CN114594272A

CN114594272A - Products and methods for detecting beta-amyloid

Info

Publication number: CN114594272A
Application number: CN202210490820.8A
Authority: CN
Inventors: 张博文; 康志云; 王怡; 郑新越; 杨志云; ***; 王月丹; 王铁山
Original assignee: Beijing First Biochemical Pharmaceutical Co ltd
Current assignee: Beijing First Biochemical Pharmaceutical Co ltd
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-06-07
Anticipated expiration: 2042-05-07
Also published as: CN114594272B

Abstract

The present application provides products for the detection of beta-amyloid in a biological sample and/or for the diagnosis of alzheimer's disease comprising reagents for determining the level of beta-amyloid in said biological sample. The present application also provides a method for in vitro detection of beta-amyloid in a biological sample comprising determining the level of beta-amyloid in said biological sample using the product described above.

Description

Products and methods for detecting amyloid beta

Technical Field

The present application relates to the field of biochemistry and antibodies, and more specifically to products for the detection of beta-amyloid in biological samples and/or for the diagnosis of alzheimer's disease.

Background

Alzheimer's Disease (AD) is a progressive neurological Disease that impairs thinking, memory and independence, leading to early infancy, which is currently not prevented, delayed or prevented, creating an increasingly serious global health crisis. The preclinical stage involves all invisible changes in the brain (such as plaque accumulation) occurring years before symptoms appear, with the development of disease and the increasing need for patient care in daily activities, mild AD patients can still take care of their lives despite beginning to forget the location of familiar words or objects; progressing to moderate, patients become more forgetful, more difficult to perform routine tasks, and experience changes in personality and behavior; patients with severe AD can no longer respond to their environment, can not talk, and can not control exercise or control stool.

The etiology of AD is still unclear, and the Amyloid beta protein (a β) cascade hypothesis suggests that a β protein metabolic imbalance, oligomers and fibers formed by aggregation and formation are deposited in the brain, and the deposits form senile plaques, which cause increase of neuroinflammation and brain lesions such as neuronal apoptosis, and are important factors inducing alzheimer's disease. A β has a molecular weight of about 4kDa, is a neurotoxic protein produced by twice cleavage of transmembrane Amyloid Precursor Protein (APP) by β and γ secretases, is formed by protein misfolding, can exist as monomers, oligomers, fibrils and insoluble fibers, deposits in the brain with synaptic damage and neuronal death.

Although there is no cure for AD, some FDA-approved drugs, such as cholinesterase inhibitors (increasing the amount of the neurotransmitter acetylcholine in the brain, promoting intercellular signaling), NMDA receptor antagonists (altering brain cell signaling), can delay disease progression, but these drugs still have inadequate efficacy, especially in moderate to severe AD patients, and still fail to meet the clinical needs of the patients. In addition, some drugs, although sometimes able to delay the deterioration of the mental capacity of some patients, can cause serious side effects. Therefore, patients need more effective drugs.

In recent years, research on treating AD by taking Abeta as a drug target has been greatly advanced, namely, in 7.6.2021, the U.S. Food and Drug Administration (FDA) announces approval of a new Alzheimer disease drug, namely, Aducamab (Aducanumab, trade name, Aduhelm), developed by Bohai Jian (Biogen) company to be marketed. Adalimumab represents the first approved treatment for alzheimer's disease with the most common adverse effects (at least 10% higher than placebo incidence): ARIA edema, headache, ARIA-H micro-bleeding, ARIA-H surface iron deposition and falls.

Studies have shown that immunotherapy designed to remove a β from the brain has produced positive results in animal models of AD, and thus detection of a β protein is of great importance for the diagnosis, intervention and treatment of AD.

Therefore, the development of A beta (particularly A beta 1-42 peptide and/or A beta 1-40 peptide) targeting antibodies has certain significance for laboratory research and clinical diagnosis of AD.

Disclosure of Invention

In a first aspect, the present application provides a product for the detection of β -amyloid in a biological sample and/or for the diagnosis of alzheimer's disease comprising a reagent for determining the level of β -amyloid in said biological sample.

In some embodiments of the first aspect, the product is a kit, a test strip, a test card, or a microfluidic device.

In some embodiments of the first aspect, the agent comprises an antibody or antigen-binding portion thereof that specifically binds to β -amyloid, said antibody or antigen-binding portion thereof comprising a heavy chain variable region, said heavy chain variable region being HCDR1, HCDR2 and/or HCDR 3. In some embodiments of the first aspect, the HCDR1 sequence comprises the amino acid sequence TSGMGVS (SEQ ID NO: 1). In some embodiments of the first aspect, the HCDR2 sequence comprises amino acid sequence HIYWDDDKRYNPSLKR (SEQ ID NO: 2). In some embodiments of the first aspect, the HCDR3 sequence comprises amino acid sequence SRGYVYYASGFAY (SEQ ID NO: 3).

In some embodiments of the first aspect, the antibody or antigen-binding portion thereof that specifically binds β -amyloid further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2, and/or LCDR3 sequence.

In some embodiments of the first aspect, the LCDR1 sequence comprises amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 5). In some embodiments of the first aspect, the LCDR2 sequence comprises the amino acid sequence KVSNRFS (SEQ ID NO: 6). In some embodiments of the first aspect, the LCDR3 sequence comprises amino acid sequence FQGSFVPLT (SEQ ID NO: 7).

In some embodiments of the first aspect, the heavy chain variable region of the antibody or antigen-binding portion thereof that specifically binds β -amyloid comprises the amino acid sequence set forth in SEQ ID No. 4 or an amino acid sequence having at least 80% homology thereto.

In some embodiments of the first aspect, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO 4.

In some embodiments of the first aspect, the light chain variable region of the antibody or antigen-binding portion thereof that specifically binds β -amyloid comprises the amino acid sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 80% homology thereto.

In some embodiments of the first aspect, the light chain variable region has the amino acid sequence set forth in SEQ ID NO 8.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody that specifically binds to amyloid beta is set forth in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region of the antibody that specifically binds to amyloid beta is set forth in SEQ ID NO. 8.

In some embodiments of the first aspect, the antigen binding moiety is selected from the group consisting of: fab fragment, Fab 'fragment, F (ab')₂A fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody.

In some embodiments of the first aspect, the antibody that specifically binds to β -amyloid is a murine antibody.

In some embodiments of the first aspect, the antibody that specifically binds to β -amyloid is a monoclonal antibody.

In some embodiments of the first aspect, the antibody or antigen-binding portion thereof that specifically binds to β -amyloid specifically binds to primate β -amyloid. In some embodiments of the first aspect, the antibody or antigen-binding portion thereof that specifically binds to amyloid-beta specifically binds to human amyloid-beta. In some embodiments of the first aspect, the antibody or antigen-binding portion thereof that specifically binds to beta-amyloid specifically binds to the A β 1-40 subtype or the A β 1-42 subtype of human beta-amyloid. In some embodiments of the first aspect, the antibody or antigen-binding portion thereof that specifically binds to amyloid beta specifically binds to the A β 1-42 subtype of human amyloid beta.

In some embodiments of the first aspect, the biological sample is cerebrospinal fluid, brain interstitial fluid, urine, blood, or serum.

In some embodiments of the first aspect, the product further comprises a detectable label, a substrate, a reference standard, a diluent, a wash solution, or any combination thereof.

In some embodiments of the first aspect, the product further comprises product instructions for use.

In a second aspect, the present application provides a method for the in vitro detection of β -amyloid in a biological sample, comprising determining the β -amyloid level in said biological sample using the product of the first aspect.

Drawings

FIG. 1 shows the EC of anti-beta-amyloid monoclonal antibody 6C8-1B9₅₀Value determination curves.

Detailed Description

The most common subtypes of A beta in a human body are A beta 1-40 and A beta 1-42, in human cerebrospinal fluid and blood, the content level of the A beta 1-40 is 10 times and 1.5 times higher than that of the A beta 1-42 respectively, and the A beta 1-42 has stronger toxicity and is easier to aggregate, so that a core of A beta precipitation is formed, and neurotoxicity effect is caused. Under pathological conditions, the generation of A beta in the brain of an AD patient is 4-10 times that of the AD patient under normal conditions, a large amount of A beta is gathered to form neuritic plaques (senile plaques), and the formation of the senile plaques causes the death of a large amount of cells in the hippocampus, so that the two prolonged amino acids not only increase the hydrophobicity of the A beta and enable the A beta to be more easily gathered, but also improve the stability of the aggregates, and the A beta can be selectively deposited in amyloid plaques at an early stage.

In AD patients, changes in the concentration of a β occur in the cerebrospinal fluid (CSF), the pathological changes of which are, in turn, brain a β accumulation, increased CSF, decreased hippocampal and gray matter volume, decreased glucose metabolism, memory impairment and dementia. A β can be produced by a variety of cells, circulating in the blood, cerebrospinal fluid and brain interstitial fluid, mostly bound to chaperone molecules, and a few in a free state. A β can be expressed not only in brain cells but also in neurons, astrocytes and microglia, and also in peripheral organs and tissues such as liver, kidney, pancreas, spleen and other organs, and various blood and endothelial cells.

The A beta antibody can depolymerize in vitro aggregated A beta, so that the toxic effect of the A beta antibody is reduced, and when the A beta antibody is used for treating a transgenic AD mouse, the number of senile plaques in the brain of a model mouse can be reduced, and the learning and memory capacity of the mouse can also be improved, so that the preparation and application of the antibody aiming at the A beta and different fragments thereof have positive influence on the diagnosis, treatment and research of related diseases.

The present application provides products for the detection of beta-amyloid in a biological sample and/or for the diagnosis of alzheimer's disease. In a preferred embodiment, the product of the present application comprises an antibody or antigen-binding portion thereof that specifically binds to beta-amyloid. The present application also provides methods for detecting amyloid-beta in a biological sample in vitro.

In order to make the present application easily understood, certain terms used herein are first defined.

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by a disulfide bond, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises a domain (CL 1). The VH and VL regions can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) into which conserved regions known as Framework Regions (FRs) are interspersed. In some embodiments, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of the entire antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: fab fragment, F (ab')₂Fragments, Fd fragments, Fv fragments, single chain Fv (scfv) molecules, single domain antibodies, dAb fragments, and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic a hypervariable region of an antibody. The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and the like. For example, the Fd fragment herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the VL and VH domains in a single arm of the antibody; dAb fragments (Ward) et al.Nature 1989, 341: 544-546) consists of a VH domain.

In the field of the artIt is well known to the skilled person that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR sequences for VH or VL, namely Kabat definition and Chothia definition, see for example Kabat et al., “Sequences of Proteins of Immunological Interest”，National Institutes of Health, Bethesda，MD. (1991); Al-Lazikani et al.J Mol Biol 273:927-948 (1997); and Martin et al.Proc. Natl. Acad. Sci. USA 86: 9268-. For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2, and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2, and LCDR3, respectively.

The CDR region sequences in the variable region sequences can be analyzed in a variety of ways for the variable region sequences of a given antibody, such as can be determined using the online software Abysis (http:// www.abysis.org /).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with at least two times greater affinity than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding to two or more antigens associated with its sequence. For example, an antibody of the invention can specifically bind to beta-amyloid in humans and non-humans (e.g., mice or non-human primates).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations in a small number of individuals.

As used herein, the term "murine antibody" refers to any antibody in which all constant domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.

Suitable techniques that may additionally be used in the antibody method include amyloid-beta based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein a column, or any combination of these techniques. The β -amyloid antibody isotype can be determined using an ELISA assay, e.g., human Ig can be identified using anti-human Ig adsorbed by murine Ig.

Beta-amyloid suitable for antibody production can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Forms of beta-amyloid suitable for generating an immune response include the subtypes A beta 1-40 and A beta 1-42 of beta-amyloid. Additional forms of β -amyloid include β -amyloid expressing cells, preparations or cell extracts or fractions containing β -amyloid, partially purified β -amyloid.

As used herein, the term "homology" is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and the introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art. As used herein, "at least 80% homology" means any value of 80% to 100% homology, e.g., 85%, 90%, 95%, 99%, etc.

As used herein, the term "EC₅₀The value "means the half maximal Effect Concentration (EC) of 50% of the maximum effect₅₀) And refers to the concentration that causes 50% of the maximum effect.

In some embodiments of the first aspect, the product can be a kit, a test strip (e.g., a colloidal gold immunochromatographic test strip), a test card, a microfluidic test device, or the like.

In some embodiments of the first aspect, the agent comprises an antibody, or antigen-binding portion thereof, that specifically binds β -amyloid protein comprising a heavy chain variable region comprising HCDR1, HCDR2 and/or HCDR 3.

Table 1 below illustrates CDR, heavy chain variable region, and light chain variable region amino acid sequences suitable for use in the antibodies disclosed herein. In certain embodiments, the anti- β -amyloid antibody or antigen-binding portion thereof comprises an HCDR1, HCDR2, and/or HCDR3 sequence independently selected from any one of the HCDR1, HCDR2, and/or HCDR3 sequences shown in table 1. In certain embodiments, an anti-beta-amyloid antibody of the present application may further comprise LCDR1, LCDR2, and/or LCDR3, independently selected from any one of the LCDR1, LCDR2, and/or LCDR3 sequences shown in table 1. For example, an anti- β -amyloid antibody of the present application may comprise a heavy chain variable region as shown in table 1, optionally in combination or pairing with a light chain variable region as shown in table 1.

Table 1: serial numbers of amino acid sequences of heavy chain CDRs, light chain CDRs, heavy chain variable regions, and light chain variable regions of an exemplary anti-beta-amyloid antibody 6C8-1B9

In some embodiments of the first aspect, the amino acid sequence of HCDR1 of the antibodies or antigen-binding portions thereof disclosed herein is set forth in SEQ ID No. 1. In some embodiments of the first aspect, the amino acid sequence of HCDR2 is set forth in SEQ ID NO 2. In some embodiments of the first aspect, the amino acid sequence of HCDR3 is set forth in SEQ ID NO 3.

The antibodies or antigen-binding portions thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

In some embodiments of the first aspect, the amino acid sequence of LCDR1 of the antibodies or antigen-binding portions thereof disclosed herein is set forth in SEQ ID No. 5. In some embodiments of the first aspect, the amino acid sequence of LCDR2 is set forth in SEQ ID No. 6. In some embodiments of the first aspect, the amino acid sequence of LCDR3 is set forth in SEQ ID No. 7.

In some specific embodiments of the first aspect, the heavy chain variable region of the antibodies, or antigen-binding portions thereof, disclosed herein is at least 80% homologous, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence set forth in SEQ ID No. 4. In some particular embodiments of the first aspect, the amino acid sequence of the variable region of the antibody heavy chain is as set forth in SEQ ID NO 4.

In some specific embodiments of the first aspect, the light chain variable region of the antibodies, or antigen-binding portions thereof, disclosed herein is at least 80% homologous, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence set forth in SEQ ID No. 8. In some specific embodiments of the first aspect, the amino acid sequence of the antibody light chain variable region is as set forth in SEQ ID NO 8.

In some embodiments of the first aspect, the heavy chain variable region or the light chain variable region of the antibodies disclosed herein can be substituted, deleted, or added with at least one amino acid based on the respective corresponding specific amino acid sequences listed above, and the resulting variants still retain the activity of binding to β -amyloid.

In certain embodiments, the number of amino acid substitutions, deletions or additions described above is any number between 1 and 30 or between 1 and 30, preferably between 1 and 20, more preferably between 1 and 10. In preferred embodiments, the sequence variant differs from the original amino acid sequence by substitutions, deletions and/or additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by a substitution, deletion or addition of about 1, 2, 3, 4 or 5 amino acids. In particular embodiments, the amino acid substitution is a conservative substitution.

In some embodiments of the first aspect, the antibody, or antigen-binding portion thereof, disclosed herein is a murine antibody.

In a preferred embodiment, the antibody disclosed herein is antibody 6C8-1B9, wherein the amino acid sequence of the heavy chain variable region of antibody 6C8-1B9 is set forth in SEQ ID NO:4 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.

In some embodiments of the first aspect, the nucleic acid molecule encoding the antibody heavy chain variable region amino acid sequence comprises the nucleotide sequence set forth in SEQ ID NO 9. In some embodiments of the first aspect, the nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence of the heavy chain variable region of the antibody is as set forth in SEQ ID No. 9. In some embodiments of the first aspect, the nucleotide sequence set forth in SEQ ID NO 9 encodes the heavy chain variable region amino acid sequence of the antibody set forth in SEQ ID NO 4.

In some embodiments of the first aspect, the nucleic acid molecule encoding the antibody light chain variable region amino acid sequence comprises the nucleotide sequence set forth in SEQ ID NO. 10. In some embodiments of the first aspect, the nucleotide sequence of the nucleic acid molecule encoding the antibody light chain variable region amino acid sequence is set forth in SEQ ID No. 10. In some embodiments of the first aspect, the nucleotide sequence set forth in SEQ ID NO. 10 encodes the light chain variable region amino acid sequence of the antibody set forth in SEQ ID NO. 8.

In some embodiments of the first aspect, the antigen binding moiety disclosed herein is selected from the group consisting of: fab fragment, Fab 'fragment, F (ab')₂A fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody.

The antibodies, or antigen binding portions thereof, disclosed herein are capable of specifically binding to beta-amyloid. In some embodiments of the first aspect, the antibody, or antigen-binding portion thereof, specifically binds to primate beta-amyloid, or beta-amyloid of a species that has high homology to primate beta-amyloid. In some specific embodiments of the first aspect, the antibody or antigen-binding portion thereof specifically binds to human beta-amyloid, e.g., the A β 1-40 subtype or the A β 1-42 subtype of human beta-amyloid, preferably the A β 1-42 subtype.

In some more specific embodiments of the first aspect, the antibodies disclosed herein are anti-human β -amyloid monoclonal antibodies. The type and subtype of amyloid-beta antibody can be determined by any means known in the art. Generally, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype.

In some embodiments of the first aspect, the product may further comprise a detectable label, such as colloidal gold, a chemiluminescent label, a fluorescent label, a nanoparticle label, and the like.

In some embodiments of the first aspect, the product may further comprise other reagents for detection, such as enzyme or colloidal gold labeled antigens or antibodies, substrates, reference standards, dilutions, washing solutions, and the like.

In some embodiments of the first aspect, the product may further comprise reagents for processing a biological sample for detection, which may be cerebrospinal fluid, brain interstitial fluid, urine, blood or serum.

In some embodiments of the first aspect, the article of manufacture may further comprise instructions for use of the article of manufacture.

Methods of making and purifying antibodies or antigen-binding portions thereof that specifically bind to beta-amyloid are also provided.

In some embodiments, after inoculation of the animal with the amyloid-beta antigen, antibodies and/or antibody-producing cells can be obtained from within the animal. Antibody-producing immortalized cell lines can be prepared from cells isolated from immunized animals. Following immunization, the animal is killed, lymph node and/or spleen B cells are immortalized, treated with an oncogenic compound and a mutagenic compound, and fused with immortalized cells (e.g., myeloma cells) to inactivate tumor suppressor genes. When myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Screening of immortalized cells using beta-amyloid, a portion thereof or cells expressing beta-amyloid. In a preferred embodiment, the primary screening is performed using enzyme-linked immunosorbent assay (ELISA). Cells, such as hybridomas, that produce anti-beta-amyloid antibodies are selected for cloning and further screened for desired characteristics, including good growth, high antibody production, and desirable antibody characteristics. Screening, cloning and amplification methods for hybridomas are well known to those of ordinary skill in the art. In some embodiments, the immunized animal is a non-human animal, wherein the splenic B cells are fused with a myeloma cell line from the same species as the non-human animal. In some embodiments, the immunized animal is a Balb/c mouse.

In some embodiments, the anti-amyloid beta antibody disclosed herein can be isolated and purified using affinity chromatography, and the anti-amyloid beta antibody bound to the affinity column can be eluted using conventional methods, such as high salt buffers, pH changes, and the like, depending on the characteristics of the affinity column used.

In some embodiments, antibody purification can be performed using a Protein G affinity chromatography column.

In the present description and claims, the words "comprise", "comprises" and "comprising" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The above disclosure generally describes the present application and the following examples are presented to further illustrate the present application and should not be construed as limiting the present application. The invention discloses products for detecting beta-amyloid levels in a biological sample and/or for diagnosing alzheimer's disease and methods for in vitro detection of beta-amyloid levels in a biological sample and/or diagnosis of alzheimer's disease. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. Those skilled in the art can implement and use the invention by making modifications, or appropriate alterations and combinations, of the methods and applications described herein without departing from the spirit, scope, and content of the invention.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to the embodiments.

Examples

Example 1 materials and methods

1.1 reagents and instruments

1.1.1 cell culture

1.1.2 ELISA detection

1.2 animals

EXAMPLE 2 preparation of monoclonal antibodies

2.1 animal immunization

Emulsifying beta-amyloid (Abeta 1-42 subtype) immunogen and adjuvant according to the volume ratio of 1:1, and performing multi-point immunization injection subcutaneously on Balb/c mice (10 mice), wherein the immunization scheme is shown in Table 2:

TABLE 2

2.2 potency assay

The ascites of 10 immunized mice are respectively diluted into different concentrations by physiological saline, and the titer is measured by an enzyme-linked immunosorbent assay (ELISA) method, which comprises the following specific steps:

1. coating: diluting beta-amyloid (Abeta 1-42 subtype) immunogen with coating buffer PBS, adding the diluted immunogen into an ELISA (enzyme-linked immunosorbent assay) enzyme-linked plate at 0.1 mu g/hole, and standing overnight at 4 ℃;

2. and (3) sealing: washing with physiological saline for 3 times, adding confining liquid, incubating at 37 deg.C for 1.5 hr, and adding blocking liquid at 200 μ l/hole;

3. adding 100 μ l of sample to be tested into the microporous plate, incubating for 45 min at 37 ℃, washing the plate for 3 times, and drying;

4. adding 100 μ l enzyme-labeled antibody, diluting with PBS 5000 times, incubating at 37 deg.C for 45 min, washing plate for 3 times, and drying;

5. adding 100 mul of TMB color development solution, reacting for 5 min at room temperature, adding stop solution, and reading on an enzyme-linked immunosorbent assay (ELISA) instrument;

the negative control was physiological saline. The ELISA titer test results are shown in Table 3:

TABLE 3

2.3 cell fusion and selection

And selecting splenocytes of a #1 Balb/C mouse with the best immune response to perform cell fusion with SP2/0 mouse myeloma cells, using beta-amyloid (Abeta 1-42 subtype) as a detection antigen package plate, and obtaining a positive hybridoma cell strain 6C8-1B9 which stably secretes a specific antibody through multiple screening and multiple stability tests. The cell line 6C8-1B9 was deposited in the China general microbiological culture Collection center (CGMCC, the institute for microbiology of China academy of sciences, 3, Nacio No. 1, Beijing, Chaoyang, China) at 2022, 2.17.d by Beijing first Biochemical pharmaceutical Co., Ltd in CGMCC, with the accession number of CGMCC 45042.

2.4 amplification culture

Culturing the positive cell strain 6C8-1B9 in a T75 culture flask, freezing and preserving seeds and preparing ascites;

the ascites preparation procedure is as follows: sensitizing a mouse by using liquid paraffin, simultaneously culturing hybridoma cells, performing mouse intraperitoneal injection on the cultured hybridoma cells, observing the growth state of ascites of the mouse, and collecting the ascites in time;

the generation marking-limited dilution 6-hole culture is considered as the first generation, one generation is added every passage, the screened cells are expanded into a T75 culture bottle for culture, the cells are frozen and preserved in 80 percent, and the cells are frozen and preserved for 3 times;

freezing and storing liquid: 90% FBS +10% DMSO.

2.5 cell cryopreservation and Resuscitation

2.5.1 cell Resuscitation

Culture medium: 10% FBS +1% PS + DMEM (high glucose type);

1. preparing 15 ml of sterile centrifuge tubes in a safety cabinet, and adding 9 ml of basic culture medium for later use;

2. rapidly thawing the frozen cells in a 37 ℃ water bath;

3. transferring the thawed cells into a 15 ml centrifuge tube by using a 1 ml gun in a biological safety cabinet, and centrifuging for 5 min at 1000 r/min;

4. the supernatant was discarded, and the pellet was resuspended in complete medium and cultured in a T75 flask.

2.5.2 cell cryopreservation

Freezing and storing liquid: 6:3:1(60% FBS +30% basal medium +10% DMSO) or 90% FBS +10% DMSO;

1. cells (with round state, bright state and cell confluency of 80% -90%) cultured by a T75 culture bottle are placed in a 50 ml sterile centrifuge tube at 800 r/min-1000 r/min for centrifugation for 5 min;

2. discarding the supernatant, taking 3 ml of the frozen stock solution for quick suspension, subpackaging into 2 frozen tubes, placing into a gradient frozen stock box, and storing in a refrigerator at-80 ℃;

3. the next day from-80 ℃ freezer to liquid nitrogen.

2.6 antibody purification

And purifying the selected immune ascites by Protein G affinity chromatography to obtain an antibody IgG which is marked as an antibody 6C8-1B9, wherein the heavy chain constant region is an IgG2B subtype, and the light chain constant region is a kappa subtype. Before purification, a preliminary test was performed using ascites dilution, and the negative control was normal saline. The ELISA assay results are shown in Table 4:

TABLE 4

2.7 ELISA detection of purified antibodies

1. Coating the ELISA plate with antigen (bs-0107P), 2. mu.g/ml, 0.2. mu.g/well;

2. the purified antibody was diluted in the following proportions: 1:500, 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, and 1: 2048000;

3. the secondary antibody is diluted with goat anti-mouse IgG labeled by horseradish peroxidase at a ratio of 1: 5000;

4. the color developing agent is tetramethyl benzidine;

5. the detection wavelength is as follows: measuring the OD value at 450 nm;

the negative control was physiological saline. The results of ELISA were shown in FIG. 1 and Table 5, and the EC of antibody 6C8-1B9₅₀The value was 19.59 pM.

TABLE 5

It will be understood that, although the invention herein has been described in the foregoing specific forms, these inventions are not to be limited to the particulars described in these specific forms. It will be obvious to those skilled in the art that various equivalent changes may be made in the technical features of the invention involved therein without departing from the spirit of the invention described in the present application, and these changes should be construed as being within the scope of the invention.

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<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Arg

1 5 10 15

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ser Arg Gly Tyr Val Tyr Tyr Ala Ser Gly Phe Ala Tyr

1 5 10

<210> 4

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Ser Trp Ile Arg Gln Thr Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Thr His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser

50 55 60

Leu Lys Arg Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Leu Val

65 70 75 80

Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Ser Arg Gly Tyr Val Tyr Tyr Ala Ser Gly Phe Ala Tyr

100 105 110

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 5

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Phe Gln Gly Ser Phe Val Pro Leu Thr

1 5

<210> 8

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Val Leu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp His Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Phe Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 9

<211> 369

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttcactgagc acttctggaa tgggtgtgag ttggattcgt 120

cagacttcag gaaagggtct ggagtggctg acacacattt actgggatga tgacaagcgc 180

tataacccat ccctgaagag gcggctcaca atctccaagg atacctccag aaacctggtt 240

ttcctcaaga tcaccagtgt ggacactgca gatactgcca catacttctg tgctcgaagt 300

cgggggtatg tttactacgc ctccgggttt gcttactggg gcccagggac tcttgtcact 360

gtctctgca 369

<210> 10

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatgttttgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga ccacgtctcc 60

atctcttgca gatctagtca gagccttgta catagtaatg gaaacaccta tttagaatgg 120

tacttgcaga aagcaggcca gtctccaaac ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcagggt cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc atttgttccg 300

ctcacgttcg gtgctgggac caagctggag ctgaaa 336

Claims

1. A product for detecting β -amyloid in a biological sample and/or for diagnosing alzheimer's disease comprising an agent for determining the level of β -amyloid in said biological sample.

2. The product of claim 1, wherein the product is a kit, test strip, test card, or microfluidic device.

3. The article of manufacture of claim 1, wherein the agent comprises an antibody or antigen-binding portion thereof that specifically binds to β -amyloid;

preferably, the antibody or antigen-binding portion thereof comprises the heavy chain variable region HCDR1, HCDR2 and/or HCDR3, wherein the HCDR1 sequence comprises the amino acid sequence TSGMGVS (SEQ ID NO: 1); the HCDR2 sequence comprises amino acid sequence HIYWDDDKRYNPSLKR (SEQ ID NO: 2); and/or the HCDR3 sequence comprises amino acid sequence SRGYVYYASGFAY (SEQ ID NO: 3);

more preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 80% homology with the above sequence; most preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO 4.

4. The product of claim 3, wherein the antibody, or antigen-binding portion thereof, further comprises a light chain variable region comprising an LCDR1, LCDR2, and/or LCDR3 sequence, wherein the LCDR1 sequence comprises amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 5); the LCDR2 sequence comprises the amino acid sequence KVSNRFS (SEQ ID NO: 6); and/or the LCDR3 sequence comprises amino acid sequence FQGSFVPLT (SEQ ID NO: 7);

preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 80% homology with the above sequence; more preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO 8.

5. The product of claim 3, wherein

The antigen binding moiety is selected from: fab fragment, Fab 'fragment, F (ab')₂A fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody; and/or

The antibody is a murine antibody; and/or

The antibody is a monoclonal antibody.

6. The product of claim 3, wherein the heavy chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO. 4 and the light chain variable region amino acid sequence of the antibody is set forth in SEQ ID NO. 8.

7. The product of claim 1, wherein the amyloid beta is a primate amyloid beta, preferably human amyloid beta, more preferably human A β 1-40 subtype or human A β 1-42 subtype, most preferably human A β 1-42 subtype.

8. The product of claim 1, wherein the biological sample is cerebrospinal fluid, brain interstitial fluid, urine, blood, or serum.

9. The product of claim 1, further comprising a detection label, a substrate, a reference standard, a diluent, a wash solution, or any combination thereof; and/or

And (5) instructions for using the product.

10. A method for the in vitro detection of β -amyloid in a biological sample comprising determining the level of β -amyloid in said biological sample using the product of any one of claims 1-9.