rabbit-derived monoclonal antibody recognizing apo e4

Abstract

The application provides a rabbit-derived monoclonal antibody for recognizing ApoE4, and belongs to the technical field of bioengineering and biological detection. The monoclonal antibody is denoted as HZK29, and includes HZK29-B0009, HZK29-B0010 and HZK29-B0019. The rabbit-derived monoclonal antibody prepared in the application can recognize human ApoE4 with high efficiency and specificity. 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 belongs to the fields of bioengineering and biodetection, and specifically relates to rabbit-derived monoclonal antibodies that recognize ApoE4. Background Technology

[0002] Apolipoprotein E (ApoE), composed of 299 amino acids, is a glycoprotein that transports cholesterol and phospholipid molecules. It is widely distributed in free form in cerebral tissue fluid and the peripheral circulation. As a lipid transport protein, ApoE is the major ligand for the low-density lipoprotein (LDL) receptor (LDLR) and plays a role in cholesterol metabolism and cardiovascular disease.

[0003] The human APOE gene is located on chromosome 19 and has three alleles, expressing ApoE 2 (Cys112, Cys158), ApoE 3 (Cys112, Arg158), and ApoE 4 (Arg112, Arg158) proteins. Despite differences of only one or two amino acids, these three apolipoprotein isoforms exhibit significant biochemical differences in structure and function, profoundly impacting disease risk. On one hand, ApoE2 and ApoE4 increase the risk of heart disease: ApoE2 increases levels of lipoproteins that contribute to atherosclerosis (ApoE2 binds poorly to LDL receptors), while ApoE4 increases LDL levels (ApoE4 preferentially binds to triglyceride-rich very low-density lipoproteins, leading to downregulation of LDL receptors). On the other hand, genome-wide association studies (GWASs) have found that APOE4 is a major genetic risk factor for Alzheimer's disease (AD) and other neurological disorders, including adverse clinical outcomes following traumatic brain injury or stroke, frontotemporal dementia, Down syndrome, certain Parkinson's disease patients, and Lewy body disease.

[0004] Epidemiological studies have shown that ApoE4 significantly influences the incidence of Alzheimer's disease (AD). 65%-80% of AD patients carry at least one APOE4 allele. Compared to APOE3 / 3 homozygotes, APOE4 increases the risk of AD by 4-fold (1 allele) to 14-fold (2 alleles). Furthermore, each APOE4 allele (for example, patients with onset in the mid-1960s carrying 2 alleles compared to healthy individuals) can advance the age of onset by 8 years. Notably, the APOE4 gene has a high frequency, with 25% of the world's population carrying at least one APOE4 allele. In addition, the increased risk of amyloid-associated imaging abnormalities (ARIA) in individuals receiving AD disease-modifying therapy is dose-dependent, exacerbating the side effects of AD antibody therapy.

[0005] Professional genetic testing for APOE gene polymorphisms can not only accurately determine whether an individual is susceptible to Alzheimer's disease (AD), but also help comprehensively assess the examinee's genetic risk of cardiovascular and cerebrovascular diseases. APOE gene polymorphisms play a major role in individual differences leading to the development of these diseases and the effectiveness of drug treatments. Based on an individual's genetic risk level, scientific and personalized prevention recommendations are provided, and by deeply studying the etiological mechanisms, the onset and progression of AD can be effectively delayed. Regular screening helps achieve early diagnosis and treatment, thereby ultimately minimizing the potential damage caused by the disease.

[0006] Currently, APOE gene detection mainly involves extracting genomic DNA and performing PCR amplification, a relatively complex experimental procedure. With the increasing automation of testing and the growing number of samples being tested, there is an urgent clinical need for a simpler and faster method for large-scale batch processing. Summary of the Invention

[0007] In view of this, in order to at least partially solve the aforementioned technical problems, the present invention provides a rabbit-derived monoclonal antibody that recognizes ApoE4.

[0008] According to one aspect of the present invention, a rabbit-derived monoclonal antibody recognizing ApoE4 is provided, denoted as HZK29, comprising HZK29-B0009, HZK29-B0010, and HZK29-B0019; the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0009 are shown in SEQ ID NO:13-SEQ ID NO:15, respectively; the light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0009 are shown in SEQ ID NO:16-SEQ ID NO:18, respectively; the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0010 are shown in SEQ ID NO:19-SEQ ID NO:21, respectively; and the light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0010 are shown in SEQ ID NO:22-SEQ ID NO:18, respectively. As shown in NO:24; the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0019 are shown in SEQ ID NO:25-SEQ ID NO:27 respectively; the light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0019 are shown in SEQ ID NO:28-SEQ ID NO:30 respectively.

[0009] According to another aspect of the present invention, a nucleic acid molecule encoding the above-mentioned monoclonal antibody is provided.

[0010] Further, the nucleic acid molecule comprises: (1) a heavy chain sequence as shown in SEQ ID NO:7 and a light chain sequence as shown in SEQ ID NO:10; (2) a heavy chain sequence as shown in SEQ ID NO:8 and a light chain sequence as shown in SEQ ID NO:11; or (3) a heavy chain sequence as shown in SEQ ID NO:9 and a light chain sequence as shown in SEQ ID NO:12.

[0011] According to another aspect of the present invention, an expression vector comprising the above-mentioned nucleic acid molecules is provided.

[0012] In a further preferred embodiment of the present invention, the expression vector may be pPIC9K, pGAPZαA, or pCHO1.0.

[0013] According to another aspect of the present invention, a host cell comprising the above-described nucleic acid molecule or expression vector is provided.

[0014] In a further preferred embodiment of the present invention, the host cell may be Pichia pastoris, CHO cells, or HEK293 cells.

[0015] Since the ApoE4 monoclonal antibody prepared by the present invention can bind to ApoE4 with high specificity, according to another aspect of the present invention, a pharmaceutical composition, detection reagent or kit comprising the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector or host cell is provided; wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0016] According to another aspect of the present invention, a method for preparing monoclonal antibodies is provided, comprising culturing the aforementioned host cells.

[0017] In a preferred embodiment of the present invention, the above method further includes a step of purifying the ApoE4 antibody.

[0018] Since APOE4 is a major genetic risk factor for Alzheimer's disease and other neurological disorders, according to another aspect of the present invention, the use of the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector, or host cell in the preparation of pharmaceutical compositions, detection reagents, or kits is provided.

[0019] According to one embodiment of the present invention, the above-described pharmaceutical composition, detection reagent or kit is used to treat or prevent neurodegenerative diseases, diagnose or assist in the diagnosis of neurodegenerative diseases, monitor ApoE4 levels, differentiate neurodegenerative diseases, Lewy body dementia and frontotemporal degeneration, or assess the therapeutic effect and disease progression of neurodegenerative diseases.

[0020] Furthermore, neurodegenerative diseases are preferably diagnosed at an early stage, including Alzheimer's disease, Parkinson's disease, Huntington's disease, or frontotemporal dementia, with Alzheimer's disease being more preferred.

[0021] According to embodiments of the present invention, a rabbit-derived monoclonal antibody (HZK29) has been developed that can highly specifically recognize ApoE4. 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. Attached Figure Description

[0022] Figure 1 This is a flow cytometry sorting hierarchy diagram in Embodiment 1 of the present invention, wherein A is a forward scattering light-area diagram, B is a 7-aminoactinomycin D screening diagram, C is an immunoglobulin G signal diagram, and D is an allophycocyanin-area diagram.

[0023] Figure 2 This is a schematic diagram illustrating the principle of nested PCR amplification in Example 1 of the present invention;

[0024] Figure 3 This is an immunoblot image of the recombinant HZK29 antibody protein and the human cerebrospinal fluid sample ApoE4 in Example 2 of the present invention. Detailed Implementation

[0025] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0027] In this invention, the term "pharmaceutically acceptable" means a compound, substance, composition, and / or dosage form that is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the limits of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0028] The term "expression" refers to the transcription and / or translation of endogenous or exogenous genes in host cells.

[0029] The term "host cell" refers to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome.

[0030] The term "monoclonal antibody," unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites.

[0031] In realizing the concept of this invention, it was found that the operation steps for detecting the APOE gene are relatively complex, requiring a simple and convenient method for large-scale batch operation. Based on this, this invention develops a rabbit-derived monoclonal antibody that recognizes ApoE4 without requiring the extraction of genomic DNA samples, and can directly detect ApoE protein subtypes in plasma.

[0032] Example 1: Preparation of HZK29 monoclonal antibody

[0033] 1.1 Peptide Conjugation

[0034] A. Based on the target ApoE4, an immunogenic peptide SinoA12324 was designed, with the sequence CGADMEDVRGRLVQYR (SEQ ID NO:43); at the same time, a negative screening peptide SinoA12325 was designed, with the sequence CGADMEDVCGRLVQYR (SEQ ID NO:44).

[0035] The relevant reagents used are shown in Table 1.

[0036] Table 1. Reagents related to peptide conjugation

[0037]

[0038] B. The specific operating method is as follows:

[0039] (1) Take 10 mL of recombinant human papillomavirus type 16 virus-like particles (HPV-16 VLP) and filter it using a 0.45 μm aqueous syringe filter to sterilize and remove any possible aggregates. Then, measure the absorbance of the filtrate at a wavelength of 280 nm (UV280) using a UV spectrophotometer. Calculate the total mass (mg) of the carrier protein in the solution based on the standard protein quantification curve of HPV-16 VLP.

[0040] (2) Weigh a certain amount of Sulfo-SMCC and dissolve it in water to keep its concentration at 4 mg / mL; add it to the HPV 16 solution in step (1) and let it stand at room temperature (25±2℃) in the dark for 2 h.

[0041] (3) After filtration of the activated HPV 16 in step (2) through a 0.45 μm filter membrane, desalting is performed to remove excess sulfo-SMCC.

[0042] (4) Dissolve SinoA12324 in phosphate buffer solution (PBS), add an average amount of HPV 16 from step (3), and let stand at room temperature (25±2℃) in the dark for 4 h.

[0043] (5) After the coupling reaction is completed, the crude product solution of the target complex (HPV-16-SinoA12330) can be obtained. The concentration of the final product is calculated based on the amount of carrier protein initially added, and the concentration is calculated based on the amount of HPV-16 added: Concentration (mg / mL) = Amount of HPV-16 (mg) / Product volume (mL).

[0044] 1.2 Immunoassay and Serum Titer Detection

[0045] The relevant reagents used are shown in Table 2.

[0046] Table 2. Reagents related to immunoassay and serum titer detection

[0047]

[0048] The specific operating method is as follows:

[0049] Two New Zealand white rabbits were immunized, with each rabbit receiving 500 μg of immunogen per immunization. For the first immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back. Two weeks later, the same dose of immunogen was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back again. After the fourth immunization, blood was collected, and serum titers were measured using indirect ELISA to determine if a booster immunization was necessary. Once the serum titers were within acceptable limits, peripheral blood was collected for B cell sorting.

[0050] 1.3 Antigen labeling and flow cytometry sorting of B cells

[0051] The relevant reagents used are shown in Table 3.

[0052] Table 3. Reagents related to protein labeling and flow cytometry separation

[0053]

[0054] A. The phycoerythrin (PE) labeling method is as follows:

[0055] (1) Concentrate PE to 3 mg / mL, measure its UV absorbance for quantification, and then add SMCC. The N-hydroxysuccinimide (NHS) group on SMCC will react with the primary amine group on PE, and the maleimide (MAL) group at the other end will react with the thiol group (-SH) on SinoA12325.

[0056] (2) Mix the dissolved SinoA12325 with the activated PE from step (1) and react overnight at 4°C.

[0057] (3) Use an ultrafiltration tube to fully replace the sample that has been bound in step (2) to remove excess SinoA12325. Finally, measure the concentration of the purified product again by ultraviolet spectrophotometry and calculate the yield of the final PE-peptide conjugate.

[0058] B. The labeling method for allophycocyanin (APC) is as follows:

[0059] (1) Concentrate APC to 3 mg / mL, determine the accurate concentration by UV spectrophotometry, and then add SMCC. React at room temperature in the dark. The NHS group on SMCC will react with the primary amine group on APC, and the MAL group at the other end will react with the -SH group on the peptide.

[0060] (2) Mix SinoA12324 with the activated APC from step (1) thoroughly and react overnight at 4°C in the dark.

[0061] (3) Use an ultrafiltration tube to fully replace the sample that has been bound in step (2) to remove excess SinoA12324. Finally, determine the concentration of the purified product by ultraviolet spectrophotometry and calculate the final yield of APC-peptide conjugate.

[0062] C. Flow cytometry sorting of B cells:

[0063] (1) Add EDTA-anticoagulated rabbit whole blood to a centrifuge tube containing Ficoll, centrifuge, take the white membrane layer containing lymphocytes and monocytes in the middle to a new centrifuge tube, add sufficient phosphate buffer to wash the cells, centrifuge and discard the supernatant to obtain relatively pure peripheral blood mononuclear cells (PBMCs).

[0064] (2) Add IgG, IgM antibodies and fluorescently labeled proteins to the isolated PBMCs and incubate. After incubation, wash the cells with PBS 2-3 times to completely remove unbound free antibodies, and then resuspend the cells with PBS.

[0065] (3) Adjust the flow sorting liquid path, delay time, and sorting angle, and adjust the fluorescence compensation. For example... Figure 1 As shown, lymphocytes were delineated based on cell diameter (FSC) and intracellular granule complexity (SSC); cells not stained with the DNA dye 7-amino-actinomycin D (7-AAD) were delineated; cells positive for IgG antibody signals were delineated; and cells specifically binding to the target peptide SinoA12324 were delineated. Utilizing the fluorescent labeling optical characteristics, this method can rapidly distinguish different cell populations. Figure 1 The sorting strategy can gradually screen out target B cells from mixed cells, namely antigen-specific B cells that can bind to ApoE4.

[0066] 1.4 Expansion of rabbit-derived B-cell positive clones

[0067] The reagents used in cell culture are shown in Table 4.

[0068] Table 4. Cell Culture Related Reagents

[0069]

[0070] A. Cell culture methods are as follows:

[0071] Prepare mitomycin C-treated feeder cells and seed them into 96-well cell culture plates until they adhere to the plate. On the second day, inject single B cells that meet the screening criteria into wells containing feeder cells. After sorting, add culture medium containing mitogens and cytokines and incubate in a CO2 incubator for about one week. Take a portion of the culture supernatant for enzyme-linked immunosorbent assay (ELISA).

[0072] The reagents used in ELISA are shown in Table 5.

[0073] Table 5 ELISA-related reagents

[0074]

[0075] B. The ELISA method is as follows:

[0076] (1) Coating of project peptides and cross peptides, with coating concentrations of 0.1 μg / mL and 100 μL / well, respectively, and coating overnight at 4℃.

[0077] (2) Shake off the liquid in the plate and pat it dry. Add the sealing solution (300 μL / well), seal and incubate at room temperature for 1 h.

[0078] (3) Add washing solution (300 μL / well) to wash the plate twice, and pat it dry on the last time.

[0079] (4) Dilute the culture supernatant in A by 5 times with sample diluent and mix thoroughly for later use.

[0080] (5) Add the diluted supernatant to the microplate (100 μL / well), mix well and incubate at room temperature for 2 h.

[0081] (6) Add washing solution (300 μL / well) to wash the plate 3 times, and pat dry the plate on the last wash.

[0082] (7) Dilute horseradish peroxidase-labeled Goat Anti-rabbit IgG Fc / HRP at a volume ratio of 1:200, mix well and add to the microplate (100 μL / well), and incubate at room temperature for 2 h.

[0083] (8) Same as step (3).

[0084] (9) Mix solution A and solution B of the colorimetric solution at a ratio of 1:1 and add them to the microplate (200 μL / well), and incubate at room temperature in the dark for 3 min.

[0085] (10) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0086] The ELISA test results are shown in Tables 9 and 10.

[0087] 1.5 Recombinant Antibody Expression

[0088] The relevant reagents used are shown in Table 6.

[0089] Table 6. Reagents related to the construction of recombinant antibody vectors

[0090]

[0091] The specific operating method is as follows:

[0092] After lysing qualified B cells from the culture supernatant, cDNA was obtained by reverse transcription using a reverse transcription kit. The antibody heavy chain variable region sequence and light chain variable region sequence were obtained using a self-developed single B cell amplification technology. The heavy chain variable region fragment and light chain variable region fragment were constructed into expression vectors, and after sequencing verification, they were transiently expressed in HEK 293 cells.

[0093] The aforementioned independently developed single B-cell amplification technology is a nested PCR amplification method, and the schematic diagram of the amplification principle is shown below. Figure 2 As shown, primers were designed from the stable region of the antibody gene to amplify the target gene DNA. Random primers were then designed to shorten the amplification range, and the VDJ fragment was cloned.

[0094] 1.6 Antibody Purification

[0095] The relevant reagents used are shown in Table 7.

[0096] Table 7 Reagents related to antibody purification

[0097]

[0098] The specific operating method is as follows:

[0099] (1) Centrifuge the collected cell supernatant (4000 g, 30 min), collect the supernatant, and filter the supernatant using a 0.45 μM filter membrane.

[0100] (2) Select a protein A column of appropriate specifications according to the expression level and connect it to the purification system; wash with 3 column volumes (CV) of ultrapure water and replace the 25% ethanol preservation solution; equilibrate 3CV with AC binding + 1 / 5 stock buffer until the UV absorption (UV) baseline is stable.

[0101] (3) Adjust the appropriate flow rate for sample loading.

[0102] (4) Rinse with the eluent for 5-10 CV until the UV baseline is stable, at the same flow rate as the sample loading flow rate.

[0103] (5) Elute with elution buffer (AC Elution) and collect the elution peaks according to the real-time UV monitoring spectrum. Immediately add 2 M Tris-HCl buffer (pH 8.0) to the collected eluent to neutralize it and prevent the antibody from being inactivated under acidic conditions.

[0104] (6) Add 2M Tris, pH 8.0 to neutralize and elute the antibody.

[0105] (7) Use binding buffer (AC Binding) to equilibrate 3 CV to neutral.

[0106] (8) In-situ cleaning (CIP) 5 CV or more.

[0107] (9) Use AC Binding to flush with alkali until the pH at the outlet is neutral.

[0108] (10) Equilibrate the column with 25% ethanol for 2 CV and preserve the column.

[0109] The above steps allow for the capture of antibodies from the cell culture supernatant. After elution and neutralization, high-purity antibody solutions HZK29-B0009, HZK29-B0010, and HZK29-B0019 are obtained.

[0110] Example 2: Identification of HZK29 monoclonal antibody

[0111] 2.1 ELISA detection and identification

[0112] The reagents used in ELISA are shown in Table 5.

[0113] The specific operating method is as follows:

[0114] (1) Coat ApoE4 and ApoE3 proteins at concentrations of 0.1 μg / mL and 1 μg / mL, respectively. Add 100 μL of the corresponding concentration of antigen solution to each well of the microplate, seal and coat overnight at 4°C.

[0115] (2) Shake off the liquid in the plate and pat it dry. Add 2% BSA (300 μL / well), seal and incubate at room temperature for 1 h to cover the non-specific binding sites in the well.

[0116] (3) Add washing solution (300 μL / well) to wash the plate twice, and pat it dry on the last time.

[0117] (4) Dilute antibodies HZK29-B0009, HZK29-B0010 and HZK29-B0019 (primary antibody) to 0.1 μg / mL and add them to the corresponding well plates (100 μL / well). Mix well and react at room temperature for 2 h.

[0118] (5) Discard the primary antibody, add washing buffer (300 μL / well) to wash the plate 3 times, and pat dry the plate on the last wash.

[0119] (6) Dilute the horseradish peroxidase-labeled goat anti-rabbit IgG (Goat Anti-rabbit IgG Fc / HRP) secondary antibody to the recommended working concentration using antibody dilution buffer. Add 100 μL to each well, mix well, and incubate at room temperature for 1 h.

[0120] (7) Discard the secondary antibody, add washing buffer (300 μL / well) to wash the plate 3 times, and pat dry the plate on the last wash.

[0121] (8) Mix color development solution A and color development solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 20 min.

[0122] (9) Add 50 μL of stop solution to each well and immediately use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0123] The ELISA test results are shown in Tables 8-9.

[0124] Table 8. ELISA results of purified antibodies (coated with ApoE4)

[0125]

[0126] Table 9. ELISA results of purified antibodies (coated with ApoE3)

[0127]

[0128] 2.2 Identification by immunoblotting assay

[0129] The relevant reagents used are shown in Table 10.

[0130] Table 10 Antibody Detection Reagents

[0131]

[0132] The specific operating method is as follows:

[0133] (1) Collect human cerebrospinal fluid (CSF) samples and label them with patient numbers and APOE genotypes (APOE23, APOE24, APOE33, APOE34, APOE44). Resuspend ApoE2, ApoE3, and ApoE4 recombinant proteins in PBS. Add pre-chilled protein lysis buffer (containing protease inhibitors) and lyse on ice for 30 minutes. Then centrifuge at 4°C and 12,000 rpm for 20 minutes and collect the supernatant, which is the total protein sample. Determine the protein concentration using the BCA method, etc., and mix the sample with 5× loading buffer and boil at 100°C for 5 minutes to denature the protein.

[0134] (2) Prepare a 16% SDS-PAGE gel. Add the prepared protein sample to the well according to the predetermined loading amount. Add electrophoresis buffer to the electrophoresis tank, set the voltage to 120V, and electrophore for about 1 hour until the bromophenol blue indicator reaches the bottom of the gel.

[0135] (3) After electrophoresis, remove the gel and cut 0.22 μm PVDF membranes and filter paper according to the gel size. The PVDF membrane needs to be activated by soaking in methanol for 5-10 seconds. Then, place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp in sequence, ensuring that there are no air bubbles between the layers. Place the transfer clamp into the transfer tank and add pre-cooled transfer buffer. Transfer the membrane at a constant current of 240 mA for 30 minutes under ice bath conditions.

[0136] (5) Block the membrane for 1 hour at room temperature using blocking buffer.

[0137] (6) Incubate the membrane with the appropriate diluted primary antibody at 4°C overnight; at room temperature, incubate the membrane with the conjugated secondary antibody dilution in the blocking buffer for 1 hour.

[0138] (7) Mix the peroxide solution and enhancer in the ECL chemiluminescence substrate in equal volumes (500 μL each) to prepare a reaction solution. Evenly drop the reaction solution onto the surface of the PVDF membrane, ensuring the liquid covers all areas. Then, perform exposure detection in a chemiluminescence imager or dark chamber. Adjust the exposure time according to the signal intensity to obtain a clear band image, as shown in the figure. Figure 3 As shown. The immunoblotting results of rabbit B cell supernatant and its purified HZK29 antibody, and human brain protein samples are as follows. Figure 3 As shown. The ApoE recombinant protein was a synthetic protein sample, and the cerebrospinal fluid (CSF) was from patients with different genotypes. The target fragment of ApoE immunoblotting is approximately 35 kDa. The ApoE4 antibody described in this article can only detect the ApoE4 recombinant protein and the ApoE target band in the CSF of patients carrying the APOE4 allele. Therefore, the HZK29 antibody can effectively distinguish ApoE4.

[0139] The complete sequence used in this invention is as follows.

[0140] B0009-H full-length amino acid sequence (rabbit IgG1) (SEQ ID NO:1):

[0141] MGWSLILLFLVAVATRVLSQSVEESGGRLVTPGTPLTLTCTASGFSLSGYLMSWVRQAPGKGLEWIGIISSSGSTNYATWAKGRFTISRTSTTVDLKIASPTTEDTATYFCAREYGGTNYYDYYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0142] Full-length amino acid sequence of B0010-H (Rabbit IgG1) (SEQ ID NO: 2):

[0143] MGWSLILLFLVAVATRVLSQSLEESGGRLVTPGGSLTLTCTASGFSLSSYVMGWVRQAPGKGLEYIGIISSSGTTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCASGGLGPGFNIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0144] Full-length amino acid sequence of B0019-H (rabbit IgG1) (SEQ ID NO: 3):

[0145] MGWSLILLFLVAVATRVLSQEQLEESGGRLVTPGTPLTLTCTASGFSLSGYFMSWVRQAPGKGLEWIGIISSSGTTNYATWAKGRFTISRTSTTVDLKITSPTTEDTATYFCAREYGGTNYYDYYGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0146] Full-length amino acid sequence of B0009-L (rabbit IgG1) (SEQ ID NO: 4):

[0147] MGWSCIILFLVATATGVHSDVEMTQTPASVEVVVGGTVTIKCQASQSINNLLAWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQDYSTSNIDNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0148] Full-length amino acid sequence of B0010-L (rabbit IgG1) (SEQ ID NO: 5):

[0149] MGWSCIILFLVATATGVHSAQGMTQTASSVSAAVGGTVTISCQSSQSVYENNWLGWYQQKPGQPPKLLIYYASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGGYNGAIYTFGGGTEVAVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0150] Full-length amino acid sequence of B0019-L (rabbit IgG1) (SEQ ID NO: 6):

[0151] MGWSCIILFLVATATGVHSELVLTQTPASVEVPVGGTVTIKCQASQSIANLLAWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISGVECADAATYYCQQDYSSSNINNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0152] Full-length nucleotide sequence of B0009-H (rabbit IgG1) (SEQ ID NO: 7):

[0153]

[0154] B0010-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID NO:8):

[0155]

[0156] B0019-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID NO:9):

[0157]

[0158] Full-length nucleotide sequence of B0009-L (rabbit IgG1) (SEQ ID NO: 10):

[0159] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATGTTGAGATGACCCAGACTCCAGCCTCTGTGGAGGTAGTTGTGGGAGGCACAGTCACCATCAAATGCCAGGCCAGTCAGAGCATTAACAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGATTATAGTACTAGTAATATTGATAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0160] Full-length nucleotide sequence of B0010-L (rabbit IgG1) (SEQ ID NO: 11):

[0161] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCTCAAGGGATGACCCAGACTGCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATGAGAACAACTGGTTAGGCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTTCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAATGGTGCTATTTATACTTTCGGCGGAGGGACCGAGGTGGCGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0162] Full-length nucleotide sequence of B0019-L (rabbit IgG1) (SEQ ID NO: 12):

[0163] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGAGCTTGTGCTGACCCAGACTCCAGCCTCTGTGGAGGTACCTGTGGGAGGCACAGTCACCATCAAATGCCAGGCCAGTCAGAGCATTGCCAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGACTATAGTTCTAGTAATATTAATAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0164] B0009-H CDR1 amino acid sequence (SEQ ID NO:13): GFSLSSGYL; B0009-H CDR2 amino acid sequence (SEQ ID NO:14): ISSSGST; B0009-H CDR3 amino acid sequence (SEQ ID NO:15): AREYGGTNYYDYYGMDL; B0009-L CDR1 amino acid sequence (SEQ ID NO:16): QSINNL; B0009-L CDR2 amino acid sequence (SEQ ID NO:17): RAS; B0009-L CDR3 amino acid sequence (SEQ ID NO:18): QQDYSTSNIDNT; B0010-H CDR1 amino acid sequence (SEQ ID NO:19): GFSLSSYV; B0010-H CDR2 amino acid sequence (SEQ ID NO:20): ISSSGTT; B0010-H CDR3 amino acid sequence (SEQ ID NO:21): ASGGLGPGFNI; B0010-L CDR1 amino acid sequence (SEQ ID NO:22): QSVYENNW; B0010-L CDR2 amino acid sequence (SEQ ID NO:23): YAS; B0010-L CDR3 amino acid sequence (SEQ ID NO:24): AGGYNGAIYT; B0019-H CDR1 amino acid sequence (SEQ ID NO:25): GFSLSGYF; B0019-H CDR2 amino acid sequence (SEQ ID NO:26): ISSSGTT; B0019-HCDR3 amino acid sequence (SEQ ID NO:27): AREYGGTNYYDYYGMDL; B0019-L CDR1 amino acid sequence (SEQ ID NO:28): QSIANL; B0019-L CDR2 amino acid sequence (SEQ ID NO:29): RAS; B0019-L CDR3 amino acid sequence (SEQ ID NO:30): QQDYSSSNINNT.

[0165] B0009-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:31):

[0166] QSVEESGGRLVTPGTPLTLTCTASGFSLSGYLMSWVRQAPGKGLEWIGIISSSGSTNYATWAKGRFTISRTSTTVDLKIASPTTEDTATYFCAREYGGTNYYDYYGMDLWGPGTLVTVSS.

[0167] B0010-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:32):

[0168] QSLEESGGRLVTPGGSLTLTCTASGFSLSSYVMGWVRQAPGKGLEYIGIISSSGTTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCASGGLGPGFNIWGPGTLVTVSL.

[0169] B0019-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:33):

[0170] QEQLEESGGRLVTPGTPLTLTCTASGFSLSGYFMSWVRQAPGKGLEWIGIISSSGTTNYATWAKGRFTISRTSTTVDLKITSPTTEDTATYFCAREYGGTNYYDYYGMDLWGPGTLVTVSS.

[0171] B0009-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:34):

[0172] DVEMTQTPASVEVVVGGTVTIKCQASQSINNLLAWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQDYSTSNIDNTFGGGTEVVVK.

[0173] B0010-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:35):

[0174] AQGMTQTASSVSAAVGGTVTISCQSSQSVYENNWLGWYQQKPGQPPKLLIYYASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGGYNGAIYTFGGGTEVAVK.

[0175] B0019-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:36):

[0176] ELVLTQTPASVEVPVGGTVTIKCQASQSIANLLAWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISGVECADAATYYCQQDYSSSNINNTFGGGTEVVVK.

[0177] Nucleotide sequence of the variable region of B0009-H (rabbit IgG1) (SEQ ID NO: 37):

[0178] CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACTTGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATGGATCGGAATCATTAGTAGTAGTGGTAGCACGAACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACGTATTTCTGTGCCAGAGAATATGGTGGTACTAATTATTACGACTACTATGGCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCA.

[0179] Nucleotide sequence of the variable region of B0010-H (rabbit IgG1) (SEQ ID NO: 38):

[0180] CAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGTACAGCCTCTGGATTCTCCCTCAGCAGCTATGTAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATCAGTAGTAGTGGTACCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGTGGGGGCCTGGGTCCTGGCTTTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTTA.

[0181] Nucleotide sequence of the variable region of B0019-H (rabbit IgG1) (SEQ ID NO: 39):

[0182] CAGGAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTGGCTACTTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATGGATCGGAATCATTAGTAGTAGTGGTACCACGAACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGAATATGGTGGTACTAATTATTACGACTACTACGGCATGGACCTCTGGGGCCCAGGGACCCTCGTCACCGTCTCTTCA.

[0183] B0009-L variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO: 40):

[0184] GATGTTGAGATGACCCAGACTCCAGCCTCTGTGGAGGTAGTTGTGGGAGGCACAGTCACCATCAAATGCCAGGCCAGTCAGAGCATTAACAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGATTATAGTACTAGTAATATTGATAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0185] B0010-L variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO: 41):

[0186] GCTCAAGGGATGACCCAGACTGCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATGAGAACAACTGGTTAGGCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTTCTGATCTATTATGCATCCACT CTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAATGGTGCTATTTATACTTTCGGCGGAGGGACCGAGGTGGCGGTCAAA.

[0187] B0019-L variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO:42):

[0188] GAGCTTGTGCTGACCCAGACTCCAGCCTCTGTGGAGGTACCTGTGGGAGGCACAGTCACCATCAAATGCCAGGCCAGTCAGAGCATTGCCAACCTCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCA TCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGGAGTGTGCCGATGCTGCCACTTACTACTGTCACAGGACTATAGTTCTAGTAATATTAATAATACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0189] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rabbit-derived monoclonal antibody that recognizes ApoE4, characterized in that: The rabbit-derived monoclonal antibody that recognizes ApoE4 is HZK29-B0009; The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0009 are shown in SEQ ID NO:13-SEQ ID NO:15, respectively. The light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK29-B0009 are shown in SEQ ID NO:16-SEQ ID NO:18, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The HZK29-B0009 includes: The heavy chain variable region sequence shown in SEQ ID NO:31 and the light chain variable region sequence shown in SEQ ID NO:

34.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The HZK29-B0009 includes: The heavy chain sequence shown in SEQ ID NO:1 and the light chain sequence shown in SEQ ID NO:

4.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody as described in any one of claims 1 to 3.

5. An expression vector, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A method for preparing monoclonal antibodies, characterized in that, The method includes culturing the host cells as described in claim 6.

8. The use of the monoclonal antibody of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the host cell of claim 6 in the preparation of a detection reagent or detection kit; wherein the detection reagent or detection kit is used to monitor ApoE4 levels.

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