Diagnostic use of highly toxic amyloid oligomers

Abstract

The present invention relates to the diagnostic use of highly toxic amyloid oligomers. In particular, the present invention relates to the use of the new highly toxic amyloid oligomer Aβo*3F as a target for the diagnosis of early and advanced Alzheimer's disease (AD) and AD-derived mild cognitive impairment (MCI), which is specifically bound by the 3F antibody, is present in the cerebrospinal fluid (CSF), blood and / or brain tissue of AD patients and AD-derived MCI patients and whose levels differ significantly in the CSF, blood and / or brain tissue of AD patients, MCI patients and healthy elderly, is a super-toxic oligomer, is the most dominant toxic component of the Aβ oligomer mixture and has a strong pathogenic effect, playing a key role in the development and progression of AD.

Description

TECHNICAL FIELD

[0001] The present application relates to the diagnostic use of highly toxic amyloid oligomers. In particular, the present application relates to the use of the new highly toxic amyloid oligomer Aβo*3F as a target for the diagnosis of early and late Alzheimer's disease (AD) and AD-derived mild cognitive impairment (MCI). BACKGROUND

[0002] Alzheimer's disease (AD, commonly known as senile dementia) is a chronic neurodegenerative disease, and there are currently about 500 million AD patients worldwide. So far, there is no specific and effective treatment drug and means, which has brought a heavy burden to human beings. The development of AD is a long process. At the beginning, the patient has no obvious clinical symptoms, and gradually develops memory loss, personality and behavior changes. In the later stage, the neurons in the brain of AD patients die extensively, and the brain atrophies significantly, making it difficult for treatment drugs to take effect. Clinical trials in recent years have shown that early intervention for AD patients who are found early and have early warning can delay the development of AD. To achieve early treatment and intervention for AD patients, the first step is to accurately identify and diagnose AD patients. However, there is no ideal early diagnosis method and technology currently applied in clinical practice. Studies have shown that the changes in β-amyloid protein (Aβ) that induce the occurrence and development of AD have appeared 15-20 years before the appearance of clinical symptoms.

[0003] The pathological features of AD are senile plaques formed by aggregation of Aβ and neurofibrillary tangles formed by aggregation of tau protein. Aβ can aggregate into oligomers, protofibrils and mature fibrils. Aβ oligomers are the most neurotoxic aggregate forms, and the most reported forms are Aβ dimers, trimers, ADDLs, etc. Aβ oligomers can be divided into low molecular weight and high molecular weight types according to the molecular weight, and the high molecular weight oligomers have greater neurotoxicity. Aβ dimers are the smallest Aβ oligomers, and it is generally believed that Aβ dimers may be the basic unit of Aβ oligomers. The content of Aβ dimers in the brain of AD patients and AD transgenic mice is increased, and Aβ dimers are stable in SDS and strong denaturants, and have certain neurotoxicity. In addition to Aβ dimers, Aβ trimers are also considered to be the aggregation unit of various Aβ oligomers such as hexamers and dodecamers, and Aβ trimers appear early in the brain of AD patients and AD transgenic mice, but there is no significant correlation between Aβ trimers and Aβ plaque deposition, and the toxicity of Aβ trimers is still controversial. In addition, Aβ oligomers can further aggregate into spherical oligomers (ASPD) with a diameter of about 12 nm and diffusible oligomers (ADDLs) with a diameter of 5-6 nm. Both of these two forms of polymers are considered to be Aβ oligomers with unique conformation and neurotoxicity. At present, although a variety of Aβ oligomer forms have been found, it is still unknown which Aβ oligomer or which Aβ oligomers have the greatest toxicity and play a major role in the occurrence and development of AD. Moreover, the 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 oligomers, which severely limits the development of clinical detection and therapeutic preparations targeting Aβ oligomers.

[0004] A large number of studies have shown that the severity of neurodegenerative diseases is closely related to the level of amyloid oligomers in the brain of patients, and Aβ oligomers play a key role in the occurrence and development of AD by causing functional neuronal death, cognitive impairment and dementia, but Aβ oligomers are diverse and can affect the function of the central nervous system through various mechanisms. The toxicity of various Aβ oligomers is different, and there are great differences in size, conformation, aggregation mode, toxicity and time of appearance in the brain. Although many studies have shown that some Aβ oligomers, such as dimers, trimers, ADDLs, etc. can exert neurotoxic effects, but the form of Aβ oligomer that can really play a key pathogenic role is not well understood. Therefore, determining the key toxic oligomers closely related to the occurrence and development of AD can provide ideal markers for early warning and early diagnosis of AD, and also provide ideal targets for the treatment of AD.

[0005] In addition to imaging detection, specific biomarkers are the basis for clinical diagnosis and detection of AD. At present, the industry is more recognized AD diagnostic markers mainly for cerebrospinal fluid (CSF) Aβ42, Aβ40, total tau protein (T-Tau) and phosphorylated tau protein (P-Tau) and so on. Other marker proteins are also reported from time to time, such as sAPPα, sAPPβ, BACE1, Aβ oligomer, total Aβ, axon and synapse marker and so on. These CSF markers have high diagnostic accuracy, combined with other diagnostic indicators of AD, the diagnostic sensitivity and specificity of AD can reach 85-90%. Compared with CSF, blood is easy to obtain, less invasive, and is an ideal source of clinical detection. At present, a number of studies have begun to combine the content of peripheral blood proteins, lipids and metabolites to distinguish AD patients and normal population, such as the content of Aβ42 dimer on peripheral blood cell membrane, plasma gelsolin (GSN) which can depolymerize Aβ42 fiber and the main degradation enzyme MMP3 of GSN. Recently, the detection of specific components in peripheral blood such as Tau181, Tau217 and Aβ content has also become a research hotspot.

[0006] The main biochemical detection methods for AD focus on the determination of Aβ and Tau protein markers in CSF. A large number of studies have shown that during the development of normal people into early cognitive impairment (MCI) and AD patients, the level of Aβ42 monomer in CSF gradually decreases, the level of Aβ42 oligomer and T-Tau gradually increases, and the change of P-Tau / Aβ42 ratio is more obvious. However, obtaining CSF samples requires lumbar puncture, which is a major injury, and MCI patients and mild AD patients often cannot accept it. In addition, due to the non-uniformity of CSF sample pretreatment and detection methods, the detection results of different manufacturers and different laboratories are greatly different, which seriously limits the clinical application of this type of method. Compared with CSF, blood-based detection has lower cost and higher compliance. However, the content of markers that can be used for AD diagnosis and detection in blood is low, the types are few, and it is greatly affected by peripheral tissues and organs, which hinders the research and development of AD diagnosis methods. At present, the most advanced method internationally is to detect the ratio of Aβ42 to Aβ40 and the content of P-Tau181, P-Tau217 and other proteins in blood by liquid chromatography-mass spectrometry (LC-MS) and single molecule immunoassay technology (Simoa). Although these indicators in blood have certain diagnostic significance, the specificity is poor, and a single indicator cannot well distinguish the target population. The reason is that the detection of Aβ or phosphorylated Tau protein is the detection of its total amount, which contains its monomer and a variety of toxic aggregates. The monomer and many types of oligomers have small cytotoxicity, but their content is high, which seriously interferes with the detection of toxic amyloid oligomers that have a key pathogenic role, and only the toxic oligomer is directly related to the development of AD. The appearance and level of the oligomer are directly related to the pathological changes in the brain of patients, and the specific detection of this type of oligomer is more valuable and meaningful.

[0007] Therefore, there is still an urgent need in the art for high-sensitivity and high-specificity detection of the content of specific Aβ oligomers in blood for the diagnosis of AD. SUMMARY

[0008] One aspect of the present application relates to a method of diagnosing whether a subject has early and late AD or AD-derived MCI or is at risk of having AD, the method comprising the steps of:

[0009] a) optionally, providing a sample to be tested from a subject,

[0010] b) contacting a reagent for detecting the presence and / or level of Aβo*3F in the sample of the subject with the sample of the subject,

[0011] c) detecting the presence and / or level of Aβo*3F in the sample of the subject,

[0012] wherein the presence and / or level of Αβο*3F in the subject sample is indicative of the subject having AD or AD-derived MCI, wherein the Αβο*3F is an Αβ oligomer that specifically binds to a 3F antibody having light and heavy chain CDR sequences as set forth in SEQ ID NOs: 17-22, respectively, and has a molecular weight of about 588 kDa based on size exclusion chromatography (SEC) analysis.

[0013] In some embodiments, the subject is a patient suspected of having AD or AD-derived MCI, preferably the subject is a human, non-human primate, cat or dog.

[0014] In some embodiments, the diagnostic method further comprises a step of clinical neuropsychological and neuroimaging assessment, preferably the neuroimaging is Αβ-PET scan imaging and / or tau-PET. More preferably, the neuroimaging is 3F-PET scan imaging, i.e. 3F-based immunoPET imaging, for identifying and detecting the presence and / or level of Αβο*3F in the brain of the subject.

[0015] In some embodiments, the diagnostic method further comprises detecting a change in tau level in the subject sample, preferably the tau level is total tau level or phospho-tau level.

[0016] A second aspect of the present application relates to a kit for diagnosing whether a subject has early and intermediate to late AD or AD-derived MCI or is at risk of developing AD, comprising a reagent for detecting the presence and / or level of Αβο*3F in a subject sample, wherein the Αβο*3F is an Αβ oligomer that specifically binds to a 3F antibody having light and heavy chain CDR sequences as set forth in SEQ ID NOs: 17-22, respectively, and has a molecular weight of about 588 kDa based on size exclusion chromatography analysis.

[0017] In some embodiments, the subject sample is a sample taken from the subject on site at the time of the diagnosis. In some embodiments, the subject sample is a sample taken from the subject in a previous diagnostic procedure.

[0018] In some embodiments, the size exclusion chromatography analysis is performed using a Superdex 200 10 / 300 GL molecular sieve chromatography column.

[0019] In some embodiments, the Αβο*3F is present in a cerebrospinal fluid sample obtained from the subject. In some embodiments, a cerebrospinal fluid sample from the subject is analyzed using immunoprecipitation and size exclusion chromatography and the molecular weight of the Αβο*3F is determined.

[0020] In some embodiments, the method or kit involves or comprises an enrichment reagent for Aβo*3F, which 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 selected from the group consisting of a scFv, a F(ab')2, a Fab2, a Fab, a Fab', a Fv, a Fd, a dAb, a camelid antibody, a nanobody, a diabody, or a bispecific antibody. By the enrichment reagent for Aβo*3F, Aβo*3F in a sample of a subject can be effectively enriched for subsequent procedures.

[0021] 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 an Aβ aggregate, preferably the first binding agent that specifically binds to Aβo*3F or an Aβ aggregate is an antibody that specifically binds to Aβo*3F or an Aβ aggregate, 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 the group consisting of a scFv, a F(ab')2, a Fab2, a Fab, a Fab', a Fv, a Fd, a dAb, a camelid antibody, a nanobody, a diabody, or a bispecific antibody. In some embodiments, the first binding agent that specifically binds to Aβo*3F or an Aβ aggregate is a scFv or a full-length antibody that specifically binds to Aβo*3F or an Aβ aggregate.

[0022] In some embodiments, the first binding agent is linked to a detection agent that allows it to be detected.

[0023] In some embodiments, the Aβo*3F can be detected and defined with a K98R mutant of 3F antibody, the Aβ oligomer detected with the K98R mutant is consistent with the Aβ oligomer detected with the 3F antibody, both are Aβo*3F, but the K98R mutant binds to Aβo*3F with higher affinity than the 3F, and can more sensitively detect the presence or absence of Aβo*3F and can more sensitively diagnose neurodegenerative diseases such as AD.

[0024] In some embodiments, the first binding agent that specifically binds to Aβo*3F or an Aβ aggregate is a 3F antibody or a K98R mutant thereof.

[0025] In some embodiments, the agent that detects the presence and / or level of Αβ0*3F further comprises a second binding agent that 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 the group consisting of a scFv, a F(ab')2, a Fab2, a Fab, a Fab', a Fv, a Fd, a dAb, a camelid antibody, a nanobody, a diabody, or a bispecific antibody, preferably the antibody that specifically binds to the first binding agent is linked 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.

[0026] In some embodiments, the detection agent is selected from the group consisting of a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, an umbelliferone, a lissamine, a cyanine, a Texas Red, a BODIPY (Invitrogen) or an analogue thereof, a paramagnetic label, a radioactive label, a biotin, a streptavidin / biotin, an avidin / biotin, a hapten, a digoxin, a metal complex, a metal, an enzyme, a colloidal gold, or a combination thereof. In some embodiments, the detection agent is selected from the group consisting of 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.

[0027] In some embodiments, the detection is selected from the group consisting of a chemiluminescent assay, an electrochemiluminescent assay, an enzyme-linked immunosorbent assay, an immunofluorescence assay, an immunohistochemistry assay, an immunochromatography assay, a radioimmunoassay, a single molecule immunoassay technology (Simoa), a flow cytometry, a cell sorting, an immunoprecipitation assay, an immunodiffusion assay, a dot-blot assay, a Western blot, a protein chip, a positron emission tomography, and / or a single photon emission computed tomography, preferably the kit comprises reagents, materials, containers, and / or devices required for performing the detection selected from the group consisting of a chemiluminescent assay, an electrochemiluminescent assay, an enzyme-linked immunosorbent assay, an immunofluorescence assay, an immunohistochemistry assay, an immunochromatography assay, a radioimmunoassay, a single molecule immunoassay technology, a flow cytometry, a cell sorting, an immunoprecipitation assay, an immunodiffusion assay, a dot-blot assay, a Western blot, and / or a protein chip, preferably the enzyme-linked immunosorbent assay is selected from the group consisting of a direct enzyme-linked immunosorbent assay, an indirect enzyme-linked immunosorbent assay, a direct sandwich enzyme-linked immunosorbent assay, and an indirect sandwich enzyme-linked immunosorbent assay.

[0028] In some embodiments, the subject sample is selected from the group consisting of a cell, a tissue, an organ and / or a body fluid of the subject, preferably the body fluid is selected from the group consisting of 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 secretion, preferably the body fluid is selected from the group consisting of whole blood, plasma, serum and / or cerebrospinal fluid.

[0029] In some embodiments, the enrichment reagent or the first binding agent that specifically binds to Aβo*3F is attached to a solid support.

[0030] In some embodiments, the method further comprises the step of detecting a control sample, and / or the kit further comprises a control sample from a healthy subject or a subject not suffering from AD and AD-derived MCI.

[0031] In some embodiments, the antibody that specifically binds to Aβo*3F is a polyclonal antibody and / or a monoclonal antibody or an antigen-binding fragment thereof obtained by immunization against Aβo*3F, 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.

[0032] In some embodiments, the concentration of Aβo*3F in cerebrospinal fluid of healthy population is Aβ42o*3F: 80.44 ± 20.88 pg / ml, Aβ40o*3F: 24.35 ± 5.08 pg / ml; and / or the concentration of Aβo*3F in plasma of healthy population is Aβ42o*3F: 71.63 ± 36.8 pg / ml, Aβ40o*3F: 2.24 ± 0.92 pg / ml; the concentration of Aβo*3F in plasma of MCI population is Aβ42o*3F: 112.93 ± 25.02 pg / ml, Aβ40o*3F: 4.64 ± 1.43 pg / ml; the concentration of Aβo*3F in cerebrospinal fluid of AD patient population is Aβ42o*3F: 264.8 ± 42.26 pg / ml, Aβ40o*3F: 85.74 ± 10.62 pg / ml, and / or the concentration of Aβo*3F in plasma of AD patient population is Aβ42o*3F: 159.44 ± 36.8 pg / ml, Aβ40o*3F: 14.0 ± 5.59 pg / ml; based on MSD electrochemiluminescence method; or

[0033] The concentration of Aβ42o*3F in the plasma of healthy people was 68.02 ± 39.17 pg / ml based on the chemiluminescence method; the concentration of Aβ42o*3F in the plasma of MCI people was 124.5 ± 12.57 pg / ml; and the concentration of Aβ42o*3F in the plasma of AD patients 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.

[0034] In some embodiments, the sensitivity of the detection is as low as 0.5 pg / ml based on the MSD electrochemiluminescence method and the chemiluminescence method.

[0035] In some embodiments, the antibody that specifically binds to Aβo*3F is a plurality of antibodies specific for different epitopes of Aβo*3F, e.g., 2, 3, 4, 5, or more antibodies specific for 2, 3, 4, 5, or more different epitopes of Aβo*3F, respectively.

[0036] In some embodiments, the step of enriching Aβo*3F from the sample is performed using immunoprecipitation with an antibody that specifically binds to Aβo*3F.

[0037] In other words, in the previous study, the inventors used phage display technology to screen a full human single-chain antibody W20 that specifically binds to Aβ oligomers (see CN101463082A), and the 3F antibody (amino acid sequence shown in SEQ ID No. 23) is an improved form of the W20 antibody. The 3F antibody has significantly improved affinity for Aβ oligomers, can more significantly inhibit the aggregation of Aβ and the neurotoxicity induced by Aβ oligomers, and can more effectively improve the cognitive and memory functions of AD model mice and reduce the pathological changes in the brains of the mice. More significantly, the Aβ oligomers specifically recognized by the 3F antibody are super-toxic oligomers, which are the most important toxic components in the mixture of Aβ oligomers and have a strong pathogenic effect, playing a key role in the occurrence and development of AD. The super-toxic oligomers recognized by the 3F antibody exist in the CSF, blood, and / or brain tissue of AD patients and AD-derived MCI patients, and their levels in the CSF, blood, and / or brain tissue of AD patients, MCI patients, and healthy elderly people are significantly different, thus accurately distinguishing AD patients, MCI patients, and healthy elderly people. The super-toxic oligomers also exist in AD transgenic mice and are directly related to the onset of AD transgenic mice. In the present application, the super-toxic Aβ oligomers recognized by the 3F antibody are referred to as Aβo*3F, which can be separated from the mixture of Aβ oligomers by immunoprecipitation using the 3F antibody. The typical characteristics of Aβo*3F are high-molecular-weight Aβ oligomers, and based on SEC analysis, the molecular weight is about 588 kDa, the diameter is about 10 nm, it has a strong toxic effect on neurons, and its toxicity is more than 200 times higher than that of the mixture of Aβ oligomers. Aβo*3F can activate microglial cells and astrocytes and secrete a large amount of inflammatory factors. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Aβo*3F, Aβ*6E10, and Aβ-ID were prepared by immunoprecipitation.

[0039] Figure 2 The molecular weight and morphological characterization of Aβo*3F are shown:

[0040] A: ThT detection of Aβ aggregation state;

[0041] B: Dot blot detection of the binding of oligomers obtained under different incubation conditions to 3F, A11, and 6E10 antibodies;

[0042] C: Affinity of 10 μM Aβ incubated for 0-4 days to 3F antibody;

[0043] D: Western blot detection of the band distribution of sAβo*3F;

[0044] E: SEC analysis of sApo*3F molecular weight size; SEC analysis of sApo*3F and SEC standards (Superdex 200 10 / 300), SEC standards: 1. thyroglobulin (669 kDa), 2. ferritin (440 kDa), 3. aldolase (158 kDa), 4. albumin (75 kDa), 5. ovalbumin (44 kDa), 6. carbonic anhydrase (29 kDa);

[0045] F: Linear equation (Y) and correlation coefficient (r2) fitted from SEC standards molecular weight and elution volume;

[0046] G: Western blot analysis of mApo*3F band distribution;

[0047] H: SEC analysis of mApo*3F molecular weight size;

[0048] I: Western blot analysis of hApo*3F band distribution.

[0049] Figure 3 Neurotoxic effects of Aβo*3F are shown:

[0050] A: IC50 of sApoS on N2a cell toxicity;

[0051] B: IC50 of sApo*3F on N2a cell toxicity;

[0052] C: MTT assay comparing sApoS, sApo*3F and sApo-ID neurotoxicity on N2a cells;

[0053] D: IC50 of mApo*3F on N2a cell and primary neuron toxicity;

[0054] E: MTT assay comparing mApo*6E10, mApo*3F and mApo-ID cytotoxicity on primary neurons;

[0055] F: IC50 of hApo*3F on primary neuron toxicity;

[0056] G: MTT assay comparing hApo*6E10, hApo*3F and hApo-ID cytotoxicity on primary neurons.

[0057] Figure 4 Effects of Aβo*3F on cytokine expression levels in glial cells are shown:

[0058] A: Effects of mApo*3F on TNF-α, IL-1 β, IL-6 expression levels in microglial cells;

[0059] B: The effect of mApo*3F on the expression levels of TNF-a, iNos, IL-1β, IL-6 in primary astrocytes;

[0060] C: The effect of mApo*3F on the expression levels of TSP1, Gpc4 and Gpc6 in primary astrocytes.

[0061] Figure 5 The effect of mApo*3F on the cognition of mice and the damage to neurons in the brain:

[0062] A: The cognition index of mice in the novel object recognition experiment;

[0063] B: The duration of mice in the new arm in the Y maze experiment;

[0064] C: The density of dendritic spines of mouse neurons detected by Golgi staining;

[0065] D: Statistical analysis of the density of dendritic spines in C;

[0066] E: The number of neurons in the hippocampus of mice detected by Nissl staining, scale bar: 20 μm;

[0067] F, G: Statistical analysis of the number of neurons in the DG region (F) and the CA1 region (G) by Image J software.

[0068] Figure 6 The activation of glial cells in the brain of mice by mApo*3F to produce neuroinflammation:

[0069] A: The activation degree of microglia in the DG and CA1 regions of the hippocampus of mice detected by immunohistochemical experiment, scale bar: 20 μm;

[0070] B: The activation degree of astrocytes in the DG and CA1 regions of the hippocampus of mice detected by immunohistochemical experiment, scale bar: 20 μm;

[0071] C, D: Quantification of Iba-1 positive microglia in the DG region (C) and the CA1 region (D) by Image J software;

[0072] E, F: Quantification of GFAP positive astrocytes in the DG region (E) and the CA1 region (F) by Image J software;

[0073] G, H: ELISA detection of the expression levels of IL-6 (G) and IL-1β (H) in the hippocampus of mice.

[0074] Figure 7Electrochemiluminescence detection of Aβo*3F levels in CSF and plasma of AD patients: A, B: Levels of Aβ42 (A) and Aβ40 (B) in Aβo*3F isolated from CSF of AD patients and healthy controls (Con) as detected by MSD method;

[0075] C, D: Levels of Aβ42 (C) and Aβ40 (D) in Aβo*3F isolated from plasma of MCI, AD and healthy controls (Con) as detected by MSD method.

[0076] Figure 8 Chemiluminescence detection of Aβo*3F levels in plasma of AD, MCI and healthy controls.

[0077] Figure 9 Aβo*3F levels in CSF or plasma samples for AD diagnosis.

[0078] Figure 10 The affinity of the 3F antibody to Aβo*3F oligomers is further improved after mutating K to R at position 98 of the heavy chain, suggesting that this optimized single chain antibody can be used to better diagnose the presence of Aβo*3F oligomers and, in turn, to better diagnose AD. DETAILED DESCRIPTION

[0079] DEFINITIONS

[0080] The terms used in the claims and specification, unless otherwise indicated, are defined as follows.

[0081] Unless otherwise defined herein, scientific and technical terms used in connection with the methods and compositions described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used herein and the laboratory procedures are those well-known and commonly used in the art to which the application relates. The nomenclature used herein and the laboratory procedures are those well-known and commonly used in the art to which the application relates.

[0082] The methods and techniques described herein are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements through 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; 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).

[0083] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0084] Unless otherwise indicated, the following terms are to be understood to have the following meanings.

[0085] The term "at risk of having Alzheimer's disease" means having a risk of having Alzheimer's disease, e.g., family history, aging, poor lifestyle such as drinking, etc.

[0086] The term "Aβ-PET scan imaging" means beta-amyloid positron emission tomography.

[0087] The term "tau-PET scan imaging" refers to tau protein positron emission tomography.

[0088] The term "3F-PET scan imaging" refers to immunopositron emission tomography based on 3F antibody. The term "Apo*3F" refers to Aβ oligomer that specifically binds to 3F antibody, which has a molecular weight of about 588 kDa based on size exclusion chromatography (SEC) analysis, the light and heavy chain CDR sequences of which are set forth in SEQ ID NOs: 17-22, respectively.

[0089] The term "Apo*3F" refers to Aβ oligomer that specifically binds to 3F antibody, which has a molecular weight of about 588 kDa based on size exclusion chromatography (SEC) analysis, the light and heavy chain CDR sequences of which are set forth in SEQ ID NOs: 17-22, respectively.

[0090] The term "Apo*3F" refers to Aβ oligomer that specifically binds to 3F antibody, which has a molecular weight of about 588 kDa based on size exclusion chromatography (SEC) analysis, the light and heavy chain CDR sequences of which are set forth in SEQ ID NOs: 17-22, respectively.

[0091] In some embodiments, the chemiluminescent label of the present disclosure is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, isoluminol and its derivatives, acridinium ester and its derivatives.

[0092] In some embodiments, the chromophore of the present disclosure is selected from the group consisting of trispyridine ruthenium.

[0093] In some embodiments, the fluorescent label of the present disclosure is selected from the group consisting of rare earth chelates or its derivatives, isothiocyanate, rhodamine, phycobilin, phycoerythrin, phycocyanin, allophycocyanin, fluorescein isothiocyanate or Alexa Fluor dyes.

[0094] In some embodiments, the fluorescein-type label of the present disclosure is selected from the group consisting of fluorescein, luciferase such as firefly luciferase and bacterial luciferase.

[0095] In some embodiments, the paramagnetic label includes, but is not limited to, containing 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) and in particular Co +2 , CR +2 , Cr +3 , Cu +2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 , Ni +2, Ti +3 , V +3 and V +4 paramagnetic ions of Cr, Mn, Fe, Co, Ni, Cu, V and Ti.

[0096] 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.

[0097] In some embodiments, the hapten is selected from a polysaccharide or a lipid.

[0098] In some embodiments, the metal complex is selected from trispyridine ruthenium.

[0099] In some embodiments, the metal is selected from ruthenium.

[0100] In some embodiments, the enzyme of the present disclosure is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase, beta-glucuronidase, beta-D-glucosidase, urease, hexokinase, malic enzyme, glucose-6-phosphate dehydrogenase, sucrose, glucoamylase, lysozyme, sugar oxidase, heterocyclic oxidase, streptavidin-beta-D-galactopyranoside conjugate, and / or streptavidin-horseradish peroxidase conjugate.

[0101] In some embodiments, the metal complex is trispyridine ruthenium for electrochemiluminescence assay.

[0102] In some embodiments, the detection agent for biotinylated detection antibody is avidin, streptavidin-HRP, or streptavidin-beta-D-galactopyranoside (SBG). In certain embodiments, the readout of the detection agent is fluorometric or colorimetric. For example, but not limited to, tetramethylbenzidine and hydrogen peroxide can be used as the readout. In certain embodiments, if the detection agent is streptavidin-HRP, the readout can be colorimetric by using tetramethylbenzidine and hydrogen peroxide. Alternatively, in certain embodiments, resorufin beta-D-galactopyranoside can be used as the readout. For example, but not limited to, if the detection agent is SBG, the readout can be fluorometric by using resorufin beta-D-galactopyranoside.

[0103] In certain embodiments, a detection agent, such as SBG, can be used at a concentration of about 50 to about 500 pM. For example, but not by way of limitation, a detection agent can 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. In certain embodiments, a detection agent can be used at a concentration of about 100 pM to about 400 pM, for example, SBG can be used at a concentration of about 110 pM, about 155 pM, or about 310 pM. In certain embodiments, SBG can be used at a concentration of about 310 pM. In certain embodiments, a detection agent, for example, HRP, can be used at a dilution of about 1 / 10 to about 1 / 1000. For example, but not by way of limitation, a detection agent can 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. In certain embodiments, a detection agent can be used at a dilution of about 1 / 100 to about 1 / 1000, for example, HRP can be used at a dilution of about 1 / 100 or about 1 / 500. One of skill in the art can readily determine the concentration at which to use a detection agent depending on the type of detection agent, without undue experimentation.

[0104] In some embodiments, as an enzyme chromogenic substrate, for example, in the case where horseradish peroxidase (HRP) is selected as an enzyme label, as a substrate, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-l-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, m-methoxyaniline, or 3,3'-dimethoxybenzidine can be used. Also, in the case where alkaline phosphatase is selected as an enzyme label, as a substrate, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitro blue tetrazolium, or p-nitrophenyl phosphate can be used. Also, in the case where β-D-glucosidase is selected as an enzyme label, as a substrate, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be used. In addition, various enzymes and enzyme chromogenic substrates well known in the art can be used.

[0105] In certain embodiments, the methods of the present disclosure can comprise blocking the first binding agent with a blocking buffer. In certain embodiments, the blocking buffer can comprise PBS, bovine serum albumin (BSA), and / or a biocide, such as ProClin TM (Sigma-Aldrich, Saint Louis, MO). In certain embodiments, the methods can comprise a plurality of wash steps. In certain embodiments, the solution used for washing is typically a buffer (e.g., a "wash buffer"), such as, but not limited to, a PBS buffer comprising a detergent (e.g., Tween 20). For example, but not by way of limitation, the first binding agent can be washed after blocking and / or the sample can be separated from the first binding agent to remove unbound material (e.g., by washing).

[0106] In some embodiments, the diagnostic methods of the present application have a detection sensitivity of as low as about 0.5 pg / mL, such as 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, 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 greater.

[0107] In some embodiments, the kits of the present application comprise a first container comprising an enrichment reagent or a first binding agent that specifically binds to Αβ0*3F.

[0108] In some embodiments, the kits of the present application comprise a second container comprising a second binding agent that specifically binds to the first binding agent.

[0109] The term "the second binding agent specifically binds to the first binding agent" means that the second binding agent specifically binds to the antibody class of the first binding agent, such as an antibody. For example, if the first binding agent is a mouse anti-Αβ0*3F IgG, then the second binding agent can be a rabbit anti-mouse IgG.

[0110] In some embodiments, the kits of the present application comprise a third container comprising a control sample.

[0111] In some embodiments, the kits of the present application include one or more additional containers comprising other reagents, materials needed to perform the assays.

[0112] In some embodiments, the enrichment reagent or first binding agent that specifically binds to Αβ0*3F 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 the group consisting of polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, plasma substitute, cross-linked glucose, Sepharose, liposome, carboxymethylcellulose, polyacrylamide, polystyrene, pumice, filter paper, ion exchange resin, plastic film, plastic tube, poly(methyl vinyl ether / maleic acid) copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles, and the like. In addition, as other solid supports, there are cell culture plates, enzyme-linked immunosorbent assay plates, electrochemiluminescence assay plates, tubes, and polymeric films. The above-mentioned solid supports can have any shape such as spherical (microbeads), cylindrical (test tube or inner face of a well), planar (sheet, test strip), and the like.

[0113] In some embodiments, the kits of the present disclosure are kits for performing an enzyme-linked immunosorbent assay (ELISA) comprising some or all of the reagents, materials, containers, and / or devices needed to perform an ELISA. One of skill in the art knows how to prepare a corresponding ELISA kit based on the reagents of the present disclosure for detecting the presence and / or level of Αβ0*3F in a sample from a subject. In some embodiments, the ELISA kit comprises pre-coated Αβ0*3F antibodies, HRP-labeled Αβ antibodies, standards, washes, substrate, and the like.

[0114] In some embodiments, the kits of the present disclosure are kits for performing an immunofluorescence assay comprising some or all of the reagents, materials, containers, and / or devices needed to perform an immunofluorescence assay. One of skill in the art knows how to prepare a corresponding immunofluorescence assay kit based on the reagents of the present disclosure for detecting the presence and / or level of Αβ0*3F in a sample from a subject. In some embodiments, the immunofluorescence assay kit is an electrochemiluminescence immunoassay. In some embodiments, the immunofluorescence assay kit is a time-resolved immunofluorescence assay. In some embodiments, the immunofluorescence assay kit comprises an electrochemiluminescence plate pre-coated with Αβ0*3F antibodies, ruthenium-labeled Αβ antibodies, standards, read buffer, and the like.

[0115] In some embodiments, the kits of the present disclosure are kits for performing a colloidal gold method assay, which include some or all of the reagents, materials, containers, and / or devices required to perform a colloidal gold method assay. One of skill in the art knows how to prepare a corresponding colloidal gold method assay kit based on the reagents of the present disclosure for detecting the presence and / or level of Aβo*3F in a sample of a subject. In some embodiments, the colloidal gold method assay kit is a colloidal gold test strip. In some embodiments, the colloidal gold method assay kit includes a colloidal gold-labeled Aβo*3F antibody, an Aβ antibody, a control antibody, and the like.

[0116] In some embodiments, the kits of the present disclosure are kits for performing a radioimmunoassay, which include some or all of the reagents, materials, containers, and / or devices required to perform a radioimmunoassay. One of skill in the art knows how to prepare a corresponding radioimmunoassay kit based on the reagents of the present disclosure for detecting the presence and / or level of Aβo*3F in a sample of a subject. In some embodiments, the radioimmunoassay kit includes a radioisotope-labeled Aβo*3F antibody, an Aβo*3F standard, and the like.

[0117] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule that has the ability to bind to an epitope of an antigen. Naturally occurring antibodies typically comprise a tetramer, usually composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include isotypes: IgG, IgA, IgM, IgD, and IgE, whose respective heavy chains are μ, δ, γ, α, and ε. The same class of Ig can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4 for IgG, and IgA1 and IgA2 for IgA, based on differences in the amino acid composition of their hinge regions and the number and location of disulfide bonds in the heavy chains. The light chains can be classified into κ and λ based on the constant region. The antibodies of the present application can have any isotype. The choice of isotype is often determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant domains, κ or λ, can be used. If desired, the class of an antibody of the present application can be switched by known methods. For example, an antibody of the present application that is initially IgG can be class switched to an IgM antibody of the present application. In addition, class switching techniques can be used to convert one IgG subclass to another, e.g., from IgG1 to IgG2. Thus, the effector function of an antibody of the present application can be changed, e.g., to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, for various therapeutic uses, provided that the Clq binding activity of the antibody is reduced or eliminated. In some embodiments, the antibodies of the present application are IgM or IgG1, 2, 3, or 4 type antibodies. An antibody belongs to a particular isotype if its amino acid sequence is largely homologous to that of other isotypes.

[0118] In the present context, the term "antibody" is used in the broadest sense, and includes proteins comprising an antigen binding site, and encompasses both natural and artificial antibodies of various structures, including but not limited to intact antibodies and antigen binding fragments of antibodies.

[0119] "Variable regions" or "variable domains" are structural domains in the heavy or light chains of an antibody that participate in the binding of the antibody to its antigen. Each heavy chain of an antibody consists of a heavy chain variable region (hereinafter referred to as VH) and a heavy chain constant region (hereinafter referred to as CH). The heavy chain constant region typically consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (hereinafter referred to as VL) and a light chain constant region (hereinafter referred to 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 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 variable regions of the heavy and light chains contain binding regions that interact with the antigen. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) called complementarity-determining regions (CDRs), interspersed with more conserved regions called backbone regions (FRs). Each VH and VL consists of three CDR domains and four FR domains, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0120] The terms "complementarity-determining region" or "CDR region" (which can be used interchangeably with "HVR" in this document) refer to regions within the antibody variable domain that are sequence-hypervariant and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In this document, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0121] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0122] The terms "monoclonal antibody," "monoclonal antibody composition," or "monoclonal antibody compositions" refer to a preparation of antibody molecules of single molecular composition, i.e., a population of individual antibodies which are identical except for possible naturally occurring mutations that can be present in minor amounts. Conventional monoclonal antibody compositions display a single binding specificity and affinity for a particular epitope. In certain embodiments, a monoclonal antibody can be composed of more than one Fab domain, thereby increasing the specificity for more than one target. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited with respect to the manner in which they are generated (e.g., recombinant, transgenic, hybridoma, etc.).

[0123] The present application also includes "bispecific antibodies" in which the antibody of the present application is part of a bivalent or multivalent bispecific framework targeting more than one epitope (e.g., the second epitope can comprise an epitope of an active transport receptor, such that the bispecific antibody will exhibit improved transcytosis across a biological barrier, such as the blood-brain barrier). Thus, in additional embodiments, the monovalent Fab of the antibody of the present application can be linked to another Fab or scfv targeting a different protein to produce a bispecific antibody. Bispecific antibodies can have dual functionality, e.g., the therapeutic functionality conferred by the present application and a transport functionality that can bind to a receptor molecule to enhance transport across a biological barrier, such as the blood-brain barrier. The terms "diabody," "dual functional antibody," "bispecific antibody," "bispecific antibody" or "BsAb" refer to an antibody that has two different antigen binding sites, and thus can bind to two target antigens simultaneously, while performing the targeting function of an antibody, and also mediating another specialized function, the specialized function effecting molecule can also be a toxin, an enzyme, a cytokine, a radionuclide, etc., the two arms of the bispecific antibody that bind to the antigens can be from a Fab, Fv, ScFv, or dSFv, etc.

[0124] The term "antigen binding fragment of an antibody" refers to a fragment, portion, region or domain of an antibody (e.g., obtainable via cleavage, recombination, synthesis, etc.) that is capable of binding to an epitope. An antigen binding fragment can contain 1, 2, 3, 4, 5, or all 6 CDR domains of such an antibody, and can exhibit different specificity, affinity, or selectivity, while being capable of binding to the epitope. Preferably, an antigen binding fragment contains all 6 CDR domains of the antibody. An antigen binding fragment of an antibody can be part of or comprise a single polypeptide chain (e.g., an scFv), or can be part of or comprise two or more polypeptide chains (each having an amino-terminal end and a carboxyl-terminal end) (e.g., a diabody, a Fab fragment, a F(ab')2 fragment, etc.).

[0125] Examples of antigen-binding fragments encompassed by the present application include (a) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (b) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge domain; (c) a Fd fragment consisting of the VH and CHI domains; (d) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (e) a single chain Fv (scFv), a recombinant protein in which the antibody VH and VL are connected by a linker peptide using genetic engineering methods; (f) a dAb fragment (Ward et al., Nature, 341, 544-546 (1989)), which essentially consists of a VH region and is also called a domain antibody (Holt et al., Trends Biotechnol., 21(11):484-90); (g) a camelid or nanobody (Revets et al., Expert Opin Biol Ther., 5(1):111-24) and (h) an isolated complementarity determining region (CDR).

[0126] In some embodiments, the antibodies of the present application and antigen-binding fragments thereof are single chain antibodies. In some embodiments, the present application provides single chain Fv (scFv), in which the heavy and light chains in the Fv of the antibodies of the present application are connected by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues) into a single peptide chain. Methods for producing such antibodies are described in, for example, US 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.

[0127] In some embodiments, the antibodies and antigen-binding fragments thereof of the application are chimeric antibodies. The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. The present application provides variable region antigen binding sequences from human antibodies. Accordingly, the chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences from non-human antibodies, such as FR or C region sequences. Furthermore, the chimeric antibodies described herein are antibodies comprising a human variable region antigen binding sequence of one antibody class or subclass and another sequence, such as a FR or C region sequence, from another antibody class or subclass.

[0128] In some embodiments, the antibodies and antigen-binding fragments thereof of the application are humanized antibodies. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0129] In some embodiments, the antibodies and antigen-binding fragments thereof of the application are human antibodies or fully human antibodies. The terms "human antibody" or "fully human antibody" ("humAb" or "HuMab") refer to antibodies that include variable and constant domains with sequences derived from human germline immunoglobulin sequences. Human antibodies of the application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during maturation in vivo).

[0130] Variant antibodies are also included within the scope of the application. Thus, variants of the sequences recited in the application are also included within the scope of the application. Additional variants of antibody sequences having improved affinity can be obtained by using methods known in the art and are also included within the scope of the application. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the efficiency of translation of the expression system in antibody production.

[0131] Such variant antibody sequences have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more) sequence homology with the sequences recited in the application. Such sequence homology is calculated over the full length of the reference sequence (i.e., the sequence recited in the application).

[0132] The numbering of amino acid residues in the application is in accordance with 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, U.S. 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 in this invention are numbered according to the Kabat numbering system.

[0133] An antibody or its antigen-binding fragment "specifically" binds to a region of another molecule (i.e., an epitope) meaning that it reacts or binds to that epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity or cohesion than to another epitope. In some embodiments, the antibody or its antigen-binding fragment of the present invention binds to at least 10 -7 M has an affinity for binding to human amyloid protein, especially its toxic forms, such as 10. -8 M, 10 -9 M, 10 -10 M, 10 -11 M or higher. Preferably, the antibody or its antigen-binding fragment binds under physiological conditions (e.g., in vivo). Therefore, specific binding to amyloid protein, especially its toxic form, refers to the ability of the antibody or its antigen-binding fragment to bind to amyloid protein, especially its toxic form, with the aforementioned specificity and / or under such conditions. Suitable methods for determining said binding are known in the art.

[0134] In the context of antibody binding to a designated antigen, the term "binding" generally refers to binding to an antigen corresponding to approximately 10-10. -6 M or smaller K D The affinity combination of K D The antibody has an affinity for binding to non-specific antigens (e.g., BSA, casein) other than the specified antigen or closely related antigens, that is at least 10-fold lower, such as at least 100-fold lower or at least 1,000-fold lower.

[0135] As used in this article, the term "k" d (sec⁻¹ or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as k. off value.

[0136] As used herein, the term "k a " (M"1x sec"1or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction.

[0137] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing k d by k a .

[0138] As used herein, the term "K A " (M"1or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing k a by k d .

[0139] The antibodies of the present application are produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, used alone or in combination. In general, knowing the amino acid sequence of the desired sequence, one of ordinary skill in the art can readily produce the antibodies by standard techniques for producing polypeptides. For example, the antibodies can be synthesized using well-known solid phase methods, preferably using a commercially available peptide synthesizer (such as one manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies of the present application can be synthesized by recombinant DNA techniques well known in the art. For example, after incorporating the DNA sequence encoding the antibody into an expression vector and introducing the vector into a suitable eukaryotic or prokaryotic host that expresses the desired antibody, the antibody can be obtained as a DNA expression product, which can then be isolated from the host using known techniques.

[0140] The antibodies of the application, and antigen-binding fragments thereof, can be modified by the inclusion of any "suitable" number of modified amino acids and / or in conjunction with conjugated substituents. In this context, "suitable" is generally determined by the ability to at least substantially retain the amyloid selectivity and / or amyloid, especially toxic form thereof, specificity associated with the non-derivatized parent antibody. The inclusion of one or more modified amino acids can be advantageous, for example, in increasing the serum half-life of the polypeptide, decreasing the antigenicity of the polypeptide, or increasing the storage stability of the polypeptide. The modification of one or more amino acids is performed, for example, concomitantly with translation during recombinant production or post-translationally (e.g., N-linked glycosylation at N-X-S / 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-geranyl) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, and the like. References for performing amino acid modifications are routine in the art, see, e.g., Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. The modified amino acid can be, for example, selected from the group consisting of glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, or amino acids conjugated to an organic derivatizing agent.

[0141] The antibodies of the application, and antigen-binding fragments thereof, can also be chemically modified by covalent coupling to a polymer to increase their circulating half-life. Exemplary polymers, and methods of attaching them to peptides, are described in, e.g., US 4,766,106; US 4,179,337; US 4,495,285; and US 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight between about 1,000 and 40,000 D, such as between about 2,000 and 20,000 D, e.g., about 3,000 and 12,000 D).

[0142] Antibodies of the application and antigen-binding fragments thereof can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines, and insect cell lines, for example, CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (such as 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).

[0143] Antibodies of the application are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0144] The term "subject" refers to a warm-blooded animal, preferably a mammal (including humans, domestic and farm animals, zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like), and more preferably a human. In one embodiment, the subject can be a "patient," i.e., a warm-blooded animal, and more preferably a human, who is awaiting to receive or is receiving medical care or will be the subject of a medical procedure, or monitoring of disease development. In one embodiment, the subject is an adult (e.g., a subject older than 18 years of age). In another embodiment, the subject is a child (e.g., a subject younger than 18 years of age). In one embodiment, the subject is an elderly person, older than 55, 60, 65, 70, 75, 80, 85, 90 years of age, or older. In one embodiment, the subject is a male. In another embodiment, the subject is a female.

[0145] The term "healthy subject" refers to a subject who does not suffer from any disease or condition or to a subject who does not suffer from any neurodegenerative disease.

[0146] The term "subject who does not suffer from Alzheimer's disease" refers to a subject who is confirmed by regular medical procedures not to suffer from Alzheimer's disease, for example, a subject who is determined not to suffer from mild cognitive impairment (MCI) and AD dementia according to the criteria established by the National Institute on Aging and the Alzheimer's Association. In some embodiments, a subject who does not suffer from Alzheimer's disease is a healthy subject.

[0147] The technical solutions of the present application will be described in detail below through examples, which are descriptive, illustrative, and not meant to be limiting. The reagents used in the following examples, if not specifically noted, are commercially available from reagent companies such as Sigma Aldrich, Merck, and the experimental methods, if not specifically noted, can be found in textbooks such as Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

[0148] Examples

[0149] Obtaining of Aβ oligomers specifically bound to 3F in Example 1

[0150] 1.1 Experimental materials and methods

[0151] 1.1.1 Experimental materials

[0152] Protein A magnetic beads: Bio-RAD, #1614833

[0153] Protein G magnetic beads: Bio-RAD, #1614023

[0154] Protein A / G agarose gel: Abmart, #A10001S

[0155] 6E10 antibody: Biolegend, #803002

[0156] Human Aβ42 detection kit: Immuno-Biological Laboratories

[0157] 1.1.2 Main solutions

[0158] (1) 50 mM sodium hydroxide (pH 10.0);

[0159] (2) 20 mM phosphate buffer (PBS): pH 7.4;

[0160] (3) 0.1% PBST: PBS containing 0.1% Tween-20;

[0161] (4) 20 mM glycine eluent (pH 2.0);

[0162] (5) 1M Tris neutralizing solution: pH 10.0;

[0163] Note: The relevant reagents and solutions mentioned in Examples 2, 3, 4, 5 and 6 but not given source are the same as in Example 1.

[0164] 1.2 Preparation of mouse brain homogenate

[0165] APP / PS1 mice were deeply anesthetized with sodium pentobarbital and sacrificed by cardiac perfusion with ice-cold PBS containing heparin (10 U / mL) and the brains were removed. To the brain tissue, 1 mL of RIPA strong lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added, and the tissue was crushed with a Tissue Lyser II tissue grinder at a frequency of 30 Hz for 8 min. The supernatant was collected by centrifugation at 14000 rpm for 30 min at 4°C. Before immunoprecipitation, the brain homogenate was added to 100 μL Protein A / G-agarose gel and incubated at 4°C for 1 h to remove endogenous IgG in the mouse brain homogenate. Then the brain homogenate was added to Protein A magnetic beads cross-linked with APP antibody to remove endogenous APP in the mouse brain.

[0166] 1.3 Cross-linking of antibody with magnetic beads

[0167] First, the 3F and 6E10 antibodies were cross-linked with Protein A or Protein G magnetic beads, respectively, and the cross-linking steps were as follows:

[0168] (1) Take 200 μL Protein A or Protein G magnetic beads, wash three times with 2 mL of 0.1% PBST, mix the magnetic beads thoroughly each time, and then place them on a magnetic stand and discard the supernatant;

[0169] (2) Add 50 μg of 3F or 6E10 antibody to the magnetic beads, shake the reaction at room temperature for 30 min to allow the magnetic beads to bind to the antibody, and then wash three times with 0.1% PBST;

[0170] (3) Wash the magnetic beads once with cross-linking buffer (0.2M triethanolamine, pH 8.2), then dissolve 12 mg of DMP in 2 mL of cross-linking buffer and add it to the magnetic beads, shake the reaction at room temperature for 1 h. After the reaction is completed, discard the cross-linking solution, wash the magnetic beads once with 2 mL of blocking buffer (0.1M ethanolamine, pH 8.2), then add another 2 mL of blocking buffer and block at room temperature for 2 h;

[0171] (4) After blocking, wash the magnetic beads three times with PBS, then elute the magnetic beads once with 0.1M glycine (pH 2.5) for 5 min, then wash the magnetic beads three times with 0.1% PBST, and store the magnetic beads in PBST containing 0.02% NaN3 at 4°C.

[0172] 1.4 In vitro preparation of Aβo*3F

[0173] Aβ42, Aβ40 or other forms of Aβ (Biotechne) were dissolved in 1 mL of 100% hexafluoroisopropanol (HFIP), vortexed for 5 min, sonicated for 10 min on a water bath, aliquoted into EP tubes, and the solvent was evaporated overnight at -20 °C. Before use, the aliquoted Aβ treated with HFIP 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 with pre-cooled PBS to 10 μM. Centrifugation at 4 °C, 21000 g for 30-40 min, the precipitate part (about 5% of the initial volume) was discarded, and Aβ monomer was obtained. The Aβ monomer was incubated at 25 °C for 2 days, and then incubated with Protein A magnetic beads cross-linked with 3F antibody at 4 °C overnight. The next 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, eluted twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (Aβo*3F prepared in vitro).

[0174] 1.5 Isolation and preparation of Aβo*3F in APP / PS1 mouse brain homogenate and AD patient CSF

[0175] To prepare Aβos (Aβo*3F) specifically recognized by 3F, the APP / PS1 mouse brain homogenate and AD patient CSF sample (from the First Affiliated Hospital of Zhengzhou University, signed informed consent and obtained approval from the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University) were incubated with Protein A magnetic beads cross-linked with 3F antibody at 4 °C overnight. The next day, the magnetic beads were washed three times with 0.1% PBST, and then eluted with 20 mM-100 mM glycine (specifically 20 mM, pH 2.0) for 3 min, eluted twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F in the brain of APP / PS1 mice (mAβo*3F, Aβo*3F isolated in the brain of AD mice) or extracted from the CSF of AD patients (hAβo*3F, Aβo*3F isolated from the CSF of AD patients). The mixture of Aβ aggregates after 3F immunodepletion is called Aβ-ID, which is sAβ-ID (prepared in vitro), mAβ-ID (isolated and prepared in the brain of APP / PS1 mice), and hAβ-ID (isolated and prepared in the CSF of AD patients), respectively, and used as controls.

[0176] Aβ aggregate mixture Aβ*6E10 was prepared by mixing brain homogenate of APP / PS1 mice or CSF of AD patients with Protein G magnetic beads cross-linked with 6E10 antibody, reacting overnight at 4°C, then eluting twice with 20 mM-100 mM glycine (specifically 20 mM, pH 2.0) for 3-5 min (specifically 3 min), and neutralizing the eluate to pH 7 with 1 M Tris, to obtain Aβo*6E10 in brain of APP / PS1 mice (mAβo*6E10, Aβo*6E10 isolated from brain of AD mice) or extracted from CSF of AD patients (hAβo*6E10, Aβo*6E10 isolated from CSF of AD patients). Figure 1 The concentration of Aβ obtained by immunoprecipitation was determined by Aβ detection kit.

[0177] Example 2 Molecular weight size and morphological characterization of Aβo*3F

[0178] 2.1 Experimental materials and methods

[0179] 2.1.1 Experimental materials

[0180] Aβ polypeptide: Zipeep Biochemical;

[0181] Superdex 200 10 / 300 GL molecular sieve: GE Healthcare;

[0182] A11 antibody: Invitrogen, # AHB0052;

[0183] Molecular sieve protein marker: GE healthcare;

[0184] 3-8% Tris-Acetate precast gel: Invitrogen;

[0185] Carbon film supported copper mesh: Zhongke Keyi;

[0186] Uranium acetate: Zhongke Keyi;

[0187] ECL chemiluminescence kit: Pierce Biotechnology;

[0188] ThT detection reagent: Sigma-Aldrich;

[0189] Nitrocellulose membrane: Millipore.

[0190] 2.1.2 Experimental instruments

[0191] AKTA protein chromatograph: GE Healthcare Life Science, USA;

[0192] Protein electrophoresis apparatus: Bio-rad, USA;

[0193] Protein transfer system: Bio-rad, USA;

[0194] HT7700 transmission electron microscope: Hitachi, Japan;

[0195] Safire2 TM Microplate reader: Tecan Group, Switzerland;

[0196] Amersham Imager 680 imaging system: GE, USA.

[0197] 2.1.3 Main solutions

[0198] (1) 20 mM TBS buffer: weigh 2.4 g of Tris, 8 g of sodium chloride, dissolve with deionized water, adjust pH to 7.5 with dilute hydrochloric acid, and make up to 1 L.

[0199] (2) 0.1% TBST: add 1 mL of Tween-20 to 1 L of TBS, stir for 20 min.

[0200] (3) ThT solution: prepare a 100X ThT stock solution (500 mM) with 50 mM PB (phosphate buffer, pH = 6.5) solution, and dilute to 1X with 50 mM PB (pH = 6.5) when used.

[0201] (4) 5% skim milk.

[0202] (5) Electrophoresis buffer: dissolve 30 g of Tris, 144 g of glycine, and 10 g of SDS in deionized water to make up to 1 L, and use 10 times dilution.

[0203] (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 glycerol, 1 mL of 1% bromophenol blue, make up to 10 mL with deionized water.

[0204] (7) Transfer buffer: dissolve 30 g of Tris and 144 g of glycine in deionized water and make up to 1 L, and use 10 times dilution.

[0205] 2.2 Preparation and characterization of Aβ monomers, oligomers and fibers

[0206] The preparation of Aβ monomers, oligomers, and fibers was the same as in 1.4. Aβ monomers were incubated statically at 25°C for 0-4 days, and the aggregation state of Aβ was detected every 24 hours using ThT assay. The specific procedure was as follows: Aβ samples were diluted to 10 μM with PBS buffer, then 190 μL of ThT assay solution and 10 μL of the sample to be tested were thoroughly mixed in a black ELISA plate, and Safire2 was used as the assay medium. TM Fluorescence was measured using a microplate reader with an excitation wavelength of 440 nm and an emission wavelength of 480 nm. The results showed that the ThT reading of the Aβ sample gradually increased with increasing incubation time, indicating that Aβ monomers gradually aggregated into oligomers and fibrils. Figure 2 (Figure A).

[0207] Dot blot assays 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 at room temperature for 1 h. The membranes were then incubated with different detection antibodies at room temperature for 1–2 h, followed by washing three times with 0.1% TBST for 5 min each time, and then incubated with HRP-labeled secondary antibody at room temperature for 1 h. After washing three times with 0.1% TBST for 5 min each time, ECL chemiluminescence buffer was added to the membranes, and color development was performed using an Amersham Imager 680 imaging system. The size and density of the blots were quantitatively analyzed using ImageJ software. The results showed that 6E10 is an antibody that broadly binds to Aβ monomers, oligomers, and cellulose, and it can bind to 10 μM and 20 μM Aβ without significant difference in binding activity. Neither 3F nor A11 antibodies bind to Aβ monomers, and they bind to different oligomer types. 3F mainly binds to 10 μM Aβos, while A11 antibody mainly binds to 20 μM Aβos, indicating that 3F and A11 recognize different oligomer conformations. Figure 2 (See Figure B). Furthermore, statistical analysis of the affinity between 10 μM Aβos obtained from incubation for 0-4 days and 3F showed that Aβos obtained from 10 μM incubation for 2 days (sAβos, in vitro prepared Aβos) had the highest affinity for 3F. Therefore, subsequent experiments used this oligomer (sAβo*3F, in vitro prepared Aβo*3F) and 3F for immunoprecipitation. Figure 2 (Figure C).

[0208] 2.3 Molecular weight characterization of sAβo*3F

[0209] First, the molecular weight of sAβo*3F was determined using Western blot. The specific procedure is as follows: The Aβ sample was loaded into a 15% SDS-PAGE gel or a 3-8% Tris-Acetate gel. For Tris-Acetate gels, the sample was mixed with Novex gel before loading. TMTris-Glycine sample buffer was mixed. Electrophoresis was performed in Tris-Glycine electrophoresis buffer (containing 0.5 mM DTT, 1 mM ATP, and 5 mM MgCl2) at 4°C and 150 V for 4 h. For Western blot, proteins separated by SDS-PAGE or Tris–Acetate gel were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk at room temperature for 1–2 h, incubated overnight at 4°C with the detection antibody (6E10), washed three times with 0.1% TBST for 5 min each time, and reacted with the corresponding secondary antibody (HRP-labeled goat anti-mouse secondary antibody) at room temperature for 1 h. After three TBST washes, the membranes were covered with ECL chromogenic buffer, developed on an Amersham Imager 680 imaging system, and the protein bands were quantified using ImageJ software.

[0210] SDS-PAGE results showed that sAβo*3F mainly consists of two oligomers with different molecular weights: one approximately 12 kDa and the other greater than 180 kDa. However, native-PAGE results showed that sAβo*3F mainly consists of one oligomer with a molecular weight of approximately 500 kDa. The difference between the two electrophoretic results may be due to the influence of SDS. Figure 2 (D diagram).

[0211] To further determine the molecular weight of sAβo*3F, 500 μL of sAβo*3F or 100 μL of SEC marker was loaded into a Superdex 200 10 / 300GL 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 2 (E and F diagrams).

[0212] 2.4 Molecular weight and speciation characterization of mAβo*3F

[0213] Aβo*3F was extracted from the brains of APP / PS1 mice (mAβo*3F, Aβo*3F isolated from the brains of AD mice), and its molecular weight was analyzed. Consistent with the results of sAβo*3F, mAβo*3F showed two bands in SDS-PAGE, with molecular weights of 12 kDa and greater than 180 kDa, while in native-PAGE, mAβo*3F showed only one band with a molecular weight of approximately 500 kDa. Figure 2 (G diagram). SEC results show that the molecular weight of mAβo*3F is 588 kDa ( Figure 2Histochemistry). In addition, 10 μL mAβo*3F was dropped on 200 mesh copper mesh for 20 min, and then filtered paper was used to dry, and then 2% uranyl acetate was used to negative staining for 30 s, and then filtered paper was used to dry, and then air dried. The sample was examined under a transmission electron microscope (TEM, Hitachi H7700, Japan) at 120 kV working voltage at 100,000 times. The results showed that mAβo*3F was a particle with a diameter of about 10 nm Figure 2 Histochemistry).

[0214] 2.5 Molecular weight characterization of Aβo*3F in CSF of AD patients

[0215] The molecular weight size and distribution of Aβo*3F extracted from CSF of AD patients (hAβo*3F, human Aβo*3F isolated from CSF of AD patients) were consistent with the results of sAβo*3F and mAβo*3F. hAβo*3F had only one band in native-PAGE with a molecular weight of about 500 kDa, and two bands with molecular weights of 12 kDa and more than 180 kDa in SDS-PAGE Figure 3 Histochemistry).

[0216] Example 3 Detection of neurotoxicity of Aβo*3F

[0217] 3.1 Experimental materials

[0218] N2a cells: National Experimental Cell Resource Sharing Service Platform (NICR);

[0219] D-polylysine hydrobromide (PDL): Sigma-Aldrich

[0220] B27 supplement (50x): Thermo Fisher Scientific

[0221] Neurobasal TM -A medium: Gibco

[0222] 70 μm nylon cell filter screen: BD Bioscience

[0223] 3.2 Primary neuron culture

[0224] The brains of 14- to 15-day pregnant C57BL / 6 (purchased from Beijing Huafukang Bioscience Co., Ltd.) fetal mice were removed, the blood-brain membranes were stripped in HBSS (Hank's Balanced Salt Solution), and the separated hippocampus and cortex were cut into pieces with tweezers. The pieces of tissue were transferred into 15 mL centrifuge tubes, centrifuged at 600 rpm for 3 min, and 10 mL of 10-fold diluted trypsin digestion solution (containing DNase I) was added to the precipitate, which was digested at 37°C for 10 min, and gently inverted every 5 min. After the digestion was completed, the trypsin digestion reaction was terminated with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell screen to remove undigested tissue pieces. The cells were collected by centrifugation at 1200 rpm for 10 min, resuspended with DMEM medium, and plated into 12-well plates. After 2-4 h, the medium was replaced with Neurobasal medium (containing B27, 0.5 mM L-glutamine, 0.5% penicillin and streptomycin), and the cells were cultured for 7-9 days for subsequent experiments.

[0225] 3.3 Cell toxicity assay of sApo*3F

[0226] In this experiment, MTT method was mainly used to detect the toxicity of Aβ aggregates on neurons. The specific operation was as follows: N2a cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were trypsinized, centrifuged, resuspended with medium, and inoculated in a 96-well plate at about 5000 cells / 100 μL medium per well. After 12 h, the cells were treated with a series of concentration gradients of sApo*3F and sApo8, and the same 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 further 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 by MD-M5 microplate reader. The average value of six repeated wells was used for each sample and control, and each experiment was repeated three times. The cell viability was calculated by dividing the absorbance of the sample wells (background corrected) by the absorbance of the solvent wells (background corrected).

[0227] The MTT results showed that the half-inhibitory concentration (IC50) of sApo*3F on cell viability was about 0.186 nM, while the IC50 of sApo8 before immunoprecipitation was about 63.26 nM, with a difference of 340 times between the two IC50s (Figures A and B of). Figure 3 In addition, the addition of 0.5 nM sApo*3F to N2a cells caused 66% cell death, while the same concentration of sApo8 and sApo-ID had no significant neurotoxicity. When the concentration of sApo8 reached 200 nM, 70% cell toxicity was produced, while the neurotoxicity of sApo-ID could only reach 32% (Figure C of). Figure 3Figure 3D). These results indicate that sApo*3F is an oligomer with strong neurotoxicity.

[0228] 3.4 Cell toxicity assay of mApo*3F

[0229] The IC50of mApo*3F to N2a cells was about 0.111 nM, and to primary neurons was about 0.057 nM (Figure 3E). Specifically, 0.5 nM mApo*3F caused 78% of primary neurons to die, while mAβ*6E10 and mAβ-ID at the same concentration did not show significant cytotoxicity; when the concentration was increased to 200 nM, mAβ*6E10 produced 54% of neurotoxicity, while mAβ-ID had only 20% of neurotoxicity (Figure 3E). Figure 3 Figure 3 3.5 Cell toxicity assay of hApo*3F

[0230] The IC50of hApo*3F to primary neurons was about 0.076 nM (Figure 3F). Specifically, 0.2 nM hApo*3F reduced the cell viability by 68%, while hAβ*6E10 and hAβ-ID at this concentration did not have significant effect on neuron survival; when the concentration was increased to 40 nM, hAβ*6E10 reduced the cell viability by 66%, while hAβ-ID only reduced the cell viability by 40% (Figure 3G).

[0231] Figure 3 Figure 4

[0232] Example 4 Effect of Aβo*3F on cytokine expression levels of glial cells

[0233] 4.1 Experimental materials and methods

[0234] 4.1.1 Experimental materials

[0235] BV2 cells: NICR

[0236] Reverse transcription kit: Kangwei Century

[0237] UltraSYBR Mixture (Low ROX): Kangwei Century

[0238] 4.1.2 Experimental instruments

[0239] 7500 Fast Real-Time PCR Instrument: Applied Biosystems

[0240] T100 Thermal Cycler PCR Instrument: BIO-RAD ​​​​

[0241] NanoDrop Microspectrophotometer: Quawell

[0242] 4.2 Aβo*3F can increase the expression level of microglial inflammatory factors

[0243] BV2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were trypsinized, centrifuged, and resuspended in culture medium after medium, and inoculated in 12-well plates at about 5x10^5 cells / mL of medium per well. After 12 h, BV2 cells were treated with different Aβ samples, while the same volume of solvent was added to the cells as a control. After 48 h, the expression level of pro-inflammatory factors in cells was detected by fluorescence quantitative PCR (qPCR). The specific operation is as follows: the total mRNA of the cells was prepared by the conventional method and the mRNA concentration was measured using a microspectrophotometer. Reverse transcription was performed according to the reverse transcription kit instructions to obtain cDNA, and then the expression level of the target gene was detected using EasyQuick RT MasterMix. The primers used are as follows:

[0244] mTNF-α: 5'-GATTATGGCTCAGGGTCCAA-3' (SEQ ID NO: 1),

[0245] 5'-GCTCCAGTGAATTCGGAAAG-3 (SEQ ID NO: 2);

[0246] mIL-1β: 5'-CCCAAGCAATACCCAAAGAA-3' (SEQ ID NO: 3),

[0247] 5'-GCTTGTGCTCTGCTTGTGAG-3' (SEQ ID NO: 4);

[0248] mIL-6: 5'-CCGGAGAGGAGACTTCACAG-3' (SEQ ID NO: 5),

[0249] 5'-TTGCCATTGCACAACTCTTT-3' (SEQ ID NO: 6);

[0250] GAPDH: 5'-TGAATACGGCTACAGCAACA-3' (SEQ ID NO: 7),

[0251] 5'-AGGCCCCTCCTGTTATTATG-3' (SEQ ID NO: 8).

[0252] qPCR results showed that 0.3 nM mApo*3F, 200 nM mApo*6E10 and 200 nM mApo-ID could induce the expression levels of proinflammatory factors in microglia cells to increase significantly, including TNF-a, IL-6 and IL-1 b, but 0.3 nM mApo*6E10 or 0.3 nM mApo-ID had no obvious stimulating effect (FIG. 4A). Figure 4

[0253] 4.3 Primary astrocyte culture

[0254] Take 1-2 day old C57BL / 6 newborn mice, soak in 75% alcohol for disinfection, take out the brain and soak in HBSS buffer, peel off the blood brain membrane, cut the brain with tissue scissors, put it in a 15 mL centrifuge tube, centrifuge at 600 rpm for 3 min, add 10 mL of 10 times diluted trypsin digestion solution (containing DNase I) to the precipitate, digest at 37°C for 10 min, and gently invert every 5 min. After digestion, terminate the trypsin digestion reaction with 40 mL of DMEM medium (containing 10% FBS), and remove the undigested tissue blocks by passing the cell suspension through a 70 μm cell screen. Centrifuge at 1200 rpm for 10 min to collect the cells, resuspend the cells with DMEM medium, and culture the cells in a T-75 culture bottle at 37°C for 5-7 days. Replace the culture medium every 2-3 days. After 7 days of cell culture, trypsinize the cells and plate them in a 12-well plate.

[0255] 4.4 Apo*3F can stimulate the expression of inflammatory factors in astrocytes

[0256] Different Aβ samples were used to treat primary astrocytes, and qPCR was used to explore their effects on the expression levels of inflammatory factors in primary astrocytes. The specific operation steps are the same as those in 4.2. The primers used are as follows:

[0257] miNos: 5'-CACCTGGAACAGCACTCTCT-3' (SEQ ID NO: 9),

[0258] 5'-CTTTGTGCGAAGTGTCAGTG-3' (SEQ ID NO: 10);

[0259] mTNF-a: SEQ ID NO: 1 and 2;

[0260] mIL-1 b: SEQ ID NO: 3 and 4;

[0261] mIL-6: SEQ ID NO: 5 and 6;

[0262] GAPDH: SEQ ID NO: 7 and 8. ​

[0263] The results showed that 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID significantly increased the expression levels of pro-inflammatory factors in primary astrocytes, including TNF-α, IL-6, IL-1β, and iNos, but 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no significant effect on the expression levels of inflammatory factors in astrocytes. Figure 4 (Figure B).

[0264] 4.5 Aβo*3F disrupts astrocyte-mediated synapse formation.

[0265] Astrocytes can induce synapse formation by secreting synaptic kinases such as TSP1 and Gpc4 / 6. mAβo*3F was added to primary astrocytes, and changes in the expression levels of various synaptic kinases in astrocytes were detected by qPCR. The primers used are as follows:

[0266] mGpc4: 5'-CTGGAGGGTCCTTTCAACATT-3' (SEQ ID NO: 11),

[0267] 5'-GACATCAGTAACCAGTCGGTC-3' (SEQ ID NO: 12);

[0268] mGpc6: 5'-TAGTCCTGTATTGGCAGCCAC-3' (SEQ ID NO: 13),

[0269] 5'-GGCTAATGTCTATAGCAGGGAA-3' (SEQ ID NO: 14);

[0270] mTSP1: 5'-GGTAGCTGGAAATGTGGTGCGT-3' (SEQ ID NO: 15),

[0271] 5'-GCACCGATGTTTCTCCGTTGTGA-3' (SEQ ID NO: 16);

[0272] GAPDH: SEQ ID NO: 7 and 8.

[0273] The results showed that treatment with 0.3 nM mAβo*3F and 200 nM mAβ*6E10 significantly reduced the expression levels of TSP1 and Gpc4 / 6, while 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no significant effect. Only when the concentration of mAβ-ID increased to 200 nM did it reduce the expression level of Gpc4 in astrocytes. Figure 5 (Figure C).

[0274] Example 5 Effects of Aβo*3F on cognition and neuronal damage in the brain of mice

[0275] 5.1 Experimental materials and methods

[0276] 5.1.1 Experimental materials

[0277] Fast Golgi staining kit: FD NeuroTechnologies

[0278] Nissl staining kit: Sigma

[0279] 5.1.2 Experimental instruments

[0280] Brain stereotaxic injection system: Chinese Academy of Medical Sciences

[0281] Y maze apparatus: Chinese Academy of Medical Sciences

[0282] Novel object recognition apparatus: Chinese Academy of Medical Sciences

[0283] 5.2 Experimental animals

[0284] 3-month-old C57BL / 6 mice. All mice were raised in a clean room at 22 ± 2°C and 45% ± 10% humidity, and could eat and drink water at will, and were cycled according to 12 h light and 12 h darkness. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals for Public Health Services in China, and the experiments involving mice were approved by the Tsinghua University Animal Protection and Use Committee.

[0285] 5.3 Brain stereotaxic injection

[0286] 3-month-old C57BL / 6 mice were randomly divided into 6 groups of 6-8 mice each: 2.5 nM (45.5 pg) mAβo*3F, 2.5 nM (45.5 pg) mAβ*6E10, 2.5 nM (45.5 pg) mAβ-ID, 600 nM (10.9 ng) mAβ*6E10, 600 nM (10.9 ng) mAβ-ID, and control group with Tris-Gly solvent. After the mice were deeply anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg), they were fixed on a brain stereotaxic injection table, the scalp was cut along the midline to expose the skull, and the injection site was located using a stereotaxic instrument: bregma as the starting point, AP = -1.7 mm, ML = ±1.0 mm, DV = -1.5 mm. After drilling the skull along the positioning point, bilateral injection was performed, 4 μL per hemisphere, at a speed of 0.4 mL / min. After injection, the needle was left for 3 min to allow complete absorption of the sample. The surgical site was washed with sterile saline and the incision was sutured. The mice were monitored and provided with postoperative care. After 24 h of stereotaxic injection, the behavior and cognitive ability of the mice were detected, and then the mice were dissected for physiological and biochemical analysis.

[0287] 5.4 Aβo*3F severely impaired the memory ability of mice

[0288] The novel object recognition test and Y maze test were used to detect changes in the cognitive ability of mice.

[0289] 5.4.1 Novel object recognition test

[0290] The novel object recognition test is based on the spontaneous tendency of mice to interact more with new, unfamiliar objects. The experiment mainly consists of three stages:

[0291] (1) Familiar environment stage: a white box 40 cm wide x 40 cm deep x 40 cm high was prepared, and each mouse was allowed to freely explore the box for 5 min without objects in the box. 1 h later, the mice were injected with the Aβ sample;

[0292] (2) Training stage: 24 h later, two identical objects were placed in the box, and each mouse was placed in the same box as in the first stage, allowing the mouse to freely explore for 5 min. The number of times the mouse sniffed and touched each object was recorded;

[0293] (3) Test phase, after 6h training, the right object was replaced by a new object with different material, color and shape, and the mice were allowed to freely explore for another 5 min. The number of sniffing and touching of the two objects were recorded to evaluate the memory ability of the mice. Discrimination index was calculated by the following formula: (number of new object - number of old object) / (number of new object + number of old object).

[0294] The results showed that the discrimination index of the mice treated with 2.5 nM mApo*3F and 600 nM mApo*6E10 were significantly decreased compared with the control group, while the discrimination index of the mice treated with other Aβ were not significantly different compared with the control group, indicating that Apo*3F significantly reduced the memory ability of the mice (Figure 1A). Figure 5

[0295] 5.4.2 Y maze experiment

[0296] After the novel object recognition experiment, the spatial memory ability of the mice was evaluated by Y maze experiment. Y maze was composed of three arms with the same size, and each arm was 120 degrees apart. The size of each arm was 8 cm x 30 cm x 15 cm (width x length x height). The experiment mainly consisted of two phases:

[0297] (1) Training phase, the three arms were randomly named A, B and C. Arms A and B were open during the experiment, while arm C was closed during the training phase. After being placed in arm A, each mouse was allowed to freely explore arms A and B for 10 min;

[0298] (2) After 1 h of training, arm C was opened, and the mice were again placed in arm A in order. The mice were allowed to explore arms A, B and C for 5 min, and the time spent in each arm was analyzed. The whole experiment was recorded by a camera installed above the Y maze.

[0299] The results showed that the time spent in the new arm of the mice treated with 2.5 nM mApo*3F and 600 nM mApo*6E10 was significantly decreased compared with the control group. However, there was no significant difference between the mice treated with 2.5 nM mApo*6E10, 2.5 nM mApo*ID and 600 nM mApo*ID and the control group. This indicated that mApo*3F, but not mApo*6E10 or mApo-ID, caused severe memory impairment in mice at low concentrations. For mApo*6E10, only when the concentration increased to 600 nM, could it cause similar degree of memory impairment in mice (Figure 1B). Figure 5

[0300] 5.5 Apo*3F significantly reduced the density of dendritic spines in mouse neurons ​​

[0301] Brain samples from mice were stained using the FD Rapid Golgi Stain Kit. 24 h before dissection of the mice, mix solutions A and B from the kit 1 : 1 and store in the dark. After sacrifice of the mice, without perfusion, remove the brain and bisect the brain along the midline. Remove the blood from the surface of the left brain half with PBS and blot the surface of the tissue with filter paper. Then place the tissue in the pre-prepared A+B solution. Change the solution once after 24 h and store at room temperature in the dark for 2 weeks. During the incubation period, gently invert the tissue twice a week for optimal results. After 2 weeks, transfer the tissue to solution C and change the solution once after 24 h. Store at room temperature in the dark for 5 days. Then, obtain 100 pm thick cryosections using a cryostat at -22 °C. After the sections have dried naturally at room temperature, start the staining. First wash the sections twice with Milli-Q water for 4 min each time, then prepare the staining working solution and place the sections in the staining working solution for 15 min. Wash the sections twice with Milli-Q water for 5 min each time. After the staining, dehydrate the sections with 50%, 75%, 95% and 100% ethanol for 4 min each concentration step. Then place the sections in xylene and mount with neutral resin for observation. Use the 100x objective lens of the Olympus inverted microscope to observe the dendrites and dendritic spines of the neurons in the CA1 region of the hippocampus. The density of the dendritic spines was determined by analysis using the Image J software (dendritic spine density = number of dendritic spines / length of dendrites).

[0302] The density of the dendritic spines was determined by analysis using the Image J software (dendritic spine density = number of dendritic spines / length of dendrites). It was found that the density of the dendritic spines of the neurons in the CA1 region of the brain of mice treated with 2.5 nM mApo*3F and 600 nM mApo*6E10 was significantly reduced compared to the control group, but 2.5 nM mApo*6E10, 2.5 nM mApo-ID or 600 nM mApo-ID had no significant effect on the density of the dendritic spines of the neurons in the brain of mice (Figures C and D). Figure 5

[0303] 5.5 Apo*3F significantly reduces the number of neurons in the hippocampus of mice

[0304] 5.5.1 Paraffin embedding and sectioning

[0305] ​After the behavioral experiment, the mice were deeply anesthetized with sodium pentobarbital (50 mg / kg) and perfused with ice-cold PBS containing heparin (10 U / mL) through the heart, and then sacrificed by dissection. After the mouse brain was removed, it was divided into left and right hemispheres along the midline. The left hemisphere was fixed in 4% paraformaldehyde for 48 h, and then dehydrated in ethanol gradient: 50%, 70%, 80%, 90% ethanol for 1 h each. 100% ethanol for 1 h, repeated once, then 50% xylene for 30 min, repeated once. After dehydration, the tissue was soaked in paraffin for 4 h for tissue embedding. 5 μm tissue sections were obtained using a paraffin tissue section machine.

[0306] 5.5.1 Nissl staining

[0307] After the 5 μm paraffin sections were heated at 60°C for 30 min, they were deparaffinized by soaking in 100% xylene, 50% xylene, 100% alcohol for 10 min each, 75% ethanol, 50% ethanol, 30% ethanol and deionized water for 5 min each. Then according to the Nissl staining instructions, add cresyl violet solution on the section, heat at 56°C for 1 h, differentiate at room temperature for 1-3 min until the background is close to colorless, then decolorize with gradient alcohol (70%, 95% and 100%; 20 seconds each), and soak in xylene, neutral resin for mounting, then collect data and analyze using a microscope. The results show that 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly reduce the number of neurons in the CA1 and DG regions of the mouse brain, while 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID or 600 nM mAβ-ID have no significant effect on the number of neurons (Figs. E, F, G). Figure 6

[0308] Example 6 Aβo*3F activates glial cells in the mouse brain to produce neuroinflammation

[0309] 6.1 Experimental materials and methods

[0310] 6.1.1 Experimental materials

[0311] Anti-GFAP antibody: Cell Signaling Technology, #3670

[0312] Anti-Iba-1 antibody: GeneTex, #GTX101495

[0313] BCA kit: Thermo Fisher Scientific

[0314] RIPA strong lysis buffer: Thermo Fisher Scientific

[0315] Protease inhibitor: Millipore ​

[0316] Phosphatase inhibitor (100×): Solarbio

[0317] Mouse IL-1β Detection Kit: Biolegend

[0318] Mouse IL-6 Detection Kit: Biolegend

[0319] 6.1.2 Experimental Apparatus

[0320] MD-M5 ELISA reader: Molecular Devices, USA

[0321] Tissue Lyser II tissue abrasive: Qiagen

[0322] 6.1.3 Immunohistochemistry

[0323] Dewaxed 5μm paraffin sections were immersed in citrate antigen retrieval solution (3g trisodium citrate, 0.4g citric acid dissolved in deionized water, diluted to 1L), boiled in a water bath for 15min, and then cooled naturally at room temperature. After antigen retrieval, the sections were washed three times with PBS for 5min each time, then fixed with 80% (vol / vol) methanol containing 0.3% H2O2 to remove endogenous catalase. The sections were then washed three times with PBS for 5min each time, placed in 0.3% Triton X-100 penetration buffer, and penetrated at room temperature for 20min, followed by three PBS washes for 5min each time. The sections were blocked with 10% donkey serum at room temperature for 1h, then incubated overnight at 4℃ with the corresponding primary antibody. The next day, the sections were washed three times with PBS for 5min each time, then incubated with the corresponding secondary antibody at room temperature for 1h, washed three times with PBS, and then developed for observation.

[0324] 6.2 Aβo*3F can activate microglia and astrocytes.

[0325] Staining the hippocampal microglia marker Iba-1 revealed 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. However, treatment with 2.5 nM mAβ*6E10 or mAβ-ID in mice had no significant effect. Figure 6 (Figures A, C, and D). Furthermore, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly induced microglia in the DG region to enter an amoeboid state characterized by enlarged cell bodies and reduced branching, indicating a state of microglia overactivation. Figure 6Figure 6. Aβo*3F increases the expression of GFAP in the hippocampus of mice. The levels of GFAP in the hippocampus of mice injected with 2.5 nM mAβo*3F, 600 nM mAβ*6E10 and 600 nM mAβ-ID were determined by Western blotting (A) and immunohistochemistry (B, E, F). The results showed that the levels of GFAP in the hippocampus of mice injected with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 were significantly higher than those of the control group (B, E, F) (p < 0.05, n = 3). The results of immunohistochemistry showed that the astrocytes in the DG and CA1 regions of the hippocampus of mice injected with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 were activated, and the expression levels of GFAP in the hippocampus of mice injected with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 were increased (C, D). Figure 6

[0326] 6.3 Aβo*3F increases the levels of inflammatory factors in the hippocampus of mice

[0327] The levels of inflammatory factors (IL-6 and IL-1 β) in the brain of mice were determined by using IL-6 and IL-1 β ELISA kits, respectively, according to the manufacturer's instructions. Briefly, the brain homogenate was diluted and added to the ELISA plate, which was previously coated with the corresponding capture antibody. Then the corresponding detection antibody and secondary antibody were added. The absorbance was measured at 450 nm using TMB as the substrate and MD-M5 microplate reader. The results showed that the levels of IL-1 β and IL-6 in the hippocampus of mice injected with 2.5 nM mAβo*3F, 600 nM mAβ*6E10 and 600 nM mAβ-ID were significantly increased, while the levels of IL-1 β and IL-6 in the brain 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 (G, H). Figure 7

[0328] Example 7 Detection of the levels of Aβo*3F in the CSF and plasma of AD patients (MSD method)

[0329] 7.1 Experimental materials and methods

[0330] 7.1.1 Experimental materials

[0331] Aβ multi-factor detection kit (4G8): Meso Scale Diagnostics

[0332] 7.1.2 Experimental instruments

[0333] MSD-S600 electrochemiluminescence detector Meso Scale Diagnostics, USA

[0334] 7.2 Detection of the levels of Aβo*3F in the plasma and CSF of AD patients

[0335] In order to evaluate the correlation between Aβos specifically recognized by 3F (Aβo*3F) and the pathogenesis of AD patients, the levels of Aβ42 and Aβ40 contained in Aβo*3F isolated from the CSF and plasma of AD patients were tested.

[0336] ​​The human plasma and CSF samples used in this study were approved by the Ethical Review Committee of the First Affiliated Hospital of Zhengzhou University, and all subjects signed a written informed consent form before participating in the study. The basic information of the subject samples used in this study is as follows: 20 mild cognitive impairment (MCI) patients with an average age of 73.3 years (age range 57-84 years), 20 AD patients with an average age of 69.4 years (age range 52-85 years), and 20 healthy people with an average age of 70.9 years (age range 65-78 years) were collected. In addition, 7 CSF samples of AD patients with an average age of 68 years (age range 59-77 years) and 7 CSF samples of non-dementia subjects with an average age of 66.3 years (age range 48-79 years) were also collected.

[0337] To extract Aβo*3F in the plasma and CSF of AD patients and healthy elderly, first, the human plasma or CSF sample was added to 100 μL Protein A / G-agarose gel, incubated at 4°C for 1 h to remove endogenous IgG, and then the sample was incubated with Protein A magnetic beads cross-linked with 3F antibody at room temperature for 2 h. The next day, the magnetic beads were washed three times with 0.1% PBST, and then eluted with 20 mM glycine (pH 2.0) for 3 min, twice, and the eluate was neutralized to pH 7 with 1M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was detected by MSD Aβ multifactor detection kit (4G8). The results showed that the levels of Aβ42 and Aβ40 contained in Aβo*3F in the CSF and plasma of AD patients were significantly increased compared with healthy elderly Figure 7 ). In addition, the levels of Aβ42 and Aβ40 contained in Aβo*3F in the plasma of MCI patients were also significantly higher than those in the control group, and gradually increased with the development of AD pathology Figure 8 ). These results showed that the levels of Aβo*3F in the CSF and plasma of AD patients and AD-derived MCI patients were significantly higher than those in healthy elderly, and were closely related to the development trend of AD pathology, indicating that Aβo*3F can be used as a biomarker for AD diagnosis, especially early diagnosis of AD. The statistical method of one-way ANOVA with Tukey’s multiple comparison test in GraphPad Prism 8 software was used for statistics, 95% confidence interval, * represents P<0.05, indicating a statistically significant difference; ** represents P<0.01, indicating a very statistically significant difference; *** represents P<0.001, indicating a highly statistically significant difference; **** represents P<0.0001, indicating a highly statistically significant difference.

[0338] Example 8 Detection of Aβo*3F levels in plasma of AD patients and MCI patients (chemiluminescence method)

[0339] 8.1 Experimental materials and methods

[0340] 8.1.1 Experimental materials

[0341] 102 antibody: Beijing Reeleng Biotechnology Co., Ltd.

[0342] 8.1.2 Experimental instruments

[0343] MG60 pro chemiluminescence detector: Beijing Reeleng Biotechnology Co., Ltd., Beijing

[0344] 8.2 Detection of Aβo*3F levels in plasma of AD patients

[0345] To evaluate the correlation between Aβos specifically recognized by 3F (Aβo*3F) and the pathogenesis of AD patients, the levels of Aβ42 contained in Aβo*3F isolated from the plasma of AD patients were tested.

[0346] 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 a written informed consent form before participating in the study. The basic information of the subject samples used in this study is as follows: the plasma samples of 3 AD patients with an average age of 65.6 years (age range 62-75 years), 3 MCI patients with an average age of 63.3 years (age range 62-65 years), and 3 healthy people with an average age of 63.6 years (age range 61-65 years) were collected.

[0347] To extract Aβo*3F from the plasma of AD patients and healthy elderly people, first, the human plasma sample was added to 200 μL Protein A-agarose gel, incubated at room temperature for 15 min to remove endogenous IgG, and then the sample was incubated with 3F antibody-coated JSR magnetic beads at room temperature for 2 h. The next day, the magnetic beads were washed three times with 0.1% PBST, eluted with 20 mM glycine (pH 2.2) for 5 min, eluted twice, and the eluate was neutralized to pH 7 with 1M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was detected by 3F-102 detection kit. The results showed that the level of Aβ42 contained in Aβo*3F in the plasma of AD patients was significantly increased compared with that of healthy elderly people ( Figure 8 ). In addition, the level of Aβ42 contained in Aβo*3F in the plasma of MCI patients was also significantly higher than that of the control group ( Figure 9). These results show that the level of Aβo*3F in the plasma of AD and MCI patients is significantly higher than that of healthy elderly, indicating that Aβo*3F can be used as a biomarker for the diagnosis of AD, especially the early diagnosis of AD.

[0348] Example 9 Diagnosis of AD by detecting the level of Aβo*3F in CSF or plasma

[0349] The human plasma and CSF samples in Example 7 were shuffled and renumbered, and submitted to an experimenter who did not know the sample information to enrich Aβo*3F in the samples with Protein A magnetic beads cross-linked with 3F antibody according to the method of Example 7, and detect the levels of Aβ42 and Aβ40 contained in Aβo*3F in the samples with MSD Aβ multi-factor detection kit (4G8). The results show that the amount of Aβ42 and Aβ40 contained in Aβo*3F in the samples is positively correlated with the pathology of AD, and the level of Aβo*3F in human plasma and CSF samples detected by this method can significantly distinguish AD, MCI patients from healthy people Figure 10 ) samples.

[0350] Example 10 Optimization of antibodies for detecting Aβo*3F oligomers

[0351] The 100th amino acid K of 3F antibody was mutated to R (not counting the MA residues introduced for expression as a single-chain antibody, it is the 98th amino acid of the antibody, and the variant is named K98R mutant, the sequence is shown as SEQ ID NO: 24), and the binding of K98R mutant to Aβo*3F oligomers was studied. The method is as follows.

[0352] The above Aβ oligomers were coated in 96-well plates at 100 ng / well, and coated overnight at 4°C. The next day, 3% BSA was used for room temperature blocking for 2 hours, and then washed with PBST for 2 times and dried. K98R mutant and 3F were diluted, starting from 1 μg / ml, with a dilution ratio of 2, a total of 14 gradients. The diluted antibody solution was added to the ELISA plate coated with the above Aβ oligomers, and parallel duplicate wells were prepared, incubated at 37°C for 1 hour, washed with PBST for 3 times, and dried. Goat anti-human IgG-HRP was added at a dilution of 1:10000, and incubated at 37°C for 45 min, washed with PBST for 3 times, and dried. TMB color developing solution was added for color development for about 15 minutes, and then stopped with stop solution. OD 450 nm was read, and the values were analyzed and plotted with graphpad.

[0353] The results are as follows ​As shown, the experiment shows that the binding affinity of the K98R mutant to the Aβo*3F oligomer is further improved, suggesting that the higher binding force of the mutant to the Aβo*3F oligomer will help to more sensitively detect the presence of the Aβo*3F oligomer in the body of the subject, thereby more early diagnose whether the subject suffers from the neurodegeneration-related disease. The present inventors considered the reason leading to this result, and found that the 98th amino acid is also a residue of the heavy chain CDR3 of the 3F antibody when numbered according to the IMGT numbering (the heavy chain / light chain CDR sequences of the 3F antibody according to the IMGT numbering 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)). The present inventors also mutated several other variant sequences to K98R, and the results are similar (data not shown).

[0354] Equivalents

[0355] While several embodiments of the present application have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for implementing the functions and / or aspects of the application and / or for achieving the results and / or advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present application. More generally, those skilled in the art will readily appreciate that all parameters, materials, and / or configurations described herein can be varied and / or combined for

[0356] The phrase “and / or,” as used herein in the specification and in claims, should be understood to mean “either or both of” i.e., “one or the other or both” when applied to elements listed with “and / or” to indicate one, some, or all of the elements so conjoined, abutting sets of elements, and in the alternatives (as “one or the other but not both” where consecutive listing of alternatives, such as “only one of the other or both”), is intended. In addition to the existence of the elements identified by the “and / or” clause, there can exist additional elements not specifically identified within the clause, and each such additional element can be conjunctively or disjunctively present provided that the additional element (s) do not standalone create a mutually exclusive progression. As a non-limiting example, a reference to “A and / or B” when used in this specification and claims, means “A alone, B alone, or A and B.” Conversely, references in the specification and claims to “only one of A or B” or “exactly one of A or B,” or “either A or B but not both” are to be understood as an

[0357] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when used in the context of listing items, “or” or “and / or” should be understood as inclusive of one or more elements, but also optionally including more than one of the elements, and optionally, other unlisted items. Only terms clearly expressed as the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of’ shall imply exclusion of more than one of a single element or element list. In general, the term “or” as used herein shall only be understood to mean an exclusive alternative (i.e., “one or the other but not both”) when preceded by terms clearly express the exclusivity, such as “either,” “only one of,” “either / or,” or “one of the other.” “Consisting essentially of’ for use in the claims shall have its ordinary meaning in the field of patent law.

[0358] As used herein in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from among the individual elements in the list of one or more elements, but not necessarily including at least one of each and every element specifically listed within the list of one or more elements and not excluding any combinations of elements in the list of one or more elements. This definition also allows that the phrases "at least one" and "one or more" can be utilized interchangeably to indicate that at least one, or one or more, elements can be present within a list of elements. The use of at least one and of one or more should therefore not be interpreted as specific limitations but to the contrary, the inclusion of at least one and of one or more items in a list of one or more items does not exclude other additional items. By way of example, in one embodiment, "at least one of A and B" (or, equivalently, "one or more of A or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0359] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively.

[0360] The use of the terms "first," "second," "third," and the like in the description and the claims does not imply any priority or order of one claim element over another, but merely distinguishes different claim elements from each other.

Claims

1. Detection of Aβo in subject samples The use of reagents containing the presence and / or levels of 3F in the preparation of kits for methods used to diagnose whether a subject has early and mid-to-late Alzheimer's disease (AD) or AD-related mild cognitive impairment (MCI) or is at risk of having AD, wherein said methods include the following steps: a) To detect Aβo in the subject sample The presence and / or level of 3F in the reagent comes into contact with the subject's sample. b) Detect Aβo in the subject sample The presence and / or level of 3F in, Aβo in the subject sample The presence and / or level of 3F indicates that the subject has AD or AD-related MCI, wherein Aβo 3F is an Aβ oligomer that specifically binds to the 3F antibody. Based on size exclusion chromatography analysis, its molecular weight is approximately 588 kDa. The light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NO: 17-22, respectively.

2. The use according to claim 1, wherein the method includes the step of providing a sample to be tested from the subject before step a).

3. The use according to claim 1, wherein the subject is a patient suspected of having AD or AD-related MCI.

4. The use according to claim 1, wherein the subject is a human, a non-human primate, a cat, or a dog.

5. The use according to claim 1, wherein the method further comprises clinical neuropsychological and neuroimaging assessment steps.

6. The use according to claim 5, wherein the neuroimaging is Aβ-PET scanning imaging and / or tau-PET.

7. The use according to claim 1, wherein the method further comprises detecting changes in tau levels in the subject sample.

8. The use according to claim 7, wherein the tau level is the total tau level or the phosphorylated tau level.

9. The use according to any one of claims 1-8, wherein the method relates to Aβo 3F enrichment reagent, the Aβo 3F enrichment reagents include those that specifically bind Aβo 3F binder.

10. The use according to claim 9, wherein Aβo is specifically bound. The 3F binder specifically binds to Aβo. Antibody 3F.

11. The use according to claim 10, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

12. The use according to claim 11, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

13. The use according to any one of claims 1-8, wherein Aβo is detected. The presence and / or level of 3F reagents include those that specifically bind to Aβo. The first binder of 3F or Aβ aggregates.

14. The use according to claim 13, wherein Aβo is specifically bound. The first binder of 3F or Aβ aggregates is Aβo specifically bound. Antibodies against 3F or Aβ aggregates.

15. The use according to claim 14, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

16. The use according to claim 15, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

17. The use according to claim 13, wherein the first binder is connected to a detection agent that allows it to be detected.

18. The use according to claim 13, wherein Aβo is detected. The reagents present and / or at levels of 3F also include a second binder that specifically binds to the first binder.

19. The use according to claim 18, wherein the second binder is an antibody that specifically binds to the first binder.

20. The use according to claim 19, wherein the antibody that specifically binds to the first binder is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

21. The use according to claim 20, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

22. The use according to claim 19, wherein the antibody that specifically binds to the first binder is linked to a detection agent that allows it to be detected.

23. The use according to any one of claims 17-22, wherein the detection agent is selected from chemiluminescent labeling, electrochemiluminescent labeling, chromophores, fluorescent labels, paramagnetic labels, radioactive labels, avidin / biotin, haptens, digoxigenin, metal complexes, metals, enzymes, colloidal gold, or combinations thereof.

24. The use according to claim 23, wherein the fluorescent label is selected from fluorescein-type labels, umbelliferone, lissamine, cyanine, Texas Red, Ingenium's BODIPY FL-SE®, or the like.

25. The use according to claim 23, wherein the avidin / biotin is selected from biotin or streptavidin / biotin.

26. The use according to any one of claims 1-8, wherein the detection is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single-molecule immunoassay, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single-photon emission computed tomography.

27. The use according to claim 26, wherein 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.

28. The use according to any one of claims 1-8, wherein the subject sample is selected from the subject's cells, tissues, organs and / or body fluids.

29. The use according to claim 28, wherein 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.

30. The use according to claim 28, wherein the body fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

31. The use according to claim 13, wherein the specific binding of Aβo The first binder of 3F is attached to the solid support.

32. The use according to any one of claims 1-8, wherein the method further comprises the step of detecting a control sample, said control sample being derived from a healthy subject or a subject not suffering from AD.

33. The use according to claim 10, wherein the specific binding of Aβo The 3F antibody is against Aβo. Polyclonal antibodies and / or monoclonal antibodies or their antigen-binding fragments obtained by immunization with 3F.

34. The use according to claim 33, wherein 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.

35. The use according to any one of claims 1-8, wherein, Based on the MSD electrochemiluminescence method, Aβo in the cerebrospinal fluid of healthy individuals The concentration of 3F is Aβ42o 3F: 80.44±20.88 pg / ml, Aβ40o 3F: 24.35±5.08 pg / ml; and / or plasma Aβo The concentration of 3F is Aβ42o 3F: 71.63±36.8 pg / ml, Aβ40o 3F: 2.24±0.92 pg / ml; plasma Aβ0 in individuals with mild cognitive impairment. The concentration of 3F is Aβ42o 3F: 112.93±25.02 pg / ml, Aβ40o 3F: 4.64±1.43 pg / ml; Aβ0 in the cerebrospinal fluid of AD patients. The concentration of 3F is Aβ42o 3F: 264.8±42.26 pg / ml, Aβ40o 3F: 85.74±10.62 pg / ml, and / or plasma Aβo The concentration of 3F is Aβ42o 3F: 159.44±36.8 pg / ml, Aβ40o 3F: 14.0±5.59 pg / ml; or Based on chemiluminescence methods, Aβ42O in the plasma of healthy individuals The concentration of 3F was 68.02±39.17 pg / ml; the plasma Aβ42O in individuals with mild cognitive impairment was... The concentration of 3F was 124.5 ± 12.57 pg / ml; the plasma Aβ42O level in AD patients was... The concentration of 3F was 205.75 ± 50.96 pg / ml.

36. The use according to any one of claims 1-8, wherein, Based on the MSD electrochemiluminescence method and the chemiluminescence method, the sensitivity of the detection is as low as 0.5 pg / ml.

37. The use according to claim 10, wherein Aβo is specifically bound. The 3F antibody is specifically targeted at Aβo. Multiple antibodies against different epitopes of 3F.

38. The use according to claim 37, wherein the plurality of antibodies are specifically targeted at Aβo. Two, three, four, five or more different epitopes of 3F antibodies.

39. The use according to claim 9, wherein Aβo is enriched from the sample. The 3F step utilizes specific binding to Aβo The immunoprecipitation method was used to detect antibodies from 3F.

40. A kit for diagnosing whether a subject has AD or AD-related MCI or is at risk of having AD, comprising detecting Aβo in a subject sample. The presence and / or level of 3F in the reagent, wherein Aβo 3F is an Aβ oligomer that specifically binds to the 3F antibody. Based on size exclusion chromatography analysis, its molecular weight is approximately 588 kDa. The light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NO: 17-22, respectively.

41. The kit of claim 40, wherein the kit comprises Aβo 3F enrichment reagent, the Aβo 3F enrichment reagents include those that specifically bind Aβo 3F binder.

42. The kit according to claim 41, wherein Aβo is specifically bound. The 3F binder specifically binds to Aβo. Antibody 3F.

43. The kit according to claim 42, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

44. The kit according to claim 43, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

45. The kit according to claim 40, wherein Aβo is detected. The presence and / or level of 3F reagents include those that specifically bind to Aβo. The first binder of 3F or Aβ aggregates.

46. ​​The kit according to claim 45, wherein Aβo is specifically bound. The first binder of 3F or Aβ aggregates is Aβo specifically bound. Antibodies against 3F or Aβ aggregates.

47. The kit according to claim 46, wherein the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

48. The kit according to claim 47, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

49. The kit of claim 45, wherein the first binder is connected to a detection agent that allows it to be detected.

50. The kit according to claim 45, wherein Aβo is detected. The reagents present and / or at levels of 3F also include a second binder that specifically binds to the first binder.

51. The kit of claim 50, wherein the second binder is an antibody that specifically binds to the first binder.

52. The kit according to claim 51, wherein the antibody that specifically binds to the first binder is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

53. The kit according to claim 52, wherein the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, biantibody, or bispecific antibody.

54. The kit of claim 51, wherein the antibody that specifically binds to the first binder is linked to a detection agent that allows it to be detected.

55. The kit according to any one of claims 49-54, wherein the detection agent is selected from chemiluminescent labeling, electrochemiluminescent labeling, chromophore, fluorescent labeling, paramagnetic labeling, radioactive labeling, avidin / biotin, hapten, digoxigenin, metal complex, metal, enzyme, colloidal gold, or combinations thereof.

56. The kit according to claim 55, wherein the fluorescent label is selected from fluorescein-type labels, umbelliferone, lissamine, cyanine, Texas red, Ingenium's BODIPY FL-SE®, or similar products.

57. The kit according to claim 55, wherein the avidin / biotin is selected from biotin or streptavidin / biotin.

58. The kit according to claim 40, wherein the detection is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single-molecule immunoassay, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single-photon emission computed tomography.

59. The kit according to claim 58, wherein the kit comprises reagents, materials, containers and / or devices required for performing detection selected from chemiluminescence assays, electrochemiluminescence assays, enzyme-linked immunosorbent assays, immunofluorescence assays, immunohistochemistry assays, immunochromatography assays, radioimmunoassays, single-molecule immunoassays, flow cytometry, cell sorting, immunoprecipitation assays, immunodiffusion assays, dot blot assays, Western blots and / or protein chips.

60. The kit according to claim 58, wherein the enzyme-linked immunosorbent assay (ELISA) is selected from direct ELISA, indirect ELISA, direct sandwich ELISA, and indirect sandwich ELISA.

61. The kit according to claim 40, wherein the subject sample is selected from the subject's cells, tissues, organs and / or body fluids.

62. The kit according to claim 61, wherein 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.

63. The kit according to claim 61, wherein the body fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

64. The kit of claim 45, wherein the specific binding of Aβo The first binder of 3F is attached to the solid support.

65. The kit of claim 40, wherein the kit further comprises a control sample derived from a healthy subject or a subject not suffering from AD.

66. The kit of claim 42, wherein the specific binding of Aβo The 3F antibody is against Aβo. Polyclonal antibodies and / or monoclonal antibodies or their antigen-binding fragments obtained by immunization with 3F.

67. The kit according to claim 66, wherein 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.

68. The kit according to claim 40, wherein, Based on the MSD electrochemiluminescence method, Aβo in the cerebrospinal fluid of healthy individuals The concentration of 3F is Aβ42o 3F: 80.44±20.88 pg / ml, Aβ40o 3F: 24.35±5.08 pg / ml; and / or plasma Aβo The concentration of 3F is Aβ42o 3F: 71.63±36.8 pg / ml, Aβ40o 3F: 2.24±0.92 pg / ml; plasma Aβ0 in individuals with mild cognitive impairment. The concentration of 3F is Aβ42o 3F: 112.93±25.02 pg / ml, Aβ40o 3F: 4.64±1.43 pg / ml; Aβ0 in the cerebrospinal fluid of AD patients. The concentration of 3F is Aβ42o 3F: 264.8±42.26 pg / ml, Aβ40o 3F: 85.74±10.62 pg / ml, and / or plasma Aβo The concentration of 3F is Aβ42o 3F: 159.44±36.8 pg / ml, Aβ40o 3F: 14.0±5.59 pg / ml; or Based on chemiluminescence methods, Aβ42O in the plasma of healthy individuals The concentration of 3F was 68.02±39.17 pg / ml; the plasma Aβ42O in individuals with mild cognitive impairment was... The concentration of 3F was 124.5 ± 12.57 pg / ml; the plasma Aβ42O level in AD patients was... The concentration of 3F was 205.75 ± 50.96 pg / ml.

69. The kit according to claim 40, wherein, Based on the MSD electrochemiluminescence method and the chemiluminescence method, the sensitivity of the detection is as low as 0.5 pg / ml.

70. The kit according to claim 42, wherein Aβo is specifically bound. The 3F antibody is specifically targeted at Aβo. Multiple antibodies against different epitopes of 3F.

71. The kit according to claim 70, wherein the plurality of antibodies are specifically targeted at Aβo. Two, three, four, five or more different epitopes of 3F antibodies.

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