; delta: MCI or dysmnesia. Opening Aβ PET negative; real phase Aβ PET positive.

FIG. 13 Western blotting (Western blotting) to detect APP fragments in urine. Frozen human urine collected from elderly persons with or without AD was thawed, samples each containing 0.25. Mu.g protein were separated with 4-20% Tricine gel, and transferred to nitrocellulose membranes after separation. Immunoblots were boiled for 5min and detected with anti-cystatin C mab (0.1. Mu.g/mL) and enzyme-labeled anti-mouse IgG secondary antibody (0.02. Mu.g/mL). Synthetic Abeta 42 (0.1 and 0.2. Mu.g), APP/PS1 mouse brain extract (50. Mu.g) and aggregated Abeta 42 oligomer (0.1. Mu.g) were used as controls. The symbols represent: CN; AD; delta: MCI or dysmnesia. Opening Aβ PET negative; real phase Aβ PET positive.

Fig. 14. SDS-PAGE gel results of urine immunoprecipitated with W0-2 antibody in HC healthy people and AD patients. The 6 running belts in W0-2 are repeated running belts. The gel was stained with coomassie blue R250 and photographed after destaining in destaining solution. The arrow indicates the general location of the cut gel run. The gel was cut horizontally on the running belt. 20. Mu.g of protein was loaded into each channel in 20. Mu.L of sample. MlgG = mouse lgG control, 50 μg; w0-2=w0-2 antibody 20 μg; aβ42,20 μg of synthetic Aβ42; IP = immunoprecipitation reaction.

Detailed Description

In Western blotting experiments, urine samples were concentrated to 1/10 volume by lyophilization or by trichloroacetic acid (TCA) precipitation. That is, 100. Mu.L of 0.15% deoxycholate was added to 1mL of urine and left at Room Temperature (RT) for 10min. TCA (100 μl) was added and the mixture was left to stand at 4 ℃ for 1 hour and then centrifuged at 16000g centrifugal force at 4 ℃ for 40min. The supernatant was discarded and 500. Mu.L of chilled acetone was added. The sample was centrifuged at 16000g for 40min, and the dried product was dissolved in 50. Mu.L of 2 XSDS buffer and analyzed by SDS-page.

6-20% Tris-Tricine gradient gel was prepared and stacked, the samples were mixed with buffer and heated at 96℃for 5min. About 40. Mu.L of the sample mixture and SeeBlue Plus (Siemens flight) pre-stained protein standard were added to the gel. Proteins were separated by electrophoresis and then transferred to nitrocellulose membranes. Boiling in 10mm Phosphate Buffered Saline (PBS) for 5min, blocking overnight with 5% nonfat dry milk in Tris buffered saline plus Tween-20 (TBST), pH 7.2. The blots were washed several times with TBST, then TBST with 1:1000-1:25000 (previously confirmed) diluted primary antibody and 5% skim milk powder was added overnight at 4 ℃. After washing, incubation with TBST containing 1:10.about.000 to 1:30.000 diluted HRP-conjugated secondary antibody and 5% nonfat milk powder for 1-2 hours. After washing, blot images were captured with Gel Doc equipment using ultrasensitive ECL reagent. First, western blotting was used to detect synthetic Abeta with or without TCA/acetone treatment. The results show that neither the results treated (TCA precipitated) nor the control (urine) affected the detection of aβ (fig. 1). Furthermore, as aβ loading increases (> 30 ng), aβ dimers (-8 KD) are increasingly visible. Further increases in aβ loading above 250ng, the presence of tetramers (. About.16 KD) was observed (fig. 1). Subsequently, aggregated aβ dissolved in PBS or urine was detected, and no significant difference in aggregated aβ was observed in PBS or urine at low protein levels (20 ng or less), indicating that urine is unlikely to affect aβ aggregation naturally occurring (fig. 2). To ensure that the system was able to detect APP, brain extracts of 3 APP/PS1 transgenic mice were prepared and tested using Western blotting. Multiple protein bands containing APP fragments or oligomers were observed, with APP total length (-100 KD) being most prominent, followed by a possible C-terminal fragment (CTF) 99 band (-14 KD) (fig. 3). Notably, no larger aβ oligomers than tetramers have been found (fig. 1-3).

Next, 5 cognitive normal healthy controls negative for aβ PET scan (centileoid score < 15), 5 samples clinically diagnosed with AD, aβ PET scan centileoid score >25, and urine from 2 frontotemporal leaf dementia patients were collected. Urine is concentrated 10 times by lyophilization or TCA precipitation. Western blotting analysis (clone W0-2) was performed using Abeta antibody. All samples had 28KD bands, but AD and FTD patients showed strong staining, while healthy controls had weaker staining. The other 14-16KD band is only visible in part of AD and FTD patients. There were no significant differences in different processes such as freeze-drying or TAC/acetone precipitation (fig. 4).

To verify the results, the present invention used a different aβ antibody, named aducanaumab. In addition to the 28KD and 14-16KD bands found with the W0-2 antibody, two additional polymer bands were found by aducanaumab, one at 56KD and the other at 68KD (FIG. 5). These two bands were evident in AD and FTD patients, but appeared very weak in HC samples (fig. 5). Also, there was no significant difference in the results of lyophilizing urine or TCA precipitating urine protein (fig. 5).

Another aβ antibody was used to further verify this finding. Western blotting was performed on the same samples, and was performed using Abeta antibodies purchased from Bioeast Biotech (Boyue organism, hangzhou, china) and QK-02 developed by I company. The detection result was similar to the W0-2 result (FIG. 6).

Using rabbit anti-APP C-terminal polyclonal antibodies, we found APP-C-terminal fragment (CTF) 99 in AD and FTD patients and CTF83 in two FTD patients, one of which showed very strong staining (FIG. 7). The results indicate that the 14-16KD bands previously recognized by the W0-2, aducaniumab and Bioeast antibodies are more likely to be CTF99 and CTF83 bands than the Abeta oligomers. Using a murine anti-APP N-terminal monoclonal antibody (clone 22C 11), the 28KD and 56KD bands were found in FTD and AD, and the color development was very weak in HC samples (FIG. 8). APP with a full length of about-100 KD is found in one HC, one AD and one FTD. Some AD patients also seen a-68 KD band (FIG. 8). The above results confirm that APP is present in urine and APP content in AD and FTD patients is significantly higher than in HC group results. The present invention further examined the presence of APP in unconcentrated frozen urine samples in 42 cognitive normal control groups (CN, n=19), MCI patients (n=15) and AD patients (n=8). Detection kit by BCA (Pierce) ^TM Race-fly) was measured for protein concentration in each urine sample, 250ng of protein was separated with 4-20% tricine gel and transferred to nitrocellulose membrane. With Abeta antibody W0-2 (FIG. 9)) And aducanaumab (fig. 10), APP N-terminal anti-22C 11 antibody (fig. 11), APP C-terminal anti-Ab 369 antibody (fig. 12). Meanwhile, the polymerized Abeta, the APP/PS1 mouse brain extract and the synthesized Abeta 42 are used as controls. These results confirm the presence of measurable APP and fragments thereof in urine. Antibodies to Cystatin C, a cysteine protease inhibitor, were also used to detect these urine samples in order to balance the loading (fig. 13).

To further verify the results of immunoblotting Western blotting, a mass spectrometer was used to identify APP fragments in urine. In the simple step, 210mL of double distilled water was heated to boiling, and 2mL of 1% chloroauric acid was added. The mixture was allowed to boil for an additional 2min, then 1.5mL of 1% trisodium citrate was added. The stirred solution was heated continuously until it turned clear red. Then, stirring was continued for 5min. After the solution cooled to room temperature, more water was added to bring the final volume to 200 ml. The quality of the gold solution was checked by 400-700nm spectroscopy. The peak wavelength was 520nm and the particle size was estimated to be 20nm. The peak width is about 1-2 nm, and the peak outer diameter is larger than 1.

With 0.1. 0.1M K ₂ CO ₃ The pH of the solution was adjusted to ph=8.5. anti-Abeta antibody (W0-2 clone) was slowly added to a final concentration of 10. Mu.g/mL, stirred slowly for 10min, and polyvinylpyrrolidone (PVP, average MW 10,000, sigma) was added to a final concentration of 0.02%. Finally, a 0.1% polyethylene glycol (PEG, average MW 20,000, sigma) solution was added. The mixture was centrifuged at 14000G for 40min at 4 ℃. After centrifugation, the supernatant was removed and resuspended in 15mL of water. Urine samples from 5 cognitive normal control groups and 5 AD patients were mixed so that the final volume of each group was 10mL. The urine sample was centrifuged at 14000G for 40min at 4 ℃. The supernatant was taken and the pH was adjusted to 7.6 with 500mM potassium borate buffer. Urine samples (1 mL) were mixed with 5mL of W0-2 antibody coated colloidal gold. The mixture was placed on a stirrer at 4 ℃ overnight. Then centrifuged at 14000G for 30min at 4 ℃. The supernatant was discarded and washed 3 times with 1mL of PBS (14000G, 30min at 4 ℃). After discarding the supernatant, 120. Mu.L of 1 Xtricine buffer was added and the precipitant was boiled at 95℃for 5min. Protein concentration was determined by BCA protein method and 20 μg protein was added to 16.5% pre-prepared Tricine gel. The gel was run at 70mA for 45min. In different waysW-02 antibody (20. Mu.g) and Abeta.42 monomer were added as controls. After electrophoresis was completed, 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 results showed that unique bands were present in AD urine samples (fig. 14). The positive control for the aβ42 monomer was the expected 4.5KD size. However, a weak band was also observed at-7 KD on the gels of HC and AD samples, indicating that the monomer formed a small amount of aβ42 dimer. The mice lgG channel as expected showed clear non-specific binding, especially in AD urine sample gels, confirming that the IP used in this experiment was effective, and the results were valid.

The unique bands of urine from these AD samples (-4,7,14,28,56 and 68 KD) were split and protein sequenced by Mass Spectrometry (MS). Protein Blast detection was performed on UniProtKB and Swiss-Prot databases using this sequence with a 100% APP positive rate and an E value of 1X 10 ^-6 . The sequence identified by Mass Spectrometry (MS) is "LVFFAEDVGSNK" at residues 688-699 of APP, which is processed to be part of A.beta.40 and A.beta.42.

Different sequences from APP in the WYFDVTEGK test were found in the-68 kD protein band. Protein Blast detection was performed on UniProtKB and Swiss-Prot databases using WYFDVTEGK with a positive rate of 100% for APP and an E value of 1X 10 ^-5 . The mass spectrum identified sequences are located at residues 307-315 of APP, which are the soluble portion of APP after processing.

The gold nanoparticles coated with the anti-aβ antibodies immunoprecipitated protein bands were only visible in urine of AD patients, while no protein bands were seen in urine of healthy control group, suggesting that APP metabolite profile of AD patients is different from healthy people. This observation is consistent with Western blotting results, which show that the number of APP fragments in the patients is high. The APP fragment thus has a high potential to make it a novel biomarker for early diagnosis of AD or other types of dementia.

Claims

1. Use of an antibody for detecting a protein biomarker panel comprising a plurality of amyloid precursor protein fragments as protein biomarkers, the sequences of the amyloid precursor protein fragments being shown in SEQ ID No.1 to SEQ ID No.7, for the preparation of a kit for diagnosing AD, MCI and other types of senile dementia.

2. The use of claim 1, wherein the diagnosis is the detection of the set of protein biomarkers from a biological sample derived from a human, and wherein the step of detecting the set of protein biomarkers comprises performing an in vitro assay; the biological sample is urine.

3. The use of claim 1, wherein the antibody is an antibody in which one of the sequences of the amyloid precursor protein is an antigen.

4. The use of claim 3, wherein the antibodies comprise anti-aβ antibodies, anti-amyloid precursor protein antibodies and anti-Cystatin C antibodies.

5. The use of claim 4, wherein the aβ antibody comprises a murine anti-human aβ monoclonal antibody, an anti-human aβ autoimmune antibody, an N-terminal antibody directed against amyloid precursor protein, and an antibody directed against the C-terminal end of amyloid precursor protein.

6. The use of claim 1, wherein the set of protein biomarkers is detected by western blotting.

7. A protein biomarker panel for diagnosing AD, MCI and other types of senile dementia, characterized in that the protein biomarker panel comprises a plurality of amyloid precursor protein fragments as protein biomarkers, the sequences of which are shown in SEQ ID No.1 to SEQ ID No. 7.

8. The set of protein biomarkers of claim 7, wherein the diagnosis is detection of the set of protein biomarkers from a biological sample from a human, and wherein the step of detecting the set of protein biomarkers comprises performing an in vitro detection; the biological sample is urine.

9. A method for diagnosing AD, MIC and other types of senile dementia, characterized in that the method is to detect a protein biomarker panel comprising a plurality of amyloid precursor protein fragments as protein biomarkers in a biological sample, the sequences of the amyloid precursor protein fragments being shown in SEQ ID No.1 to SEQ ID No. 7.

10. The application of early screening and auxiliary diagnosis of AD, MIC and other senile dementia types by detecting the fragment of amyloid precursor protein in urine; the fragment 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.