CN118459582B

CN118459582B - Anti-pyroglutamic acid modified beta amyloid antibody and application thereof

Info

Publication number: CN118459582B
Application number: CN202410926229.1A
Authority: CN
Inventors: 李德彬; 宋锐; 苏立燕; 龙丽梅; 薛玉婷; 种笑妍
Original assignee: Suzhou Inshore Protein Technology Co ltd
Current assignee: Suzhou Inshore Protein Technology Co ltd
Priority date: 2024-07-11
Filing date: 2024-07-11
Publication date: 2024-11-15
Anticipated expiration: 2044-07-11
Also published as: CN118459582A

Abstract

The invention relates to the field of antibodies, in particular to an anti-pyroglutamic acid modified beta amyloid antibody and application thereof. The CDR of the heavy chain variable region in the anti-pyroglutamic acid modified amyloid beta antibody provided by the invention comprises CDR-H1 with an amino acid sequence shown as SEQ ID No.2, CDR-H2 with an amino acid sequence shown as SEQ ID No.3 and CDR-H3 with an amino acid sequence shown as SEQ ID No. 4; the CDR of the light chain variable region in the anti-Abeta _pE3‑42 antibody comprises CDR-L1 with the amino acid sequence shown as SEQ ID No.6, CDR-L2 with the amino acid sequence shown as SEQ ID No.7 and CDR-L3 with the amino acid sequence shown as SEQ ID No. 8. The anti-pyroglutamic acid modified beta amyloid antibody provided by the invention has high affinity, can specifically bind with brain slices of an Alzheimer disease transgenic mouse model (5 xFAD), and has higher affinity than the anti-Abeta _p3‑42 antibody in the prior art.

Description

Anti-pyroglutamic acid modified beta amyloid antibody and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-pyroglutamic acid modified beta amyloid antibody and application thereof.

Background

Beta-amyloid (aβ) contains 39 to 43 amino acids and has a molecular weight of about 4kDa and is a fragment hydrolyzed from the amyloid precursor protein beta-Amyloid Precursor Protein (APP). It can be produced by a variety of cells, circulating in blood, cerebrospinal fluid and cerebral interstitial fluid, most of aβ being bound to chaperonin molecules, and a few being present in a free state. Aβ is highly conserved in evolutionary processes and plays a key role in maintaining central nervous system homeostasis. Aβ has two main types of aβ _1-40、Aβ_1-42 and other rare types: aβ _1-42 is a β lamellar structure, is highly hydrophobic, is easily deposited, and is more easily aggregated, thereby forming a core for Aβ precipitation, and has neurotoxicity. Normally 90% of the human body is present in a type of aβ _1-40, with only small amounts of aβ _1-42 present. Due to factors such as inheritance, the ratio of Abeta _1-42/Aβ_1-40 in the brain of patients with Alzheimer's Disease (AD) is unbalanced, and Abeta _1-42 is increased. The produced aβ monomers aggregate into a variety of forms such as aβ oligomers, aβ fibrils, aβ fibrous high aggregates, and the like, and can change conformation into plaques, mediating AD disease through a variety of mechanisms. Increased aβ _1-42 deposits in the brain to form the core of senile plaques, which can activate microglial cells, induce inflammatory reactions, activate apoptosis pathways, mediate apoptosis, promote abnormal phosphorylation of Tau protein by activating protein kinase, and the like. These pathological changes in turn promote increased aβ production and abnormal deposition, producing a cascade of positive feedback amplification effects, ultimately leading to reduced neurons, abnormal transmitters, and triggering clinical cognitive and behavioral symptoms. A few species of beta amyloid (aβ), such as aβ _1–43 and pyroglutamates aβ _3–42, are involved in the initiation of the aβ aggregation process, closely related to the etiology of alzheimer's disease.

Extracellular plaques of beta amyloid (aβ) and neurofibrillary tangles formed by Tau protein are histopathological features of Alzheimer's Disease (AD). Plaques contain aβ fibrils assembled from monomeric and oligomeric intermediates, which are prognostic indicators of AD, and oligomers are the major toxic form of aβ. The n-terminal truncated, pyroglutamyl (pE) form of aβ is closely related to AD, is more toxic than aβ _1-40、Aβ_1-42, and is considered to be a contributing factor in the pathogenesis of AD.

N-terminally truncated and pyroglutamate modified amyloid beta (Abeta) plays a major role in the development of Alzheimer's disease. This pyroglutamic acid modified amyl beta is also called Aβ _pE3-42, pE3-A, N3pG, N3pGlu. Post-translational formation of pyroglutamic acid (pGlu) at the 3 or 11 position of aβ means cyclization of the N-terminal glutamic acid residue, rendering the modified peptide resistant to degradation, more hydrophobic, and prone to aggregation. Aβ _pE3-42 is present in the amyloid deposits of alzheimer's disease and is the core of AD amyloid. Aβ _pE3-42 can co-oligomerize with excess aβ _1‐42 to form misfolded metastable Low N Oligomers (LNOS), LNOS is more cytotoxic to cultured neurons than similar LNOS produced by aβ _1‐42 alone, and can promote the production of new LNOS. Due to the unique ability of aβ _pE3-42, it has become a new potential drug target for AD.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an anti-pyroglutamic acid modified amyloid β (aβ _pE3-42) antibody and its use for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides an anti-aβ _pE3-42 antibody comprising a heavy chain variable region and a light chain variable region, the anti-aβ _pE3-42 antibody having one or more of the following technical features:

1) The complementarity determining region of the heavy chain variable region comprises CDR-H1 having the amino acid sequence shown in SEQ ID No. 2;

2) The complementarity determining region of the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 3;

3) The complementarity determining region of the heavy chain variable region comprises CDR-H3 having the amino acid sequence shown in SEQ ID No. 4;

4) The complementarity determining region of the light chain variable region comprises CDR-L1 having the amino acid sequence shown in SEQ ID No. 6;

5) The complementarity determining region of the light chain variable region comprises CDR-L2 having the amino acid sequence shown in SEQ ID No. 7;

6) The complementarity determining region of the light chain variable region comprises CDR-L3 having the amino acid sequence shown in SEQ ID No. 8.

Preferably, the complementarity determining region of the heavy chain variable region of the anti-Abeta _pE3-42 antibody comprises CDR-H1 with an amino acid sequence shown as SEQ ID No.1, CDR-H2 with an amino acid sequence shown as SEQ ID No.2 and CDR-H3 with an amino acid sequence shown as SEQ ID No. 3.

The invention also provides an isolated polynucleotide encoding one or more of the following peptide fragments:

1) Heavy chain variable regions in the anti-aβ _pE3-42 antibodies as described above;

2) A light chain variable region of an anti-aβ _pE3-42 antibody as described above;

3) An anti-aβ _pE3-42 antibody as described above.

The present invention also provides a nucleic acid construct comprising the aforementioned isolated polynucleotide.

The invention also provides an isolated engineered cell comprising the aforementioned nucleic acid construct or the aforementioned polynucleotide.

The invention also provides the use of the anti-Abeta _pE3-42 antibody, the polynucleotide or the nucleic acid construct in the preparation of a disease diagnosis, prevention or treatment product.

The invention also provides a detection kit which contains the anti-Abeta _pE3-42 antibody.

As described above, the anti-aβ _pE3-42 antibody and the use thereof of the present invention have the following beneficial effects:

according to the invention, the anti-Abeta _pE3-42 antibody with high affinity is obtained by immunizing a mouse with Abeta _pE3-42 polypeptide, the obtained anti-Abeta _pE3-42 antibody can be specifically combined with brain slices of an Alzheimer's disease transgenic mouse model (5 xFAD), and the affinity is higher than that of the anti-Abeta _pE3-42 antibody in the prior art.

Drawings

FIG. 1 is a graph showing the results of detection of Abeta murine antibody antigen discrimination in the present invention.

FIG. 2 is a graph showing the results of antigen discrimination detection of Abeta humanized antibodies of the present invention.

FIG. 3 is a graph showing the results of ELISA detection of the affinity of Abeta antibody to Abeta _pE3-42 polypeptide protein in the present invention.

FIG. 4 is a graph showing the results of immunohistochemical detection of Abeta humanized antibody in the present invention.

Detailed Description

The invention provides an anti-pyroglutamic acid modified beta amyloid (Abeta _pE3-42) antibody, wherein the anti-Abeta _pE3-42 antibody comprises a heavy chain variable region and a light chain variable region, and the anti-Abeta _pE3-42 antibody has one or more of the following technical characteristics:

In some embodiments, the complementarity determining region of the heavy chain variable region of the anti-Abeta _pE3-42 antibody comprises CDR-H1 having the amino acid sequence shown as SEQ ID No.2, CDR-H2 having the amino acid sequence shown as SEQ ID No.3 and CDR-H3 having the amino acid sequence shown as SEQ ID No. 4; or, the complementarity determining region of the heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No.6, CDR-H2 with an amino acid sequence shown as SEQ ID No.7 and CDR-H3 with an amino acid sequence shown as SEQ ID No. 8.

In some embodiments, the complementarity determining region of the heavy chain variable region in the anti-Abeta _pE3-42 antibody comprises CDR-H1 having the amino acid sequence shown as SEQ ID No.2, CDR-H2 having the amino acid sequence shown as SEQ ID No.3 and CDR-H3 having the amino acid sequence shown as SEQ ID No.4, and the complementarity determining region of the light chain variable region comprises CDR-L1 having the amino acid sequence shown as SEQ ID No.6, CDR-L2 having the amino acid sequence shown as SEQ ID No.7 and CDR-L3 having the amino acid sequence shown as SEQ ID No. 8.

In some embodiments, the anti-aβ _pE3-42 antibody is a monoclonal antibody.

In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-aβ _pE3-42 antibody comprises:

a) An amino acid sequence shown as one of SEQ ID No.1 and 11; or (b)

B) An amino acid sequence having 80% or more identity to the amino acid sequence shown in one of SEQ ID Nos. 1 and 11 and having the function of the amino acid sequence defined in a).

Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown as one of SEQ ID No.1 and 11 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminal and/or C-terminal, and has the amino acid sequence function shown as one of SEQ ID No.1 and 11. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity with one of SEQ ID nos. 1, 11.

In some embodiments, the amino acid sequence of the light chain variable region of the anti-aβ _p3-42 antibody comprises:

c) An amino acid sequence as shown in one of SEQ ID No.5 and 12; or (b)

D) An amino acid sequence having 80% or more identity to the amino acid sequence shown in one of SEQ ID Nos. 5 and 12 and having the function of the amino acid sequence defined in c).

Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown as one of SEQ ID No.5 and 12 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminal and/or C-terminal, and has the amino acid sequence function shown as one of SEQ ID No.5 and 12. The amino acid sequence in d) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity to one of SEQ ID nos. 5, 12.

In some embodiments, the anti-aβ _pE3-42 antibody comprises a peptide fragment having the amino acid sequence of the heavy chain variable region as shown in SEQ ID No.1 and the amino acid sequence of the light chain variable region as shown in SEQ ID No. 5; or, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.11 and the amino acid sequence of the light chain variable region is shown as peptide fragment shown as SEQ ID No. 12.

In some embodiments, the anti-aβ _pE3-42 antibody further comprises a heavy chain constant region, i.e., an hIgG1 constant region, having the amino acid sequence shown as SEQ ID No. 9.

In some embodiments, the anti-Abeta _pE3-42 antibody further comprises a light chain constant region, i.e., a kappa chain constant region, having the amino acid sequence shown as SEQ ID No. 10.

In some embodiments, the anti-aβ _pE3-42 antibody further comprises a secretion signal peptide. In particular, the secretion signal peptide is derived from DsbA, pelB, ompA, tolB, malE, lpp, torA or HylA signal peptide. Wherein the secretion signal peptide directs the anti-aβ _pE3-42 antibody to the periplasmic space of the host cell.

In some embodiments, the anti-aβ _pE3-42 antibody comprises a histidine tag. The histidine tag or poly-histidine tag is a sequence of 2 to 20 histidine residues attached to an anti-aβ _pE3-42 antibody. The histidine tag comprises 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues.

In some embodiments, the anti-aβ _pE3-42 antibody is an anti-human aβ _pE3-42 antibody or an anti-murine aβ _pE3-42 antibody.

1) The heavy chain variable region of the aforementioned anti-aβ _pE3-42 antibody;

2) The light chain variable region of the aforementioned anti-aβ _pE3-42 antibody;

3) The aforementioned anti-aβ _pE3-42 antibody.

In some embodiments, the nucleic acid construct is constructed from the isolated polynucleotide inserted into a multiple cloning site of an expression vector. Expression vectors may be transformed, transduced or transfected into host cells to express the genetic material elements carried thereby. The construct is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC), phages such as lambda phage or M13 phage, animal viruses, and the like. The viral vector comprises: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-vacuolated viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including, but not limited to, viral particles, liposomes, or protein shells.

In the present invention, the engineered cell can be obtained by introducing the nucleic acid construct into a host cell, or a cell having the exogenous polynucleotide integrated in the genome is the engineered cell.

Any cell suitable for expression of the expression vector may be used as a host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell, etc.; or lower eukaryotic cells such as yeast cells, etc.; or higher eukaryotic cells such as mammalian cells, etc. The host cells include a number of cell types such as prokaryotic cells like E.coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like S2 Drosophila cells or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

In some embodiments, the disease is alzheimer's disease.

In some embodiments, the prophylactic or therapeutic product is an antibody drug.

The invention also provides a method of treating alzheimer's disease comprising administering to a subject an effective amount of the aforementioned anti-aβ _p3-42 antibody.

In the present invention, the term "complementarity determining region" or "CDR" generally refers to a region of an antibody that is spatially complementary to an epitope. Variability in antibodies is typically not evenly distributed throughout the variable region of the antibody, and both the heavy and light chain variable regions of a monoclonal antibody typically have 3 hypervariable regions (hypervariable region, HVR) that are typically complementary to an epitope in spatial structure, so the hypervariable regions are also known as complementarity determining regions (complementarity determining region, CDRs), i.e., the heavy chain variable regions typically comprise three complementarity determining regions, i.e., HCDR1, HCDR2, and HCDR3, and the light chain variable regions typically comprise three complementarity determining regions, i.e., LCDR1, LCDR2, and LCDR3.

In the present invention, the term "monoclonal antibody" generally refers to a population of antibodies that are substantially identical (except for a few naturally occurring mutations that may be present). Monoclonal antibodies are typically directed against specific determinants on the antigen.

In the present invention, the term "identity" means: if two or more sequences have the same nucleotide or amino acid length and order, then the sequences are identical. The percent identity generally describes the degree to which two sequences are identical, i.e., generally describes the percent of nucleotides at the sequence positions that correspond to the same nucleotides of a reference sequence. To determine the degree of identity (percent identity), the sequences to be compared are typically of the same length, i.e., the length of the longest sequence of the sequences to be compared. The examples are: the first sequence consisting of 8 nucleotides is 80% identical to the second sequence consisting of 10 nucleotides comprising the first sequence. It is thus understood that in the context of the present invention, sequence identity preferably relates to the percentage of nucleotides or amino acids of a sequence having the same position in two or more sequences of the same length. Specifically, the "identity%" of two amino acid sequences or two nucleotide sequences may be determined by comparing the sequences to each other for optimal comparison purposes and comparing the amino acids or nucleotides at the corresponding positions, e.g., gaps may be introduced in either sequence for optimal alignment with the other sequence. The null is generally considered to be a non-identical location regardless of its actual location therein. An "optimal alignment" is generally the alignment of two sequences that yields the highest percentage of identity. The percent identity is determined by the number of identical nucleotides in the compared sequences, i.e.,% identity=number of identical positions/total number of positions×100. Other known mathematical algorithms can also be used by those skilled in the art to determine the percent identity between two sequences.

In the present invention, the term "plasmid backbone" is generally a circular DNA molecule or linear DNA molecule that can autonomously replicate and express an inserted gene of interest in a cell. The backbone plasmid may contain regulatory sequences such as promoters, replicons, transcription and translation initiation and termination codons. The backbone plasmid is typically ligated to the gene of interest to form an intact expression vector that can express a particular yield in the cell.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The nucleotide or amino acid sequence information used in the present invention is as follows:

SEQ ID No.1：

QVQLQQPGAELVRPGASVKLSCKTSGYTFTRYWINWVKQRPGQGLEWIGNIYPSDSYSNYNHKFKDKATLTVDKSSSTAYMQLSSSTSEDSAVYYCTREGGRGEVYWGQGTTLTVSS

SEQ ID No.5：

DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKIYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPYTFGGGTKLEIKR

SEQ ID No.9：

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID No.10：

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No.11：

QVQLQESGPGLVKPSETLSLTCTTSGYTFTRYWINWVRQPPGKGLEWIGNIYPSDSYSNYNHKFKDRATLSVDKSKNQASLNLDSVSAADTAIYYCTREGGRGEVYWGKGSTVTVSS

SEQ ID No.12：

DVQMTQSPSSLSASVGDRVTITCKSSQSLLYSNGKIYLNWLQQKPGKAPKRLIYLVSKLDSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCVQGTHFPYTFGPGTKVDIKR

Example 1 preparation of anti-Abeta _pE3-42 antibody

To generate antibodies to human aβ _pE3-42, balb/c female mice that were immunized 6-8 weeks with N-terminally truncated and pyroglutamate-modified human amyloid β peptide 3-42 (aβ _pE3-42) antigen pre-treated overnight at 37 ℃ to form aggregates, the mice were immunized by standard polypeptide immunization procedures, spleens were harvested after 3 times and fusion with SP2/0 cells was completed to generate hybridomas using a BTX electrofusion system. Hybridoma supernatants were assayed for cross-reactivity with aβ _pE3-42 polypeptide coated ELISA plate and aβ _1-40、Aβ_1-42 polypeptide using an indirect ELISA format. The positive clone only reacted with the Abeta _pE3-42 polypeptide coated ELISA plate and did not react with Abeta _1-40、Aβ_1-42 polypeptide. The selected candidate clone is used for modulating the related antibody sequence through a specific gene amplification technology.

VH (amino acid sequence shown in SEQ ID No. 1) and VL (amino acid sequence shown in SEQ ID No. 5) of the prepared antibody sequence a42Ta are respectively constructed on eukaryotic expression vectors containing a constant region of hgg 1 (amino acid sequence shown in SEQ ID No. 9) -kappa (amino acid sequence shown in SEQ ID No. 10), VH and VL are co-transfected into fresh 293F cells, the supernatant is collected after 37 days of culture, and protein a column is used for purification, thus obtaining antibody proteins corresponding to a42 Ta.

The CDRs sequence information in this example is as follows (IMGT definition, single column as follows):

TABLE 1 CDR sequences of murine anti-Abeta _pE3-42 antibodies

Name of the name	Sequence(s)	SEQ ID NO:
			CDR1	GYTFTRYW	2
CDR2	IYPSDSYS	3
			CDR3	TREGGRGEVY	4
CDR1	QSLLYSNGKIY	6
			CDR2	LVS	7
CDR3	VQGTHFPYT	8

Construction of humanized anti-aβ _pE3-42 antibodies expression:

The VH and VL of the full length antibody a42Ta were selected for humanization to yield the humanized antibody a42TaA. The VH (amino acid sequence shown as SEQ ID No. 11) and VL (amino acid sequence shown as SEQ ID No. 12) of the humanized antibody sequence A42TaA are respectively constructed on a eukaryotic expression vector containing a hIgG1 kappa constant region, the VH and the VL are co-transfected into a freeform 293F cell, the culture is carried out for 37 days, the supernatant is collected, and protein A column is used for purification, so that the antibody protein corresponding to A42TaA is obtained.

The antibody used in this example can be obtained by introducing it into cells by homologous recombination after chemical synthesis or decompilation into a nucleotide sequence on the basis of known amino acid sequences.

Example 2A beta antibody antigen discrimination detection

This example mainly demonstrates the affinity detection of aβ antibodies with aβ three antigens (aβ _pE3-42、Aβ_1-42、Aβ_1-40), respectively. Specifically, abeta _pE3-42、Aβ_1-42、Aβ_1-40 antigen is coated with 3ug/ml by enzyme-linked immunosorbent assay (ELISA), antibody is diluted by 5 times for 8 gradients at 20ug/ml concentration, finally anti-human IgG (Fc specific) -peroxidase antibody goat anti (sigma, A0170) is added for incubation, TMB is added for color development, 1M HCl is added for stopping reaction, and then an ELISA reader is used for OD450 reading. The results are plotted as S-curves in fig. 1,2, 3 and table 2. The results show that both aβ antibodies exhibit specificity for the aβ _pE3-42 polypeptide and that 3 antibodies have similar affinities to the aβ _pE3-42 polypeptide.

TABLE 2 ELISA detection of Abeta antibodies against Abeta _pE3-42 polypeptide protein affinity EC50 display

Anti-Aβ_p3-42	A42Ta	A42TaA	Donepezil antibody
				EC50(ug/mL)	0.006792	0.005796	0.005401

EXAMPLE 3A beta humanized antibody immunohistochemical detection

This example shows mainly binding of aβ antibodies to brain slices of the transgenic mouse model of alzheimer's disease (5 xFAD). Specifically, taking 3-5 mu m brain coronary tissue slices for conventional dewaxing and hydration; placing the slices into citric acid antigen retrieval liquid, placing in a microwave oven for antigen retrieval (medium fire for 8min to boiling, stopping fire for 8min for heat preservation, and medium and low fire for 7 min), wherein the whole process needs to prevent excessive evaporation of the retrieval liquid and cut the slices; placing the slices into a 3% hydrogen peroxide solution to cover all tissues, and incubating for 25min at room temperature in a dark place to block endogenous peroxidase; after the slices are slightly dried, a histochemical pen is used for circling around the tissues, 10% BSA blocking solution is dripped, and the room temperature is closed for 30min; gently throwing away the sealing liquid, dripping a primary antibody working liquid on the slice, and placing the slice in a wet box for incubation at 4 ℃ overnight; after the sections were slightly spun dry, peroxidase-labeled goat anti-human IgG (Fc specific) secondary antibody working solution (Sigma, cat#a0170) was added dropwise and incubated at room temperature for 50 min. Dripping DAB color development working solution on the tissue, dyeing about 5min (grasping the color development degree under the mirror), and washing the slice with water to terminate the color development; hematoxylin dye counterstain 3min, rinsing with water. Then, the mixture is differentiated into 5 s by using hydrochloric acid alcohol differentiation liquid, washed by water, and finally, blued by bluing liquid and washed by tap water. Sequentially soaking in 75%, 85%, absolute ethanol I, absolute ethanol II, and n-butanol each 5min, and gradient ethanol dehydrating. And the xylene I and the xylene II are transparent and respectively 5min, and the sealing sheet is dried. The result of the staining is shown in FIG. 4. The results show that the brown yellow spots displayed by the dyed A42TaA have more colors and have darker colors compared with the polynaphthalamus antibody, and show stronger binding capacity.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. An anti-aβ _pE3-42 antibody, wherein the anti-aβ _pE3-42 antibody comprises a heavy chain variable region and a light chain variable region, and wherein the anti-aβ _pE3-42 antibody has the following technical characteristics:

1) The amino acid sequence of the complementarity determining region CDR-H1 of the heavy chain variable region is shown in SEQ ID No. 2;

2) The amino acid sequence of the complementarity determining region CDR-H2 of the heavy chain variable region is shown in SEQ ID No. 3;

3) The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region is shown in SEQ ID No. 4;

4) The amino acid sequence of the complementarity determining region CDR-L1 of the light chain variable region is shown in SEQ ID No. 6;

5) The amino acid sequence of the complementarity determining region CDR-L2 of the light chain variable region is shown in SEQ ID No. 7;

6) The amino acid sequence of CDR-L3 of the light chain variable region is shown in SEQ ID No. 8.

2. The anti-aβ _pE3-42 antibody of claim 1, wherein said anti-aβ _pE3-42 antibody further comprises one or more of the following features:

a) The amino acid sequence of the heavy chain variable region in the anti-Abeta _pE3-42 antibody is shown as SEQ ID No.1, and the amino acid sequence of the light chain variable region in the anti-Abeta _pE3-42 antibody is shown as SEQ ID No. 5;

b) The amino acid sequence of the heavy chain variable region in the anti-Abeta _pE3-42 antibody is shown as SEQ ID No.11, and the amino acid sequence of the light chain variable region in the anti-Abeta _pE3-42 antibody is shown as SEQ ID No. 12;

c) The anti-Abeta _pE3-42 antibody contains a heavy chain constant region with an amino acid sequence shown as SEQ ID No. 9;

d) The anti-Abeta _pE3-42 antibody also comprises a light chain constant region with an amino acid sequence shown as SEQ ID No. 10.

3. An isolated polynucleotide encoding the anti-aβ _pE3-42 antibody of any of claims 1-2.

4. A nucleic acid construct characterized in that, the nucleic acid construct comprising the polynucleotide of claim 3.

5. An isolated engineered cell comprising the nucleic acid construct of claim 4 or the polynucleotide of claim 3.

6. Use of an anti-aβ _pE3-42 antibody of any of claims 1-2, a polynucleotide of claim 3 or a nucleic acid construct of claim 4 in the preparation of a diagnostic product for alzheimer's disease.

7. A test kit comprising an anti-aβ _pE3-42 antibody of any of claims 1-2.