A rabbit-derived a beta 1-40 monoclonal antibody, a preparation method and application thereof

Abstract

The application belongs to the field of bioengineering and biological detection, and discloses a rabbit-derived A beta 1-40 monoclonal antibody, a pharmaceutical composition, a detection reagent or a kit containing the monoclonal antibody, and application of the monoclonal antibody in preparation of a pharmaceutical composition, a detection reagent or a kit.The rabbit-derived monoclonal antibody prepared in the application can specifically recognize human A beta 40 with high efficiency.Compared with mice or rats, the rabbit-derived monoclonal antibody in the application has a simpler structure, higher affinity and is easier to be humanized.

Description

Technical Field

[0001] This invention relates to a rabbit-derived Aβ1-40 monoclonal antibody, its preparation method, and its application, belonging to the fields of bioengineering and biodetection. Background Technology

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

[0003] β-amyloid (Aβ) is a hydrophobic polypeptide encoded by a gene on human chromosome 21. It has a molecular weight of approximately 4 kDa, contains 39 to 43 amino acids, and has a β-sheet structure. Aβ is produced from amyloid precursor protein (APP), which is hydrolyzed by β and γ secretases. These secretases cleave APP to produce Aβ polypeptides of varying lengths, the most common being Aβ40 and Aβ42.

[0004] Under pathological conditions, APP is first hydrolyzed by β-secretase 1 (BACE1) to generate sAPPβ and a 99-amino acid-long C99 peptide linking the cell membrane. Subsequently, the C99 peptide is acted upon by γ-secretase, ultimately producing Aβ. These Aβ polypeptides circulate in the blood, cerebrospinal fluid, and brain interstitium, existing in a bound or free state with chaperone proteins. The Aβ cascade hypothesis posits that an imbalance between the overproduction and clearance of Aβ is one of the key causes of AD. Aβ accumulation leads to synaptic dysfunction, reduced neuronal plasticity, altered cellular energy metabolism, oxidative stress, and mitochondrial dysfunction, thereby disrupting intracellular calcium balance. In particular, the formation, aggregation, and deposition of Aβ42 can induce significant neurotoxicity; its oligomers can directly affect long-term enhanced responses and synaptic function in the hippocampus, leading to neurotransmitter imbalances and neurological dysfunction.

[0005] Aβ antibody therapy is based on early intervention in Alzheimer's disease (AD), but its clinical application faces many challenges, especially the difficulty in early diagnosis. Current AD diagnostic methods include cognitive function scales 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 highly invasive nature of lumbar puncture, the high requirements and cost of PET equipment, these methods are difficult to widely apply clinically, especially in the early diagnostic stage.

[0006] Recent studies have shown that highly sensitive detection technologies can detect AD-related biomarkers, such as Aβ40 or Aβ42, in blood. However, current Aβ40 antibodies have insufficient sensitivity, and batch-to-batch variations in monoclonal antibody preparation directly affect the batch-to-batch reproducibility of test kits. Because β-amyloid protein in blood has a small molecular weight and is extremely rare, these issues lead to reduced sensitivity and accuracy of detection kits. Therefore, the development and application of an anti-β-amyloid protein antibody is urgently needed. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention has developed a rabbit-derived monoclonal antibody that highly specifically recognizes Aβ40 without binding to Aβ42 or other related peptides. Compared to mouse or rat antibodies, rabbit-derived monoclonal antibodies are easier to humanize due to their greater genetic diversity, simple immunoglobulin structure, and high affinity. Furthermore, this invention has also developed a corresponding detection kit based on MSD technology.

[0008] Therefore, the present invention provides a rabbit-derived Aβ1-40 monoclonal antibody, wherein...

[0009] (a) The heavy chain variable region of the monoclonal antibody contains the following CDR sequence:

[0010] (i) GFSLNNYY (as shown in SEQ ID NO:25),

[0011] (ii) IYGNGGT (as shown in SEQ ID NO: 26)

[0012] (iii) ARGGL (as shown in SEQ ID NO: 27), and

[0013] (b) The light chain variable region of the monoclonal antibody contains the following CDR sequence:

[0014] (i) QSVYNKNN (as shown in SEQ ID NO: 28),

[0015] (ii) EAS (as shown in SEQ ID NO: 29),

[0016] (iii) QGGYDCSRADCNV (as shown in SEQ ID NO: 30); or

[0017] (c) The heavy chain variable region of the monoclonal antibody contains the following CDR sequence:

[0018] (i) GIDFNSNV (as shown in SEQ ID NO:31),

[0019] (ii) IFSTGIA (as shown in SEQ ID NO: 32)

[0020] (iii) ARSGI (as shown in SEQ ID NO: 33), and

[0021] (d) The light chain variable region of the monoclonal antibody contains the following CDR sequence:

[0022] (i) QSVYKSNY (as shown in SEQ ID NO: 34),

[0023] (ii) DAS (as shown in SEQ ID NO: 35),

[0024] (iii) QGGYDCSAADCNV (as shown in SEQ ID NO: 36); or

[0025] (e) The heavy chain variable region of the monoclonal antibody contains the following CDR sequence:

[0026] (i) GIGFNSDT (as shown in SEQ ID NO:37),

[0027] (ii) IITSGSA (as shown in SEQ ID NO: 38)

[0028] (iii) ARSGI (as shown in SEQ ID NO: 39), and

[0029] (f) The light chain variable region of the monoclonal antibody contains the following CDR sequence:

[0030] (i) QSVYNNNR (as shown in SEQ ID NO: 40),

[0031] (ii) YTS (as shown in SEQ ID NO: 41),

[0032] (iii) QGGYDCAIADCNV (as shown in SEQ ID NO: 42).

[0033] In a preferred embodiment of the present invention, the monoclonal antibody comprises any of the heavy chain variable region sequences shown in SEQ ID NO:1-3 and any of the light chain variable region sequences shown in SEQ ID NO:4-6.

[0034] 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.

[0035] In a preferred embodiment of the present invention, the monoclonal antibody further includes a heavy chain constant region and a light chain constant region.

[0036] In a further preferred embodiment of the present invention, the heavy chain constant region comprises any of the sequences shown in SEQ ID NO:7-9, and the light chain constant region comprises any of the sequences shown in SEQ ID NO:10-12.

[0037] 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.

[0038] In a preferred embodiment of the present invention, the monoclonal antibody comprises any of the heavy chain variable region sequences shown in SEQ ID NO:1-3 and any of the heavy chain constant region sequences shown in SEQ ID NO:7-9, or comprises any of the light chain variable region sequences shown in SEQ ID NO:4-6 and any of the light chain constant region sequences shown in SEQ ID NO:10-12.

[0039] 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 heavy chain constant region sequence shown in SEQ ID NO:7, and comprises the light chain variable region sequence shown in SEQ ID NO:4 and the light chain constant region sequence shown in SEQ ID NO:10; or comprises the heavy chain variable region sequence shown in SEQ ID NO:2 and the heavy chain constant region sequence shown in SEQ ID NO:8, and comprises the light chain variable region sequence shown in SEQ ID NO:5 and the light chain constant region sequence shown in SEQ ID NO:11; or comprises the heavy chain variable region sequence shown in SEQ ID NO:3 and the heavy chain constant region sequence shown in SEQ ID NO:9, and comprises the light chain variable region sequence shown in SEQ ID NO:6 and the light chain constant region sequence shown in SEQ ID NO:12.

[0040] In a preferred embodiment of the present invention, the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:43-45 and the light chain sequence shown in SEQ ID NO:46-48.

[0041] 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 the heavy chain sequence shown in SEQ ID NO:44 and the light chain sequence shown in SEQ ID NO:47, or the heavy chain sequence shown in SEQ ID NO:45 and the light chain sequence shown in SEQ ID NO:48.

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

[0043] In another aspect, the present invention provides a nucleotide sequence encoding the monoclonal antibody described herein.

[0044] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding the heavy chain variable region CDR sequence shown in any of SEQ ID NO:25-27 and a nucleotide sequence encoding the light chain variable region CDR sequence shown in any of SEQ ID NO:28-30; or comprises a nucleotide sequence encoding the heavy chain variable region CDR sequence shown in any of SEQ ID NO:31-33 and a nucleotide sequence encoding the light chain variable region CDR sequence shown in any of SEQ ID NO:34-36; or comprises a nucleotide sequence encoding the heavy chain variable region CDR sequence shown in any of SEQ ID NO:37-39 and a nucleotide sequence encoding the light chain variable region CDR sequence shown in any of SEQ ID NO:40-42.

[0045] In a further preferred embodiment of the present invention, the nucleotide sequence comprises any of the nucleotide sequences shown in SEQ ID NO:49-51 and any of the nucleotide sequences shown in SEQ ID NO:52-54; or comprises any of the nucleotide sequences shown in SEQ ID NO:55-57 and any of the nucleotide sequences shown in SEQ ID NO:58-60; or comprises any of the nucleotide sequences shown in SEQ ID NO:61-63 and any of the nucleotide sequences shown in SEQ ID NO:64-66.

[0046] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain variable region shown in any of SEQ ID NO:1-3 and a nucleotide sequence encoding a light chain variable region shown in any of SEQ ID NO:4-6.

[0047] In a further preferred embodiment of the present invention, the nucleotide sequence comprises any of the nucleotide sequences shown in SEQ ID NO:13-15 and any of the nucleotide sequences shown in SEQ ID NO:16-18.

[0048] 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 alternatively 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.

[0049] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain constant region shown in any of SEQ ID NO:7-9 and a nucleotide sequence encoding a light chain constant region shown in any of SEQ ID NO:10-12.

[0050] In a further preferred embodiment of the present invention, the nucleotide sequence comprises any of the nucleotide sequences shown in SEQ ID NO:19-21 and any of the nucleotide sequences shown in SEQ ID NO:22-24.

[0051] 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 the nucleotide sequence shown in SEQ ID NO:20 and the nucleotide sequence shown in SEQ ID NO:23, or the nucleotide sequence shown in SEQ ID NO:21 and the nucleotide sequence shown in SEQ ID NO:24.

[0052] In a preferred embodiment of the present invention, the nucleotide sequence comprises a nucleotide sequence encoding a heavy chain shown in any of SEQ ID NO:43-45 and a nucleotide sequence encoding a light chain shown in any of SEQ ID NO:46-48.

[0053] In a further preferred embodiment of the present invention, the nucleotide sequence comprises any of the nucleotide sequences shown in SEQ ID NO:67-69 and any of the nucleotide sequences shown in SEQ ID NO:70-72.

[0054] 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 the nucleotide sequence shown in SEQ ID NO:68 and the nucleotide sequence shown in SEQ ID NO:71, or the nucleotide sequence shown in SEQ ID NO:69 and the nucleotide sequence shown in SEQ ID NO:72.

[0055] In the most preferred embodiment of the present invention, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:67 and the nucleotide sequence encoding the light chain is shown in SEQ ID NO:70; or the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:68 and the nucleotide sequence encoding the light chain is shown in SEQ ID NO:71; or the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:69 and the nucleotide sequence encoding the light chain is shown in SEQ ID NO:72.

[0056] Another aspect of the present invention provides an expression vector comprising the nucleotide sequence described herein.

[0057] Another aspect of the present invention provides a host cell comprising the nucleotide sequence described in the present invention or the expression vector described in the present invention.

[0058] Another aspect of the present invention provides pharmaceutical compositions, detection reagents, or kits comprising the monoclonal antibodies, nucleotide sequences, expression vectors, or host cells described in the present invention.

[0059] In a preferred embodiment of the invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0060] Another aspect of the present invention provides a method for preparing the monoclonal antibody described herein, comprising culturing the host cell described herein.

[0061] In a preferred embodiment of the present invention, the method further includes a step of purifying the Aβ1-40 antibody.

[0062] In another aspect, the present invention provides the use of the monoclonal antibody, the nucleotide sequence, the expression vector, or the host cell described herein in the preparation of pharmaceutical compositions, detection reagents, or kits.

[0063] In a preferred embodiment of the present invention, the pharmaceutical composition, diagnostic reagent or kit is used to treat or prevent neurodegenerative diseases, diagnose or assist in the diagnosis of neurodegenerative diseases, monitor Aβ40 levels, differentiate neurodegenerative diseases from other forms of disease, or evaluate the treatment efficacy and disease progression of neurodegenerative diseases.

[0064] In a further preferred embodiment of the invention, the neurodegenerative disease is an early-diagnosed neurodegenerative disease, and other forms of the disease include Lewy body dementia and frontotemporal degeneration, while neurodegenerative diseases include Alzheimer's disease.

[0065] In a preferred embodiment of the present invention, the monoclonal antibody is used in combination with another drug or reagent.

[0066] Beneficial effects

[0067] This invention utilizes rabbit monoclonal antibodies to develop antibodies that recognize Aβ40. Compared to mice or rats, rabbits have greater genetic diversity, therefore rabbit monoclonal antibodies have a broader antibody spectrum, simpler immunoglobulin structures, higher affinity, and are easier to humanize.

[0068] Furthermore, the Aβ1-40 monoclonal antibody prepared in this invention can bind to the Aβ40 peptide with high specificity, without binding to Aβ42 or other related peptides, thereby enabling the development of more specific detection kits for related body fluid markers. Attached Figure Description

[0069] Figure 1 This is an immunofluorescence image of rabbit serum.

[0070] The antibody binding capacity in the serum of five-immune rabbits was tested. The primary antibody, β-Amyloid (1-40)(D8Q71) Rabbit mAb #12990 1:200, was used as a positive control. The experimental group was E24870-5 five-immune serum 1:10. The secondary antibody was Anti-Rabbit IgG, HRP-linked Antibody CST 1:3000.

[0071] Aβ plaques were observed to bind in the brains of 8-month-old 5×FAD mice, but their specificity for different Aβ fragments could not be demonstrated.

[0072] Figure 2 This is an immunoblot image of the antibody test protein.

[0073] Western blot experiments were performed using Aβ40 / Aβ42 standards, with Anti-β-Amyioid, 1-40 Antibody (1-40) used as a positive control.

[0074] It can be seen that all three antibodies have significant Aβ40 / Aβ42 specificity.

[0075] Figure 3 A speckled pattern for Aβ1-40 testing.

[0076] The antibodies 6F3, 10E6, and 11D12 of this invention were tested using a variety of Aβ peptides listed on the left to coat the NC membrane, with Anti-β-Amyoid 1-16 Antibody (6E10), Anti-beta Amyloid 1-40 Antibody (1-40), and Anti-beta Amyloid 1-42 Antibody (1-42) as positive controls.

[0077] All three antibodies were found to have excellent Aβ40 specificity.

[0078] Figure 4 This is a graph from a routine ELISA test.

[0079] The sera from rabbits E24868, E24869, and E24870, both from the fourth and fifth immunizations, showed titers against the positive detectant (biotin-GLMVGGVV); however, the sera from the same three rabbits showed no titers against the negative detectant (biotin-GAIIGLMVG+biotin-GGVVIA) and did not bind.

[0080] Figure 5 This is a diagram showing the cell sorting results.

[0081] Twenty-six positive clones were selected and identified as positive detection peptides. Among them, 13 clones did not cross-recognize the negative selection peptide mixture, namely 4E10, 5G3, 6F3, 6G2, 6G3, 8D8, 9C2, 10E6, 11C2, 11C8, 11C12, 11D12, and 11F4.

[0082] Figure 6 This is a detection diagram for the LEM stage.

[0083] Nine clones that specifically recognized WG-01914M-2 and did not cross-recognize WG-01914M-4 and WG-01915M-2 were identified as 4E10, 6F3, 6G3, 8D8, 10E6, 11C2, 11C8, 11D12, and 11F4; one clone, 9C2, lost its function.

[0084] Figure 7 Image of homologous recombinant antibody detection.

[0085] All three antibodies, 6F3, 10E6, and 11D12, were able to recognize positive detectants but not negative detectants. Detailed Implementation

[0086] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0087] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0088] Example 1: Preparation of Aβ1-40 monoclonal antibody

[0089] 1.1 Peptide Design and Animal Immunization

[0090] A. Immunogen polypeptide sequence information was designed based on the target Aβ40, as follows:

[0091] (1) Immunogen, CGLMVGGVV, conjugated with KLH and then immunized, 9 mg, purity >90%;

[0092] (2) Screening of the original, biotin-GLMVGGVV, 3mg, purity >95%;

[0093] (3) Additional negative detectant, biotin-GLMVGGVVIA, 3mg, purity >95%.

[0094] B. The specific reagents used are shown in Table 1.

[0095] Table 1. Reagents used in this invention

[0096]

[0097] C. The immunization process is as follows:

[0098] (1) Before immunization, negative serum from 6 rabbits was collected for background screening, and 3 rabbits were selected for immunization;

[0099] (2) Immunize three selected rabbits with the immunogen CGLMVGGVV conjugated with KLH.

[0100] (3) On day 0, negative serum was collected, 4 ml / rabbit. First immunization: 400 μg antigen + Freund's complete adjuvant was injected subcutaneously at multiple points.

[0101] Day 7, second immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0102] Day 21, third immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0103] Day 42, fourth immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0104] On day 49, 4 ml of serum was collected after the fourth immunization for testing;

[0105] Day 63, fifth immunization: multiple subcutaneous injections (SQ) of 200 μg antigen + Freund's incomplete adjuvant;

[0106] On day 70, 4 ml of serum was collected after the fifth immunization and tested.

[0107] 1.2 Polyclonal Antiserum Detection

[0108] A. The testing process is as follows:

[0109] (1) Pre-coating: Neutravidin, 2 μg / ml, pre-coated (384-well plate), 25 μl / well, overnight at 4°C;

[0110] (2) ELISA screening: positive detectant, biotin-GLMVGGVV; negative detectant, biotin-GAIIGLMVG+biotin-GGVVIA; 1 μg / mL, 25 μl / well, incubate at room temperature for 1 h;

[0111] (3) Blocking solution: 50 μl / well, incubate at room temperature for 1 h;

[0112] (4) Primary antibody: serum 1:1000 as the starting concentration, 3-fold dilution, 8 gradients, 25 μl / well, incubated at room temperature for 1 h;

[0113] (5) Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L), incubated at room temperature for 1 h;

[0114] (6) Color development: TMB, 25 μl / well, develop color at room temperature in the dark for 3 min;

[0115] NC: Diluent Buffer.

[0116] B. The test results are as follows:

[0117] The sera from rabbits E24868, E24869, and E24870, both from their fourth and fifth immunizations, all showed titers against the positive detectant (biotin-GLMVGGVV); the sera from rabbits E24868, E24869, and E24870, both from their fourth and fifth immunizations, showed no titers against the negative detectant (biotin-GAIIGLMVG+biotin-GGVVIA) and did not bind. Results are attached. Figure 4 .

[0118] 1.3 Flow Cytometry Sorting of B Cells

[0119] A. Sorting reagents are shown in Table 1.

[0120] B. The sorting process is as follows:

[0121] (1) Flow cytometry cell sorting

[0122] Flow cytometry was used to sort cells using biotin-GLMVGGVV, and antigen-specific single B cells were collected from 8×96 plates.

[0123] (2) First round of testing

[0124] The first round of testing was conducted on the collected 8×96 boards;

[0125] Routine ELISA testing was performed on the original biotin-GLMVGGVV.

[0126] (3) Collect positive clones

[0127] Based on the ELISA OD values ​​from the first round of testing, 134 positive clones were selected for routine ELISA testing.

[0128] For negative screening, a mixed peptide biotin-GAIIGLMVG+biotin-GGVVIA was used.

[0129] C. The sorting results and their detection are shown below:

[0130] (1) Testing process

[0131] Neutravidin: 2 μg / ml, pre-coated (384-well plate), 25 μl / well, overnight at 4°C;

[0132] ELISA screening agents: positive detectant, biotin-GLMVGGVV; negative detectant, biotin-GAIIGLMVG+biotin-GGVVIA; 1 μg / mL, 25 μl / well, coating (384-well plate), overnight at 4°C;

[0133] Blocking solution: 50 μl / well, incubate at room temperature for 1 h;

[0134] B cell supernatant: 10 μL of B cell supernatant + 15 μL of diluent, 25 μL / well, incubate at room temperature for 1 h;

[0135] Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L), incubated at room temperature for 1 hour;

[0136] Color development: TMB, 25 μl / well, develop at room temperature in the dark for 3 min;

[0137] PC: Positive Control, corresponding to positive serum of rabbit number E24870-5;

[0138] NC: Negetive Control, cell culture medium.

[0139] (2) Test results

[0140] 26 positive clones identified the original polypeptide being detected (test results are attached). Figure 5Among them, 13 non-cross-recognition negative selection precursor mixed peptides were identified as 4E10, 5G3, 6F3, 6G2, 6G3, 8D8, 9C2, 10E6, 11C2, 11C8, 11C12, 11D12, and 11F4.

[0141] 1.4 Amplification and Expression of Positive Clones

[0142] A. Construction of the Molecular Cloning-LEM Expression Module

[0143] Based on the experimental data obtained in the cell screening phase, 10 positive clones were selected, and a linear expression module (LEM) was constructed using up to 1 ml of cell supernatant per clone (typically containing >5 μg / ml of recombinant antibody). The 10 selected positive clones were then retested.

[0144] Routine ELISA testing: for positive detection of the original biotin-GLMVGGVV;

[0145] Routine ELISA assay: targeting negative selectors, mixed peptides: biotin-GAIIGLMVG + biotin-GGVVIA;

[0146] B. ELISA detection of culture supernatant

[0147] Nine clones specifically identified WG-01914M-2 and did not cross-recognize WG-01914M-4 and WG-01915M-2: 4E10, 6F3, 6G3, 8D8, 10E6, 11C2, 11C8, 11D12, and 11F4. One clone, 9C2, lost its function (see appendix for details). Figure 6 ).

[0148] 1.5 Recombinant Antibody Expression

[0149] A. The recombinant antibody expression process is as follows:

[0150] Three positive clones were selected, and recombinant expression vectors were constructed. Antibodies were expressed and purified in small quantities, and Protein A affinity purified to obtain recombinant antibodies with a concentration of >100 μg / clone.

[0151] The purified antibody was used to verify the function of the three selected positive clones.

[0152] Routine ELISA testing: targeting the positive detectant, biotin-GLMVGGVV;

[0153] Routine ELISA assay: targeting negative selectors, mixed peptides: biotin-GAIIGLMVG + biotin-GGVVIA;

[0154] B. Detection of homologous recombination antibodies

[0155] The three antibodies that were able to identify the positive detector were 6F3, 10E6, and 11D12 (test results are attached). Figure 7 ).

[0156] Example 2: Identification of Aβ1-40 monoclonal antibody

[0157] 2.1 Immunofluorescence identification

[0158] A. Experimental Methods

[0159] (1) Tissue preparation: Fresh mouse brain tissue was taken out at -80℃, placed in a 5ml centrifuge tube, soaked in 4% PFA at 4℃ for 24h, and allowed to stand. The tissue was then removed from the PFA, washed once with PBS, and placed in a 5ml centrifuge tube in 15% sucrose at 4℃ for about 24h to allow it to settle. The tissue was then removed from the 15% sucrose and placed in a 5ml centrifuge tube in 30% sucrose at 4℃ for about 24h to allow it to settle.

[0160] (2) Frozen sectioning: pre-cooled the microtome at OT-20℃ and CT-25℃, and cut coronal sections to 30µm;

[0161] (3) Draw circles with the tissue, wash with PBS 3 times × 5 min;

[0162] (4) Permeability: 0.5% Triton X-100 / 1xPBSRT for 15 min;

[0163] (5) Blocking: 5% NGS / 0.1% Triton X-100 / 1xPBSRT 60min;

[0164] (6) Antibody incubation: Incubate the membrane with an appropriately diluted primary antibody overnight at 4°C; incubate the membrane with a fluorescent secondary antibody dilution in blocking buffer for 1.5 hours at room temperature; PBS 3×5 min;

[0165] (7) Filming the cover.

[0166] B. Experimental Results

[0167] In the initial stage of the experiment, immunofluorescence assays were performed on the serum of the immunized rabbits, and the results are shown in the attached figure. Figure 1 As shown, recognition of Aβ plaques was observed only in the pentathioneous serum of E24870 rabbits. Therefore, it was decided to select this rabbit for pulse immunization, followed by spleen removal and B cell sorting for subsequent experiments.

[0168] 2.2 Identification of Immunoprotein Precipitation

[0169] A. Experimental Methods

[0170] (1) Sample preparation: Dissolve the standard in 1% NH4OH and dilute with PBS to 4 μg / ml;

[0171] (2) Sample loading: Load the sample into the wells of a 16% SDS-PAGE gel and purify the protein with 40 ng.

[0172] (3) Electrophoresis: 90V for 30min, then run at 120V.

[0173] (4) Transfer: 0.22um PVDF membrane, 200mA, transfer for 30min;

[0174] (5) Blocking: Block the membrane with blocking buffer for 1 hour at room temperature or overnight at 4°C;

[0175] (6) Antibody incubation: Incubate the membrane with an appropriately diluted primary antibody overnight at 4°C; incubate the membrane with a conjugated secondary antibody dilution in blocking buffer for 1 hour at room temperature;

[0176] (7) Color development: Add equal volumes (500uL each) of peroxide and reinforcing agent to the surface of PVDF film until the liquid covers the surface, and then develop in a developer.

[0177] B. Experimental Results

[0178] Immunoprotein precipitation assays were performed on B cell supernatants and their purified antibodies from E24870 rabbits, as shown in the attached figure. Figure 2 As shown. Standard samples were selected to identify the specificity of the three clones 6F3, 10E6, and 11D12. It was found that all three could effectively distinguish between Aβ40 and Aβ42.

[0179] 2.3 Identification by Spot Imprint Test

[0180] A. Experimental Methods

[0181] (1) Membrane preparation: NC membranes are used for DotBlot detection. Cut the NC membrane into strips 6.5cm wide.

[0182] (2) Sample preparation: Dissolve the standard in 1% NH4OH and dilute with PBS to 4 μg / ml;

[0183] (3) Peptide coating: Peel off the blue protective paper on the membrane, use a pipette to draw 2 μL of peptide dilution solution and place it in the center of a 1 cm × 1 cm square grid on the membrane; after spotting, place the NC membrane in a 37 °C oven for 30 min.

[0184] (4) Blocking: Block the membrane at room temperature for 1 hour using blocking buffer;

[0185] (5) Antibody incubation: Incubate the membrane with an appropriately diluted primary antibody at 4°C overnight; incubate the membrane with a conjugated secondary antibody dilution in blocking buffer for 1 hour at room temperature;

[0186] (6) Color development: Add equal volumes (500uL each) of peroxide and reinforcing agent to the surface of the PVDF film. Once the liquid covers the surface, develop the film in a developer.

[0187] B. Experimental Results

[0188] To further demonstrate the specificity of the antibody, a dot blot assay was performed on the B cell supernatant of E24870 rabbits and its purified antibody, as shown in the attached figure. Figure 3 As shown.

[0189] Six different 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 showed significant specificity for Aβ40 and Aβ42.

[0190] All sequences used in this invention are shown in Table 2.

[0191] Table 2. Sequences used in this invention

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] The above description is merely a preferred embodiment of the present invention, and the present invention should not be limited to the content disclosed in this embodiment and the accompanying drawings. Any equivalent or modified embodiments made without departing from the spirit of the present invention 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 CDR1-3 sequences shown in (i)-(iii) respectively: (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 CDR1-3 sequences shown in (i)-(iii) respectively: (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).

2. A rabbit-derived Aβ1-40 monoclonal antibody, characterized in that: (c) The heavy chain variable region of the monoclonal antibody comprises the CDR1-3 sequences shown in (i)-(iii) respectively: (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 CDR1-3 sequences shown in (i)-(iii) respectively: (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).

3. A rabbit-derived Aβ1-40 monoclonal antibody, characterized in that: (e) The heavy chain variable region of the monoclonal antibody comprises the CDR1-3 sequences shown in (i)-(iii) respectively: (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 CDR1-3 sequences shown in (i)-(iii) respectively: (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).

4. The monoclonal antibody according to claim 1, wherein 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.

5. The monoclonal antibody according to claim 2, wherein 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.

6. The monoclonal antibody according to claim 3, wherein 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.

7. The monoclonal antibody according to any one of claims 1-6, further comprising a heavy chain constant region and a light chain constant region.

8. The monoclonal antibody according to claim 1 or 4, wherein 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.

9. The monoclonal antibody according to claim 2 or 5, wherein the monoclonal antibody 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.

10. The monoclonal antibody according to claim 3 or 6, wherein the monoclonal antibody 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.

11. The monoclonal antibody according to claim 8, wherein the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:1 and the heavy chain constant region sequence shown in SEQ ID NO:7, and the light chain variable region sequence shown in SEQ ID NO:4 and the light chain constant region sequence shown in SEQ ID NO:

10.

12. The monoclonal antibody according to claim 9, wherein the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:2 and the heavy chain constant region sequence shown in SEQ ID NO:8, and the light chain variable region sequence shown in SEQ ID NO:5 and the light chain constant region sequence shown in SEQ ID NO:

11.

13. The monoclonal antibody according to claim 10, wherein the monoclonal antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:3 and the heavy chain constant region sequence shown in SEQ ID NO:9, and the light chain variable region sequence shown in SEQ ID NO:6 and the light chain constant region sequence shown in SEQ ID NO:

12.

14. The monoclonal antibody according to claim 11, wherein the heavy chain sequence of the monoclonal antibody is shown in SEQ ID NO:43 and the light chain sequence is shown in SEQ ID NO:

46.

15. The monoclonal antibody according to claim 12, wherein the heavy chain sequence of the monoclonal antibody is shown in SEQ ID NO:44 and the light chain sequence is shown in SEQ ID NO:

47.

16. The monoclonal antibody according to claim 13, wherein the heavy chain sequence of the monoclonal antibody is shown in SEQ ID NO:45 and the light chain sequence is shown in SEQ ID NO:

48.

17. A nucleic acid encoding the monoclonal antibody of any one of claims 1-16.

18. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region CDR sequence shown in SEQ ID NO:25-27 and a nucleic acid sequence encoding the light chain variable region CDR sequence shown in SEQ ID NO:28-30.

19. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region CDR sequence shown in SEQ ID NO:31-33 and a nucleic acid sequence encoding the light chain variable region CDR sequence shown in SEQ ID NO:34-36.

20. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region CDR sequence shown in SEQ ID NO:37-39 and a nucleic acid sequence encoding the light chain variable region CDR sequence shown in SEQ ID NO:40-42.

21. The nucleic acid according to claim 18, comprising the nucleic acid sequences shown in SEQ ID NO:49-51 and SEQ ID NO:52-54.

22. The nucleic acid according to claim 19, comprising the nucleic acid sequences shown in SEQ ID NO:55-57 and SEQ ID NO:58-60.

23. The nucleic acid according to claim 20, comprising the nucleic acid sequences shown in SEQ ID NO:61-63 and SEQ ID NO:64-66.

24. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO:1 and a nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:

4.

25. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO:2 and a nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:

5.

26. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO:3 and a nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:

6.

27. The nucleic acid according to claim 25, comprising the nucleic acid sequence shown in SEQ ID NO:14 and the nucleic acid sequence shown in SEQ ID NO:

17.

28. The nucleic acid according to claim 26, comprising the nucleic acid sequence shown in SEQ ID NO:15 and the nucleic acid sequence shown in SEQ ID NO:

18.

29. The nucleic acid according to claim 18, comprising a nucleic acid sequence encoding the heavy chain constant region shown in SEQ ID NO:7 and a nucleic acid sequence encoding the light chain constant region shown in SEQ ID NO:

10.

30. The nucleic acid of claim 19, comprising a nucleic acid sequence encoding the heavy chain constant region shown in SEQ ID NO:8 and a nucleic acid sequence encoding the light chain constant region shown in SEQ ID NO:

11.

31. The nucleic acid according to claim 20, comprising a nucleic acid sequence encoding the heavy chain constant region shown in SEQ ID NO:9 and a nucleic acid sequence encoding the light chain constant region shown in SEQ ID NO:

12.

32. The nucleic acid according to claim 29, comprising the nucleic acid sequence shown in SEQ ID NO:19 and the nucleic acid sequence shown in SEQ ID NO:

22.

33. The nucleic acid according to claim 30, comprising the nucleic acid sequence shown in SEQ ID NO:20 and the nucleic acid sequence shown in SEQ ID NO:

23.

34. The nucleic acid according to claim 31, comprising the nucleic acid sequence shown in SEQ ID NO:21 and the nucleic acid sequence shown in SEQ ID NO:

24.

35. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain shown in SEQ ID NO:43 and a nucleic acid sequence encoding the light chain shown in SEQ ID NO:

46.

36. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain shown in SEQ ID NO:44 and a nucleic acid sequence encoding the light chain shown in SEQ ID NO:

47.

37. The nucleic acid according to claim 17, comprising a nucleic acid sequence encoding the heavy chain shown in SEQ ID NO:45 and a nucleic acid sequence encoding the light chain shown in SEQ ID NO:

48.

38. The nucleic acid according to claim 35, comprising the nucleic acid sequence shown in SEQ ID NO:67 and the nucleic acid sequence shown in SEQ ID NO:

70.

39. The nucleic acid according to claim 36, comprising the nucleic acid sequence shown in SEQ ID NO:68 and the nucleic acid sequence shown in SEQ ID NO:

71.

40. The nucleic acid according to claim 37, comprising the nucleic acid sequence shown in SEQ ID NO:69 and the nucleic acid sequence shown in SEQ ID NO:

72.

41. An expression vector comprising the nucleic acid of any one of claims 17-40.

42. A host cell comprising the nucleic acid of any one of claims 17-40 or the expression vector of claim 41.

43. A pharmaceutical composition comprising any one of the monoclonal antibodies of claims 1-16, any one of the nucleic acids of claims 17-40, the expression vector of claim 41, or the host cell of claim 42.

44. The pharmaceutical composition of claim 43, further comprising a pharmaceutically acceptable carrier.

45. A detection reagent comprising any one of the monoclonal antibodies of claims 1-16, any one of the nucleic acids of claims 17-40, the expression vector of claim 41, or the host cell of claim 42.

46. ​​A kit comprising any one of the monoclonal antibodies of claims 1-16, any one of the nucleic acids of claims 17-40, the expression vector of claim 41, or the host cells of claim 42.

47. A method for preparing a monoclonal antibody according to any one of claims 1-16, comprising culturing the host cell according to claim 42.

48. The method of claim 47, further comprising the step of purifying the Aβ1-40 antibody.

49. The use of the monoclonal antibody of any one of claims 1-16, the nucleic acid of any one of claims 17-40, the expression vector of claim 41, or the host cell of claim 42 in the preparation of a pharmaceutical composition, a detection reagent, or a kit, wherein the pharmaceutical composition, detection reagent, or kit is used for the diagnosis or auxiliary diagnosis of Alzheimer's disease.

50. The use of the monoclonal antibody of any one of claims 1-16, the nucleic acid of any one of claims 17-40, the expression vector of claim 41, or the host cell of claim 42 in the preparation of a pharmaceutical composition, a detection reagent, or a kit, wherein the pharmaceutical composition, detection reagent, or kit is used to monitor Aβ40 levels.

51. The application according to claim 49 or 50, wherein the monoclonal antibody is used in combination with another agent or reagent.

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