Use in the diagnosis of highly toxic amyloid protein oligomers

Abstract

The present invention provides the use of a novel, highly toxic amyloid protein oligomer, Aβo*3F, as a target for diagnosing early-stage and middle-stage Alzheimer's disease (AD) and mild cognitive impairment (MCI) resulting from AD. Aβo*3F specifically binds to 3F antibody and is present in the cerebrospinal fluid (CSF), blood, and / or brain tissue of AD patients and MCI patients resulting from AD. Its levels differ significantly between the CSF, blood, and / or brain tissue of AD patients, MCI patients, and healthy elderly individuals. Aβo*3F is an extremely toxic oligomer, the most predominant toxic component in the Aβ oligomer mixture, and has potent pathogenic effects, playing an important role in the development and progression of AD.

Description

[Technical Field]

[0001] The present invention relates to the use of highly toxic amyloid protein oligomers in diagnosis. Specifically, the present invention relates to the use of a novel highly toxic amyloid protein oligomer, Aβo*3F, as a target in the diagnosis of early and middle-to-late stages of Alzheimer's disease (AD) and mild cognitive impairment (MCI) resulting from AD. [Background technology]

[0002] Alzheimer's disease (AD, commonly known as senile dementia) is a chronic neurodegenerative disease. Approximately 50 million people worldwide currently suffer from AD. To date, there are no specific and effective treatments or treatments available, which has already placed a heavy burden on humanity. AD progression is relatively slow. Initially, patients exhibit no obvious clinical symptoms, but gradually progress to memory loss and personality and behavioral changes. In the later stages, AD patients experience widespread neuronal death, significant cerebral atrophy, and treatment becomes less effective. Recent clinical trials have shown that early detection and early intervention in AD patients can slow the progression of AD. However, to achieve early treatment and intervention for AD patients, timely and accurate differential diagnosis and diagnosis are essential. However, ideal early diagnostic methods and technologies currently in clinical use are lacking. Research has shown that changes in amyloid beta protein (Aβ), which trigger the onset and progression of AD, appear 15 to 20 years before clinical symptoms appear.

[0003] The pathological hallmarks of AD are the formation of senile plaques, which are aggregates of Aβ, and neurofibrillary tangles, which are aggregates of tau protein, in the brain. Aβ can aggregate into oligomers, protofibrils, and mature fibrils. Aβ oligomers are the most neurotoxic aggregate form, and many forms, such as Aβ dimers, trimers, and amyloid beta-derived diffusible ligands (ADDLs), have been reported. Aβ oligomers are divided into two types, low molecular weight and high molecular weight, based on molecular weight, with high molecular weight oligomers exhibiting greater neurotoxicity. Aβ dimers are the smallest Aβ oligomers and are generally believed to be the basic building block of Aβ oligomers. Aβ dimers are elevated in the brains of AD patients and AD transgenic mice, are stable to SDS and strong denaturing agents, and exhibit some degree of neurotoxicity. In addition to Aβ dimers, Aβ trimers are also thought to be the aggregation unit of multiple Aβ oligomers, such as hexamers and dodecamers. Aβ trimers appear early in the brains of AD patients and AD transgenic mice, but there is no significant correlation between Aβ trimers and Aβ plaque deposition, and their toxicity remains unclear. Furthermore, Aβ oligomers can further aggregate to form spherical oligomers (amylospheroids, ASPDs) with a diameter of approximately 12 nm and diffusible oligomers (ADDLs) with a diameter of 5–6 nm. Both of these polymer forms are thought to be Aβ oligomers with unique conformations and neurotoxicity. Although multiple Aβ oligomer forms have been identified, it is still unknown which specific type or types of Aβ oligomers are most toxic and play a key role in the development and progression of AD. Furthermore, methods for specifically detecting oligomers, especially toxic oligomers, are very limited, and it is particularly difficult to obtain antibodies that specifically bind to toxic amyloid protein oligomers, which seriously limits the clinical detection of Aβ oligomers and the development of therapeutic agents.

[0004] A large body of research has shown that the severity of neurodegenerative diseases is closely related to the levels of amyloid protein oligomers in patients' brains. Aβ oligomers play a key role in the development and progression of AD by causing functional neuron death, cognitive impairment, and dementia. However, there are numerous types of Aβ oligomers, which can affect central nervous system function through multiple mechanisms. Various Aβ oligomers vary in toxicity, with significant differences in size, conformation, aggregation morphology, toxicity, and time to brain appearance. While numerous studies have shown that some Aβ oligomers, such as dimers, trimers, and ADDLs, can exert neurotoxic effects, our understanding of the Aβ oligomer forms that exert truly significant pathogenic effects remains limited. Therefore, identifying key toxic oligomers closely related to the development and progression of AD could provide ideal markers for early warning and diagnosis of AD, as well as pinpoint ideal targets for AD treatment.

[0005] In addition to imaging detection, specific biomarkers are the basis for clinical diagnosis and detection of AD. Currently, the industry's most widely accepted diagnostic markers for AD are Aβ42, Aβ40, total tau protein (T-tau), and phosphorylated tau protein (P-tau) in cerebrospinal fluid (CSF). Other marker proteins, such as sAPPα, sAPPβ, BACE1, Aβ oligomers, total Aβ, axonal markers, and synaptic markers, have also been reported. These CSF markers have high diagnostic accuracy, and when combined with other AD diagnostic indicators, the diagnostic sensitivity and specificity for AD can reach 85-90%. Compared to CSF, blood is an ideal sample source for clinical detection due to its ease of acquisition and minimal invasiveness. Currently, several studies have begun to distinguish AD patients from healthy individuals by combining peripheral blood protein, lipid, and metabolite content, such as the content of Aβ42 dimers in peripheral blood cell membranes, plasma gelsolin (GSN), which can depolymerize Aβ42 fibrils, and MMP3, the main degradation enzyme of GSN.Recently, detecting the content of specific components in peripheral blood, such as Tau181, Tau217, and Aβ, has also become a focus of research.

[0006] The mainstream biochemical detection methods for AD mainly focus on measuring Aβ and tau protein markers in CSF. Extensive research has shown that as healthy individuals develop early cognitive impairment (MCI) and AD, the levels of Aβ42 monomers in CSF gradually decrease, while the levels of Aβ42 oligomers and T-tau gradually increase. However, the change in the ratio of P-tau to Aβ42 is even more pronounced. However, obtaining CSF samples requires lumbar puncture, which is traumatic and often unacceptable for MCI and mild AD patients. Furthermore, inconsistent CSF sample preparation and detection methods result in significant discrepancies between results from different manufacturers and laboratories, seriously limiting the clinical widespread adoption and application of such methods. Compared to CSF, blood-based detection has lower costs and higher compliance. However, the low content and limited variety of markers applicable to AD diagnosis and detection in blood, as well as the significant influence from peripheral tissues and organs, have hindered the development of AD diagnostic methods. Currently, the most advanced international method is to use liquid chromatography-mass spectrometry (LC-MS) and single-molecule immunoassay (Simoa) to detect the ratio of Aβ42 to Aβ40 in blood and the content of proteins such as P-Tau181 and P-Tau217. While these blood indicators have some diagnostic value, they lack specificity and cannot effectively distinguish between subjects. This is because the detection of Aβ or phosphorylated Tau protein only detects the total amount, which includes monomers and various aggregates with different toxicity. While monomers and many types of oligomers have low cytotoxicity, their high content significantly contributes to the overall Aβ level, seriously hindering the detection of highly toxic and pathogenic amyloid protein oligomers. Only highly toxic oligomers are directly related to the onset and development of AD, and their appearance and levels are directly and accurately correlated with pathological changes in the brain of patients. Therefore, specific detection of these oligomers is of greater value and significance.

[0007] Therefore, there remains a strong demand in this field for a method for detecting the content of specific Aβ oligomers in blood with high sensitivity and specificity and for use in diagnosing AD. Summary of the Invention

[0008] One aspect of the present invention relates to a method for diagnosing whether a subject has early, middle, or late stage AD, or MCI resulting from AD, or whether a subject is at risk for AD, said method comprising: a) optionally providing a subject sample to be detected; b) contacting a sample from a subject with a reagent that detects the presence and / or level of Aβo*3F in said sample from a subject; c) detecting the presence and / or level of Aβo*3F in a sample from said subject; Here, the presence and / or level of Aβo*3F in the subject's sample indicates that the subject is suffering from AD or MCI resulting from AD, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and has a molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC), and the light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NO:17-22, respectively.

[0009] In some embodiments, the subject is a patient suspected of suffering from AD or MCI resulting from AD, and preferably the subject is a human, a non-human primate, a cat, or a dog.

[0010] In some embodiments, the diagnostic method further comprises evaluating the subject by clinical neuropsychology and neuroimaging, preferably the neuroimaging is Aβ-PET scanning imaging and / or tau-PET. More preferably, the neuroimaging is 3F-PET scanning imaging, i.e., 3F-based immunoPET imaging, for identifying and detecting the presence and / or level of Aβo*3F in the subject's brain.

[0011] In some embodiments, the diagnostic method further comprises detecting an alteration in the level of tau in the subject's sample, preferably the level of tau is the level of total tau or the level of phosphorylated tau.

[0012] A second aspect of the present invention relates to a kit for diagnosing whether a subject has early, intermediate, or late stage AD, or MCI resulting from AD, or whether a subject is at risk of developing AD, the kit comprising a reagent for detecting the presence and / or level of Aβo*3F in a sample from the subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and has a molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography, and the light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are set forth in SEQ ID NOs: 17-22, respectively.

[0013] In some embodiments, the subject sample is a sample obtained from the subject at the time of said diagnosis, hi some embodiments, the subject sample is a sample obtained during a previous diagnosis of the subject.

[0014] In some embodiments, the size exclusion chromatography analysis is performed using a Superdex 200 10 / 300 GL molecular sieve column. In some embodiments, Aβo*3F is present in a sample of cerebrospinal fluid obtained from a subject. In some embodiments, the sample of cerebrospinal fluid from the subject is analyzed by immunoprecipitation and size exclusion chromatography to determine the molecular weight of Aβo*3F.

[0015] In some embodiments, the method or kit relates to or includes an enrichment reagent for Aβo*3F, the enrichment reagent for Aβo*3F comprising a binding agent that specifically binds to Aβo*3F. Preferably, the binding agent that specifically binds to Aβo*3F is an antibody that specifically binds to Aβo*3F. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, bibody, or bispecific antibody. The enrichment reagent for Aβo*3F effectively enriches Aβo*3F in a subject's sample, allowing it to be used in subsequent programs.

[0016] In some embodiments, the reagent for detecting the presence and / or level of Aβo*3F comprises a first binding agent that specifically binds to Aβo*3F or Aβ aggregates. Preferably, the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is an antibody that specifically binds to Aβo*3F or Aβ aggregates. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from an scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, bibody, or bispecific antibody. In some embodiments, the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is an scFv or a full-length antibody that specifically binds to Aβo*3F or Aβ aggregates.

[0017] In some embodiments, the first binding agent is conjugated to a detection agent that allows for its detection.

[0018] In some embodiments, the Aβo*3F can be detected and defined using the K98R mutant of the 3F antibody. Upon detection, the Aβ oligomers detected by the K98R mutant are identical to the Aβ oligomers detected by the 3F antibody, and both are Aβo*3F. However, the binding affinity between the K98R mutant and Aβo*3F is higher than that of 3F, allowing for more sensitive detection of the presence or absence of Aβo*3F and more sensitive diagnosis of neurodegenerative diseases such as AD.

[0019] In some embodiments, the first binding agent that specifically binds Aβo*3F or Aβ aggregates is the 3F antibody or its K98R mutant.

[0020] In some embodiments, the reagent for detecting the presence and / or level of Aβo*3F further comprises a second binding agent, which specifically binds to the first binding agent, preferably the second binding agent is an antibody that specifically binds to the first binding agent, preferably the antibody that specifically binds to the first binding agent is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof, more preferably the antigen-binding fragment is selected from an scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camelid antibody, nanobody, bibody, or bispecific antibody, preferably the antibody that specifically binds to the first binding agent is conjugated to a detection agent that allows its detection. In some embodiments, the antibody that specifically binds to the first binding agent is a full-length antibody.

[0021] In some embodiments, the detection agent is selected from a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, umbelliferone, Lissamine, cyanine, Texas Red, BODIPY FL-SE® (Invitrogen) or an analog thereof, a paramagnetic label, a radioactive label, biotin, streptavidin / biotin, avidin / biotin, a hapten, digoxigenin, a metal complex, a metal, an enzyme, colloidal gold, or a combination thereof. In some embodiments, the detection agent is selected from a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a radioactive label, an enzyme, or a combination thereof. In some embodiments, the detection agent is a chemiluminescent label or an electrochemiluminescent label.

[0022] In some embodiments, the detection is selected from chemiluminescence, electrochemiluminescence, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunochromatography, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation, immunodiffusion, dot blot assay, Western blot, protein chip, positron emission tomography, and / or single photon emission computed tomography; preferably, the kit includes reagents, materials, containers, and / or equipment necessary to perform a detection selected from chemiluminescence, electrochemiluminescence, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunochromatography, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation, immunodiffusion, dot blot assay, Western blot, and / or protein chip; preferably, the enzyme-linked immunosorbent assay is selected from direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay, and indirect sandwich enzyme-linked immunosorbent assay.

[0023] In some embodiments, the subject sample is selected from cells, tissues, organs and / or bodily fluids of the subject, preferably the bodily fluid is selected from whole blood, plasma, serum, cerebrospinal fluid, lymphatic fluid, saliva, synovial fluid, bronchoalveolar lavage fluid, sputum, ascites, urine, amniotic fluid, peritoneal fluid, pericardial fluid, semen and / or vaginal secretions, preferably the bodily fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

[0024] In some embodiments, the enrichment reagent or first binding agent that specifically binds Aβo*3F is attached to a solid support.

[0025] In some embodiments, the method further comprises detecting a control sample and / or the kit further comprises a control sample, wherein the control sample is derived from a healthy subject or a subject not suffering from AD and MCI as a result of AD.

[0026] In some embodiments, the antibody that specifically binds to Aβo*3F is a polyclonal antibody and / or a monoclonal antibody obtained by immunization with Aβo*3F, or an antigen-binding fragment thereof, and preferably, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a sheep antibody, or a non-human primate antibody.

[0027] In some embodiments, based on the MSD electrochemiluminescence method, the concentrations of Aβo*3F in cerebrospinal fluid of the healthy subject group are 80.44±20.88 pg / mL for Aβ42o*3F and 24.35±5.08 pg / mL for Aβ40o*3F, and / or the concentrations of Aβo*3F in plasma are 71.63±36.8 pg / mL for Aβ42o*3F and 2.24±0.92 pg / mL for Aβ40o*3F, and the concentrations of Aβo*3F in plasma of the MCI group are 71.63±36.8 pg / mL for Aβ42o*3F and 2.24±0.92 pg / mL for Aβ40o*3F, and and the concentrations of Aβo*3F in the cerebrospinal fluid of the AD patient group are Aβ42o*3F 264.8±42.26pg / mL and Aβ40o*3F 85.74±10.62pg / mL, and / or the concentrations of Aβo*3F in the plasma are Aβ42o*3F 159.44±36.8pg / mL and Aβ40o*3F 14.0±5.59pg / mL, or

[0028] Based on the chemiluminescence method, the concentration of Aβ42o*3F in plasma of the healthy subject group was 68.02±39.17 pg / mL, the concentration of Aβ42o*3F in plasma of the MCI group was 124.5±12.57 pg / mL, and the concentration of Aβ42o*3F in plasma of the AD patient group was 205.75±50.96 pg / mL. In some embodiments, the MSD electrochemiluminescence method is performed as described in Example 7 of the specification. In some embodiments, the chemiluminescence method is performed as described in Example 8 of the specification.

[0029] In some embodiments, the sensitivity of detection is as low as 0.5 pg / mL based on MSD electrochemiluminescence and chemiluminescence methods.

[0030] In some embodiments, the antibodies that specifically bind to Aβo*3F are multiple types of antibodies that specifically bind to different epitopes of Aβo*3F, for example, two, three, four, five or more types of antibodies that each specifically bind to two, three, four, five or more different epitopes of Aβo*3F.

[0031] In some embodiments, enriching Aβo*3F from the sample is carried out using immunoprecipitation with an antibody that specifically binds to Aβo*3F.

[0032] In other words, in previous research, the inventors pioneered the use of phage display technology to screen for a fully human single-chain antibody, W20 (see CN101463082A), which specifically binds to Aβ oligomers. The 3F antibody (amino acid sequence shown in SEQ ID No. 23) is an improved version of the W20 antibody, and has significantly improved affinity for Aβ oligomers compared to the W20 antibody. It can more effectively inhibit Aβ aggregation and Aβ oligomer-induced neuronal toxicity, thereby more effectively improving the cognitive and memory functions of AD model mice and reducing pathological changes in the mouse brain. More significantly, the Aβ oligomers specifically recognized by this antibody are highly toxic oligomers, the most predominant toxic component in the Aβ oligomer mixture, with potent pathogenic effects and an important role in the development and progression of AD. The highly toxic oligomers recognized by 3F are present in the cerebrospinal fluid (CSF), blood, and / or brain tissue of AD patients and AD-derived MCI patients, and their levels show marked differences in the CSF, blood, and / or brain tissue of three types of humans: AD patients, MCI patients, and healthy elderly individuals, allowing for accurate differentiation between AD patients, MCI patients, and healthy elderly individuals. These toxic oligomers are also present in AD transgenic mice and are directly related to the onset of AD transgenic mice. In the present invention, the highly toxic Aβ oligomer recognized by 3F is called AβO*3F. It can be isolated from Aβ oligomer mixtures by immunoprecipitation (using the 3F antibody). Its typical characteristics are high-molecular-weight Aβ oligomers, with a molecular weight of approximately 588 kDa and a diameter of approximately 10 nm as determined by size-exclusion chromatography (SEC). It has a highly toxic effect on neurons, more than 200 times stronger than the toxicity of the Aβ oligomer mixture. AβO*3F can activate microglia and astrocytes and secrete large amounts of proinflammatory cytokines. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram showing the production of Aβo*3F, Aβ*6E10, and Aβ-ID by immunoprecipitation.

[0034] [Figure 2] Figure 2 shows the molecular weight and morphological characteristics of Aβo*3F. Figure 2A: Detection of Aβ aggregation state by ThT; Figure 2B: Detection of the binding status of oligomers obtained under different incubation conditions with 3F, A11, and 6E10 antibodies by dot blot; Figure 2C: Affinity of Aβos obtained by incubation with 10 μM Aβ for 0 to 4 days with 3F antibody; Figure 2D: Detection of the band distribution of sAβo*3F by Western blot; Figure 2E: Analysis of the molecular weight of sAβo*3F by size exclusion chromatography (SEC); SEC analysis of sAβo*3F and SEC standard (Superdex 200) 10 / 300), SEC standards: 1. Thyroglobulin (669 kDa), 2. Ferritin (440 kDa), 3. Aldolase (158 kDa), 4. Conalbumin (75 kDa), 5. Ovalbumin (44 kDa), 6. Carbonic anhydrase (29 kDa); Figure 2F: Linear equation (Y) and correlation coefficient (r2) fitted based on the molecular weight and elution volume of SEC standards; Figure 2G: Detection of band distribution of mAβo*3F by Western blot; Figure 2H: Analysis of molecular weight of mAβo*3F by SEC; Figure 2I: Detection of band distribution of hAβo*3F by Western blot.

[0035] [Figure 3]Figure 3 shows the toxic effects of Aβo*3F on neuronal cells. Figure 3A: IC50 of sAβos toxicity on N2a cells; Figure 3B: IC50 of sAβo*3F toxicity on N2a cells; Figure 3C: Comparison of the neurotoxicity of sAβos, sAβo*3F, and sAβ-ID on N2a cells by MTT assay; Figure 3D: IC50 of mAβo*3F toxicity on N2a cells and primary neurons; Figure 3E: Comparison of the cytotoxicity of mAβ*6E10, mAβo*3F, and mAβ-ID on primary neurons by MTT assay; Figure 3F: IC50 of hAβo*3F toxicity on primary neurons; Figure 3G: Comparison of the cytotoxicity of hAβ*6E10, hAβo*3F, and hAβ-ID on primary neurons by MTT assay.

[0036] [Figure 4] These figures show the effects of mAβo*3F on the expression levels of cytokines in glial cells. Figure 4A shows the effects of mAβo*3F on the expression levels of TNF-α, IL-1β, and IL-6 in microglia; Figure 4B shows the effects of mAβo*3F on the expression levels of TNF-α, iNos, IL-1β, and IL-6 in primary astrocytes; Figure 4C shows the effects of mAβo*3F on the expression levels of TSP1, Gpc4, and Gpc6 in primary astrocytes.

[0037] [Figure 5] Figure 5 shows the effects of Aβo*3F on cognition and neuronal damage in mice. Figure 5A shows the cognitive scores of each group of mice in the novel object recognition experiment; Figure 5B shows the duration of time in the novel arm of each group of mice in the Y-maze experiment; Figure 5C shows the dendritic spine density of mouse neurons detected by Golgi staining; Figure 5D shows statistical analysis of the dendritic spine density in Figure 5C; Figure 5E shows the number of hippocampal neurons detected by Ziehl-Neelsen staining (scale bar: 20 μm); Figure 5F and G show statistical analysis of the number of neurons in the DG (F) and CA1 (G) regions using Image J software.

[0038] [Figure 6]Figure 6 shows that Aβo*3F activates glial cells in the mouse brain and induces neuroinflammation. Figure 6A: Immunohistochemical detection of microglial activation in the DG and CA1 regions of the mouse hippocampus (scale bar: 20 μm). Figure 6B: Immunohistochemical detection of astrocyte activation in the DG and CA1 regions of the mouse hippocampus (scale bar: 20 μm). Figures 6C and 6D: Quantification of the area of Iba-1-positive microglia in the DG (C) and CA1 (D) regions using Image J software. Figures 6E and 6F: Quantification of the area of GFAP-positive astrocytes in the DG (E) and CA1 (F) regions using Image J software. Figures 6G and 6H: ELISA detection of IL-6 (G) and IL-1β (H) expression levels in the mouse hippocampus.

[0039] [Figure 7] 7 shows the detection of Aβo*3F levels in CSF and plasma of AD patients by electrochemiluminescence. Figures 7A and 7B: Detection of corresponding Aβ42 (A) and Aβ40 (B) levels in Aβo*3F isolated from CSF of AD patients and a healthy control group (Con) by MSD; Figures 7C and 7D: Detection of corresponding Aβ42 (C) and Aβ40 (D) levels in Aβo*3F isolated from plasma of mild cognitive impairment (MCI), AD, and a healthy control group (Con) by MSD.

[0040] [Figure 8] FIG. 1 shows the detection of Aβo*3F levels in plasma from AD patients, MCI patients, and healthy subjects by a chemiluminescence method.

[0041] [Figure 9] FIG. 1 shows that detecting levels of Aβo*3F in CSF or plasma samples can be used to diagnose AD.

[0042] [Figure 10]This figure shows that the binding affinity to Aβo*3F oligomers is further improved after K at position 98 of the heavy chain of the 3F antibody is mutated to R, suggesting that this optimized single-chain antibody can be used to better diagnose the presence of Aβo*3F oligomers and thus better diagnose AD. DETAILED DESCRIPTION OF THE INVENTION

[0043] Definition: Unless otherwise stated, terms used in the claims and specification are defined as follows: Unless otherwise defined herein, scientific and technical terms used in connection with the methods and compositions of the invention described herein shall have the ordinary meanings understood by those of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature used in connection with and the following techniques are those well known and commonly used in the art: biochemistry, immunology, enzymology, molecular and cell biology, microbiology, genetics, and polypeptide chemistry described herein.

[0044] Unless otherwise specified, the methods and techniques described herein are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed herein. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements until 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford University Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0045] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise indicated, the following terms shall be understood to have the following meanings: The term "at risk of developing Alzheimer's disease" refers to the risk of developing Alzheimer's disease, including, for example, family history, aging, and poor lifestyle habits such as drinking alcohol.

[0046] The term "Aβ-PET scanning imaging" refers to positron emission tomography of the β-amyloid protein. The term "tau-PET scanning imaging" refers to positron emission tomography of the Tau protein.

[0047] The term "3F-PET scanning imaging" refers to immunopositron emission tomography based on the 3F antibody. The term "Aβo*3F" refers to an Aβ oligomer that specifically binds to the 3F antibody, and has a molecular weight of approximately 588 kDa based on size exclusion chromatography (SEC) analysis. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively.

[0048] The term "Aβ42o*3F" refers to the portion of Aβ42 oligomers in Aβo*3F. The term "Aβ40o*3F" refers to the portion of Aβ40 oligomers in Aβo*3F. In some embodiments, the chemiluminescent label of the present invention is selected from alkaline phosphatase, horseradish peroxidase, isoluminol and its derivatives, and acridinium esters and its derivatives.

[0049] In some embodiments, the chromophores of the present invention are selected from tris(bipyridine)ruthenium. In some embodiments, the fluorescent labels of the present invention are selected from rare earth chelates or derivatives thereof, isothiocyanates, rhodamines, phycobilins, phycoerythrins, phycocyanins, allophycocyanins, fluorescein isothiocyanates, or AlexaFluor dyes.

[0050] In some embodiments, the fluorescein-based labels of the present invention are selected from fluorescein, fluorescein enzymes, such as, for example, Firefly Luciferase and bacterial luciferases.

[0051] In some embodiments, the paramagnetic label is selected from the group consisting of aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (Mo), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), vanadium (V), and zirconium (Zi), particularly Co. +2 , C.R. +2 , Cr +3 , Cu +2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 , Ni +2 , Ti +3 , V +3 and V +4 Paramagnetic ionic compounds include, but are not limited to, paramagnetic ionic compounds containing:

[0052] In some embodiments, the radiolabel is selected from technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulfur-35, deuterium, tritium, rhenium-186, rhenium-188, and yttrium-90.

[0053] In some embodiments, the hapten is selected from a polysaccharide or a lipid. In some embodiments, the metal complex is selected from tris(bipyridine)ruthenium.

[0054] In some embodiments, the metal is selected from ruthenium. In some embodiments, the enzyme of the present invention is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, β-glucuronidase, β-D-glucosidase, urease, hexokinase, malic enzyme, glucose-6-phosphate dehydrogenase, sucrase, glucoamylase, lysozyme, glucose oxidase, heterocyclic oxidase, streptavidin-β-D-galactopyranose conjugate, and / or streptavidin-horseradish peroxidase conjugate.

[0055] In some embodiments, tris(bipyridine)ruthenium is used in electrochemiluminescence assays.

[0056] In some embodiments, the biotinylated detection agent used to detect the antibody is avidin, streptavidin-HRP, or streptavidin-β-D-galactopyranose (SBG). In some embodiments, the readout of the detection agent is fluorometric or colorimetric. For example, but not limited to, readout can be performed using tetramethylbenzidine and hydrogen peroxide. In some embodiments, when the detection agent is streptavidin-HRP, readout can be performed by colorimetry using tetramethylbenzidine and hydrogen peroxide. Alternatively, in some embodiments, resorufin β-D-galactopyranoside can be used for readout. For example, but not limited to, when the detection agent is SBG, readout can be performed by fluorometric measurement using resorufin β-D-galactopyranoside.

[0057] In some embodiments, the detection agent, such as SBG, may be used at a concentration of about 50 to about 500 pM. For example, the detection agent may be used at a concentration of about 50 to about 100 pM, about 50 to about 150 pM, about 50 to about 200 pM, about 50 to about 250 pM, about 50 to about 300 pM, about 50 to about 350 pM, about 50 to about 400 pM, about 50 to about 450 pM, about 100 to about 500 pM, about 150 to about 500 pM, about 200 to about 500 pM, about 250 to about 500 pM, about 300 to about 500 pM, about 350 to about 500 pM, about 400 to about 500 pM, about 450 to about 500 pM, about 100 to about 400 pM, or about 200 to about 400 pM, but is not limited thereto. In some embodiments, the detection agent may be used at a concentration of about 100 pM to about 400 pM. For example, SBG may be used at a concentration of about 110 pM, about 155 pM, or about 310 pM. In some embodiments, SBG may be used at a concentration of about 310 pM. In some embodiments, the detection agent, such as HRP, may be used at a dilution of about 1 / 10 to about 1 / 1000. For example, the detection agent may be used at a dilution of about 1 / 10 to about 1 / 100, about 1 / 10 to about 1 / 500, about 1 / 100 to about 1 / 1000, or about 1 / 500 to about 1 / 1000, but is not limited thereto. In some embodiments, the detection agent may be used at a dilution of about 1 / 100 to about 1 / 1000, for example, HRP may be used at a dilution of about 1 / 100 or about 1 / 500. Those skilled in the art can easily determine the concentration of detection agent to use, depending on the type of detection agent, without more than routine experimentation.

[0058] In some embodiments, when horseradish peroxidase (HRP) is selected as the enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, dimethoxyaniline, or 3,3'-dimethoxybenzidine can be used as the substrate. When alkaline phosphatase is selected as the enzyme label, a solution containing 5-bromo-4-chloro-3-indolylphosphate, nitroblue tetrazolium, or p-nitrophenol phosphate can be used as the substrate. When β-D-glucosidase is selected as the enzyme label, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be used as the substrate. Additionally, various enzymes and enzyme-coloring substrates known in the art can be used.

[0059] In some embodiments, the method can include blocking the first binding agent with a blocking buffer. In some embodiments, the blocking buffer can be, for example, a blocking buffer such as ProClin TM The blocking agent may comprise PBS, such as PBS (Sigma-Aldrich, Saint Louis, MO), bovine serum albumin (BSA), and / or a biocide. In some embodiments, the method may comprise multiple washing steps. In some embodiments, the solution for washing is typically a buffer (e.g., a "washing buffer"), such as, but not limited to, a PBS buffer containing a stain release agent (e.g., Tween 20). For example, but not limited to, after blocking, the first binding agent can be washed and / or the sample can be isolated from the first binding agent (e.g., by washing) to remove unbound material.

[0060] In some embodiments, the diagnostic methods of the present invention have a detection sensitivity down to about 0.5 pg / mL, e.g., about 1.0 pg / mL, 1.5 pg / mL, 2.0 pg / mL, 2.5 pg / mL, 3.0 pg / mL, 3.5 pg / mL, 4.0 pg / mL, 4.5 pg / mL, 5.0 pg / mL, 5.5 pg / mL, 6.0 pg / mL, 7.5 pg / mL, 8.0 pg / mL, 8.5 pg / mL, 9.0 pg / mL, Examples include 9.5 pg / mL, 10.0 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 30 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 250 pg / mL, 500 pg / mL, 750 pg / mL, 1000 pg / mL, 1500 pg / mL, 2000 pg / mL, 3000 pg / mL, 5000 pg / mL or more.

[0061] In some embodiments, the kits of the invention comprise a first container comprising an enrichment reagent or first binding agent that specifically binds Aβo*3F. In some embodiments, the kits of the invention include a second container containing a second binding agent that specifically binds to the first binding agent.

[0062] The term "a second binding agent specifically binds to said first binding agent" refers to the second binding agent specifically binding to the antibody class of the first binding agent, e.g., antibody. For example, if the first binding agent is mouse anti-Aβo*3F IgG, the second binding agent may be rabbit anti-mouse IgG.

[0063] In some embodiments, the kits of the invention comprise a third container that contains a control sample. In some embodiments, the kits of the present invention include one or more other containers that contain other reagents and materials necessary to carry out the detection.

[0064] In some embodiments, the enrichment reagent, the first binding agent, or the second binding agent that specifically binds to the first binding agent is attached to a solid support. In some embodiments, the solid support is selected from polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, plasma substitute, cross-linked glucose, Sepharose, liposomes, carboxymethylcellulose, polyacrylamide, polystyrene, porphyry, paper filters, ion exchange resins, plastic films, plastic tubes, poly(methyl vinyl ether / maleic acid) copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, metals, glass, glass beads, or magnetic particles. Other solid supports include cell culture plates, enzyme-linked immunosorbent assay plates, electrochemiluminescence assay plates, tubes, and polymer membranes. The solid supports may have any shape, such as spheres (microbeads), cylinders (the inner surface of a test tube or well), or flat surfaces (sheets, test strips).

[0065] In some embodiments, the kit of the present invention is a kit for performing an enzyme-linked immunosorbent assay (ELISA) and includes some or all of the reagents, materials, containers, and / or equipment necessary for performing the ELISA. It would be clear to one skilled in the art how to prepare a corresponding ELISA kit based on the reagents of the present invention for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the ELISA kit includes a pre-coated Aβo*3F antibody, an HRP-labeled Aβ antibody, a standard, a washing solution, a substrate, etc.

[0066] In some embodiments, the kit of the present invention is a kit for performing an immunofluorescence assay and includes some or all of the reagents, materials, containers, and / or devices necessary for performing the immunofluorescence assay. It will be clear to one skilled in the art how to prepare a corresponding immunofluorescence assay kit based on the reagents of the present invention for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the immunofluorescence assay kit is an electrochemiluminescence immunoassay kit. In some embodiments, the immunofluorescence assay kit is a time-resolved immunofluorescence assay kit. In some embodiments, the immunofluorescence assay kit includes an electrochemiluminescence plate pre-coated with an Aβo*3F antibody, a ruthenium-labeled Aβ antibody, a standard, a read buffer, etc.

[0067] In some embodiments, the kit of the present invention is a kit for performing a measurement using the colloidal gold method, and includes some or all of the reagents, materials, containers, and / or devices necessary for performing the measurement using the colloidal gold method. It will be clear to those skilled in the art how to prepare a corresponding colloidal gold method measurement kit based on the reagent of the present invention for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the colloidal gold method measurement kit is a colloidal gold test strip. In some embodiments, the colloidal gold method measurement kit includes a colloidal gold-labeled Aβo*3F antibody, an Aβ antibody, a control antibody, etc.

[0068] In some embodiments, the kit of the present invention is a kit for performing a radioimmunoassay and includes some or all of the reagents, materials, containers, and / or devices necessary for performing the radioimmunoassay. It would be clear to one skilled in the art how to prepare a corresponding radioimmunoassay kit based on the reagents of the present invention for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the radioimmunoassay kit includes a radioisotope-labeled Aβo*3F antibody, an Aβo*3F standard, etc.

[0069] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule capable of binding to an epitope of an antigen. Naturally occurring antibodies typically comprise a tetramer and are usually composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the isotypes IgG, IgA, IgM, IgD, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig molecules of the same class may be divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. For example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4 subtypes, and IgA may be divided into IgA1 and IgA2 subtypes. Light chains are divided into κ and λ chains by the constant region. The antibodies of the present invention may have any isotype. The choice of isotype is usually determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region kappa or lambda can be used. If necessary, the class of the antibody of the present invention can be converted using known methods. For example, the class of an initial IgG antibody of the present invention can be converted to an IgM antibody of the present invention. Note that class conversion techniques can also be used to convert an IgG subclass to another subclass, for example, IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be converted by isotype switching to, for example, an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, and if the C1q binding activity of the antibody of the present invention is reduced or eliminated, it can be used for various therapeutic purposes. In some embodiments, the antibody of the present invention is an IgM, IgG1, IgG2, IgG3, or IgG4 antibody. An antibody belongs to a particular isotype if its amino acid sequence is nearly identical to that of another isotype.

[0070] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, complete antibodies and antigen-binding fragments of antibodies.

[0071] A "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), and the heavy chain constant region is usually composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The variable regions of the heavy and light chains are typically responsible for antigen recognition, while the constant regions of the heavy and light chains can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain binding regions that interact with antigens. The VH and VL regions may be further subdivided into hypervariable regions (HVRs) called "complementarity-determining regions (CDRs)," with more conserved regions called "framework regions" (FRs) intervening between them. Each VH and VL is composed of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0072] The terms "complementarity determining region" or "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") refer to the regions of an antibody variable domain that form highly variable sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to an epitope of an antigen. Herein, the three CDRs of a heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of a light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0073] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different numbering schemes will be different. Therefore, when an antibody is defined using a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose so-called CDR boundaries differ from the specific CDR boundaries defined in the present invention by applying a different scheme (e.g., a different numbering scheme rule or combination).

[0074] The terms "monoantibody," "monoclonal antibody," or "monoclonal antibody composition" refer to a preparation of antibody molecules of a single molecular composition, derived from an essentially homogenous antibody population; i.e., the population comprising the individual antibodies is identical except for any naturally occurring mutations that may be present. A typical monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. In some embodiments, a monoclonal antibody may be composed of two or more Fab domains, thereby increasing its specificity for two or more targets. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0075] The present invention further includes "bispecific antibodies," in which the antibodies of the present invention are part of a bivalent or multivalent bispecific framework targeting more than one epitope (e.g., the second epitope can include an epitope of an active transport receptor, thereby providing the bispecific antibody with improved cell transport across biological barriers, such as the blood-brain barrier). Thus, in another embodiment, a monovalent Fab of an antibody of the present invention can be linked to a Fab or scfv targeting another, different protein to produce a bispecific antibody. Bispecific antibodies can have dual functions, for example, a therapeutic function conferred by the present invention and the ability to enhance transport across biological barriers, such as the blood-brain barrier, by binding to a receptor molecule. The terms "biantibody," "bifunctional antibody," "bispecific antibody," "bispecific antibody," or "BsAb" refer to an antibody having two distinct antigen-binding sites, capable of simultaneously binding to two target antigens and mediating another specialized function while simultaneously exerting its targeting function. The mediated specific function effector molecule may be a toxin, enzyme, cytokine, radionuclide, etc., and the two antigen-binding arms of the bispecific antibody may each be derived from Fab, Fv, ScFv, dSFv, etc.

[0076] The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody (which may be obtained, for example, by truncation, recombinantly, synthetically, etc.) that is capable of binding to an epitope. An antigen-binding fragment may comprise one, two, three, four, five, or all six CDR regions of such an antibody and may exhibit different specificities, affinities, or selectivities while still being capable of binding to the epitope. Preferably, the antigen-binding fragment comprises all six CDR domains of the antibody. An antigen-binding fragment of an antibody may be part of or comprises a single polypeptide chain (e.g., an scFv), or may be part of or comprises two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., a bibody, an Fab fragment, an F(ab')2 fragment, etc.).

[0077] Examples of antigen-binding fragments included in the present invention include: (a) Fab' or Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (b) F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge domain; (c) Fd fragment, which is composed of the VH and CH1 domains; (d) Fv fragment, which is composed of the VL and VH domains of one arm of an antibody; (e) single-chain antibody (single chain Fv, scFv), which is a recombinant protein in which antibody VH and VL are linked by a connecting peptide segment by genetic engineering techniques; (f) dAb fragment (Ward et al., Nature, 341, 544-546 (1989)), which is essentially composed of the VH domain and is also called domain antibody (Holt et al., Trends Biotechnol., 2i(ll):484-90); (g) camelid antibody or nanobody (Revets et al., Expert Opin Biol. Ther., 5(l):111-24) and (h) isolated complementarity determining regions (CDRs).

[0078] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are single-chain antibodies. In some embodiments, the present invention provides single-chain Fvs (scFvs), in which the heavy and light chains in the Fv of an antibody of the present invention are linked by a flexible peptide (typically about 10, 12, 15, or more amino acid residues) to form a single peptide chain. Methods for producing such antibodies are described, for example, in U.S. Pat. No. 4,946,778; Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer-Verlag, New York, pp. 269-315 (1994); Bird et al., Science, 242, 423-426 (1988); Huston et al., PNAS USA 85, 5879-5883 (1988); and McCafferty et al., Nature, 348, 552-554 (1990). Single-chain antibodies are monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

[0079] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are chimeric antibodies. The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such an antibody, so long as the desired biological activity is exhibited. The present invention provides variable region antigen-binding sequences derived from human antibodies. Therefore, the chimeric antibodies primarily focused on herein include antibodies having one or more human antigen-binding sequences (e.g., CDRs) and one or more sequences derived from a non-human antibody, such as FR or C region sequences. Note that the chimeric antibody described herein refers to an antibody containing human variable region antigen-binding sequences of one antibody class or subclass and other sequences, such as FR or C region sequences, derived from another antibody class or subclass.

[0080] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized antibodies. The term "humanized antibody" refers to an antibody in which CDR sequences from another mammalian species, such as a mouse species, have been grafted onto human framework sequences, in which additional framework region modifications can be made.

[0081] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are human or fully human antibodies. The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") includes antibodies having variable and constant regions derived from human species-based immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human species-based immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or during gene rearrangement, or by somatic mutation in vivo).

[0082] Mutant antibodies are also included within the scope of the present invention. Therefore, variants of the sequences listed herein are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art, and these variants are also included within the scope of the present invention. For example, amino acid substitution can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency of expression systems for antibody production.

[0083] The sequences of such variant antibodies have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed herein, where such sequence identity is calculated over the full length of the reference sequence (i.e., the sequence listed herein).

[0084] The amino acid residues herein are numbered according to IMGT (registered trademark) (the international ImMunoGeneTics information system) (registered trademark) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, United States Department of Health and Human Services; Chothia, C. & Lesk, AM, (1987), Canonical Structures For The Hypervariable Domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the amino acid residues herein are numbered according to the Kabat numbering system.

[0085] An antibody or antigen-binding fragment thereof "specifically" binds to a region of another molecule (i.e., an epitope) if it reacts or binds to that epitope more frequently, more rapidly, with a longer duration, and / or with greater affinity than it does to other epitopes. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least 10 -7 M, e.g. 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody or antigen-binding fragment thereof binds to human amyloid protein, particularly its toxic form, with an affinity of M or greater. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specific binding to amyloid protein, particularly its toxic form, refers to the antibody or antigen-binding fragment thereof's ability to bind to amyloid protein, particularly its toxic form, with the above-mentioned specificity and / or under such conditions. Suitable methods for determining such binding are known in the art.

[0086] In the context of antibody binding to a designated antigen, the term "binding" typically refers to binding of an antibody to a designated antigen within about 10 -6M or lower K D This means that the molecule binds with an affinity corresponding to K D is at least 10-fold, such as at least 100-fold, at least 1,000-fold lower than the affinity of the antibody's binding to a nonspecific antigen other than the designated antigen or a closely related antigen (e.g., BSA, casein).

[0087] As used herein, the term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value. As used herein, the term "k a " (M-1 x sec-1 or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction.

[0088] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and k d k a This is obtained by dividing by As used herein, the term "K A " (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction, and k a k d This is obtained by dividing by

[0089] Antibodies of the present invention can be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, used alone or in combination. Typically, the amino acid sequence of the desired sequence is known, and one of skill in the art can readily produce the antibody using standard techniques for producing polypeptides. For example, these antibodies can be synthesized by known solid-phase methods, preferably using commercially available peptide synthesizers (e.g., those manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, a DNA sequence encoding the antibody can be incorporated into an expression vector and the vector introduced into a suitable eukaryotic or prokaryotic host for expression of the desired antibody, resulting in the antibody as a DNA expression product, which can then be isolated from the host using known techniques.

[0090] The antibodies and antigen-binding fragments thereof of the present invention can be modified by including any "suitable" number of modified amino acids and / or by coupling with coupling substituents. In such cases, "suitable" is typically determined by the ability to at least essentially retain amyloid protein, particularly its toxic form, selectivity and / or specificity relative to the underivatized parent antibody. Inclusion of one or more modified amino acids can contribute, for example, to an increase in polypeptide serum half-life, a decrease in polypeptide antigenicity, or improved polypeptide storage stability. Modifications to one or more amino acids can be made, for example, co-translationally during recombinant production, or post-translationally (e.g., N-linked glycosylation at NXS / T motifs during mammalian cell expression), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenylated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotin-acylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References for performing amino acid modifications are well known in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. Modified amino acids may be selected from, for example, glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotin-acylated amino acids, amino acids conjugated to a lipid moiety, or amino acids conjugated to an organic derivatizing agent.

[0091] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent conjugation to a polymer to increase their circulating half-life. Exemplary polymers and methods for linking them to peptides are described in, e.g., U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. Exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000-40,000 D, e.g., about 2,000-20,000 D, e.g., about 3,000-12,000 D).

[0092] The antibodies and antigen-binding fragments thereof of the present invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13, the contents of which are incorporated herein by reference).

[0093] Antibodies of the invention and culture medium are suitably isolated by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0094] The term "subject" refers to a warm-blooded animal, preferably a mammal (human, domestic and farm animals, zoo animals, sporting or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), more preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting admission, receiving medical care, or the subject of a medical program or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is elderly, over 55, 60, 65, 70, 75, 80, 85, 90 years of age, or older. In one embodiment, the subject is male. In another embodiment, the subject is female.

[0095] The term "healthy subject" refers to a subject who is not suffering from any disease or condition or who is not suffering from any neurodegenerative disease. The term "Alzheimer's disease-free subject" refers to a subject who has been confirmed as free of Alzheimer's disease through a formal medical program, e.g., a subject who does not have mild cognitive impairment (MCI) or AD dementia, as determined based on criteria established by the National Institute on Aging and the Alzheimer's Disease Association. In some embodiments, a subject who does not have Alzheimer's disease is a healthy subject.

[0096] The following examples are provided to illustrate the technical solutions of the present invention, but these examples are illustrative and illustrative and are not limiting. Unless otherwise noted, all reagents used in the following examples can be easily purchased from reagent companies such as Sigma-Aldrich and Merck. Unless otherwise noted, the test methods can be found in textbooks such as Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

[0097] Example Example 1: Obtaining Aβ oligomers that specifically bind to 3F 1.1 Experimental materials and methods 1.1.1 Experimental materials Protein A magnetic beads: Bio-RAD, #1614833. Protein G magnetic beads: Bio-RAD, #1614023. Protein A / G agarose gel: Abmart, #A10001S. 6E10 antibody: Biolegend, #803002. Human-derived Aβ42 detection kit: Immuno-Biological Laboratories.

[0098] 1.1.2 Main solutions (1) 50 mM sodium hydroxide (pH 10.0). (2) 20 mM phosphate buffer solution (PBS): pH 7.4. (3) 0.1% PBST: PBS containing 0.1% Tween-20. (4) 20 mM glycine eluate (pH 2.0). (5) 1M Tris neutralization solution: pH 10.0. Note: Relevant reagents and solutions mentioned in Examples 2, 3, 4, 5 and 6, but whose origin is not indicated, are the same as in Example 1.

[0099] 1.2 Preparation of mouse brain homogenate APP / PS1 mice were deeply anesthetized with pentobarbital sodium and injected intracardially with ice-cold PBS containing 10 U / mL heparin. The mice were then sacrificed and their brains were removed. The brain tissue was then homogenized in 1 mL of RIPA strong lysis agent (containing protease and phosphatase inhibitors) using a Tissue Lyser II tissue homogenizer at 30 Hz for 8 min, then centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected. Prior to immunoprecipitation, the brain homogenate was loaded onto 100 μL of protein A / G-agarose gel and incubated at 4°C for 1 h to remove endogenous IgG. The brain homogenate was then loaded onto protein A magnetic beads cross-linked with an APP antibody to remove endogenous APP.

[0100] 1.3 Cross-linking of antibodies with magnetic beads First, the 3F and 6E10 antibodies were cross-linked to protein A or protein G magnetic beads, respectively. The specific cross-linking steps are as follows: (1) 200 μL of Protein A or Protein G magnetic beads were taken and washed three times with 2 mL of 0.1% PBST, with the magnetic beads being thoroughly mixed each time to homogenize them, and then placed on a magnetic shelf. The supernatant was discarded. (2) 50 μg of 3F or 6E10 antibody was added to the magnetic beads, and the mixture was shaken at room temperature for 30 minutes to allow the magnetic beads and antibody to bind sufficiently, after which the mixture was washed three times with 0.1% PBST. (3) The magnetic beads were washed once with crosslinking buffer (0.2 M triethanolamine, pH 8.2). Then, 12 mg of DMP was dissolved in 2 mL of crosslinking buffer, added to the magnetic beads, and incubated at room temperature for 1 hour. After the reaction was complete, the crosslinking solution was discarded, and the beads were washed once with 2 mL of blocking buffer (0.1 M ethanolamine, pH 8.2). Then, another 2 mL of blocking buffer was added and the beads were incubated at room temperature for 2 hours. (4) After blocking, the magnetic beads were washed three times with PBS, then washed once with 0.1 M glycine (pH 2.5), and allowed to react for 5 minutes. They were then washed three times with 0.1% PBST, immersed in PBST containing 0.02% NaN3, and stored at 4°C.

[0101] 1.4 In vitro production of Aβo*3F One mg of Aβ42, Aβ40, or other forms of Aβ (Naka Peptide Biochemicals, Inc.) was dissolved in 1 mL of 100% hexafluoroisopropanol (HFIP), vortexed for 5 min, sonicated in a water bath for 10 min, dispensed into EP tubes, allowed to evaporate overnight, and stored at -20°C. Before use, the HFIP-treated Aβ aliquot was dissolved in 50 mM NaOH at a concentration of 1 mg / mL, vortexed for 3–5 min, sonicated for 1 min, and then diluted to 10 μM with pre-chilled PBS. The solution was centrifuged at 21,000 xg for 30–40 min at 4°C, and the precipitate (approximately 5% of the initial volume) was discarded to obtain Aβ monomers. The Aβ monomers were incubated at 25°C for 2 days under static conditions and then incubated overnight at 4°C with protein A magnetic beads cross-linked with 3F antibody. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20-100 mM glycine (pH 2.0) for 3-5 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (in vitro produced Aβo*3F).

[0102] 1.5 Isolation and preparation of Aβo*3F in APP / PS1 mouse brain homogenates and CSF of AD patients To prepare Aβo*3F specifically recognized by 3F (Aβo*3F), APP / PS1 mouse brain homogenate and CSF samples from AD patients (derived from the First Affiliated Hospital of Zhengzhou University, with signed informed consent and approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University) were incubated overnight at 4 °C with Protein A magnetic beads crosslinked with 3F antibodies. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F from APP / PS1 mouse brain (mAβo*3F, Aβo*3F isolated from AD mouse brain) or Aβo*3F extracted from AD patient CSF (hAβo*3F, Aβo*3F isolated from AD patient cerebrospinal fluid). The mixtures of Aβ aggregates after 3F immunodepletion, called Aβ-ID, were used as controls: sAβ-ID (produced in vitro), mAβ-ID (produced by isolation from the brains of APP / PS1 mice), and hAβ-ID (produced by isolation from CSF of AD patients).

[0103] Aβ*6E10, a mixture of Aβ aggregates, was prepared by mixing APP / PS1 mouse brain homogenate or AD patient CSF with protein G magnetic beads cross-linked with 6E10 antibody and incubating overnight at 4°C. The mixture was then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 to 5 min (specifically, 3 min). This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβ*6E10 from APP / PS1 mouse brain (mAβ*6E10, Aβ*6E10 isolated from AD mouse brain) or Aβ*6E10 extracted from AD patient CSF (hAβ*6E10, Aβ*6E10 isolated from AD patient CSF) (Figure 1). The concentration of Aβ obtained by immunoprecipitation was measured using an Aβ detection kit.

[0104] Example 2 Characterization of the molecular weight and morphology of Aβo*3F 2.1 Experimental materials and methods 2.1.1 Experimental materials Aβ polypeptide: Nakapeptide Biochemical Co., Ltd. Superdex 200 10 / 300 GL molecular sieve: GE Healthcare. A11 antibody: Invitrogen, #AHB0052. Molecular sieve protein marker: GE healthcare. 3–8% Tris-Acetate precast gels: Invitrogen. Copper mesh with carbon support film: Beijing Zhongke Kegyi Co., Ltd. Uranyl acetate: Beijing Zhongkeke Yi Co., Ltd. ECL Chemiluminescence Kit: Pierce Biotechnology. ThT detection reagent: Sigma-Aldrich. Nitrocellulose membrane: Millipore.

[0105] 2.1.2 Experimental equipment AKTA Protein Chromatography: GE Healthcare Life Science, USA. Protein electrophoresis equipment: Bio-rad, USA. Protein membrane transfer system: Bio-rad, USA. HT7700 transmission electron microscope: Hitachi, Japan. Safire2 TM Microplate reader: Tecan Group, Switzerland. Amersham Imager 680 imaging system: GE, USA.

[0106] 2.1.3 Main solutions (1) 20 mM TBS buffer solution: 2.4 g of Tris and 8 g of sodium chloride were weighed and dissolved in deionized water, and the pH was adjusted to 7.5 with dilute hydrochloric acid, and the solution was made up to 1 L. (2) 0.1% TBST: 1 mL of Tween-20 was added to 1 L of TBS and stirred for 20 min. (3) ThT solution: A 100X ThT stock solution (500 μm) was prepared in 50 mM PB (phosphate buffer, pH = 6.5) solution, and diluted to 1X with 50 mM PB (pH = 6.5) before use. (4) 5% skim milk. (5) Electrophoresis buffer: 30 g of Tris, 144 g of glycine, and 10 g of SDS were dissolved, and the solution was diluted to 1 L with deionized water and diluted 10 times before use. (6) 5x non-reducing loading buffer (10 mL): 2 mL of 10% SDS, 0.6 mL of 1 M Tris-HCl (pH 6.8), 5 mL of glycerin, and 1 mL of 1% bromophenol blue were taken and made up to 10 mL with deionized water. (7) Membrane transfer buffer: 30 g of Tris and 144 g of glycine were dissolved in deionized water, made up to 1 L, and diluted 10-fold before use.

[0107] 2.2 Preparation and characterization of Aβ monomers, oligomers, and fibrils Aβ monomers, oligomers, and fibrils were prepared as described in 1.4. The Aβ monomers were incubated at 25°C for 0-4 days under static conditions, and the Aβ aggregation state was detected every 24 hours using ThT. The specific procedure is as follows: Aβ samples were diluted to 10 μM with PBS buffer, and then 190 μL of ThT detection solution and 10 μL of test sample were thoroughly mixed in a black ELISA plate to homogenize, and then the plate was subjected to Safire 2 TM Fluorescence was measured using a microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. As can be seen from the results, with increasing incubation time, the ThT readings of the Aβ samples gradually increased, indicating the gradual aggregation of Aβ monomers into oligomers and fibrils (Figure 2A).

[0108] Dot blot experiments were used to evaluate the binding of different antibodies to Aβ monomers and aggregates. Aβ samples were spotted onto nitrocellulose membranes and blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with different detection antibodies for 1–2 h at room temperature, then washed three times with 0.1% TBST for 5 min each, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After three washes with 0.1% TBST for 5 min each, ECL chemiluminescence solution was added to the membranes, and the staining was developed using an Amersham Imager 680 imaging system. Quantitative analysis of blot size and optical density was performed using Image J software. The results show that 6E10 is an antibody that broadly binds to Aβ monomers, oligomers, and fibrils, and can bind to 10 μM and 20 μM Aβ without significant differences in binding. Neither 3F nor A11 antibodies bound to Aβ monomers. Furthermore, they bound to different types of oligomers. The 3F antibody primarily bound to 10 μM Aβos, whereas the A11 antibody primarily bound to 20 μM Aβos. This indicates that the 3F and A11 antibodies recognize oligomers with different conformations (Figure 2B). Furthermore, statistical analysis of the affinity of 3F for 10 μM Aβos obtained after 0–4 days of incubation revealed that the affinity of 3F for Aβos obtained after 2 days of incubation at 10 μM (sAβos, in vitro-produced Aβos) was highest. Therefore, in subsequent experiments, we immunoprecipitated this oligomer (sAβo*3F, in vitro-produced Aβo*3F) with 3F (Figure 2C).

[0109] 2.3 Molecular weight characterization of sAβo*3F First, the molecular weight of sAβo*3F was detected by Western blotting. The specific procedure is as follows: Aβ samples were loaded onto a 15% SDS-PAGE gel or a 3-8% Tris-Acetate gel. For Tris-Acetate gels, samples were loaded onto Novex gels before loading. TMThe mixture was mixed with Tris-glycine sample buffer. Electrophoresis was performed in Tris-glycine electrophoresis buffer (containing 0.5 mM DTT, 1 mM ATP, and 5 mM MgCl2) at 4°C for 4 hours at 150 V. For Western blot analysis, proteins separated by SDS-PAGE or Tris-Acetate gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1–2 hours at room temperature, and then incubated overnight at 4°C with a detection antibody (6E10). The membrane was then washed three times for 5 minutes with 0.1% TBST and incubated with the corresponding secondary antibody (HRP-conjugated goat anti-mouse secondary antibody) for 1 hour at room temperature. After three washes with TBST, the membrane was covered with ECL developer and developed using an Amersham Imager 680 imaging system. Protein bands were quantified using Image J software.

[0110] As can be seen from the SDS-PAGE experiment, sAβo*3F is mainly composed of two types of oligomers with molecular weights of approximately 12 kDa and the other with molecular weights greater than 180 kDa. As can be seen from the native-PAGE results, sAβo*3F is mainly composed of oligomers with molecular weights of approximately 500 kDa. The difference in the electrophoretic results between the two types may be due to the influence of SDS (Figure 2D).

[0111] To further characterize the molecular weight of sAβo*3F, 500 μL of sAβo*3F or 100 μL of a SEC marker was loaded onto a Superdex 200 10 / 300 GL molecular sieve column connected to an AKTA pure system. The column was pre-equilibrated with Tris-Gly buffer and eluted at a flow rate of 0.5 mL / min. Comparison with the marker revealed that the molecular weight of sAβo*3F was 588 kDa (Figure 2E and F).

[0112] 2.4 Molecular weight and morphology characterization of mAβo*3F Aβo*3F was extracted from the brains of APP / PS1 mice (mAβo*3F, Aβo*3F isolated from AD mouse brains) and its molecular weight was analyzed. Consistent with the results for sAβo*3F, mAβo*3F showed two bands on SDS-PAGE, with molecular weights of 12 kDa and over 180 kDa, respectively. However, on native-PAGE, mAβo*3F showed only a single band with a molecular weight of approximately 500 kDa (Figure 2G). SEC analysis revealed that the molecular weight of mAβo*3F was 588 kDa (Figure 2H). Additionally, 10 μL of Aβo*3F was dropped onto a 200-mesh copper mesh and allowed to adsorb for 20 min, dried on filter paper, and negatively stained with 2% uranyl acetate for 30 s. The mesh was then dried on filter paper and air-dried. The samples were examined by a transmission electron microscope (TEM, Hitachi H7700, Japan) at an operating voltage of 120 kV and a magnification of 100,000. As can be seen from the results, mAβo*3F was a particle with a diameter of approximately 10 nm (Figure 2H).

[0113] 2.5 Molecular weight characterization of Aβo*3F in CSF of AD patients The molecular weight and distribution of Aβo*3F extracted from the CSF of AD patients (hAβo*3F, Aβo*3F isolated from the cerebrospinal fluid of AD patients) were consistent with the results for sAβo*3F and mAβo*3F. hAβo*3F showed only one band with a molecular weight of approximately 500 kDa in native-PAGE, but showed two bands with molecular weights of 12 kDa and over 180 kDa in SDS-PAGE (Figure 2, Panel I).

[0114] Example 3: Detection of Aβo*3F neurotoxicity 3.1 Experimental materials N2a cells: National Experimental Cell Resources Sharing Service Platform (NICR). Poly-D-lysine hydrobromide (PDL): Sigma-Aldrich. B27 Supplement (50x): Thermo Fisher Scientific. Neurobasal TM -A medium: Gibco. 70 μm cell nylon screen: BD Bioscience.

[0115] 3.2 Primary neuron culture The brains of C57BL / 6 (purchased from Beijing Huafukang Biotechnology Co., Ltd.) mouse fetuses (14-15 days pregnant) were removed, and the meninges were peeled off with HBSS (Hank's Balanced Salt Solution). The isolated hippocampus and cortex were finely minced with tweezers. The crushed tissue mass was transferred to a 15 mL centrifuge tube and centrifuged at 600 rpm for 3 min. 10 mL of 10-fold diluted trypsin digestion solution (containing DNase I) was added to the pellet and digested at 37 °C for 10 min, followed by 5 min. After 10 min, the cells were collected by centrifugation at 1200 rpm for 10 min, and then resuspended in DMEM medium and plated onto a 12-well plate. After 2–4 h, the medium was replaced with Neurobasal medium (containing B27, 0.5 mM L-glutamine, 0.5% penicillin and streptomycin). The culture was continued for 7–9 days before use in subsequent experiments.

[0116] 3.3 Detection of sAβo*3F cytotoxicity This experiment primarily utilized the MTT assay to detect the toxicity of Aβ aggregates to neurons. The specific procedure was as follows: N2a cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were digested with trypsin, centrifuged, resuspended in medium, and then seeded into a 96-well plate at approximately 5,000 cells per well in 100 μL of medium. After 12 h, the cells were treated with a gradient series of sAβo*3F and sAβos. At the same time, an equal volume of solvent was added to the cells as a control. After 72 h, 25 μL of 5 mg / mL MTT was added to each well. After 3 h of incubation at 37°C, the medium was aspirated and 150 μL of DMSO was added. The absorbance at 570 nm and 630 nm was measured using an MD-M5 microplate reader. The average of six wells was used for each sample and control, and each experiment was repeated three times. Cell vitality was calculated by dividing the absorbance (background corrected) of the sample-added wells by the absorbance (background corrected) of the solvent-added wells.

[0117] As can be seen from the MTT results, the 50% inhibitory concentration (IC50) of sAβo*3F on cell viability was approximately 0.186 nM, whereas the IC50 of sAβos before immunoprecipitation was approximately 63.26 nM, a 340-fold difference between the two (Figure 3A and B). Furthermore, when 0.5 nM of sAβo*3F was added to N2a cells, it induced 66% cell death, whereas the same concentrations of sAβos and sAβ-ID had no significant neurotoxic effects. When sAβos reached 200 nM, it induced 70% cytotoxicity, whereas the neurotoxicity of sAβ-ID at this time was only 32% (Figure 3C). These results demonstrate that sAβo*3F is a potent oligomer with neurotoxicity.

[0118] 3.4 Detection of mAβo*3F cytotoxicity The IC50 of mAβo*3F for N2a cells was approximately 0.111 nM, as detected by the MTT assay, and the IC50 for primary neuronal cells was approximately 0.057 nM (Figure 3D). Specifically, 0.5 nM of mAβo*3F was able to induce 78% cell death in primary neurons, whereas the same concentrations of mAβ*6E10 and mAβ-ID had no obvious cytotoxicity. When the concentration was increased to 200 nM, mAβ*6E10 was able to induce 54% neurotoxicity, whereas the neurotoxicity of mAβ-ID was only 20% (Figure 3E).

[0119] 3.5 Detection of cytotoxicity of hAβo*3F The IC50 of hAβo*3F on primary neurons was approximately 0.076 nM as detected by the MTT assay (Figure 3F). Specifically, when 0.2 nM of hAβo*3F was added to primary neurons, it was able to reduce cell vitality by 68%, whereas hAβ*6E10 and hAβ-ID had no significant effect on neuronal survival at this concentration. When the concentration was increased to 40 nM, hAβ*6E10 reduced cell vitality by 66%, whereas hAβ-ID reduced cell vitality by only 40% (Figure 3G).

[0120] Example 4 Effect of Aβo*3F on the expression levels of cytokines in glial cells 4.1 Experimental materials and methods 4.1.1 Experimental materials BV2 cells: NICR. Reverse transcription kit: Kangwei Century Co., Ltd. UltraSYBR Mixture (Low ROX): Kangwei Century Co., Ltd. 4.1.2 Experimental equipment 7500 Fast real-time quantitative PCR instrument: Applied Biosystems. T100 Thermal Cycler PCR Instrument: BIO-RAD. NanoDrop Microvolume Spectrophotometer: Quawell.

[0121] 4.2 Aβo*3F can increase the expression levels of inflammatory cytokines in microglia. BV2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were digested with trypsin, centrifuged, resuspended in the medium, and then plated at approximately 5 × 10 cells per well in a 12-well plate. 5 The cells were seeded at 1000 cells / mL in medium. After 12 h, BV2 cells were treated with different Aβ samples; at the same time, the same volume of solvent was added to the cells as a control. After 48 h, the expression levels of intracellular proinflammatory cytokines (proinflammatory factors) were detected by fluorescent quantitative PCR (qPCR). The specific procedures were as follows: Total cellular mRNA was prepared using standard methods, and the mRNA concentration was measured using a microspectrophotometer. After reverse transcription according to the reverse transcription kit's instructions to obtain cDNA, the expression levels of the target genes were detected using EasyQuick RT MasterMix. The primers used were as follows:

[0122] mTNF-α:5'-GATTATGGCTCAGGGTCCAA-3' (SEQ ID NO:1), 5'-GCTCCAGTGAATTCGGAAAG-3(SEQ ID NO:2); mIL-1β:5'-CCCAAGCAATACCCAAAGAA-3' (SEQ ID NO:3), 5'-GCTTGTGCTCTGCTTGTGAG-3'(SEQ ID NO:4); mIL-6:5'-CCGGAGAGGAGACTTCACAG-3' (SEQ ID NO:5), 5'-TTGCCATTGCACAACTCTTT-3'(SEQ ID NO:6); GAPDH:5'-TGAATACGGCTACAGCAACA-3'(SEQ ID NO:7), 5'-AGGCCCCTCCTGTTATTATG-3' (SEQ ID NO:8).

[0123] As can be seen from the qPCR results, 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID were able to induce a significant increase in the expression levels of microglial inflammatory cytokines, including TNF-α, IL-6, and IL-1β, while 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID showed no obvious stimulatory effect (Figure 4A).

[0124] 4.3 Primary astrocyte culture One to two day old C57BL / 6 mouse neonates were taken and disinfected by immersion in 75% alcohol. The cerebrum was then removed and immersed in HBSS buffer. The meninges were peeled off and the cerebrum was finely cut with tissue scissors. The cerebrum was placed in a 15 mL centrifuge tube and centrifuged at 600 rpm for 3 minutes. 10 mL of 10-fold diluted trypsin digestion solution (containing DNase I) was added to the pellet and incubated at 37°C. The cells were digested for 10 min, gently inverting several times every 5 min. After digestion, the trypsin digestion reaction was stopped with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell strainer to remove undigested tissue clumps. The cells were collected by centrifugation at 1200 rpm for 10 min, resuspended in DMEM medium, plated into T-75 culture bottles, and cultured at 37°C in DMEM medium for 5–7 days. The medium was changed every 2–3 days. After 7 days of culture, the cells were digested with trypsin and plated into 12-well plates for use.

[0125] 4.4 Aβo*3F can stimulate the expression of inflammatory cytokines in astrocytes. Primary astrocytes were treated with different Aβ samples and their effects on the expression levels of inflammatory cytokines in primary astrocytes were examined by qPCR. The specific procedure was the same as in 4.2. The primers used were as follows:

[0126] miNos:5'-CACCTGGAACAGCACTCTCT-3'(SEQ ID NO:9), 5'-CTTTGTGCGAAGTGTCAGTG-3'(SEQ ID NO:10); mTNF-α: SEQ ID NO: 1 and 2; mIL-1β: SEQ ID NO: 3 and 4; mIL-6: SEQ ID NO: 5 and 6; GAPDH: SEQ ID NO:7 and 8.

[0127] As can be seen from the results, 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID could significantly enhance the expression levels of inflammatory cytokines, including TNF-α, IL-6, IL-1β, and iNos, in primary astrocytes, whereas 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no obvious effect on the expression levels of inflammatory cytokines in astrocytes (Figure 4B).

[0128] 4.5 Aβo*3F disrupts astrocyte-mediated synaptogenesis. Astrocytes can induce synapse formation by secreting synaptic factors such as TSP1 and Gpc4 / 6. mAβo*3F was added to primary astrocytes, and changes in the expression levels of several synaptic factors in astrocytes were detected by qPCR. The primers used were as follows:

[0129] mGpc4:5'-CTGGAGGGTCCTTTCAACATT-3' (SEQ ID NO:11), 5'-GACATCAGTAACCAGTCGGTC-3'(SEQ ID NO:12); mGpc6:5'-TAGTCCTGTATTGGCAGCCAC-3' (SEQ ID NO:13), 5'-GGCTAATGTCTATAGCAGGGAA-3'(SEQ ID NO:14); mTSP1:5'-GGTAGCTGGAAATGTGGTGCGT-3' (SEQ ID NO:15), 5'-GCACCGATGTTCTCCGTTGTGA-3'(SEQ ID NO:16); GAPDH: SEQ ID NO:7 and 8.

[0130] As can be seen from the results, treatment of cells with 0.3 nM mAβo*3F and 200 nM mAβ*6E10 significantly reduced the expression levels of TSP1 and Gpc4 / 6, whereas treatment with 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no obvious effect. Only increasing the concentration of mAβ-ID up to 200 nM was able to reduce the expression level of Gpc4 in astrocytes (Figure 4C).

[0131] Example 5: Effects of Aβo*3F on cognition in mice and damaging effects on neurons in the brain 5.1 Experimental materials and methods 5.1.1 Experimental materials Rapid Golgi staining kit: FD NeuroTechnologies. Ziehl-Neelsen staining kit: Solarbio. 5.1.2 Experimental equipment Brain stereotactic injection system: Chinese Academy of Medical Sciences. Y-maze equipment: Chinese Academy of Medical Sciences. Novel object recognition device: Chinese Academy of Medical Sciences.

[0132] 5.2 Experimental animals Three-month-old C57BL / 6 mice were housed in a clean room at 22±2°C and 45%±10% humidity, with free access to food and water and a 12-hour light / 12-hour dark cycle. All animal experiments were performed in accordance with the "Guidelines for the Care and Use of Laboratory Animals of the China Public Health Service." All mouse experiments were approved by the Tsinghua University Animal Care and Use Committee.

[0133] 5.3 Stereotactic injection into the brain Three-month-old C57BL / 6 mice were randomly divided into six groups of 6–8 mice each: one group injected with 2.5 nM (45.5 pg) mAβo*3F, one group injected with 2.5 nM (45.5 pg) mAβ*6E10, one group injected with 2.5 nM (45.5 pg) mAβ-ID, one group injected with 600 nM (10.9 ng) mAβ*6E10, one group injected with 600 nM (10.9 ng) mAβ-ID, and a control group injected with Tris-Gly vehicle. Mice were deeply anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) and then secured to a stereotaxic brain injection platform. The skin over the brain was incised along the midline to expose the skull. The injection site was positioned using a stereotaxic instrument at AP = -1.7 mm, ML = ±1.0 mm, and DV = -1.5 mm from the bregma. After a craniotomy along the stereotaxic point was performed with a skull drill, 4 μL of solution was injected into each half of the brain bilaterally at a rate of 0.4 mL / min. After the injection, the needle was left in place for 3 min to allow complete absorption of the sample. The surgical site was irrigated with sterile saline and the incision was sutured. After surgery, the mice were monitored and provided with postoperative care. 24 h after stereotaxic injection, behavioral and cognitive performance were assessed, and the mice were then dissected for physiological and biochemical analyses.

[0134] 5.4 Aβo*3F severely impairs memory ability in mice Novel object recognition experiments and Y-maze experiments are used to detect changes in cognitive abilities of mice.

[0135] 5.4.1 Novel object recognition experiment The novel object recognition experiment was designed based on the spontaneous tendency of mice to show more interaction with unfamiliar novel objects. The experiment was divided into three main stages. (1) During the familiarization phase, a white box measuring 40 cm wide x 40 cm deep x 40 cm high was prepared. When there was no object in the box, each mouse was allowed to freely explore the box for 5 min. After 1 h, the mice were injected with Aβ samples. (2) In the training phase, after 24 h, two identical objects were placed in the box, and each mouse was placed in the same box as in the first phase. Each mouse was allowed to freely explore the box for 5 min. The number of times the mouse sniffed or touched each object was recorded. (3) In the test phase, after 6 hours of training, the object on the right side was replaced with a new object that was different in material, color, and shape. Each mouse was allowed to explore freely for 5 minutes, and the number of times the mouse smelled and touched the two objects was recorded to detect the mouse's memory ability. The recognition index was calculated using the formula: (number of times for new object - number of times for old object) / (number of times for new object + number of times for old object). As can be seen from the results, the recognition index for novel objects of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group, whereas the recognition index for novel objects of mice in other Aβ-treated groups showed no significant difference compared to the control group, indicating that Aβo*3F significantly reduced the memory ability of mice (Figure 5A).

[0136] 5.4.2 Y-maze experiment After the novel object recognition experiment, the spatial memory ability of the mice was detected by a Y-maze experiment. The Y-maze consisted of three identical arms, with a 120-degree angle between each arm. The dimensions of each arm were 8 cm x 30 cm x 15 cm (width x length x height). The experiment was mainly divided into two stages. (1) In the training phase, three arms were randomly named A, B, and C. Arms A and B were open throughout the experimental phase, while arm C was closed as a new arm during the training phase. After each mouse entered arm A, it was allowed to freely explore arms A and B for 10 min. (2) After 1 h of training, the C arm was opened, and the mice were placed in the A arm again, one after the other. The mice were allowed to explore the A, B, and C arms for 5 min, and the time spent in the three arms was analyzed. All testing procedures were recorded by a camera installed above the Y-maze.

[0137] As can be seen from the results, the duration in the novel arm of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group. In contrast, there was no significant difference in the duration of the novel arm in mice injected with 2.5 nM mAβ*6E10, 2.5 nM mAβ*ID, or 600 nM mAβ*ID compared to the control group. mAβo*3F, but not mAβ*6E10 or mAβ-ID, was able to induce severe memory impairment in mice at low concentrations, whereas mAβ*6E10 was only able to induce similar memory impairment at concentrations up to 600 nM (Figure 5B).

[0138] 5.5 Aβo*3F significantly reduces dendritic spine density in mouse neurons Mouse brain samples were stained using the FD Rapid Golgi Stain Kit. 24 h before dissection, solutions A and B in the kit were mixed 1:1 and stored in a dark place. After sacrifice, the brain was removed without perfusion and divided along the midline into left and right hemispheres. Blood on the surface of the left hemisphere was washed with PBS, and the solution on the surface of the tissue was removed with filter paper. The tissue was then placed in the pre-prepared A+B solution. The solution was changed once after 24 h and left at room temperature for two weeks, protected from light. For best results, the tissue was gently inverted twice a week during the immersion period. After two weeks, the tissue was transferred to solution C, the solution was changed once after 24 h, and left at room temperature for five days, protected from light. Then, 100 μm-thick frozen sections were obtained using a freezing microtome at -22°C. The sections were allowed to air dry at room temperature before staining began. First, sections were washed twice with Milli-Q water for 4 min each. Then, a working staining solution was prepared. The sections were placed in the working staining solution for 15 min and then washed twice with Milli-Q water for 5 min each. After staining, sections were dehydrated in 50%, 75%, 95%, and 100% ethanol, each for 4 min. The sections were then placed in xylene, sealed with neutral resin, and observed. Dendrites and dendritic spines of neurons in the hippocampal CA1 region were observed using a 100x objective lens on an Olympus inverted microscope. Dendritic spine density was determined using Image J software analysis (dendritic density = number of dendritic spines / dendritic length).

[0139] We calculated the dendritic spine density and found that the dendritic spine density of neurons in the CA1 region of the brains of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group, whereas 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no obvious effect on the dendritic spine density of neurons in the brains of mice (Figure 5C, D).

[0140] 5.5 Aβo*3F significantly reduces the number of hippocampal neurons in mice 5.5.1 Paraffin embedding and sectioning After the behavioral experiments, mice were deeply anesthetized with pentobarbital sodium (50 mg / kg) and intracardially injected with ice-cold PBS containing heparin (10 U / mL). The mice were then sacrificed and dissected. The cerebrum was removed and separated into left and right hemispheres along the midline. The left hemisphere was immersed in 4% paraformaldehyde for 48 hours, then dehydrated in gradients of 50%, 70%, 80%, and 90% ethanol for 1 hour each. The tissue was then dehydrated once in 100% ethanol for 1 hour, followed by 50% xylene for 30 minutes. After dehydration, the tissue was embedded in paraffin for 4 hours. 5 μm tissue sections were obtained using a paraffin tissue slicer.

[0141] 5.5.1 Ziehl-Neelsen staining Five-micrometer paraffin sections were heated at 60°C for 30 minutes, then dewaxed by immersion in 100% xylene, 50% xylene, and 100% alcohol for 10 minutes each, followed by immersion in 75% ethanol, 50% ethanol, 30% ethanol, and deionized water for 5 minutes each. Following the instructions for Ziehl-Neelsen staining, sections were stained with cresol purple solution and heated at 56°C for 1 hour. After differentiation at room temperature for 1–3 minutes until the background was nearly colorless, sections were destained in graded alcohols (70%, 95%, and 100%; 20 seconds each). The sections were then immersed in xylene and sealed with neutral resin. Data were then collected and analyzed microscopically. As can be seen from the results, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly reduced the number of neurons in the CA1 and DG regions of the mouse brain, whereas 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no significant effect on the number of neurons (Figure 5E, F, G).

[0142] Example 6 Aβo*3F activates glial cells and induces neuroinflammation in the mouse brain 6.1 Experimental materials and methods 6.1.1 Experimental materials Anti-GFAP antibody: Cell Signaling Technology, #3670. Anti-Iba-1 antibody: GeneTex, #GTX101495. BCA kit: Thermo Fisher Scientific. RIPA strong lysis solution: Solarbio. Protease inhibitors: Millipore. Phosphatase inhibitor (100x): Solarbio. Mouse IL-1β detection kit: Biolegend. Mouse IL-6 detection kit: Biolegend.

[0143] 6.1.2 Experimental equipment MD-M5 microplate reader: Molecular Devices, USA. Tissue Lyser II tissue disrupter: Qiagen. 6.1.3 Immunohistochemistry After dewaxing, 5 μm paraffin sections were immersed in citrate antigen repair solution (3 g trisodium citrate and 0.4 g citric acid dissolved in deionized water and made up to 1 L), boiled in a water bath for 15 min, and then allowed to cool at room temperature. After antigen repair, the sections were washed three times with PBS for 5 min each, and then fixed in 80% (vol / vol) methanol containing 0.3% HO to remove endogenous catalase. After three 5-min washes with PBS, the sections were placed in 0.3% Triton X-100 permeabilization solution for 20 min at room temperature, and then washed three times with PBS for 5 min each. The sections were blocked with 10% donkey serum at room temperature for 1 h, then incubated overnight at 4°C with the corresponding primary antibody. On the second day, the sections were washed three times with PBS for 5 min each, and then the corresponding secondary antibody was added and incubated at room temperature for 1 h. After washing three times with PBS, the sections were developed and observed.

[0144] 6.2 Aβo*3F can activate microglia and astrocytes We stained for Iba-1, a marker of hippocampal microglia, and found that 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly activated microglia in the DG and CA1 regions of the hippocampus, increasing Iba-1 expression levels in microglia, but had no significant effect on mice treated with 2.5 nM mAβ*6E10 or mAβ-ID (Figure 6A, C, D). Furthermore, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly induced microglia in the DG region to adopt an amoeboid state, characterized by enlarged cell bodies and reduced branching, indicating that microglia were in an overactivated state (Figure 6A). We stained for GFAP, a marker of astrocytes in the mouse hippocampus, and found that intracerebral injection of 2.5 nM mAβo*3F and 600 nM mAβ*6E10 activated astrocytes in the hippocampal DG and CA1 regions and increased the expression level of hippocampal GFAP (Figure 6, B, E, F).

[0145] 6.3 Aβo*3F increases the levels of inflammatory cytokines in the hippocampus of mouse brains The levels of inflammatory cytokines (IL-6 and IL-1β) in mouse brains were detected using IL-6 and IL-1β ELISA kits, respectively, according to the manufacturer's instructions. Briefly, brain homogenates were diluted to the specified fold and added to ELISA plates. The plates were pre-coated with the corresponding capture antibodies, followed by the addition of the corresponding detection and secondary antibodies. TMB was used as the substrate, and the absorbance was measured at 450 nm using an MD-M5 microplate reader. Results showed that injection of 2.5 nM mAβo*3F, 600 nM mAβ*6E10, and 600 nM mAβ-ID significantly increased IL-1β and IL-6 levels in the hippocampus, whereas the brain levels of mice injected with 2.5 nM mAβ*6E10 or 2.5 nM mAβ-ID were not significantly different from those of the control group (Figure 6G, H).

[0146] Example 7 Detection of Aβo*3F levels in CSF and plasma of AD patients (MSD method) 7.1 Experimental materials and methods 7.1.1 Experimental materials Aβ Multifactorial Detection Kit (4G8): Meso Scale Diagnostics. 7.1.2 Experimental equipment MSD-S600 electrochemiluminescence detection instrument: Meso Scale Diagnostics, USA.

[0147] 7.2 Detection of Aβo*3F levels in plasma and CSF of AD patients To evaluate the correlation between Aβos (Aβo*3F), which is specifically recognized by 3F, and the pathogenesis of AD patients, we measured the levels of Aβ42 and Aβ40 contained in Aβo*3F isolated from CSF and plasma of AD patients.

[0148] Both the human plasma and CSF samples used in this study were approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University, and all subjects signed written informed consent before participating in the study. Basic information about the subject samples used in this study is as follows: Plasma samples were collected from 20 mild cognitive impairment (MCI) patients with a mean age of 73.3 years (age range 57-84 years), 20 AD patients with a mean age of 69.4 years (age range 52-85 years), and 20 healthy subjects with a mean age of 70.9 years (age range 65-78 years). Additionally, CSF samples were collected from seven AD patients with a mean age of 68 years (age range 59-77 years) and seven non-demented subjects with a mean age of 66.3 years (age range 48-79 years).

[0149] To extract Aβo*3F from plasma and CSF of AD patients and healthy elderly subjects, human plasma or CSF samples were first added to 100 μL of protein A / G-agarose gel and incubated at 4°C for 1 h to remove endogenous IgG. The sample and the protein A magnetic beads crosslinked with 3F antibodies were then incubated at room temperature for 2 h. On day 2, the magnetic beads were washed three times with 0.1% PBST and eluted with 20 mM glycine (pH 2.0) for 3 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was detected using the MSD Aβ Multifactor Detection Kit (4G8). The results show that the levels of Aβ42 and Aβ40 contained in Aβo*3F in CSF and plasma of AD patients were significantly increased compared to healthy elderly subjects (Figure 7). Furthermore, the levels of Aβ42 and Aβ40 contained in Aβo*3F in the plasma of MCI patients were significantly higher than those of controls, and these levels gradually increased with the progression of AD pathology (Figure 7C and 7D). These results demonstrate that Aβo*3F levels in the CSF and plasma of AD patients and AD-derived MCI patients are significantly higher than those of healthy elderly individuals and are closely correlated with the progression of AD pathology. Therefore, Aβo*3F can be used as a biomarker for AD diagnosis, especially for early diagnosis. Statistics were performed using GraphPad Prism 8 software, combining one-way ANOVA and Tukey's multiple comparison test. 95% confidence intervals were used. * indicates a statistically significant difference (P<0.05). ** indicates a highly statistically significant difference (P<0.01). *** indicates a highly statistically significant difference (P<0.001). **** indicates P<0.0001, indicating a highly statistically significant difference.

[0150] Example 8 Detection of Aβo*3F levels in plasma of AD and MCI patients (chemiluminescence method) 8.1 Experimental Materials and Methods 8.1.1 Experimental materials 102 Antibody: Beijing Rejing Biotechnology Co., Ltd. 8.1.2 Experimental equipment MG60 pro chemiluminescence detector: Beijing Thermal Viewing Biotechnology Co., Ltd., Beijing.

[0151] 8.2 Detection of Aβo*3F levels in plasma of AD patients To assess the relationship between Aβos (Aβo*3F), which is specifically recognized by 3F, and the pathogenesis of AD patients, we measured the levels of Aβ42 contained in Aβo*3F isolated from the plasma of AD patients.

[0152] Both the human plasma and CSF samples used in this study were approved by the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University, and all subjects signed written informed consent before participating in the study. Basic information about the subject samples used in this study is as follows: Plasma samples were collected from three AD patients with a mean age of 65.6 years (age range 62-75 years), three MCI patients with a mean age of 63.3 years (age range 62-65 years), and three healthy subjects with a mean age of 63.6 years (age range 61-65 years).

[0153] To extract Aβo*3F from the plasma of AD patients and healthy elderly subjects, human plasma samples were first added to 200 μL of protein A-agarose gel and incubated at room temperature for 15 min to remove endogenous IgG. The sample and JSR magnetic beads coated with 3F antibody were then incubated at room temperature for 2 h. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20 mM glycine (pH 2.2) for 5 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was detected using the 3F-102 detection kit. As can be seen from the results, the levels of Aβ42 contained in Aβo*3F in the plasma of AD patients were significantly higher than those in healthy elderly subjects (Figure 8). Furthermore, the levels of Aβ42 contained in Aβo*3F in the plasma of MCI patients were significantly higher than those in the control group (Figure 8). These results demonstrate that the plasma Aβo*3F levels of AD and MCI patients are significantly higher than those of healthy elderly individuals, and that Aβo*3F can be used as a biomarker for AD diagnosis, especially for the early diagnosis of AD.

[0154] Example 9 Diagnosis of AD by detecting the level of Aβo*3F in CSF or plasma The human plasma and CSF samples from Example 7 were shuffled and renumbered, and given to an unacquainted experimenter. Aβo*3F was enriched in the samples using protein A magnetic beads cross-linked with 3F antibody according to the method of Example 7. The levels of Aβ42 and Aβ40 contained in the Aβo*3F in the samples were detected using the MSD Aβ Multifactor Detection Kit (4G8). The results showed that the amounts of Aβ42 and Aβ40 contained in the Aβo*3F in the samples were positively correlated with AD pathology. This method was able to detect the levels of Aβo*3F in human plasma and CSF samples, and significantly distinguish between samples from AD and MCI patients and healthy controls (Figure 9).

[0155] Example 10 Optimization of antibodies for detecting Aβo*3F oligomers The 100th amino acid, K, of the 3F antibody was mutated to R (the 98th amino acid in the antibody sequence if the MA residue introduced for expression as a single-chain antibody is not taken into account; this mutant was designated the K98R mutant, and its sequence is shown in SEQ ID NO: 24), and the binding of the K98R mutant to Aβo*3F oligomers was examined. The method of use was as follows.

[0156] The above Aβ oligomers were coated onto a 96-well plate at 100 ng / well and left overnight at 4°C. The next day, the plate was blocked with 3% BSA for 2 hours at room temperature, washed twice with PBST, and then tapped dry. The K98R mutant and 3F were diluted 14 times starting from 1 μg / mL. The diluted antibody solution was added to the ELISA plate coated with the above Aβ oligomers, and the plate was incubated in parallel in multiple wells at 37°C for 1 hour, washed three times with PBST, and tapped dry. Goat anti-human IgG-HRP diluted 1:10,000 was added, and the plate was incubated for 45 minutes at 37°C, washed three times with PBST, and tapped dry. TMB color development solution was added for approximately 15 minutes, and the color was stopped with stop solution, followed by OD analysis. 450The data was read in nm and plotted and analyzed using graphpad.

[0157] The results are shown in Figure 10. The experiment shows that the binding affinity of the K98R mutant to Aβo*3F oligomers is further improved, and the higher binding strength of this mutant to Aβo*3F oligomers contributes to more sensitive detection of the presence of Aβo*3F oligomers in the subject's body, thereby enabling earlier diagnosis of whether the subject is suffering from a neurodegenerative disease. The present inventors investigated the cause of this result and found that the 98th amino acid is also a residue in the heavy chain CDR3 of the 3F antibody when numbered according to IMGT numbering (the heavy and light chain CDR sequences of the 3F antibody when numbered according to IMGT are CDR-H1:GFTFSSYA (SEQ ID NO:25), CDR-H2:ISNLGLTT (SEQ ID NO:26), CDR-H3:AKTTSRFDY (SEQ ID NO:27), CDR-L1:QSISSY (SEQ ID NO:28), CDR-L2:KAS, CDR-L3:QNSAVRPVT (SEQ ID NO:29) respectively). The present inventors also simultaneously performed the K98R mutation on the sequences of several other mutants and obtained similar results (data not shown).

[0158] Equivalent Scheme While various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of numerous other means and / or structures for achieving the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and all such variations and / or modifications are intended to be included within the scope of the present invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are exemplary, and that the actual parameters, materials, and / or settings will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalent schemes to the specific embodiments of the present invention described herein. Thus, it should be understood that the foregoing embodiments and examples are presented merely by way of example, and that the invention may be practiced otherwise than as specifically described and claimed, within the scope of the appended claims and their equivalents. Any combination of two or more such features, systems, articles, materials, and / or methods, if not mutually inconsistent, is within the scope of the present invention.

[0159] The term "and / or," as used in the present specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjoined in some cases but not in other cases. In addition to the elements specifically identified by the term "and / or," other elements may optionally be present, whether associated with those specifically identified elements or not, unless expressly indicated otherwise. Thus, as a non-limiting example, when used in conjunction with an open term such as "comprising," a reference to "A and / or B" can, in one embodiment, refer to A without B (optionally including elements other than B), in another embodiment, refer to B without A (optionally including elements other than A), in yet another embodiment, include both A and B (optionally including other elements), and so forth.

[0160] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or," as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but including more than one, including a plurality of elements and optionally other items not listed. When used in the claims, terms clearly indicating the contrary, such as "only one of" or "exactly one of," "consisting of" refers to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally, as used herein, the term "or" should only be understood as indicating an exclusive alternative scheme (i.e., "one or the other, but not both") when preceded by terms of exclusion, such as "either," "one of," "only one of," or "exactly one." "Consisting essentially of" when used in the claims should have its ordinary meaning in the field of patent law.

[0161] As used herein and in the claims, when referring to a list of one or more elements, the term "at least one" should be understood to mean at least one element selected from any one or more elements of the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the term "at least one" refers, may optionally be present, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B" or equivalently "at least one of A and / or B") can mean, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); etc.

[0162] In the claims and the above specification, all conjunctions such as "comprising," "including," "with," "comprises," "containing," "relating to," "having," etc. are understood to be open, i.e., to mean including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0163] The use of ordinal terms, such as "first," "second," and "third," in the claims to modify the claim elements themselves does not imply any priority, precedence, or order of one claim element relative to another claim element, or the chronological order in which actions are performed in a method, but is used solely as a tag to distinguish one claim element with a certain name from another element with the same name (although used in ordinal number terms).

Claims

1. a) optionally providing a sample to be detected from a subject; b) contacting a sample from a subject with a reagent that detects the presence and / or level of Aβo*3F in said sample from a subject; c) detecting the presence and / or level of Aβo*3F in a sample from said subject; The presence and / or level of Aβo*3F in the sample from the subject indicates that the subject suffers from Alzheimer's disease (AD) or mild cognitive impairment (MCI) resulting from AD, wherein the Aβo*3F is an Aβ oligomer that specifically binds to a 3F antibody, and has a molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC), and the light chain CDR sequences and heavy chain CDR sequences of the 3F antibody are set forth in SEQ ID NOs: 17-22, respectively. A method for diagnosing whether a subject has early, middle, or late stage Alzheimer's disease (AD) or mild cognitive impairment (MCI) resulting from AD, or whether a subject is at risk of developing AD.

2. The subject is a patient suspected of suffering from AD or MCI resulting from AD, and preferably the subject is a human, a non-human primate, a cat, or a dog. The method of claim 1.

3. The method further comprises the steps of evaluating the patient by clinical neuropsychology and neuroimaging, preferably the neuroimaging is Aβ-PET scanning imaging and / or tau-PET.

3. The method according to claim 1 or 2.

4. The method further comprises detecting an alteration in the level of tau in the subject's sample, wherein preferably the level of tau is the level of total tau or the level of phosphorylated tau.

4. The method according to any one of claims 1 to 3.

5. a reagent for detecting the presence and / or level of Aβo*3F in a sample from a subject; The Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and its molecular weight is approximately 588 kDa based on size exclusion chromatography analysis. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively. A kit for diagnosing whether a subject is suffering from AD or MCI resulting from AD, or whether a subject is at risk of suffering from AD.

6. Relating to or comprising an enrichment reagent for Aβo*3F, The enrichment reagent for Aβo*3F comprises a binding agent that specifically binds to Aβo*3F. Preferably, the binding agent that specifically binds to Aβo*3F is an antibody that specifically binds to Aβo*3F. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is a scFv, F(ab') 2 , Fab 2 , Fab, Fab', Fv, Fd, dAb, camelid antibody, nanobody, bibody or bispecific antibody The method of any one of claims 1 to 4 or the kit of claim 5.

7. The reagent for detecting the presence and / or level of Aβo*3F comprises a first binding agent that specifically binds to Aβo*3F or Aβ aggregates, preferably the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is an antibody that specifically binds to Aβo*3F or Aβ aggregates, preferably the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof, more preferably the antigen-binding fragment is an scFv, F(ab') 2 , Fab 2 , Fab, Fab', Fv, Fd, dAb, camelid antibody, nanobody, bibody or bispecific antibody 7. The method or kit of any one of claims 1 to 6.

8. The first binding agent binds to a detection agent that allows its detection.

8. The method or kit of claim 7.

9. The reagent for detecting the presence and / or level of Aβo*3F further comprises a second binding agent; The second binding agent specifically binds to the first binding agent, preferably the second binding agent is an antibody that specifically binds to the first binding agent, preferably the antibody that specifically binds to the first binding agent is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof, more preferably the antigen-binding fragment is an scFv, F(ab') 2 , Fab 2 , Fab, Fab', Fv, Fd, dAb, camelid antibody, nanobody, bibody or bispecific antibody, preferably the antibody that specifically binds to said first binding agent is conjugated to a detection agent that allows its detection.

8. The method or kit of claim 7.

10. The detection agent is selected from a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, umbelliferone, Lissamine, cyanine, Texas Red, BODIPY FL-SE® (Invitrogen) or an analog thereof, a paramagnetic label, a radioactive label, biotin, streptavidin / biotin, avidin / biotin, a hapten, digoxigenin, a metal complex, a metal, an enzyme, colloidal gold, or a combination thereof.

10. The method or kit according to claim 8 or 9.

11. The detection is selected from chemiluminescence, electrochemiluminescence, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunochromatography, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation, immunodiffusion, dot blot assay, Western blot, protein chip, positron emission tomography and / or single photon emission computed tomography, preferably the kit comprises reagents, materials, containers and / or equipment necessary to perform a detection selected from chemiluminescence, electrochemiluminescence, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, immunochromatography, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation, immunodiffusion, dot blot assay, Western blot and / or protein chip, preferably the enzyme-linked immunosorbent assay is selected from direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay and indirect sandwich enzyme-linked immunosorbent assay.

11. The method or kit of any one of claims 1 to 10.

12. The subject sample is selected from cells, tissues, organs and / or body fluids of the subject, preferably the body fluid is selected from whole blood, plasma, serum, cerebrospinal fluid, lymph, saliva, synovial fluid, bronchoalveolar lavage fluid, sputum, ascites, urine, amniotic fluid, peritoneal fluid, pericardial fluid, semen and / or vaginal secretions, preferably the body fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

12. A method or kit according to any one of claims 1 to 11.

13. The first binding agent that specifically binds Aβo*3F is attached to a solid support.

13. The method or kit of any one of claims 7 to 12.

14. The method further comprises detecting a control sample, and / or the kit further comprises a control sample, the control sample being derived from a healthy subject or a subject not affected by AD.

14. A method or kit according to any one of claims 1 to 13.

15. The antibody that specifically binds to Aβo*3F is a polyclonal antibody and / or a monoclonal antibody obtained by immunization with Aβo*3F, or an antigen-binding fragment thereof, and preferably, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a sheep antibody, or a non-human primate antibody.

15. The method or kit of any one of claims 6 to 14.

16. Based on the MSD electrochemiluminescence method, the concentrations of Aβo*3F in the cerebrospinal fluid of the healthy subject group were 80.44±20.88 pg / mL for Aβ42o*3F and 24.35±5.08 pg / mL for Aβ40o*3F, and / or the concentrations of Aβo*3F in the plasma were 71.63±36.8 pg / mL for Aβ42o*3F and 2.24±0.92 pg / mL for Aβ40o*3F, and the concentrations of Aβo*3F in the plasma of the mild cognitive impairment group were 1. and the concentrations of Aβo*3F in the cerebrospinal fluid of the AD patient group are Aβ42o*3F 264.8±42.26pg / mL and Aβ40o*3F 85.74±10.62pg / mL, and / or the concentrations of Aβo*3F in the plasma are Aβ42o*3F 159.44±36.8pg / mL and Aβ40o*3F 14.0±5.59pg / mL, or Based on the chemiluminescence method, the concentration of Aβ42o*3F in plasma of the healthy subject group was 68.02±39.17 pg / mL, the concentration of Aβ42o*3F in plasma of the mild cognitive impairment group was 124.5±12.57 pg / mL, and the concentration of Aβ42o*3F in plasma of the AD patient group was 205.75±50.96 pg / mL.

16. A method or kit according to any one of claims 1 to 15.

17. Based on MSD electrochemiluminescence and chemiluminescence methods, the sensitivity of the detection is as low as 0.5 pg / mL 17. A method or kit according to any one of claims 1 to 16.

18. The antibody that specifically binds to Aβo*3F is a plurality of types of antibodies that specifically bind to different epitopes of Aβo*3F, for example, two, three, four, five or more types of antibodies that each specifically bind to two, three, four, five or more different epitopes of Aβo*3F.

18. A method or kit according to any one of claims 7 to 17.

19. The step of enriching Aβo*3F from the sample is carried out using immunoprecipitation with an antibody that specifically binds to Aβo*3F.

7. The method or kit of claim 6.

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