Rabbit-derived A beta 1-40 monoclonal antibody, preparation method and application

Abstract

The invention belongs to the field of biological engineering and biological detection, discloses a rabbit-derived Abeta1-40 monoclonal antibody, and a pharmaceutical composition, a detection reagent or a kit containing the monoclonal antibody, and also discloses an application of the monoclonal antibody in preparation of the pharmaceutical composition, the detection reagent or the kit. The rabbit monoclonal antibody prepared by the invention can efficiently and specifically recognize human A beta 40. Compared with mice or rats, the rabbit-derived monoclonal antibody disclosed by the invention is simpler in structure, has higher affinity and is more easily humanized.

Description

Technical Field

[0001] The invention relates to a rabbit-derived Aβ 1-40 monoclonal antibody, a preparation method and application thereof, and belongs to the field of bioengineering and biodetection. Background Art

[0002] Alzheimer's disease (AD) is a common degenerative disease of the central nervous system. Its important pathological characteristics include deposition of β-amyloid protein (Aβ) plaques, neurofibrillary tangles formed by phosphorylated tau protein, and neuronal damage and loss. AD patients usually experience memory loss and cognitive decline for up to 10 to 15 years, which not only brings a heavy burden to the patients' families, but also has an impact on society.

[0003] Amyloid β (Aβ) is a hydrophobic polypeptide encoded by a gene on human chromosome 21, with a molecular weight of about 4KDa, containing 39 to 43 amino acids and a β-pleated sheet structure. The production of Aβ originates from the protein amyloid precursor protein (APP), which is hydrolyzed by β and γ secretases, which cut APP to produce Aβ polypeptides of different lengths, the most common of which are Aβ40 and Aβ42.

[0004] Under pathological conditions, APP is first hydrolyzed by β-secretase 1 (BACE1) to generate sAPPβ and a 99-amino acid C99 peptide segment connected to the cell membrane. Then, the C99 peptide segment is acted upon by γ-secretase to eventually produce Aβ. These Aβ polypeptides circulate in the blood, cerebrospinal fluid, and brain interstitium, and exist in a state bound to chaperone proteins or in a free state. The Aβ cascade hypothesis holds that excessive production and imbalance of Aβ clearance are one of the key causes of AD. The accumulation of Aβ can lead to loss of synaptic function, reduce neuronal plasticity, change cellular energy metabolism, cause oxidative stress response and mitochondrial dysfunction, and thus disrupt intracellular calcium ion balance. In particular, the formation, aggregation, and deposition of Aβ42 can cause significant neurotoxicity, and its oligomers can directly affect the long-term potentiation response and synaptic function in the hippocampus, leading to neurotransmitter disorders and neurological dysfunction.

[0005] According to the World Health Organization's forecast, by 2050, one in every 85 people in the world will suffer from AD. Therefore, it is an urgent task to implement intervention treatment, reduce the burden of AD disease, and improve people's physical and mental health. As the main cause of dementia, Alzheimer's disease (AD) has greatly affected the health and quality of life of the elderly. With the aging of the population, the number of AD patients in China has reached tens of millions and is increasing year by year. The number of AD patients worldwide has also exceeded 50 million. Therefore, it has become an urgent task to carry out intervention and treatment of AD, reduce the health burden brought by AD, and improve the physical and mental health of the public.

[0006] Aβ antibody therapy is based on early intervention of AD, but its clinical application faces many challenges, especially the difficulty of early diagnosis of AD. Current AD diagnostic methods include cognitive function scale tests and pathological feature analysis based on biological specimens. The diagnostic framework covers features such as Aβ deposition, neurofibrillary tangles formed by phosphorylated tau protein, and neurodegenerative damage. Due to the high invasiveness of lumbar puncture, high requirements for PET equipment and high cost, these methods are difficult to be widely used in clinical practice, especially in the early diagnosis stage.

[0007] The latest research shows that AD-related biomarkers, such as Aβ40 or Aβ42, can be detected in the blood through highly sensitive detection technology. However, the current Aβ40 antibody is not sensitive enough, and the batch-to-batch differences in the preparation of monoclonal antibodies will directly affect the batch-to-batch reproducibility of the test kit. Since the molecular weight of amyloid-β in the blood is small and its content is extremely rare, the above problems will lead to reduced sensitivity and accuracy of the test kit. Therefore, the development of an anti-amyloid-β antibody and its application are urgent. Summary of the invention

[0008] In response to the above technical problems, the present invention has developed a rabbit monoclonal antibody that can highly specifically recognize Aβ40 without binding to Aβ42 or other related peptides. Compared with mouse or rat antibodies, rabbit monoclonal antibodies are easier to humanize due to their greater genetic diversity, simple immunoglobulin structure and high affinity. In addition, the present invention has also developed a corresponding detection kit based on MSD technology.

[0009] To this end, the present invention provides a rabbit-derived Aβ1-40 monoclonal antibody, wherein

[0010] (a) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0011] (i) GFSLNNYY (as shown in SEQ ID NO: 25),

[0012] (ii) IYGNGGT (as shown in SEQ ID NO: 26)

[0013] (iii) ARGGL (as shown in SEQ ID NO: 27), and

[0014] (b) The light chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0015] (i) QSVYNKNN (as shown in SEQ ID NO: 28),

[0016] (ii) EAS (as shown in SEQ ID NO: 29),

[0017] (iii) QGGYDCSRADCNV (as shown in SEQ ID NO: 30); or

[0018] (c) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0019] (i) GIDFNSNV (as shown in SEQ ID NO: 31),

[0020] (ii) IFSTGIA (as shown in SEQ ID NO: 32)

[0021] (iii) ARSGI (as shown in SEQ ID NO: 33), and

[0022] (d) the light chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0023] (i) QSVYKSNY (as shown in SEQ ID NO: 34),

[0024] (ii) DAS (as shown in SEQ ID NO: 35),

[0025] (iii) QGGYDCSAADCNV (as shown in SEQ ID NO: 36); or

[0026] (e) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0027] (i) GIGFNSDT (as shown in SEQ ID NO: 37),

[0028] (ii) IITSGSA (as shown in SEQ ID NO: 38)

[0029] (iii) ARSGI (as shown in SEQ ID NO: 39), and

[0030] (f) The light chain variable region of the monoclonal antibody comprises the following CDR sequence:

[0031] (i) QSVYNNNR (as shown in SEQ ID NO: 40),

[0032] (ii) YTS (as shown in SEQ ID NO:41),

[0033] (iii) QGGYDCAIADCNV (as shown in SEQ ID NO: 42).

[0034] In a preferred embodiment of the present invention, the monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 1-3, and a light chain variable region sequence shown in any one of SEQ ID NOs: 4-6.

[0035] In a further preferred embodiment of the present invention, the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:1 and the light chain variable region sequence shown in SEQ ID NO:4; or the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:2 and the light chain variable region sequence shown in SEQ ID NO:5; or the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:3 and the light chain variable region sequence shown in SEQ ID NO:6.

[0036] In a preferred embodiment of the present invention, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

[0037] In a further preferred embodiment of the present invention, the heavy chain constant region comprises a sequence shown in any one of SEQ ID NOs: 7-9, and the light chain constant region comprises a sequence shown in any one of SEQ ID NOs: 10-12.

[0038] In a more preferred embodiment of the present invention, the monoclonal antibody comprises the heavy chain constant region sequence shown in SEQ ID NO:7 and the light chain constant region sequence shown in SEQ ID NO:10; or comprises the heavy chain constant region sequence shown in SEQ ID NO:8 and the light chain constant region sequence shown in SEQ ID NO:11; or comprises the heavy chain constant region sequence shown in SEQ ID NO:9 and the light chain constant region sequence shown in SEQ ID NO:12.

[0039] In a preferred embodiment of the present invention, the monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 1-3 and a heavy chain constant region sequence shown in any one of SEQ ID NOs: 7-9, or comprises a light chain variable region sequence shown in any one of SEQ ID NOs: 4-6 and a light chain constant region sequence shown in any one of SEQ ID NOs: 10-12.

[0040] In a further preferred embodiment of the present invention, the monoclonal antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:1 and a heavy chain constant region sequence as shown in SEQ ID NO:7, and a light chain variable region sequence as shown in SEQ ID NO:4 and a light chain constant region sequence as shown in SEQ ID NO:10; or comprises a heavy chain variable region sequence as shown in SEQ ID NO:2 and a heavy chain constant region sequence as shown in SEQ ID NO:8, and a light chain variable region sequence as shown in SEQ ID NO:5 and a light chain constant region sequence as shown in SEQ ID NO:11; or comprises a heavy chain variable region sequence as shown in SEQ ID NO:3 and a heavy chain constant region sequence as shown in SEQ ID NO:9, and a light chain variable region sequence as shown in SEQ ID NO:6 and a light chain constant region sequence as shown in SEQ ID NO:12.

[0041] In a preferred embodiment of the present invention, the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NOs: 43-45 and the light chain sequence shown in SEQ ID NOs: 46-48.

[0042] In a further preferred embodiment of the present invention, the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:43 and the light chain sequence shown in SEQ ID NO:46, or comprises the heavy chain sequence shown in SEQ ID NO:44 and the light chain sequence shown in SEQ ID NO:47, or comprises the heavy chain sequence shown in SEQ ID NO:45 and the light chain sequence shown in SEQ ID NO:48.

[0043] In a more preferred embodiment of the present invention, the heavy chain sequence of the monoclonal antibody is as shown in SEQ ID NO:43, and the light chain sequence is as shown in SEQ ID NO:46; or the heavy chain sequence is as shown in SEQ ID NO:44, and the light chain sequence is as shown in SEQ ID NO:47; or the heavy chain sequence is as shown in SEQ ID NO:45, and the light chain sequence is as shown in SEQ ID NO:48.

[0044] Another aspect of the present invention provides a nucleotide sequence encoding the monoclonal antibody of the present invention.

[0045] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 25-27 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 28-30; or comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 31-33 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 34-36; comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 37-39 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 40-42.

[0046] In a further preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 49-51 and a nucleotide sequence as shown in any one of SEQ ID NOs: 52-54; or comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 55-57 and a nucleotide sequence as shown in any one of SEQ ID NOs: 58-60; or comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 61-63 and a nucleotide sequence as shown in any one of SEQ ID NOs: 64-66.

[0047] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain variable region shown in any one of SEQ ID NOs: 1-3 and a nucleotide sequence encoding a light chain variable region shown in any one of SEQ ID NOs: 4-6.

[0048] In a further preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 13-15 and comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16-18.

[0049] In a more preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:13 and the nucleotide sequence shown in SEQ ID NO:16, or comprises the nucleotide sequence shown in SEQ ID NO:14 and the nucleotide sequence shown in SEQ ID NO:17, or comprises the nucleotide sequence shown in SEQ ID NO:15 and the nucleotide sequence shown in SEQ ID NO:18.

[0050] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain constant region as shown in any one of SEQ ID NOs: 7-9 and a nucleotide sequence encoding a light chain constant region as shown in any one of SEQ ID NOs: 10-12.

[0051] In a further preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 19-21 and comprises the nucleotide sequence shown in any one of SEQ ID NOs: 22-24.

[0052] In a more preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:19 and the nucleotide sequence shown in SEQ ID NO:22, or comprises the nucleotide sequence shown in SEQ ID NO:20 and the nucleotide sequence shown in SEQ ID NO:23, or comprises the nucleotide sequence shown in SEQ ID NO:21 and the nucleotide sequence shown in SEQ ID NO:24.

[0053] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain shown in any one of SEQ ID NOs: 43-45 and a nucleotide sequence encoding a light chain shown in any one of SEQ ID NOs: 46-48.

[0054] In a further preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 67-69 and comprises the nucleotide sequence shown in any one of SEQ ID NOs: 70-72.

[0055] In a more preferred embodiment of the present invention, the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO:67 and the nucleotide sequence shown in SEQ ID NO:70, or comprises the nucleotide sequence shown in SEQ ID NO:68 and the nucleotide sequence shown in SEQ ID NO:71, or comprises the nucleotide sequence shown in SEQ ID NO:69 and the nucleotide sequence shown in SEQ ID NO:72.

[0056] In the most preferred embodiment of the present invention, the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:67, and the nucleotide sequence encoding the light chain sequence is as shown in SEQ ID NO:70, or the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:68, and the nucleotide sequence encoding the light chain sequence is as shown in SEQ ID NO:71, or the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:69, and the nucleotide sequence encoding the light chain sequence is as shown in SEQ ID NO:72.

[0057] Another aspect of the present invention provides an expression vector comprising the nucleotide sequence of the present invention.

[0058] Another aspect of the present invention provides a host cell comprising the nucleotide sequence of the present invention or the expression vector of the present invention.

[0059] Another aspect of the present invention provides a pharmaceutical composition, a detection reagent or a kit comprising the monoclonal antibody of the present invention, the nucleotide sequence of the present invention, the expression vector of the present invention, or the host cell of the present invention.

[0060] In a preferred embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0061] Another aspect of the present invention provides a method for preparing the monoclonal antibody of the present invention, comprising culturing the host cell of the present invention.

[0062] In a preferred embodiment of the present invention, the method further comprises the step of purifying the Aβ1-40 antibody.

[0063] In another aspect, the present invention provides use of the monoclonal antibody, the nucleotide sequence, the expression vector or the host cell of the present invention in the preparation of a pharmaceutical composition, a detection reagent or a kit.

[0064] In a preferred embodiment of the present invention, the pharmaceutical composition, detection reagent or kit is used to treat or prevent neurodegenerative diseases, diagnose or assist in the diagnosis of neurodegenerative diseases, monitor Aβ40 levels, differentially diagnose neurodegenerative diseases and other forms of diseases, or evaluate the therapeutic effect and disease progression of neurodegenerative diseases.

[0065] In a further preferred embodiment of the present invention, the neurodegenerative disease is an early-diagnosed neurodegenerative disease, other forms of the disease include Lewy body dementia and frontotemporal lobar degeneration, and the neurodegenerative disease includes Alzheimer's disease.

[0066] In a preferred embodiment of the invention, the monoclonal antibody is used in combination with another pharmaceutical agent or reagent.

[0067] Beneficial Effects

[0068] The present invention develops antibodies that recognize Aβ40 using rabbit monoclonal antibodies. Compared with mice or rats, rabbits have greater genetic diversity, so rabbit monoclonal antibodies have a wider antibody spectrum, a simpler immunoglobulin structure, a higher affinity, and are easier to humanize.

[0069] In addition, the Aβ1-40 monoclonal antibody prepared by the present invention can bind to the Aβ40 polypeptide with high specificity, but not to Aβ42 or other related peptides, and thereby develop a more specific related body fluid marker detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the immunofluorescence image of the experimental rabbit serum.

[0071] Used to test the antibody binding ability in the five-rabbit rabbit serum, where the primary antibody: β-Amyloid (1-40) (D8Q71) Rabbit mAb #12990 1:200 as a positive control; the experimental group is E24870-5 five-rabbit serum 1:10; secondary antibody: Anti-Rabbit lgG, HRP-linked Antibody CST 1:3000.

[0072] Binding to Aβ plaques in the brain of 8-month-old 5×FAD mice was observed, but its specificity for different Aβ fragments could not be demonstrated.

[0073] Figure 2 Western blot for antibody testing.

[0074] Aβ40 / Aβ42 standards were used for WB experiments, and Anti-β-Amyioid, 1-40 Antibody (1-40) was used as a positive control group.

[0075] It can be seen that all three antibodies have significant Aβ40 / Aβ42 specificity.

[0076] Figure 3 Dot blot image for Aβ1-40 assay.

[0077] The various Aβ polypeptides listed on the left were coated on the NC membrane, and Anti-β-Amyoid, 1-16Antibody (6E10), Anti-beta Amyloid 1-40Antibody (1-40), Anti-betaAmyloid 1-42Antibody (1-42) were used as positive controls to test the antibodies 6F3, 10E6, and 11D12 of the present invention.

[0078] It was found that all three antibodies had excellent Aβ40 specificity.

[0079] Figure 4 This is a routine ELISA test chart.

[0080] The sera from four or five immune systems of the three rabbits E24868, E24869, and E24870 all had titers against the positive detection source (biotin-GLMVGGVV); the sera from four or five immune systems of the three rabbits E24868, E24869, and E24870 had no titers and did not bind to the negative detection source (biotin-GAIIGLMVG+biotin-GGVVIA).

[0081] Figure 5 This is a diagram of cell sorting results.

[0082] Twenty-six positive clones were selected and recognized the original positive detection polypeptide, of which 13 did not cross-recognize the original negative screening mixed polypeptide, namely 4E10, 5G3, 6F3, 6G2, 6G3, 8D8, 9C2, 10E6, 11C2, 11C8, 11C12, 11D12, and 11F4.

[0083] Figure 6 This is the LEM stage detection diagram.

[0084] The nine clones that specifically recognized WG-01914M-2 and did not cross-recognize WG-01914M-4 and WG-01915M-2 were 4E10, 6F3, 6G3, 8D8, 10E6, 11C2, 11C8, 11D12, and 11F4; one clone had lost its function, specifically 9C2.

[0085] Figure 7 Homologous recombination antibody detection diagram.

[0086] The three antibodies 6F3, 10E6, and 11D12 were able to recognize the positive detection antigen but not the negative detection antigen. DETAILED DESCRIPTION

[0087] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0088] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0089] Example 1: Preparation of Aβ1-40 monoclonal antibody

[0090] 1.1 Peptide design and animal immunization

[0091] A. Design immunogen peptide sequence information based on target Aβ40, as follows:

[0092] (1) Immunogen, CGLMVGGVV, coupled with KLH, 9 mg, purity >90%;

[0093] (2) Screening source, biotin-GLMVGGVV, 3 mg, purity >95%;

[0094] (3) Additional negative test substance, biotin-GLMVGGVVIA, 3 mg, purity >95%.

[0095] B. The specific reagents used are shown in Table 1.

[0096] Table 1 Related reagents used in the present invention

[0097]

[0098]

[0099] C. The immunization process is as follows:

[0100] (1) Before immunization, negative sera from 6 rabbits were taken for background screening, and 3 rabbits were selected for immunization;

[0101] (2) Using the immunogen CGLMVGGVV, coupled with KLH, immunize three selected rabbits;

[0102] (3) On day 0, negative serum was collected, 4 ml / rabbit, and the first immunization was performed: multiple subcutaneous injections (SQ) of 400 μg antigen + Freund's complete adjuvant;

[0103] On day 7, the second immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0104] On day 21, the third immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0105] On day 42, the fourth immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0106] On day 49, 4 ml of serum after four immunizations was collected and tested;

[0107] On day 63, the fifth immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0108] On day 70, 4 ml of serum after five immunizations was collected and tested.

[0109] 1.2 Polyclonal antibody detection

[0110] A. The testing process is as follows:

[0111] (1) Pre-coating: Neutravidin, 2 μg / ml, pre-coating (384-well plate), 25 μl / well, 4°C overnight;

[0112] (2) ELISA screening: positive detection source, biotin-GLMVGGVV; negative detection source, biotin-GAIIGLMVG+biotin-GGVVIA; 1 μg / mL, 25 μl / well, incubation at room temperature for 1 h;

[0113] (3) Blocking solution: 50 μl / well, incubate at room temperature for 1 h;

[0114] (4) Primary antibody: serum 1:1000 as the starting concentration, 3-fold dilution, 8 gradients, 25 μl / well, incubation at room temperature for 1 h;

[0115] (5) Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L), incubate at room temperature for 1 h;

[0116] (6) Color development: TMB, 25 μl / well, room temperature in the dark for 3 min;

[0117] NC: Dilution Buffer.

[0118] B. The test results are as follows:

[0119] The sera from the four immune and five immune sera of the three rabbits E24868, E24869, and E24870 all had titers against the positive detection source (biotin-GLMVGGVV); the sera from the four immune and five immune sera of the three rabbits E24868, E24869, and E24870 had no titers and did not bind to the negative detection source (biotin-GAIIGLMVG+biotin-GGVVIA). The results are shown in the attached Figure 4 .

[0120] 1.3 Flow cytometry sorting of B cells

[0121] A. The sorting reagents are shown in Table 1.

[0122] B. The sorting process is as follows:

[0123] (1) Flow cytometry

[0124] Flow cytometry sorting was performed using biotin-GLMVGGVV, and antigen-specific single B cells were collected in 8×96 plates.

[0125] (2) First round of testing

[0126] The first round of testing was performed on the collected 8 × 96 plates;

[0127] Routine ELISA testing was performed on the positive detection source biotin-GLMVGGVV.

[0128] (3) Collecting positive clones

[0129] According to the ELISA OD values ​​of the first round of testing, 134 positive clones were selected for routine ELISA testing;

[0130] For negative screening, a mixed peptide biotin-GAIIGLMVG+biotin-GGVVIA was used.

[0131] C. Sorting results and test results are as follows:

[0132] (1) Testing process

[0133] Neutravidin: 2 μg / ml, pre-coated (384-well plate), 25 μl / well, overnight at 4°C;

[0134] ELISA screening agent: positive detection agent, biotin-GLMVGGVV; negative detection agent, biotin-GAIIGLMVG+biotin-GGVVIA; 1 μg / mL, 25 μl / well, coating (384-well plate), overnight at 4°C;

[0135] Blocking solution: 50 μl / well, incubate at room temperature for 1 h;

[0136] B cell supernatant: 10 μL of B cell supernatant + 15 μL of diluent, 25 μl / well, incubate at room temperature for 1 h;

[0137] Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L), incubate at room temperature for 1 h;

[0138] Color development: TMB, 25 μl / well, color development at room temperature in the dark for 3 min;

[0139] PC: Positive Control, corresponding to the positive serum of rabbit number E24870-5;

[0140] NC: Negetive Control, cell culture medium.

[0141] (2) Test results

[0142] There are 26 positive clones that recognize the original peptide (see the attached test results). Figure 5 ), among which 13 strains did not cross-recognize the original mixed peptides of negative screening, namely 4E10, 5G3, 6F3, 6G2, 6G3, 8D8, 9C2, 10E6, 11C2, 11C8, 11C12, 11D12, and 11F4.

[0143] 1.4 Positive clone expansion and expression

[0144] A. Molecular cloning-construction of LEM expression module

[0145] Based on the experimental data obtained in the cell screening stage, 10 positive clones were selected to construct the linear expression module LEM, with a maximum of 1 ml of cell supernatant / clone (usually containing > 5 μg / ml recombinant antibody). The 10 selected positive clones were tested again.

[0146] ELISA routine test: for positive detection of original biotin-GLMVGGVV;

[0147] ELISA routine test: for negative screening, mixed peptides: biotin-GAIIGLMVG + biotin-GGVVIA;

[0148] B. ELISA test of culture supernatant

[0149] The 9 clones that specifically recognized WG-01914M-2 and did not cross-recognize WG-01914M-4 and WG-01915M-2 were 4E10, 6F3, 6G3, 8D8, 10E6, 11C2, 11C8, 11D12, and 11F4; 1 clone lost its function, specifically 9C2 (see Appendix for details). Figure 6 ).

[0150] 1.5 Recombinant Antibody Expression

[0151] A. The process of recombinant antibody expression is as follows:

[0152] Three positive clones were selected, recombinant expression vectors were constructed, antibodies were expressed and purified in small quantities, and protein A affinity purification was performed to obtain recombinant antibodies of >100μg / clone.

[0153] The function of the purified antibodies was verified for the 3 selected positive clones.

[0154] ELISA routine test: for the positive detection source, biotin-GLMVGGVV;

[0155] ELISA routine test: for negative screening, mixed peptides: biotin-GAIIGLMVG + biotin-GGVVIA;

[0156] B. Homologous recombination antibody detection

[0157] The three antibodies that can recognize the positive detection source are 6F3, 10E6, and 11D12 (see the attached test results). Figure 7 ).

[0158] Example 2: Identification of Aβ1-40 monoclonal antibodies

[0159] 2.1 Immunofluorescence identification

[0160] A. Experimental Methods

[0161] (1) Tissue preparation: Fresh mouse brain tissue was taken out from -80℃, placed in a 5 ml centrifuge tube, and immersed in 4% PFA at 4℃ for 24 h. The tissue was taken out from PFA, washed once with PBS, and immersed in 15% sucrose at 4℃ in a 5 ml centrifuge tube until it sank to the bottom for about 24 h. The tissue was taken out from 15% sucrose, and immersed in 30% sucrose at 4℃ in a 5 ml centrifuge tube until it sank to the bottom for about 24 h.

[0162] (2) Cryosectioning: microtome OT-20℃, CT-25℃ precooling, coronal sectioning, 30um;

[0163] (3) Histochemical circle drawing, wash with PBS 3 times × 5 min;

[0164] (4) Permeabilization: 0.5% Triton X-100 / 1xPBS RT 15 min;

[0165] (5) Blocking: 5% NGS / 0.1% TritonX-100 / 1xPBSRT 60 min;

[0166] (6) Antibody incubation: Incubate the membrane with appropriately diluted primary antibodies at 4°C overnight; incubate the membrane with fluorescent secondary antibody dilution in blocking buffer for 1.5 h at room temperature; PBS 3×5 min;

[0167] (7) Seal film photography.

[0168] B. Experimental Results

[0169] At the beginning of the experiment, the sera of the immunized rabbits were tested by immunofluorescence test. The results are shown in the attached Figure 1 As shown in Figure 2, only the five-immune serum of E24870 rabbit was observed to recognize Aβ plaques. Therefore, it was decided to select this rabbit for pulse immunization and then remove the spleen to sort B cells for subsequent experiments.

[0170] 2.2 Immune protein precipitation identification

[0171] A. Experimental Methods

[0172] (1) Sample preparation: Dissolve the standard in 1% NH4OH and dilute to 4 μg / ml in PBS;

[0173] (2) Loading: 40 ng of purified protein was loaded into the wells of a 16% SDS-PAGE gel;

[0174] (3) Electrophoresis: 90V for 30min, 120V off;

[0175] (4) Transfer: 0.22um PVDF membrane, 200mA, transfer for 30min;

[0176] (5) Blocking: Block the membrane with blocking buffer at room temperature for 1 hour or at 4°C overnight;

[0177] (6) Antibody incubation: Incubate the membrane with appropriately diluted primary antibodies at 4°C overnight; incubate the membrane with conjugated secondary antibody dilution in blocking buffer for 1 hour at room temperature;

[0178] (7) Color development: Add equal volumes (500uL each) of peroxide and enhancer onto the surface of the PVDF membrane until the membrane is completely covered with liquid and then develop in a developer.

[0179] B. Experimental Results

[0180] For the E24870 rabbit B cell supernatant and its purified antibodies, immunoprotein precipitation tests were performed, as shown in the attached Figure 2 Standard samples were selected as samples to identify the specificity of the three clones 6F3, 10E6, and 11D12, and it was found that all three could effectively distinguish Aβ40 from Aβ42.

[0181] 2.3 Dot blot assay

[0182] A. Experimental Methods

[0183] (1) Membrane preparation: DotBlot detection uses NC membrane, which is cut into 6.5 cm wide strips;

[0184] (2) Sample preparation: Dissolve the standard in 1% NH4OH and dilute to 4 μg / ml in PBS;

[0185] (3) Peptide coating: Remove the blue protective paper on the membrane, use a pipette to absorb 2ul of peptide dilution and spot it in the center of a 1cm×1cm square grid on the membrane; after spotting, place the NC membrane in a 37℃ oven for 30min;

[0186] (4) Blocking: Block the membrane with blocking buffer at room temperature for 1 hour;

[0187] (5) Antibody incubation: Incubate the membrane with appropriately diluted primary antibodies at 4°C overnight; incubate the membrane with conjugated secondary antibody dilution in blocking buffer for 1 hour at room temperature;

[0188] (6) Color development: Add equal volumes (500uL each) of peroxide and enhancer onto the surface of the PVDF membrane until the membrane is completely covered with liquid and then develop in a developer.

[0189] B. Experimental Results

[0190] To further demonstrate the specificity of the antibodies, dot blot tests were performed on the B cell supernatant of E24870 rabbit and its purified antibodies, as shown in the attached Figure 3 shown.

[0191] Six lengths of Aβ were used as samples, and three positive control antibodies, 6E10, 1-40, and 1-42, were used. In the experimental group, all three clones had significant specificity for Aβ40 and Aβ42.

[0192] All sequences used in the present invention are shown in Table 2.

[0193] Table 2 Sequences used in the present invention

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] The above is only a preferred embodiment of the present invention, and the present invention should not be limited to the contents disclosed in the embodiment and the drawings. Any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A rabbit-derived Aβ1-40 monoclonal antibody, characterized in that: (a) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) GFSLNNYY (as shown in SEQ ID NO: 25), (ii) IYGNGGT (as shown in SEQ ID NO: 26) (iii) ARGGL (as shown in SEQ ID NO: 27), and (b) The light chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) QSVYNKNN (as shown in SEQ ID NO: 28), (ii) EAS (as shown in SEQ ID NO: 29), (iii) QGGYDCSRADCNV (as shown in SEQ ID NO: 30); or (c) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) GIDFNSNV (as shown in SEQ ID NO: 31), (ii) IFSTGIA (as shown in SEQ ID NO: 32) (iii) ARSGI (as shown in SEQ ID NO: 33), and (d) the light chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) QSVYKSNY (as shown in SEQ ID NO: 34), (ii) DAS (as shown in SEQ ID NO: 35), (iii) QGGYDCSAADCNV (as shown in SEQ ID NO: 36); or (e) The heavy chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) GIGFNSDT (as shown in SEQ ID NO: 37), (ii) IITSGSA (as shown in SEQ ID NO: 38) (iii) ARSGI (as shown in SEQ ID NO: 39), and (f) The light chain variable region of the monoclonal antibody comprises the following CDR sequence: (i) QSVYNNNR (as shown in SEQ ID NO: 40), (ii) YTS (as shown in SEQ ID NO:41), (iii) QGGYDCAIADCNV (as shown in SEQ ID NO: 42).

2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 1-3, and a light chain variable region sequence shown in any one of SEQ ID NOs: 4-6; wherein the monoclonal antibody preferably comprises a heavy chain variable region sequence shown in SEQ ID NO: 1 and a light chain variable region sequence shown in SEQ ID NO: 4, preferably comprises a heavy chain variable region sequence shown in SEQ ID NO: 2 and a light chain variable region sequence shown in SEQ ID NO: 5, and preferably comprises a heavy chain variable region sequence shown in SEQ ID NO: 3 and a light chain variable region sequence shown in SEQ ID NO:

6.

3. The monoclonal antibody according to claim 1 or 2, further comprising a heavy chain constant region and a light chain constant region.

4. The monoclonal antibody according to any one of claims 1 to 3, wherein the heavy chain constant region comprises the sequence shown in any one of SEQ ID NOs: 7 to 9, and the light chain constant region comprises the sequence shown in any one of SEQ ID NOs: 10 to 12; wherein the monoclonal antibody preferably comprises the heavy chain constant region sequence shown in SEQ ID NO: 7 and the light chain constant region sequence shown in SEQ ID NO: 10, preferably comprises the heavy chain constant region sequence shown in SEQ ID NO: 8 and the light chain constant region sequence shown in SEQ ID NO: 11, and preferably comprises the heavy chain constant region sequence shown in SEQ ID NO: 9 and the light chain constant region sequence shown in SEQ ID NO:

12.

5. The monoclonal antibody according to any one of claims 1 to 4, wherein the monoclonal antibody comprises a heavy chain variable region sequence as shown in any one of SEQ ID NOs: 1 to 3 and a heavy chain constant region sequence as shown in any one of SEQ ID NOs: 7 to 9, and a light chain variable region sequence as shown in any one of SEQ ID NOs: 4 to 6 and a light chain constant region sequence as shown in any one of SEQ ID NOs: 10 to 12; preferably comprises a heavy chain variable region sequence as shown in SEQ ID NO: 1 and a heavy chain constant region sequence as shown in SEQ ID NO: 7, and comprises a light chain variable region sequence as shown in SEQ ID NO: 4 and a light chain constant region sequence as shown in SEQ ID NO: 10; preferably comprises a heavy chain variable region sequence as shown in SEQ ID NO: 2 and a heavy chain constant region sequence as shown in SEQ ID NO: 8, and comprises a light chain variable region sequence as shown in SEQ ID NO: 5 and a light chain constant region sequence as shown in SEQ ID NO: 11; preferably comprises a heavy chain variable region sequence as shown in SEQ ID NO: 3 and a heavy chain constant region sequence as shown in SEQ ID NO: 9, and comprises a light chain variable region sequence as shown in SEQ ID NO: 6 and a light chain constant region sequence as shown in SEQ ID NO:

12.

6. The monoclonal antibody according to any one of claims 1 to 5, wherein the monoclonal antibody comprises a heavy chain sequence as shown in SEQ ID NOs: 43-45 and a light chain sequence as shown in SEQ ID NOs: 46-48; preferably comprises a heavy chain sequence as shown in SEQ ID NO: 43 and a light chain sequence as shown in SEQ ID NO: 46, preferably comprises a heavy chain sequence as shown in SEQ ID NO: 44 and a light chain sequence as shown in SEQ ID NO: 47, preferably comprises a heavy chain sequence as shown in SEQ ID NO: 45 and a light chain sequence as shown in SEQ ID NO: 48; more preferably, the heavy chain sequence is as shown in SEQ ID NO: 43, and the light chain sequence is as shown in SEQ ID NO: 46, more preferably, the heavy chain sequence is as shown in SEQ ID NO: 44, and the light chain sequence is as shown in SEQ ID NO: 47, more preferably, the heavy chain sequence is as shown in SEQ ID NO: 45, and the light chain sequence is as shown in SEQ ID NO:

48.

7. A nucleotide sequence encoding the monoclonal antibody according to any one of claims 1 to 6.

8. The nucleotide sequence according to claim 7, which comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 25-27 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 28-30, or comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 31-33 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 34-36, or comprises a nucleotide sequence encoding a heavy chain variable region CDR sequence as shown in any one of SEQ ID NOs: 37-39 and a nucleotide sequence encoding a light chain variable region CDR sequence as shown in any one of SEQ ID NOs: 40-42; preferably comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 49-51 and a nucleotide sequence as shown in any one of SEQ ID NOs: 52-54, or comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 55-57 and a nucleotide sequence as shown in any one of SEQ ID NOs: 58-60, or comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 61-63 and a nucleotide sequence as shown in any one of SEQ ID NOs: NO:64-66; more preferably comprising a nucleotide sequence encoding a heavy chain variable region as shown in any one of SEQ ID NOs:1-3 and a nucleotide sequence encoding a light chain variable region as shown in any one of SEQ ID NOs:4-6, preferably comprising a nucleotide sequence as shown in any one of SEQ ID NOs:13-15 and a nucleotide sequence as shown in any one of SEQ ID NOs:16-18, more preferably comprising a nucleotide sequence as shown in SEQ ID NO:13 and a nucleotide sequence as shown in SEQ ID NO:16, more preferably comprising a nucleotide sequence as shown in SEQ ID NO:14 and a nucleotide sequence as shown in SEQ ID NO:17, more preferably comprising a nucleotide sequence as shown in SEQ ID NO:15 and a nucleotide sequence as shown in SEQ ID NO:18;The sequence comprises a nucleotide sequence encoding a heavy chain constant region as shown in any one of SEQ ID NOs: 7-9 and a nucleotide sequence encoding a light chain constant region as shown in any one of SEQ ID NOs: 10-12, preferably comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 19-21 and comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 22-24, more preferably comprises a nucleotide sequence as shown in SEQ ID NO: 19 and comprises a nucleotide sequence as shown in SEQ ID NO: 22, more preferably comprises a nucleotide sequence as shown in SEQ ID NO: 20 and comprises a nucleotide sequence as shown in SEQ ID NO: 23, more preferably comprises a nucleotide sequence as shown in SEQ ID NO: 21 and comprises a nucleotide sequence as shown in SEQ ID NO: 24; the sequence comprises a nucleotide sequence encoding a heavy chain as shown in any one of SEQ ID NOs: 43-45 and a nucleotide sequence encoding a light chain as shown in any one of SEQ ID NOs: 46-48, preferably comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 67-69 and comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 70-72, more preferably comprises a nucleotide sequence as shown in SEQ ID NO: 67 and comprises a nucleotide sequence as shown in SEQ ID NO: NO:70, more preferably comprising the nucleotide sequence shown in SEQ ID NO:68 and the nucleotide sequence shown in SEQ ID NO:71, more preferably comprising the nucleotide sequence shown in SEQ ID NO:69 and the nucleotide sequence shown in SEQ ID NO:72; preferably the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:67 and the light chain sequence is as shown in SEQ ID NO:70, preferably the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:68 and the light chain sequence is as shown in SEQ ID NO:71, preferably the nucleotide sequence encoding the heavy chain is as shown in SEQ ID NO:69 and the light chain sequence is as shown in SEQ ID NO:

72. ; 9. An expression vector comprising the nucleotide sequence of claim 7 or 8.

10. A host cell comprising the nucleotide sequence of claim 7 or 8 or the expression vector of claim 9.

11. A pharmaceutical composition, a detection reagent or a kit comprising the monoclonal antibody according to any one of claims 1 to 6, the nucleotide sequence according to claim 7 or 8, the expression vector according to claim 9, or the host cell according to claim 10, wherein the pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier.

12. A method for preparing the monoclonal antibody according to any one of claims 1 to 6, comprising culturing the host cell according to claim 10, wherein the method preferably comprises a step of purifying the Aβ1-40 antibody.

13. Use of the monoclonal antibody according to any one of claims 1 to 6, the nucleotide sequence according to claim 7 or 8, the expression vector according to claim 9, or the host cell according to claim 10 in the preparation of a pharmaceutical composition, a detection reagent or a kit, wherein the pharmaceutical composition, the detection reagent or the kit is preferably used to treat or prevent neurodegenerative diseases, diagnose or assist in the diagnosis of neurodegenerative diseases, monitor Aβ40 levels, differentially diagnose neurodegenerative diseases and other forms of diseases, or evaluate the therapeutic effect and disease progression of neurodegenerative diseases; wherein neurodegenerative diseases are preferably early diagnosis of neurodegenerative diseases, other forms of diseases are preferably Lewy body dementia and frontotemporal lobar degeneration, and neurodegenerative diseases are more preferably Alzheimer's disease.

14. The use according to claim 13, wherein the monoclonal antibody is used in combination with another drug or agent.

Images