WO2024087430A1

WO2024087430A1 - Enzyme-linked immunosorbent assay method for quantitatively detecting urine amyloid precursor protein fragment

Info

Publication number: WO2024087430A1
Application number: PCT/CN2023/078565
Authority: WO
Inventors: 戴正乾; 顾柏俊
Original assignee: 湖南乾康科技有限公司
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2024-05-02
Also published as: CN117157527A

Abstract

An enzyme-linked immunosorbent assay method for quantitatively detecting a urine amyloid precursor protein (APP) fragment. The method comprises: after coupling phagocytosis promoting peptides (PPP) with biotin, detecting an amyloid precursor protein fragment by means of enzyme-linked immunosorbent assay, wherein the sequence of the amyloid precursor protein fragment is as shown in SEQ ID NO. 1 to SEQ ID NO. 7.

Description

An enzyme-linked immunosorbent assay for quantitatively detecting amyloid precursor protein fragments in urine

Technical Field

The present invention belongs to the field of biomedical technology, and specifically relates to an enzyme-linked immunosorbent assay method for quantitatively detecting urine amyloid precursor protein fragments. The method can be used for early screening and auxiliary diagnosis of Alzheimer's disease and mild cognitive impairment.

Background technique

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 Aβ40 and 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.

Amyloid precursor protein (APP) is a single transmembrane protein expressed at high levels in the brain and can be rapidly metabolized and degraded 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. We found that the degradation of APP is increasing in patients with sporadic AD, especially in APOEε4-negative individuals. However, except for Aβ40 and Aβ42, other APP fragments in urine have not been reported. The current method for detecting these APP fragments is mainly immunoblotting (Western blotting), but this method can only be semi-quantitative and is time-consuming and cumbersome, and is not suitable for clinical application.

Summary of the invention

The present invention aims to overcome the deficiencies of the prior art and provide a quantitative detection method for urine amyloid precursor protein fragments. The method can be used for early screening and auxiliary diagnosis of Alzheimer's disease.

In order to achieve the above object, the technical solution provided by the present invention is:

The enzyme-linked immunosorbent assay method for detecting amyloid precursor protein fragments in urine for quantitative early screening of Alzheimer's disease is to couple phagocytosis promoting peptides (PPP) with biotin, and then use enzyme-linked immunosorbent assay (ELISA) to detect amyloid precursor protein fragments. The sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.

The present invention also provides the use of a phagocytic peptide coupled to biotin in the preparation of a reagent for detecting urine amyloid precursor protein fragments for quantitative early screening of Alzheimer's disease, the sequence of the amyloid precursor protein fragments being shown as SEQ ID NO.1 to SEQ ID NO.7.

Preferably, the detection is to couple tuftsin with biotin and then use enzyme-linked immunosorbent assay to quantitatively detect urine amyloid precursor protein fragments. This method can be used for early screening and auxiliary diagnosis of AD and MCI.

The present invention will be further described below:

Previously, we discovered the presence of amyloid precursor protein (APP) fragments in human urine, and observed that these fragments were associated with Alzheimer's disease (AD) and other dementias, such as frontotemporal dementia (FTD). We developed a modified enzyme-linked immunosorbent assay (ELISA) to quantify these APP fragments in urine. In the present invention, a polypeptide with high affinity for Aβ is used as a detection reagent in the ELISA system instead of the traditional detection antibody to detect APP fragments, so that multiple APP fragments containing Aβ can be detected simultaneously, greatly enhancing the sensitivity of the method. Using this improved ELISA method, we found more APP fragments in the urine of donors with mild cognitive impairment (MCI) compared with age-matched Aβ PET scan negative cognitive normal control group (CN). Therefore, this improved ELISA-based quantitative method can be used for early screening and auxiliary diagnosis of patients with AD or other types of dementia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: ELISA for detection of APP and Aβ; ELISA plate was coated with anti-Aβ mAb (clone W02), sample was captured with biotinylated W02 and detected with enzyme-labeled streptavidin;

Figure 2: ELISA for the detection of APP and Aβ; the ELISA plate was coated with anti-Aβ monoclonal antibody (clone W02), and the captured sample was detected with biotin-labeled phagocytosis-promoting peptide (PPP), which showed that PPP had a high affinity for Aβ; detection was performed with enzyme-labeled streptavidin;

Figure 3: ELISA for detecting APP fragments and Aβ in urine; Human urine samples were diluted 1:1 in 200mM Tris buffer (pH 6.8). The ELISA plate was coated with anti-Aβ mAb (clone W-02), and the captured urine samples (U1 and U2) were detected with biotinylated phagocytosis-promoting peptide (PPP) or biotinylated W-02 mAb; and detected with enzyme-labeled streptavidin;

Figure 4: ELISA for detection of APP fragments and Aβ in urine; Human urine samples were diluted 1:1 in 200 mM Tris buffer (pH 6.8). ELISA plates were coated with anti-Aβ mAb (clone W0-2). Frozen-thawed urine samples (100 μL per well, 22 MCI patients and 61 age-matched Aβ PET scan-negative cognitive normal controls) were tested in duplicate; captured samples were detected with biotinylated PPP and then with enzyme-labeled streptavidin; *: p = 0.0347.

Detailed ways

Coat 96-well plates with 100 μL of anti-Aβ monoclonal antibody (clone W0-2, 10 μg/mL) at 4°C overnight. Wash twice with PBS and block with 0.1% casein for 3 hours at room temperature. Wash three times with TBSTP buffer (50 mM Tris-HCl, 0.137 M NaCl, 0.01% PVP, 0.05% Tween-20, pH 8.0) and store at 4°C.

To detect Aβ oligomers, the W0-2 antibody was conjugated to biotin using a biotin protein labeling kit produced by Thermo Fisher Scientific. To detect APP fragments, we conjugated tuftsin (PPP) to biotin, which has previously been shown to have a high affinity for Aβ42 (PCT/CN2019/111303, LU101972, CN111656197A).

Urine samples were diluted 3:1 with urine buffer (400 mM Tris, 20 mM EDTA, 0.2% Tween-20, 400 mM NaCl, 20 μM deferiprone, pH 6.8) and then diluted 1:1 with 100 mM Tris buffer (pH 6.8). The amount added to each well was 100 μL. The culture plate was gently shaken at room temperature for 1 hour and then washed 4 times with TBSTP. Biotin-labeled W0-2 or biotin-labeled PPP was added to the wells, incubated at room temperature for 15-30 minutes, and then washed four times. Enzyme-labeled streptavidin was added, incubated for 15 minutes and then washed. TMB substrate was added for color development, 0.5 M sulfuric acid was used to stop the color, and the OD value of each well was detected at a wavelength of 450 nm.

The present invention first tested the traditional antibody sandwich ELISA scheme, such as unlabeled W0-2-antigen-biomarker combined with W0-2. The method can detect artificially synthesized APP polypeptides (Abcam, #ab7875) (Figure 1A), but cannot detect APP fragments (residues 18-289) (Abcam, #ab124588) (Figure 1B). Similarly, it is insensitive to APP751 (>128ng/mL) (Figure 1C) and APP770 (>256ng/mL) (Figure 1D). It shows high sensitivity to Aβ oligomers (>4ng/mL) (Figure 1E). In addition, APP can be detected in brain extracts of transgenic APP/PS1 mice, but not in wild-type mice (Figure 1F), indicating high specificity for human APP.

Afterwards, the present invention tested a new ELISA protocol that used biotin-labeled PPP instead of biotin-labeled W0-2. This method could not detect synthetic APP peptides or APP fragments (residues 18-289) (Figure 2A&B). However, it could detect APP751 and APP770 at 8 ng/mL (Figure 2C&D). The method showed low sensitivity to Aβ oligomers (>32 ng/mL) (Figure 2E). Moreover, it showed a linear response to serially diluted AD urine samples (Figure 2F).

The fresh urine samples were tested using the two different methods. It was found that only biotin-labeled PPP could detect Urinary APP fragments (Figure 3), indicating that APP fragments rather than Aβ oligomers are present in urine. Therefore, we collected urine from 22 MCI patients and 61 age-matched AβPET-negative cognitive normal controls and used the new protocol to detect these frozen/thawed urine samples. The results showed that APP fragments in MCI patients were significantly higher than those in healthy controls (P=0.0347) (Figure 4).

Claims

An enzyme-linked immunosorbent assay method for quantitatively detecting amyloid precursor protein fragments in urine, characterized in that the method comprises coupling tuftsin with biotin and then detecting the amyloid precursor protein fragments using an enzyme-linked immunosorbent assay, wherein the sequences of the amyloid precursor protein fragments are shown in SEQ ID NO.1 to SEQ ID NO.7.
The use of a biotin-coupled tuftsin in the preparation of a reagent for quantitatively detecting urinary amyloid precursor protein fragments, the sequences of the amyloid precursor protein fragments being shown as SEQ ID NO.1 to SEQ ID NO.7.
The use as claimed in claim 1 is characterized in that the detection is to couple tuftsin with biotin and then use enzyme-linked immunosorbent assay to quantitatively detect urine amyloid precursor protein fragments. This method can be used for early screening and auxiliary diagnosis of AD and MCI.
The use according to claim 1 is characterized in that the method of quantitatively detecting urine amyloid precursor protein fragments by enzyme-linked immunosorbent assay after coupling tuftsin with biotin can be used for early screening and auxiliary diagnosis of AD and MCI.