CN121991218A

CN121991218A - Antibody for detecting beta-amyloid and application thereof

Info

Publication number: CN121991218A
Application number: CN202411579564.5A
Authority: CN
Inventors: 郭伟芸; 晏盼盼; 张爽
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2024-11-06
Filing date: 2024-11-06
Publication date: 2026-05-08

Abstract

本发明公开了检测β‑淀粉样蛋白的抗体及其应用，具体涉及检测阿尔茨海默症血液标志物Aβ1‑40及Aβ1‑42的检测抗体和试剂盒，该试剂盒包含三种针对β淀粉样蛋白不同抗原决定簇的抗体，基于该抗体及试剂盒能够准确的检测β‑淀粉样蛋白的存在。This invention discloses antibodies for detecting β-amyloid protein and their applications, specifically relating to detection antibodies and kits for detecting Alzheimer's disease blood markers Aβ1-40 and Aβ1-42. The kit contains three antibodies targeting different antigenic determinants of β-amyloid protein, and the presence of β-amyloid protein can be accurately detected based on these antibodies and the kit.

Description

Antibody for detecting beta-amyloid and application thereof

Technical Field

The invention relates to the technical field of immunodiagnosis, in particular to an antibody for resisting detection of beta-amyloid and application thereof.

Background

Alzheimer's Disease (AD), senile dementia, is one of the most common neurodegenerative diseases in the elderly population. The pathogenesis and etiology of AD is currently unknown, and among many hypotheses, the amyloid cascade hypothesis (amyloid cascade hypothesis) is the main theory that the overproduction or untimely elimination of β -amyloid (aβ), such as aβ1-42 or other aβ polypeptide fragments, in the brain is thought to lead to the deposition of soluble aβ oligomers and insoluble amyloid proteins in the brain forming amyloid plaques, playing an important pathological role in the course of AD. Clinically, by using amyloid positron emission tomography (positron emission tomography, PET) (Abeta-PET for short), pre-clinical patients and MCI patients without symptoms can be identified early, and the differential diagnosis accuracy of dementia patients can be improved. However, PET imaging is a non-clinical routine examination, and has a large limitation, particularly in primary hospitals where the popularity is low, accessibility is poor, and charging is expensive.

In recent years, research into Abeta as a body fluid marker of AD has been significantly progressed, with Abeta 1-40 and Abeta 1-42 being the most important. Aβ1-42 is a small 4kDa protein of about 40 amino acids that is formed after proteolytic cleavage of the transmembrane protein of the amyloid precursor. The type of sample studied initially was cerebrospinal fluid (cerebrospinal fluid, CSF), and generally the content of Abeta 1-40 in CSF is about 10 times that of Abeta 1-42. In AD patients, the concentration of Abeta 1-40 is not generally significantly changed, while the concentration of Abeta 1-42 is significantly decreased. Multiple studies demonstrated that the ratio of Abeta 1-42/Abeta 1-40 was more accurate than Abeta 1-42 alone in diagnosing AD from non-AD. Because CSF detection requires lumbar puncture operation, the sampling mode is complex and has problems of complications such as headache and the like, minimally invasive, convenient and low-cost blood detection is receiving more and more attention. The specific and sensitive overlap between clinical CSF and plasma aβ values and between the latter and aβ -PET has been widely accepted.

The immune detection method is widely applied to detection of the biomarker due to simple operation, high sensitivity and strong specificity. Common immunodetection methods include colloidal gold methods, fluorescence methods, chemiluminescence methods, and the like, which all require specific antibodies to antigens. In particular, in healthy normal humans, the concentration ranges of Aβ1-40 and Aβ1-42 in blood are 100 pg/mL-400 pg/mL and 15 pg/mL-30 pg/mL, respectively. However, since the level of Abeta 1-42 in AD patients is decreased, an immunological detection method is required to accurately and precisely quantify Abeta 1-42 at a low concentration in blood, and a raw material antibody excellent in performance is required. In addition, some immunoassays require a partner antibody to be effective, and thus the partner antibody to β -amyloid is also critical for achieving immunoassays. Currently, there is a lack of antibodies and paired antibodies on the market that have a performance advantage, which limits the accuracy and reliability of beta-amyloid detection. There is a strong need in the art for antibodies and paired antibodies that have strong binding properties and high sensitivity.

Disclosure of Invention

The application provides an antibody, which provides an important raw material source for detecting beta-amyloid and has good detection performance.

In order to achieve the above object, according to a first aspect of the present invention, there is provided an antibody for detecting β -amyloid, which comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs 19, 20, 21, 22, 45, 65 and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in any one of SEQ ID NOs 27, 47, 67.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody for detecting β -amyloid, the complementarity determining region of the antibody comprising any one of the groups (a ') to (c'):

The amino acid sequence of (a') is shown as HCDR1 shown in SEQ ID NO 1 (AYYIH) and SEQ ID NO 2 in sequence

(RIDPATGNTKYAPRLQD) or 15 (RLDPATGNTKYAPRLQD), HCDR2 shown in SEQ ID NO:3 (LYSLPVY) or 16 (IYSLPVY), and HCDR3 shown in SEQ ID NO:4 in that order

(KSSQSLLYSDAKTYLN) LCDR1, LCDR2 shown in SEQ ID NO 5 (QISRLDP), LCDR3 shown in SEQ ID NO 6 (LQGTHYPVL);

(b') the amino acid sequence is shown as HCDR1 shown in SEQ ID NO 29 (NYGMS) and SEQ ID NO 30 in sequence

(SIRSGGGRTYYSDNVKG) HCDR2 shown in SEQ ID NO:31 (YDHYSGSSDY), and LCDR1 shown in SEQ ID NO:32 (KSSQSLLDSDGKTYLN), LCDR2 shown in SEQ ID NO:33 (LVSKLDS), LCDR3 shown in SEQ ID NO:33 (WQGTHFPRT), and the amino acid sequence in that order

(C') HCDR1, SEQ ID NO 50, the amino acid sequence of which is shown in SEQ ID NO 49 (DYTMH) in order

(GINPNSGGTIYNEKFKD) HCDR2 shown in SEQ ID NO:51 (GVYDGYFY), and LCDR1 shown in SEQ ID NO:52 (RSSQSLVYSNGNTFLH), LCDR2 shown in SEQ ID NO:53 (KVSTRFSGVPDRFS), LCDR3 shown in SEQ ID NO:54 (SQTTHAPFT) in that order.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody for detecting β -amyloid, the antibody comprising a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NOS 19, 20, 21, 22, 45, 65 and a light chain variable region having an amino acid sequence shown in any one of SEQ ID NOS 27, 47, 67.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody for detecting β -amyloid, the antibody comprising a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs 23, 24, 25, 26, 46, 66 and a light chain having an amino acid sequence shown in any one of SEQ ID NOs 28, 48, 68.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody pair for detecting β -amyloid, the antibody pair comprising a coated antibody and/or a labeled antibody, the coated antibody and the labeled antibody being selected from the above antibodies, respectively.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a detection reagent or kit for detecting β -amyloid, the aforementioned reagent or kit comprising the above antibody or antibody pair.

In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a method for detecting β -amyloid, comprising:

a) Contacting the antibody, antibody pair or reagent or kit with a sample to be tested under conditions sufficient for an antibody/antigen binding reaction to form an immunocomplex, and

B) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of beta-amyloid in the test sample.

In order to achieve the above object, according to an eighth aspect of the present invention there is provided the use of an antibody, antibody pair or reagent or kit as described above for the detection of β -amyloid or for the preparation of a product for the detection of β -amyloid.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies so long as they exhibit the desired antigen-binding activity. An antigen binding fragment of an antibody is a substance comprising the CDRs of the antibody that lacks some of the amino acids present in the full-length chain, but is still capable of specifically binding to the antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab ') 2, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized bifunctional antibodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), and antibody minimal recognition units. The antigen binding fragment of the above antibody is capable of binding the same antigen as the parent antibody.

Antigen binding fragments of antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that antigen binding fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies. Antigen binding fragments may also be obtained by recombinant genetic techniques also known to those skilled in the art or by synthesis, for example, by an automated peptide synthesizer such as that sold by Applied BioSystems and the like.

In the present disclosure, the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated.

In some embodiments, the invention provides an antibody pair comprising at least two antibodies that bind to β -amyloid.

In some embodiments, the first antibody comprises at least one antibody that binds β -amyloid, and the second antibody comprises at least one antibody that binds β -amyloid.

In some embodiments, the first antibody is only one antibody and the second antibody is only one antibody.

In the present invention, the terms "comprising," "including," and "containing" are open-ended terms that include the teachings to which the present invention pertains, but do not exclude other aspects.

In the present invention, the term "optionally" generally means that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

In a first aspect, embodiments of the present invention provide an antibody for detecting β -amyloid, the antibody comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region having amino acid sequences as shown in any one of SEQ ID NOs 19, 20, 21, 22, 45, 65, and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region having amino acid sequences as shown in any one of SEQ ID NOs 27, 47, 67.

In alternative embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above are defined by any one or a combination of systems Kabat, chothia, IMGT, abM or contacts.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is denoted by HCDR, the 3 CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3, the light chain complementarity determining region is denoted by LCDR, and the 3 CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3. In the present invention, the term "antibody pair" means that a plurality of antibodies are used in pairs, and may be two antibodies, three antibodies, four antibodies and more, at least two antibodies.

CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below with the labels in the different documents being slightly different. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

TABLE 1 CDR definition ¹

CDR	Kabat	AbM²	IMGT	Chothia
					HCDR1	H31~H35³	H26~H35³	H26~H33..5⁵	H26~H32..34⁴
HCDR2	H50~H65	H50~H58	H51~H57	H52~H56
					HCDR3	H95~H102	H95~H102	H93~H102	H95~H102
LCDR1	L24~L34	L24~L34	L27~L32	L24~L34
					LCDR2	L50~L56	L50~L56	L50~L51	L50~L56
LCDR3	L89~L97	L89~L97	L89~L97	L89~L97

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ CDR-H1 ends at position 35 if both H35A and H35B are absent, CDR-H1 ends at position 35A if only H35A is present, and CDR-H1 ends at position 35B if both H35A and H35B are present.

⁴ CDR-H1 ends at bit 32 if both H35A and H35B are absent, at bit 33 if only H35A is present, and at bit 34 if both H35A and H35B are present.

⁵ CDR-H1 ends at position 33 if both H35A and H35B are absent, CDR-H1 ends at position 34 if only H35A is present, and CDR-H1 ends at position 35 if both H35A and H35B are present.

According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a Contact system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.

In a second aspect, embodiments of the present invention provide an antibody for detecting β -amyloid, wherein the complementarity determining region of the antibody comprises any one of (a ')to (c'):

(C') an amino acid sequence of HCDR1 shown in SEQ ID NO. 49 (DYTMH), HCDR2 shown in SEQ ID NO. 50 (GINPNSGGTIYNEKFKD), HCDR3 shown in SEQ ID NO. 51 (GVYDGYFY), and an amino acid sequence of LCDR1 shown in SEQ ID NO. 52 (RSSQSLVYSNGNTFLH), LCDR2 shown in SEQ ID NO. 53 (KVSTRFSGVPDRFS), LCDR3 shown in SEQ ID NO. 54 (SQTTHAPFT) in this order.

According to an embodiment of the present invention, the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

The antibodies of the invention also include framework regions, in the present invention, "framework region" or "FR" region includes both heavy and light chain framework regions, meaning regions of the antibody heavy and light chain variable regions other than CDRs, wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework regions can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.

In alternative embodiments, the first antibody, second antibody or antibody of the first aspect, second aspect further has a framework region indicated by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR 4.

In an alternative embodiment, the framework region of the first antibody is selected from any one of (a) to (c):

(a) HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO 7 to SEQ ID NO 10, and LFR1, LFR2, LFR3, LFR4 having amino acid sequences shown in SEQ ID NO 11 to SEQ ID NO 14, or an amino acid sequence having at least 80% identity to the respective framework region sequences, and

(B) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NO. 55 to SEQ ID NO. 58 in sequence, and LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 59 to SEQ ID NO. 62 in sequence, or the amino acid sequences with at least 80% identity with the sequences of the framework regions;

(c) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NO. 35 to SEQ ID NO. 38, and the amino acid sequences are shown as LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 39 to SEQ ID NO. 42, or the amino acid sequences with at least 80% identity with the sequences of the framework regions.

In the present invention, the term "identity" percent refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. The alignment of the percentage of amino acid sequence identity can be performed in a variety of ways known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA, among others, as known in the art.

In other embodiments, each framework region amino acid sequence of a first antibody, a second antibody, or an antibody provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region described above.

In a third aspect, embodiments of the present invention provide an antibody for detecting β -amyloid, the antibody comprising a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs 19, 20, 21, 22, 45, 65 and a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs 27, 47, 67.

In alternative embodiments, the heavy chain variable region and the light chain variable region amino acid sequences of the above antibodies are selected from any one of the following combinations:

Combination of two or more kinds of materials	Heavy chain variable region	Light chain variable region
			A	SEQ ID NO:19	SEQ ID NO:27
A1	SEQ ID NO:20	SEQ ID NO:27
			A2	SEQ ID NO:21	SEQ ID NO:27
A3	SEQ ID NO:22	SEQ ID NO:27
			C	SEQ ID NO:65	SEQ ID NO:67
B	SEQ ID NO:45	SEQ ID NO:47

In an alternative embodiment, the antibody of the first, second and third aspects further comprises a constant region.

In alternative embodiments, the constant regions comprise heavy chain constant regions and/or light chain constant regions.

In alternative embodiments, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, camel, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.

In an alternative embodiment, the constant region is derived from a mouse.

In alternative embodiments, the heavy chain constant region of the above antibody is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, igD, or a combination of constant regions, and/or the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions, which may be selected from the group consisting of lambda 1, lambda 2, lambda 3, and lambda 4 subtypes.

In alternative embodiments, the heavy chain constant region comprises CH1 of IgG1, hinge region of IgG1, CH2 of IgM, CH3 of IgM, and/or CH4 of IgM.

In alternative embodiments, the antibody comprises a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO. 17 and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO. 18, or an amino acid sequence having at least 80% identity to each of the constant regions.

In some embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NOS: 17, 18) described above.

In this context, the division of the variable and constant region sequences is referred to the IMGT division method, see Lefranc,the international ImMunoGeneTics database.Nucl.Acids Res.,29(1):207-209(2001).DOI:10.1093/nar/29.1.207.PMID:11125093. Martinez-Jean C.and Bosc N. OrEhrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire.the international ImMunoGenetics informationHttp:// www.imgt.org.Created:16/03/2011.Version:17/01/2020. OrEhrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire.the international ImMunoGenetics informationHttp:// www.imgt.org.Created:16/03/2011.Version:17/01/2020. The variable region divided by different methods may have a partial amino acid difference from the variable region C-terminal or constant region N-terminal divided by IMGT, and other methods known in the art may be used to divide the variable region or constant region within the scope of the present invention.

In a fourth aspect, embodiments of the present invention provide an antibody for detecting β -amyloid, the antibody comprising a heavy chain having an amino acid sequence as set forth in any one of SEQ ID NOs 23, 24, 25, 26, 46, 66 and a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs 28, 48, 68.

In alternative embodiments, the heavy and light chains of the above antibodies are selected from any one of the following combinations:

Combination of two or more kinds of materials	Heavy chain variable region	Light chain variable region
			A’	SEQ ID NO:23	SEQ ID NO:28
A1’	SEQ ID NO:24	SEQ ID NO:28
			A2’	SEQ ID NO:25	SEQ ID NO:28
A3’	SEQ ID NO:26	SEQ ID NO:28
			C’	SEQ ID NO:66	SEQ ID NO:68
B’	SEQ ID NO:46	SEQ ID NO:48

In a fifth aspect, embodiments of the present invention provide an antibody pair for detecting β -amyloid, the pre-antibody pair comprising a coated antibody and/or a labeled antibody, the coated antibody and the labeled antibody being selected from the antibodies of the first, second, third and fourth aspects, respectively.

In an alternative embodiment, the coated antibody includes a first antibody selected from the group consisting of the (C ') group, the (C) group, and the (C') group antibody, and a second antibody selected from the group consisting of the (a ') group, the (A1) group, the (A2) group, the (A3) group, the (A1') group, the (A2 ') group, and the (A3') antibody.

In an alternative embodiment, the aforementioned labeled antibody is selected from the group (B '), an antibody of group (B), or an antibody of group (B').

The meaning of the reagent and the meaning of the kit in the present application may be regarded as equivalent to each other.

In alternative embodiments, the labeled antibodies described above are labeled with a detectable label and/or a binding partner.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the detectable label is selected from, but not limited to, metal ions, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, electron dense labels, adamantane, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the above-described fluorescent dye is selected from, but not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), etc., or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc., or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc., or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc., or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), etc., respectively.

In alternative embodiments, the enzyme is selected from, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope is selected from the group consisting of, but not limited to ²¹²Bi、¹³¹I、¹¹¹In、⁹⁰Y、¹⁸⁶Re、²¹¹At、¹²⁵I、¹⁸⁸Re、¹⁵³Sm、²¹³Bi、³²P、⁹⁴mTc、⁹⁹mTc、²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、¹²¹Sn、¹⁶¹Tb、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷⁷Lu、¹⁷²Lu and ¹⁸ F.

In alternative embodiments, the chemiluminescent reagent is selected from the group consisting of, but not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lothecine and its derivatives, and peroxyoxalate and its derivatives.

In an alternative embodiment, the label is an acridinium ester.

In alternative embodiments, the nanoparticle-based labels described above are selected from, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloid is selected from, but is not limited to, colloidal metals, colloidal carbons, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the above-described colloidal metals are selected from, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the labeled antibodies are labeled with a detectable label via a binding partner.

After the antibody is labeled with the label, the immune response characteristics of the antibody are not affected, and the activity of the label is not changed.

In alternative embodiments, the coated antibodies are attached to a solid phase and/or a binding partner.

In alternative embodiments, the solid phase is selected from microspheres, latex particles, microfluidic chips, magnetic beads, microwell plates, or nitrocellulose membranes.

In an alternative embodiment, the antibody conjugate further comprises a solid support coupled to the antibody.

In alternative embodiments, the solid support is selected from, but not limited to, magnetic microspheres, plastic microspheres, latex particulates, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In an alternative embodiment, the solid phase is a magnetic bead.

In alternative embodiments, the binding partner comprises biotin/avidin or biotin derivative/avidin.

In the present invention, the term "binding partner" refers to a pair of molecules capable of binding to each other by non-covalent interactions. Such binding is generally specific and may be temporary or form more stable complexes.

In the present invention, the binding partner may be bound to the label or the solid phase in various binding forms by physical adsorption, electrostatic adsorption or covalent binding, and such binding/conjugation methods are known to those skilled in the art.

In a sixth aspect, embodiments of the present invention provide a reagent or kit for detecting β -amyloid, the reagent or kit comprising an antibody or antibody pair as described above.

In some embodiments, the above-described kits are used for detecting β -amyloid in a sample from a subject, the above-described kits comprising the above-described antibody pair or antibody.

The antibody pairs or antibodies of some embodiments or examples of the invention are capable of binding to beta-amyloid, and therefore, reagents or kits comprising the antibody pairs or antibodies are effective for the qualitative or quantitative detection of beta-amyloid. The reagent or the kit provided by the invention can be used for detection of specific binding performance of beta-amyloid and antibodies thereof, such as immunoblotting, immunoprecipitation and the like. As previously mentioned, the antibodies of the invention have improved beta-amyloid binding activity, affinity, stability or specificity, and thus reagents or kits comprising the antibodies have improved detection sensitivity, specificity and/or reduced false negatives.

The kit of the invention comprises a beta-amyloid detection reagent card (test strip).

In alternative embodiments, antibodies can be labeled on detectable labels such as fluorescent microspheres using immunofluorescence techniques, and the principle of double antibody sandwich is used to detect beta-amyloid in a subject, e.g., a patient.

In alternative embodiments, the invention can utilize antibodies that bind to different amino acid fragments to identify multiple positions on an antigen, reducing the risk of missed detection, and increasing the detection rate.

In alternative embodiments, the invention provides a method of detecting beta-amyloid, a detection kit, and a method of making. The kit has improved sensitivity and specificity. In some embodiments, the antibodies of the invention may be monoclonal antibodies or polyclonal antibodies.

In alternative embodiments, the antibody pairs or antibodies of the invention may be prepared using methods known in the art.

In alternative embodiments, antigen-immunized animals that can include the amino acid fragments described herein produce an antibody pair or antibody of the invention. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) can be coupled to immunoreactive substances (e.g., epitope peptides). Carrier proteins may include proteins or polypeptides, which may function as carriers for immunogens. These types of polypeptides include albumin, serum proteins, globulins, lens proteins, lipoproteins and/or fragments thereof.

In alternative embodiments, immunoreactive substances (e.g., but not limited to, beta-amyloid amino acid fragments) may be used to produce antibodies having affinity for beta-amyloid.

In alternative embodiments, any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc. may be used to detect the effect, such as binding activity and/or cross-reactivity, of an antibody of the invention on or with an antibody.

In alternative embodiments, the assay may include, for example, ELISA, FACS binding assay, biacore, competitive binding assay, and the like.

In alternative embodiments, the kits of the invention comprise reagents suitable for performing an immunoassay.

In alternative embodiments, the kits of the invention may be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme linked antibody binding assays or methods.

In a seventh aspect, embodiments of the present invention provide a method of detecting β -amyloid, comprising:

a) Contacting the antibody or reagent with a sample to be tested under conditions sufficient for an antibody/antigen binding reaction to form an immune complex, and

In order to achieve the above object, according to an eighth aspect of the present invention, there is provided the use of the above antibody or reagent for detecting β -amyloid or for preparing a product for detecting β -amyloid.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

Based on long-term creative research on beta-amyloid, the inventor discovers antibodies capable of meeting the detection requirement of beta-amyloid. Antibody preparation, activity identification and performance testing are shown in the examples.

Example 1 antibody preparation

Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART ^TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation. Three hybridoma cell lines (named 7F3, 5H8 and 9D7 respectively) secreting the beta-amyloid monoclonal antibody are the existing hybridoma cell lines, and are recovered for later use.

(1) Antibody Gene production

MRNA is extracted from two hybridoma cell strains secreting beta-amyloid monoclonal antibodies respectively, DNA products are obtained through an RT-PCR method, the products are subjected to an A adding reaction by rTaq DNA polymerase and then are inserted into a pMD-18T vector to be transformed into DH5 alpha competent cells, heavy chain and light chain gene clones are taken respectively after bacterial colonies grow out, and 4 clones are sent to a gene sequencing company for sequencing.

(2) Sequence analysis of beta-amyloid antibody variable region genes

The gene sequences obtained by sequencing were analyzed in a kabat antibody database and analyzed using VNTI 11.5.5 software to determine that the amplified genes were correct for both heavy and light chain primer pairs. Through software analysis, single-point saturation mutation is carried out on the CDR region of the heavy chain variable region of 7F3 to obtain mutant sequences, which are named 7F3-1, 7F3-2 and 7F3-3, and the gene sequences amplified by the other two strains of cells are 5H8 and 9D7

(3) Construction of recombinant antibody expression plasmids

pcDNA^TM3.4Vector is a constructed eukaryotic expression vector of recombinant antibody, which has been introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, 3.4A expression vector, and VL and VH gene specific primers with HindIII, ecoRI enzyme cutting sites and protecting bases on two ends, and heavy chain gene segment and light chain gene segment are amplified by PCR amplification method.

The heavy chain and light chain gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the heavy chain gene and the light chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmid DNA of the heavy chain and the light chain are respectively obtained.

2. Recombinant antibody production

Resuscitate HEK293 cells in advance, subculture to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml, the cell density to reach the concentration of selected antibodies and cell viability to be more than 95%, centrifugally clean the cells, re-dissolve the cells with a culture medium, simultaneously adjust the cell density to 3.4×10 ⁶ cells/ml, re-dissolve the cells with the culture medium, and simultaneously serve as a cell dilution. The dilutions of the 6 plasmid DNAs and transfection reagents in step (3) above were prepared separately using medium. Adding transfection reagent diluent into plasmid DNA diluent, mixing, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing, sampling, counting, recording and observing activity of transfected cells, and culturing in a 35 ℃ constant temperature incubator at a rotation speed 120rmp and a CO ₂ content of 8%.

3. Indirect ELISA method for detecting recombinant cell strain expression supernatant

1) Coating A.beta.1-40 protein and A.beta.1-42 protein (from Fipeng organism) were coated at a concentration of 0.125ug/ml, and at 4℃overnight.

2) PBST was washed twice, patted dry, blocked with 120 ul/well of 20% bovine serum, incubated for 1h at 37 ℃.

3) Cell supernatants were diluted to different concentrations with 20% bovine serum, supernatants of 7F3, 7F3-1, 7F3-2, 7F3-3, 5H8 were added to the Abeta 1-40 protein coated ELISA plates, and supernatants of 9D7 were added to Abeta 1-42 protein coated ELISA plates and incubated at 37℃for 30min.

4) PBST was washed 5 times, patted dry, 100 ul/well with goat anti-rabbit IgG-HRP (5000 fold dilution of 1% casein), and incubated at 37℃for 30min.

5) PBST is washed 5 times, the mixture is patted dry, 50ul of solution A and 50ul of solution B are added, the mixture is incubated for 10min in dark, 50ul of stop solution is added, and the result is shown in Table 2. The results show that the supernatant antibodies of 7F3, 7F3-1, 7F3-2, 7F3-3 and 5H8 can be effectively combined with the Abeta 1-40 protein, and the supernatant antibody of 9D7 can be effectively combined with the Abeta 1-42 protein, and the combination activity is stronger.

TABLE 2 Indirect ELISA method cell supernatant binding Activity data

4. Antibody supernatant purification

The supernatant of the recombinant expression antibody was subjected to affinity purification by using a protein A affinity column, and the obtained antibodies were designated as 7F3 Rmb1, 7F3Rmb2, 7F3 Rmb3, 7F3 Rmb4, 5H8Rmb, and 9D7Rmb, and the sequences of the heavy chain (H) and the light chain (L) of the above antibodies are shown in Table 3.

TABLE 3 antibody sequences

Antibody name	Heavy chain variable region	Light chain variable region
			7F3 Rmb1	SEQ ID NO:23	SEQ ID NO:28
7F3 Rmb2	SEQ ID NO:24	SEQ ID NO:28
			7F3 Rmb3	SEQ ID NO:25	SEQ ID NO:28
7F3 Rmb4	SEQ ID NO:26	SEQ ID NO:28
			9D7Rmb	SEQ ID NO:66	SEQ ID NO:68
5H8Rmb	SEQ ID NO:46	SEQ ID NO:48

Example 2 Performance test

This example shows exemplary performance test data for a portion of an antibody.

The antibodies 7F3 Rmb1, 7F3 Rmb2, 7F3 Rmb3, 7F3 Rmb4 and 9D7Rmb obtained above are respectively used as coating antibodies, and the 5H8Rmb antibody is used as a labeling antibody, so that different experimental groups are formed for experiments, and the experimental processes are as follows:

1. Antibody coating magnetic bead process:

10mg of the carboxyl magnetic beads were washed 3 times with MES (100 mM MES, pH 5.5) buffer. The MES buffer resuspended the beads. EDC with a final concentration of 1mg/mL is added, mixed evenly by a 25 ℃ shaking table, fully mixed and reacted for 30min. After magnetic separation, the supernatant was discarded, MES buffer was added to resuspend the beads, antibodies 7F3 Rmb1, 7F3 Rmb2, 7F3 Rmb3, 7F3 Rmb4, 9D7 Rmbl were added, and the mixture was mixed with a 25℃shaker and reacted for 120min. Magnetically separating, discarding the supernatant, adding Tris buffer solution for resuspension, and preserving at 2-8 ℃ for standby, wherein the final concentration is 10mg/mL of solid content.

2. Acridinium ester labeling antibody procedure:

Antibody 5H8Rmb was replaced with Zeba desalting column (10K MWCO) to PBS (100 mM PB,50mM sodium chloride, pH 8.0). Acridinium esters were formulated in 4mM solution in DMSO. The antibody solution and acridine ester solution were mixed in a 1:10 molar ratio and reacted at 25 ℃ for 2 hours. Desalting to remove excessive reagent, adding glycerol with final concentration of 50%, and storing at-20deg.C.

3. And (3) the on-machine testing process:

The magnetic beads coated by the antibodies in the step (1) are respectively diluted to 0.5mg/mL by using a diluent (20 mM PB,150mM sodium chloride, 1% BSA, pH 7.2) as a magnetic bead working solution, the acridine ester modified antibodies in the step (2) are diluted to 1 mug/mL as an acridine ester working solution, detection is carried out on a full-automatic chemiluminescence immunoassay instrument of Kaishan i2910 model, a double antibody sandwich method is adopted, namely, 50 mu L of a sample, 50 mu L of a magnetic particle working solution and 50 mu L of an acridine ester working solution are sequentially added into the instrument, evenly mixed and incubated for 15 minutes, a reaction mixture is washed after incubation, a pre-excitation solution and an excitation solution are added, relative luminous intensity (RELATIVE LIGHT Units, RLU) is detected, and corresponding concentration values are calculated according to a four-parameter fitting mode.

Remarks:

The sample comprises:

1) Blank sample without test protein

2) Samples containing different concentrations of Abeta 1-40 protein and samples containing different concentrations of Abeta 1-42 protein (a high value sample near the upper limit of a linear interval and a low value sample near the lower limit of the linear interval or a zero concentration sample are mixed to form samples with different concentrations)

3) Random Abeta 1-40 protein clinical samples collected in laboratory

4) Beta amyloid 1-40 (Abeta 1-40), beta amyloid 1-41 (Abeta 1-41) and beta amyloid 1-43 (Abeta 1-43) (from the Fit organism)

4. Detection result

(1) Different clinical sample detection results

The laboratory collects 20 cases of random Abeta 1-40 protein clinical samples, and uses the experimental steps to detect and compare the differences between different antibodies. The results of the antibodies 7F3 Rmb1, 7F3 Rmb2, 7F3 Rmb3, 7F3 Rmb4 paired with antibody 5H8Rmb to detect A.beta.1-40 protein clinical samples are shown in Table 4. The results show that the antibodies 7F3 Rmb1, 7F3 Rmb2, 7F3 Rmb3 and 7F3 Rmb4 have smaller pairing detection performance difference with the antibody 5H8Rmb, and can effectively detect clinical samples of the Abeta 1-40 proteins with different concentrations.

TABLE 4 detection results of Abeta 1-40 protein clinical samples

(2) Blank limit

The assay was performed according to the experimental procedure in example 2 using antibodies 7f3 Rmb1, 9D7Rmb, control antibody 6E8 (source gold, cat No. V28702) paired with 5H8 Rmb.

And repeatedly testing a blank sample without the protein to be tested for 20 times, calculating the average value (M) and Standard Deviation (SD) of the blank sample, obtaining an RLU value corresponding to M+2SD, and taking the RLU value into a linear equation to obtain a corresponding concentration value, namely a blank limit. The results are shown in Table 5, and show that the blank of the pairing detection of 9D7Rmb and 5H8Rmb is 0.20pg/mL, and the blank of the pairing detection of 7F3 Rmb1 and 5H8Rmb is 0.13pg/mL, which are superior to the control antibody.

TABLE 5 detection results of blank samples without protein to be tested

(3) Repeatability of

The measurement was repeated 20 times for each of 2 samples of plasma containing Abeta 1-42 protein, and the mean value M and Standard Deviation (SD) of the 20 measurements were calculated to obtain the Coefficient of Variation (CV). The detection results are shown in Table 6, and the results show that the pairing detection CV value of 9D7Rmb and 5H8Rmb is lower, and the repeatability is superior to that of the control antibody.

TABLE 6 detection results of Abeta 1-42 protein-containing plasma samples

(4) Linear range

Repeating the test for 3 times on samples containing the Abeta 1-40 protein and the Abeta 1-42 protein with different concentrations respectively to obtain luminescence values, recording the measurement results of the samples, calculating the average value of 3 measurement values of the samples, performing linear fitting on the average value of the measured concentrations and the theoretical concentration by using a least square method, and calculating a linear correlation coefficient (r). Sample detection results of the different concentrations of Abeta 1-40 protein are shown in Table 7, and sample detection results of the different concentrations of Abeta 1-42 protein are shown in Table 8. The result shows that the detection linear range of the pairing of the 9D7Rmb and the 5H8Rmb is 3-2000 pg/mL, the detection linear range of the pairing of the 7F3 Rmb1 and the 5H8Rmb is 5-2000 pg/mL, and the linear range is wide, so that the clinical requirements are met.

TABLE 7 detection results of Abeta 1-40 protein samples at different concentrations

TABLE 8 detection results of Abeta 1-42 protein samples at different concentrations

(5) Cross-reactivity:

as shown in Table 9, the results show that the reagent composed of the antibodies 9D7Rmb and 5H8Rmb binds very weakly to amyloid beta 1-40 (Abeta 1-40), amyloid beta 1-41 (Abeta 1-41) and amyloid beta 1-43 (Abeta 1-43), and has low cross-reactivity and good specificity.

TABLE 9 Cross reaction results

The partial amino acid sequences involved in the present application are shown in Table 10:

TABLE 10 amino acid sequence listing

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibody for detecting β-amyloid protein, characterized in that,

The antibody comprises three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, in the heavy chain variable region as shown in any of SEQ ID NO:19, 20, 21, 22, 45, and 65, and three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, in the light chain variable region as shown in any of SEQ ID NO:27, 47, and 67.

Optionally, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody for detecting β-amyloid protein, characterized in that the complementarity-determining region of the antibody comprises any one of (a’) to (c’):

(a’) HCDR1 as shown in SEQ ID NO:1 (AYYIH), HCDR2 as shown in SEQ ID NO:2 (RIDPATGNTKYAPRLQD) or 15 (RLDPATGNTKYAPRLQD), HCDR3 as shown in SEQ ID NO:3 (LYSLPVY) or 16 (IYSLPVY), and LCDR1 as shown in SEQ ID NO:4 (KSSQSLLYSDAKTYLN), LCDR2 as shown in SEQ ID NO:5 (QISRLDP), and LCDR3 as shown in SEQ ID NO:6 (LQGTHYPVL);

(b’) The amino acid sequences are as shown in SEQ ID NO:29 (NYGMS) for HCDR1 and SEQ ID NO:30, respectively.

HCDR2 as shown in (SIRSGGGRTYYSDNVKG), HCDR3 as shown in SEQ ID NO:31 (YDHYSGSSDY), and LCDR1 as shown in SEQ ID NO:32 (KSSQSLLDSDGKTYLN), LCDR2 as shown in SEQ ID NO:33 (LVSKLDS), and LCDR3 as shown in SEQ ID NO:33 (WQGTHFPRT); and

(c’) The amino acid sequences are as shown in SEQ ID NO:49(DYTMH) for HCDR1 and SEQ ID NO:50, respectively.

HCDR2 as shown in SEQ ID NO: 51 (GVYDGYFY), HCDR3 as shown in SEQ ID NO: 51 (GVYDGYFY), and LCDR1 as shown in SEQ ID NO: 52 (RSSQSLVYSNGNTFLH), LCDR2 as shown in SEQ ID NO: 53 (KVSTRFSGVPDRFS), and LCDR3 as shown in SEQ ID NO: 54 (SQTTHAPFT).

3. The antibody according to claim 1 or 2, characterized in that the antibody further comprises the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

Optionally, HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from any group of (a) to (c):

(a) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or the amino acid sequences having at least 80% identity with the sequences of each of the frame regions.

(b) The amino acid sequences HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:55 to SEQ ID NO:58, and the amino acid sequences LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:59 to SEQ ID NO:62; or amino acid sequences having at least 80% identity with the sequences of the respective frame regions; and

(c) The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:35 to SEQ ID NO:38, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:39 to SEQ ID NO:42; or the amino acid sequences have at least 80% identity with the sequences of each of the frame regions.

4. An antibody for detecting β-amyloid protein, characterized in that the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO:19, 20, 21, 22, 45, 65, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO:27, 47, 67;

Optionally, the amino acid sequences of the heavy chain variable region and the light chain variable region shown are selected from any one of (A) to (C):

(A) The heavy chain variable region with an amino acid sequence as shown in any of SEQ ID NO:19, 20, 21, 22, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:27;

(B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:45, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:47; and

(C) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:65, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:67.

5. The antibody according to any one of claims 1 to 4, characterized in that the antibody further comprises a constant region;

Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

Optionally, the species source of the constant region is mice.

Optionally, the constant region includes a heavy chain constant region and/or a light chain constant region;

Optionally, the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, or a combination of multiple constant regions; and/or; the light chain constant region is selected from κ-type or λ-type light chain constant regions;

Optionally, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM and/or CH4 of IgM;

Optionally, the antibody includes the following constant region:

The amino acid sequence of the heavy chain constant region as shown in SEQ ID NO:17; and the amino acid sequence of the light chain constant region as shown in SEQ ID NO:18; or an amino acid sequence having at least 80% identity with each of the constant regions.

6. An antibody for detecting β-amyloid protein, characterized in that the antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 23, 24, 25, 26, 46, 66, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 28, 48, 68;

Optionally, the amino acid sequences of the heavy and light chains shown are selected from any group of (A’) to (C’):

(A’) Heavy chain with amino acid sequence as shown in any of SEQ ID NO:23, 24, 25, 26, and light chain with amino acid sequence as shown in SEQ ID NO:28;

(B’) The heavy chain with the amino acid sequence as shown in SEQ ID NO:46, and the light chain with the amino acid sequence as shown in SEQ ID NO:48; and

(C’) The heavy chain with an amino acid sequence as shown in SEQ ID NO:66, and the light chain with an amino acid sequence as shown in SEQ ID NO:68.

7. An antibody pair for detecting β-amyloid protein, characterized in that the antibody pair comprises a coating antibody and/or a labeled antibody; the coating antibody and the labeled antibody are respectively selected from the antibodies according to any one of claims 1 to 6;

Optionally, the coating antibody comprises a first antibody and a second antibody, wherein the first antibody is selected from the antibody group (c’) of claim 2, the antibody group (C) of claim 4, or the antibody group (C’) of claim 6; and the second antibody is selected from the antibody group (a’) of claim 2, the antibody group (A) of claim 4, or the antibody group (A’) of claim 6.

Optionally, the labeled antibody is selected from the group of antibodies in claim 2 (b’), the group of antibodies in claim 4 (B), or the group of antibodies in claim 6 (B’).

Optionally, the labeled antibody is labeled with a detectable marker and/or binding partner;

Optionally, the detectable marker is selected from metal ions, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent markers, electron-dense markers, adamantane, and nanoparticle markers;

Optionally, the marker is an acridine ester;

Optionally, the coated antibody is linked to a solid-phase carrier and/or a binding coupler.

Optionally, the solid phase is selected from microspheres, magnetic beads, latex particles, microfluidic chips, plates, or membranes;

Optionally, the solid phase is selected from magnetic beads;

Optionally, the binding coupler includes a biotin/avidin protein or a biotin derivative/avidin derivative protein.

8. A detection reagent or kit for detecting β-amyloid protein, characterized in that the reagent or kit comprises the antibody according to any one of claims 1 to 6 or the antibody pair according to claim 7.

9. A method for detecting β-amyloid protein, characterized in that it comprises:

a) Under conditions sufficient to induce an antibody/antigen binding reaction, the antibody of any one of claims 1 to 6, the antibody pair of claim 7, or the reagent or kit of claim 8 is contacted with the sample to be tested to form an immune complex; and

b) Detect the presence of the immune complex, the presence of which indicates the presence of β-amyloid protein in the test sample.

10. Use of the antibody of any one of claims 1 to 6, the antibody pair of claim 7, or the reagent or kit of claim 9 in the detection of β-amyloid protein or in the preparation of products for the detection of β-amyloid protein.