rabbit monoclonal antibody recognizing apo e

Abstract

The application provides a rabbit-derived monoclonal antibody for recognizing ApoE, and belongs to the technical field of bioengineering and biological detection.The monoclonal antibody is denoted as HZK31, and includes HZK31-B0002, HZK31-B0005, HZK31-B0007 and HZK31-B0011.The rabbit-derived monoclonal antibody prepared in the application can recognize human ApoE 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 ApoE. Background Technology

[0002] Apolipoprotein E (ApoE) is a glycoprotein composed of 299 amino acids with a molecular weight of 34 kDa. It is widely distributed in free form in cerebral tissue fluid and peripheral circulation. As a lipid transport protein, ApoE plays an important role in cholesterol metabolism, cardiovascular disease, and the development of neurodegenerative diseases.

[0003] In the central nervous system, astrocytes, microglia, and vascular wall cells can all secrete ApoE. Under the action of ATP family transporters ABCA1 and ABCG1, cholesterol and phospholipid molecules combine with ApoE to form lipoprotein particles, which are then transported to neurons via receptors such as the low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein 1 (LRP1). In the peripheral circulation, the liver is the main source of ApoE.

[0004] 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.

[0005] Epidemiological studies have shown a close correlation between plasma ApoE levels and diseases such as hypertriglyceridemia and insulin resistance. Notably, research has found that plasma ApoE levels follow the order APOE2 > APOE3 > APOE4. Because AD patients have a higher APOE4 gene frequency, their plasma ApoE levels are actually lower than those in the normal population. Therefore, detecting plasma ApoE levels can play an auxiliary role in disease risk prediction and clinical diagnosis. This demonstrates the promising application potential of developing novel ApoE antibodies. Summary of the Invention

[0006] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides a rabbit-derived monoclonal antibody that recognizes ApoE.

[0007] According to one aspect of the present invention, a rabbit-derived monoclonal antibody for recognizing ApoE is provided, denoted as HZK31, comprising HZK31-B0002, HZK31-B0005, HZK31-B0007, and HZK31-B0011; the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK31-B0002 are shown in SEQ ID NO:17-SEQ ID NO:19, respectively; the light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK31-B0002 are shown in SEQ ID NO:20-SEQ ID NO:22, respectively; the heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK31-B0005 are shown in SEQ ID NO:23-SEQ ID NO:25, respectively; the light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK31-B0005 are shown in SEQ ID NO:23-SEQ ID NO:25, respectively. The CDR1, CDR2, and CDR3 sequences of the heavy chain variable regions of HZK31-B0007 are shown in SEQ ID NO:26-SEQ ID NO:28; the CDR1, CDR2, and CDR3 sequences of the light chain variable regions of HZK31-B0007 are shown in SEQ ID NO:32-SEQ ID NO:34; the CDR1, CDR2, and CDR3 sequences of the heavy chain variable regions of HZK31-B0011 are shown in SEQ ID NO:35-SEQ ID NO:37; and the CDR1, CDR2, and CDR3 sequences of the light chain variable regions of HZK31-B0011 are shown in SEQ ID NO:38-SEQ ID NO:40.

[0008] According to another aspect of the present invention, a nucleic acid molecule encoding the above-mentioned monoclonal antibody is provided.

[0009] Further, the nucleic acid molecule comprises: (1) the sequences shown in SEQ ID NO:9 and SEQ ID NO:13; (2) the sequences shown in SEQ ID NO:10 and SEQ ID NO:14; (3) the sequences shown in SEQ ID NO:11 and SEQ ID NO:15; or (4) the sequences shown in SEQ ID NO:12 and SEQ ID NO:16.

[0010] According to another aspect of the present invention, an expression vector comprising the above-mentioned nucleic acid molecules is provided.

[0011] In a further preferred embodiment of the present invention, the expression vector may be pPIC9K, pGAPZαA, or pCHO1.0.

[0012] According to another aspect of the present invention, a host cell comprising the above-described nucleic acid molecule or expression vector is provided.

[0013] In a further preferred embodiment of the present invention, the host cell may be Pichia pastoris, CHO cells, or HEK293 cells.

[0014] Since the ApoE monoclonal antibody prepared by the present invention can bind to ApoE 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.

[0015] According to another aspect of the present invention, a method for preparing monoclonal antibodies is provided, comprising culturing the aforementioned host cells.

[0016] In a preferred embodiment of the present invention, the above method further includes a step of purifying the ApoE antibody.

[0017] Since the plasma ApoE level in AD patients is lower than that in normal individuals, detecting plasma ApoE levels can play an auxiliary role in disease risk prediction and clinical diagnosis. Therefore, according to another aspect of the present invention, the above-mentioned monoclonal antibody, nucleotide sequence, expression vector, or host cell is provided for use in the preparation of pharmaceutical compositions, detection reagents, or kits.

[0018] According to one embodiment of the present invention, the above-described pharmaceutical composition, detection reagent or kit is used to diagnose or assist in the diagnosis of neurodegenerative diseases, monitor ApoE4 levels, differentiate neurodegenerative diseases, Lewy body dementia and frontotemporal degeneration, or assess the treatment effect and disease progression of neurodegenerative diseases.

[0019] 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.

[0020] The invention utilizes rabbit monoclonal antibodies to develop HZK31 antibodies that recognize ApoE. Compared to mice or rats, rabbits have greater genetic diversity, so rabbit monoclonal antibodies have a broader antibody spectrum, simpler immunoglobulin structures, higher affinity, and are easier to humanize. Attached Figure Description

[0021] 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.

[0022] Figure 2 This is a schematic diagram illustrating the principle of nested PCR amplification in Example 1 of the present invention;

[0023] Figure 3 This is an ApoE immunoblot image of HZK31 antibody and human cerebrospinal fluid sample in Example 2 of the present invention. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The term "expression" refers to the transcription and / or translation of endogenous or exogenous genes in host cells.

[0028] 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.

[0029] 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.

[0030] In the process of realizing the concept of this invention, it was discovered that detecting plasma ApoE levels can play an auxiliary role in disease risk prediction and clinical diagnosis. Based on this, this invention developed a rabbit-derived monoclonal antibody that identifies ApoE.

[0031] Example 1: Preparation of HZK31 monoclonal antibody

[0032] 1.1 Immunoassay and Serum Titer Detection

[0033] The relevant reagents used are shown in Table 1.

[0034] Table 1. Reagents related to immunoassay and serum titer detection

[0035]

[0036] The specific operating method is as follows:

[0037] 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 by indirect ELISA to determine whether a booster immunization was needed. Once the serum titers were within acceptable limits, peripheral blood was collected from the rabbits for B cell sorting.

[0038] 1.2 Antigen labeling and flow cytometry sorting of B cells

[0039] The relevant reagents used are shown in Table 2.

[0040] Table 2. Reagents related to protein labeling and flow cytometry separation

[0041]

[0042] A. The phycoerythrin (PE) labeling method is as follows:

[0043] (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 the polypeptide.

[0044] (2) Mix the dissolved ApoE3 protein with the activated PE from step (1) and react overnight at 4°C.

[0045] (3) Use an ultrafiltration tube to fully replace the sample that has been bound in step (2) to remove excess ApoE3 protein. Finally, measure the concentration of the purified product again by ultraviolet spectrophotometry and calculate the yield of the final PE-peptide conjugate.

[0046] B. The labeling method for allophycocyanin (APC) is as follows:

[0047] (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.

[0048] (2) Mix the ApoE4 protein with the activated APC from step (1) and react overnight at 4°C in the dark.

[0049] (3) Use an ultrafiltration tube to fully replace the sample that has been bound in step (2) to remove excess ApoE4 protein. Finally, determine the concentration of the purified product by ultraviolet spectrophotometry and calculate the final yield of APC-peptide conjugate.

[0050] C. Flow cytometry sorting of B cells:

[0051] (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).

[0052] (2) After the PBMCs are separated, IgG, IgM antibodies and fluorescently labeled proteins are added and incubated. After incubation, the cells are washed with PBS 2-3 times to completely remove unbound free antibodies, and then the cells are resuspended with PBS.

[0053] (3) Adjust the flow sorting liquid path, delay time, and sorting angle; adjust fluorescence compensation; and follow the instructions. Figure 1 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; IgG antibody-positive cells were delineated; and cells specifically binding to ApoE were delineated. 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.

[0054] 1.3 Expansion of rabbit-derived B-cell positive clones

[0055] The reagents used in cell culture are shown in Table 3.

[0056] Table 3 Reagents related to cell culture

[0057]

[0058] A. Cell culture methods are as follows:

[0059] 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).

[0060] The reagents used in ELISA are shown in Table 4.

[0061] Table 4 ELISA-related reagents

[0062]

[0063] B. The ELISA method is as follows:

[0064] (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℃.

[0065] (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.

[0066] (3) Add washing solution (300 μL / well) to wash the plate twice, and pat it dry on the last time.

[0067] (4) Dilute the culture supernatant in A by 5 times with sample diluent and mix thoroughly for later use.

[0068] (5) Add the diluted supernatant to the microplate (100 μL / well), mix well and incubate at room temperature for 2 h.

[0069] (6) Add washing solution (300 μL / well) to wash the plate 3 times, and pat dry the plate on the last wash.

[0070] (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.

[0071] (8) Same as step (3).

[0072] (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.

[0073] (10) Add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0074] The ELISA test results are shown in Tables 8-10.

[0075] 1.4 Recombinant Antibody Expression

[0076] The relevant reagents used are shown in Table 5.

[0077] Table 5. Reagents related to the construction of recombinant antibody vectors

[0078]

[0079] The specific operating method is as follows:

[0080] 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 by using a self-developed single B cell amplification technology. The heavy chain variable region sequence and light chain variable region fragment were constructed into expression vectors, and after being verified by sequencing, they were transduced into HEK 293 cells for transient expression.

[0081] Nested PCR amplification yields variable region fragments of the antibody heavy and light chains. A schematic diagram illustrating the amplification principle is shown below. Figure 2 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.

[0082] 1.5 Antibody Purification

[0083] The relevant reagents used are shown in Table 6.

[0084] Table 6 Reagents related to antibody purification

[0085]

[0086] The specific operating method is as follows:

[0087] (1) The collected cell supernatant was centrifuged using a benchtop centrifuge at 4000 g for 30 min. The supernatant was collected after centrifugation and filtered using a 0.45 μM filter membrane.

[0088] (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.

[0089] (3) Adjust the appropriate flow rate for sample loading.

[0090] (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.

[0091] (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.

[0092] (6) Add 2M Tris, pH 8.0 to neutralize and elute the antibody.

[0093] (7) Use binding buffer (AC Binding) to equilibrate 3 CV to neutral.

[0094] (8) In-situ cleaning (CIP) 5 CV or more.

[0095] (9) Use AC Binding to flush with alkali until the pH at the outlet is neutral.

[0096] (10) Equilibrate the column with 25% ethanol for 2 CV and preserve the column.

[0097] The antibodies can then be captured from the cell culture supernatant through the above steps. After elution, neutralization and other steps, high-purity antibody solutions HZK31-B0002, HZK31-B0005, HZK31-B0007 and HZK31-B0011 can be obtained.

[0098] Example 2: Identification of HZK31 monoclonal antibody

[0099] 2.1 ELISA detection and identification

[0100] The reagents used in ELISA are shown in Table 4.

[0101] The specific operating method is as follows:

[0102] (1) Coat ApoE2, ApoE3 and ApoE4 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.

[0103] (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.

[0104] (3) Add washing solution (300 μL / well) to wash the plate twice, and pat it dry on the last time.

[0105] (4) Dilute antibodies HZK31-B0002, HZK31-B0005, HZK31-B0007 and HZK31-B0011 (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.

[0106] (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.

[0107] (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.

[0108] (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.

[0109] (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.

[0110] (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.

[0111] The test results are shown in Tables 7-9.

[0112] Table 7. ELISA results of purified antibodies (coated with ApoE2)

[0113]

[0114] Table 8. ELISA results of purified antibodies (coated with ApoE3)

[0115]

[0116] Table 9. ELISA results of purified antibodies (coated with ApoE4)

[0117]

[0118] 2.2 Identification by immunoblotting assay

[0119] The relevant reagents used are shown in Table 10.

[0120] Table 10 Antibody Detection Reagents

[0121]

[0122] The specific operating method is as follows:

[0123] (1) Take cerebrospinal fluid (CSF) samples from human brain tissue, and label them with the patient number and APOE genotype (APOE23, APOE24, APOE33, APOE34, APOE44). 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.

[0124] (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.

[0125] (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.

[0126] (5) Block the membrane for 1 hour at room temperature using blocking buffer.

[0127] (6) Incubate the membrane with the appropriate diluted primary antibody at 4°C overnight; incubate the membrane with the conjugated secondary antibody dilution in the blocking buffer for 1 hour at room temperature.

[0128] (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 HZK31 antibody, and human brain protein samples are as follows. Figure 3 As shown. According to Figure 3 The cerebrospinal fluid (CSF) samples were from patients with different genotypes. The ApoE immunoblot target fragment is approximately 35 kDa, and the antibody described in this article can detect the ApoE target band in the CSF of patients carrying different APOE alleles. Therefore, the HZK31 antibody can effectively distinguish ApoE.

[0129] The relevant sequences in this application are as follows.

[0130] B0002-H full-length amino acid sequence (rabbit IgG1) (SEQ ID NO:1):

[0131] MGWSLILLFLVAVATRVLSQSLEESEGGLFKPTDTLTLTCTVSGFSLISYGVTWVRQAPGNGLEWIAFISTGGSTYYASWAKSRSTITRKTNLNTVTLKMTSLTAADTATYFCARGFTNSYIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0132] Full-length amino acid sequence of B0005-H (rabbit IgG1) (SEQ ID NO:2):

[0133] MGWSLILLFLVAVATRVLSQSVKESEGGLFKPTETLTLTCTVSGIDLSSYGVTWVRQAPGNGLEYIGTIGGSGSTYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCARLYNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0134] Full-length amino acid sequence of B0007-H (rabbit IgG1) (SEQ ID NO: 3):

[0135] MGWSLILLFLVAVATRVLSQEQLEESGGRLVKPDETLTLTCTVSGLSLSRNAITWVRQAPGKGLQYIGIISKDGYTFYANWAKGRFTISKTSTTVDLKITTPTTEDTATYFCVRWNFSSDNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0136] Full-length amino acid sequence of B0011-H (rabbit IgG1) (SEQ ID NO: 4):

[0137] MGWSLILLFLVAVATRVLSQQQLEESGGRLVKPDETLTLTCTVSGLSLSSNAITWVRQAPGKGLEYIGIISNNGYTFYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRWNYSSDNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*.

[0138] Full-length amino acid sequence of B0002-L (rabbit IgG1) (SEQ ID NO: 5):

[0139] MGWSCIILFLVATATGVHSAAVLTQTPSPVSAAVGGTVSISCQSSQNVYSNDYLSWYQQKPGQRPKLLISETSTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSGNILTFGGGTEVLVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0140] Full-length amino acid sequence of B0005-L (rabbit IgG1) (SEQ ID NO: 6):

[0141] MGWSCIILFLVATATGVHSAVVLTQTPSSVSAAVGGTVTINCQSSQSVYSNNYLSWYQQKPGQPPKLLIYKASTLPSGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGDYGCDPADCYNFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0142] Full-length amino acid sequence of B0007-L (Rabbit IgG1) (SEQ ID NO: 7):

[0143] MGWSCIILFLVATATGVHSDVVMTQTPASVEVAVGGTVTIKCQASEDIESYLAWYQQKPGQRPKLLIYKASTLASGVPSRFKGSGSGTQYTLTISGVQCDDAATYYCQQGYIYNDVDNVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0144] Full-length amino acid sequence of B0011-L (Rabbit κ light chain) (SEQ ID NO: 8):

[0145] MGWSCIILFLVATATGVHSAQVMTQTPASVEVAVGGTVTINCRASEDIESYLAWYQQKPGQRPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQQGYTYNDVDNVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0146] Full-length nucleotide sequence of B0002-H (Rabbit IgG1) (SEQ ID NO: 9):

[0147]

[0148] B0005-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID NO:10):

[0149]

[0150] B0007-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID NO:11):

[0151]

[0152] B0011-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID NO:12):

[0153]

[0154] Full-length nucleotide sequence of B0002-L (rabbit IgG1) (SEQ ID NO: 13):

[0155] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCAGCCGTGCTGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAGTAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTCCGAAACATCCACTCTGGCATCTGGGGTCCCATCGCGGTTTAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGGTAATATTCTTACTTTTGGCGGAGGGACCGAGGTGCTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0156] Full-length nucleotide sequence of B0005-L (rabbit IgG1) (SEQ ID NO: 14):

[0157] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCCGTCGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAGTAACAACTACTTATCCTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACAAGGCTTCCACTCTGCCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTATGGTTGTGATCCTGCTGATTGTTATAATTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0158] Full-length nucleotide sequence of B0007-L (rabbit IgG1) (SEQ ID NO: 15):

[0159] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATGTTGTGATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTGAAAGCTATTTAGCCTGGTATCAGCAGAAACCAGGACAGCGTCCCAAGCTCCTGATCTACAAGGCTTCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTATATTTATAATGATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0160] Full-length nucleotide sequence of B0011-L (rabbit IgG1) (SEQ ID NO: 16):

[0161] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGCCCAAGTGATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCGGGCCAGTGAGGACATTGAAAGCTATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAAGGCTTCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTATACTTATAATGATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0162] B0002-H CDR1 amino acid sequence (SEQ ID NO:17): GFSLISYG; B0002-H CDR2 amino acid sequence (SEQ ID NO:18): ISTGGST; B0002-H CDR3 amino acid sequence (SEQ ID NO:19): ARGFTNSYI; B0002-LCDR1 amino acid sequence (SEQ ID NO:20): QNVYSNDY; B0002-L CDR2 amino acid sequence (SEQ ID NO:21): ETS; B0002-L CDR3 amino acid sequence (SEQ ID NO:22): AGGYSGNILT; B0005-H CDR1 amino acid sequence (SEQ ID NO:23): GIDLSSYG; B0005-H CDR2 amino acid sequence (SEQ ID NO:24): IGSGST; B0005-HCDR3 amino acid sequence (SEQ ID NO:26): ARLYNI; B0005-L CDR1 amino acid sequence (SEQ ID NO:17): GFSLISYG; B0002-H CDR2 amino acid sequence (SEQ ID NO:18): ISTGGST; B0002-H CDR3 amino acid sequence (SEQ ID NO:26): ARLYNI; B0005-L CDR1 amino acid sequence (SEQ ID NO:17): GFSLISYG; B0002-H CDR2 amino acid sequence (SEQ ID NO:24): IGSGST; B0005-HCDR3 amino acid sequence (SEQ ID NO:26): ARLYNI; NO:26): QSVYSNNY; B0005-L CDR2 amino acid sequence (SEQ ID NO:27): KAS; B0005-L CDR3 amino acid sequence (SEQ ID NO:28): LGDYGCDPADCYN; B0007-H CDR1 amino acid sequence (SEQ ID NO:29): GLSLSRNA; B0007-HCDR2 amino acid sequence (SEQ ID NO:30): ISKDGYT; B0007-H CDR3 amino acid sequence (SEQ ID NO:31): VRWNFSSDNI; B0007-L CDR1 amino acid sequence (SEQ ID NO:32): EDIESY; B0007-L CDR2 amino acid sequence (SEQ ID NO:33): KAS; B0007-L CDR3 amino acid sequence (SEQ ID NO:34): QQGYIYNDVDNV; B0011-HCDR1 amino acid sequence (SEQ ID NO:26): QSVYSNNY; B0005-L CDR2 amino acid sequence (SEQ ID NO:27): KAS; B0005-L CDR3 amino acid sequence (SEQ ID NO:28): LGDYGCDPADCYN; B0007-H CDR1 amino acid sequence (SEQ ID NO:29): GLSLSRNA; B0007-HCDR2 amino acid sequence (SEQ ID NO:30): ISKDGYT; B0007-H CDR3 amino acid sequence (SEQ ID NO:34): QQGYIYNDVDNV; B0011-HCDR1 amino acid sequence (SEQ ID NO:26): QSVYSNNY; B0005-L CDR2 amino acid sequence (SEQ ID NO:27): KAS; B0005-L CDR3 amino acid sequence (SEQ ID NO:34): QQGYIYNDVDNV; B0011-HCDR1 amino acid sequence (SEQ ID NO:28): QSVYSNNY; B0005-L CDR3 amino NO:35): GLSLSSNA; B0011-H CDR2 amino acid sequence (SEQ ID NO:36): ISNNGYT; B0011-H CDR3 amino acid sequence (SEQ ID NO:37): VRWNYSSDNI; B0011-L CDR1 amino acid sequence (SEQ ID NO:38): EDIESY; B0011-L CDR2 amino acid sequence (SEQ ID NO:39): KAS; B0011-L CDR3 amino acid sequence (SEQ ID NO:40): QQGYTYNDVDNV.

[0163] B0002-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:41):

[0164] QSLEESEGGLFKPTDTLTLTCTVSGFSLISYGVTWVRQAPGNGLEWIAFISTGGSTYYASWAKSRSTITRKTNLNTVTLKMTSLTAADTATYFCARGFTNSYIWGPGTLVTVSS.

[0165] B0005-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:42):

[0166] QSVKESEGGLFKPTETLTLTCTVSGIDLSSYGVTWVRQAPGNGLEYIGTIGGSGSTYYASWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCARLYNIWGPGTLVTVSS.

[0167] B0007-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:43):

[0168] QEQLEESGGRLVKPDETLTLTCTVSGLSLSRNAITWVRQAPGKGLQYIGIISKDGYTFYANWAKGRFTISKTSTTVDLKITTPTTEDTATYFCVRWNFSSDNIWGPGTLVTVSS.

[0169] B0011-H variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:44):

[0170] QQQLEESGGRLVKPDETLTLTCTVSGLSLSSNAITWVRQAPGKGLEYIGIISNNGYTFYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRWNYSSDNIWGPGTLVTVSS.

[0171] B0002-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:45):

[0172] AAVLTQTPSPVSAAVGGTVSISCQSSQNVYSNDYLSWYQQKPGQRPKLLISETSTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSGNILTFGGGTEVLVK.

[0173] B0005-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:46):

[0174] AVVLTQTPSSVSAAVGGTVTINCQSSQSVYSNNYLSWYQQKPGQPPKLLIYKASTLPSGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGDYGCDPADCYNFGGGTEVVVK.

[0175] B0007-L variable region amino acid sequence (rabbit IgG1) (SEQ ID NO:47):

[0176] DVVMTQTPASVEVAVGGTVTIKCQASEDIESYLAWYQQKPGQRPKLLIYKASTLASGVPSRFKGSGSGTQYTLTISGVQCDDAATYYCQQGYIYNDVDNVFGGGTEVVVK.

[0177] B0011-L variable region amino acid sequence (rabbit κ light chain) (SEQ ID NO:48):

[0178] AQVMTQTPASVEVAVGGTVTINCRASEDIESYLAWYQQKPGQRPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQQGYTYNDVDNVFGGGTEVVVK.

[0179] B0002-H variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO:49):

[0180] CAGTCGTTGGAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTTATTAGTTATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAGTGGATCGCATTCATTAGTACTGGTGGTAGCACATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAAGACCAACCTGAACACGGTGACTCTGAAGATGACCAGTCTGACAGCCGCGGACACGGCCACATATTTCTGTGCGAGAGGATTTACTAATTCTTATATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA。

[0181] B0005-H variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO: 50):

[0182] CAGTCGGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGAGACCCTGACACTCACCTGCACAGTCTCTGGAATTGACCTCAGTAGCTATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATATATCGGAACCATTGGTGGTAGTGGTAGCACATACTACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACGAGAACACGGTGACTCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGACTCTATAATATTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA。

[0183] B0007-H variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO: 51):

[0184] CAGGAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCAAGCCTGACGAAACCCTGACACTCACCTGCACCGTCTCTGGATTATCCCTCAGTCGCAATGCAATAACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAATACATCGGAATCATTAGTAAAGATGGTTACACCTTCTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACTTCGACCACGGTGGATCTGAAAATCACCACTCCGACAACCGAGGACACGGCCACCTATTTCTGTGTCAGATGGAATTTTAGTAGTGACAATATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA.

[0185] Nucleotide sequence of B0011-H variable region (rabbit IgG1) (SEQ ID NO: 52):

[0186] CAGCAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCAAGCCTGACGAAACCCTGACACTCACCTGCACCGTCTCTGGATTATCCCTCAGTAGCAATGCAATAACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAGTAATAATGGTTACACCTTCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACTTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGTCAGATGGAATTATAGTAGTGACAATATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA.

[0187] Nucleotide sequence of B0002-L variable region (rabbit IgG1) (SEQ ID NO: 53):

[0188] GCAGCCGTGCTGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAATGTTTATAGTAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTCCGAAACATCCACTCTGGCATCTGGGGTCCCATCGCGGTTTAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTGGTAATATTCTTACTTTTGGCGGAGGGACCGAGGTGCTCGTCAAA.

[0189] Nucleotide sequence of B0005-L variable region (rabbit IgG1) (SEQ ID NO: 54):

[0190] GCCGTCGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAGTAACAACTACTTATCCTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACAAGGCTTCCACTCTGCCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTATGGTTGTGATCCTGCTGATTGTTATAATTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0191] Nucleotide sequence of B0007-L variable region (rabbit IgG1) (SEQ ID NO: 55):

[0192] GATGTTGTGATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGGACATTGAAAGCTATTTAGCCTGGTATCAGCAGAAACCAGGACAGCGTCCCAAGCTCCTGATCTACAAGGCTTCCACTCTGGCA TCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTATATTTATAATGATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0193] B0011-L variable region nucleotide sequence (rabbit IgG1) (SEQ ID NO:56):

[0194] GCCCAAGTGATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCGGGCCAGTGAGGACATTGAAAGCTATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTACAAGCTTCCACTCTGGCA TCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAACAGGGTTATACTTATAATGATGTTGATAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA.

[0195] 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 ApoE, characterized in that: The monoclonal antibody is HZK31-B0002; The heavy chain variable region CDR1, CDR2, and CDR3 sequences of HZK31-B0002 are shown in SEQ ID NO:17-SEQ ID NO:19, respectively. The light chain variable regions CDR1, CDR2, and CDR3 sequences of HZK31-B0002 are shown in SEQ ID NO:20-SEQ ID NO:22, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The HZK31-B0002 includes: The heavy chain variable region sequence shown in SEQ ID NO:41 and the light chain variable region sequence shown in SEQ ID NO:

45.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The HZK31-B0002 includes: The heavy chain sequence shown in SEQ ID NO:1 and the light chain sequence shown in SEQ ID NO:

5.

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 pharmaceutical composition, a diagnostic reagent, or a diagnostic kit, characterized in that, The pharmaceutical composition, detection reagent or detection kit comprises any one of the monoclonal antibodies 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; The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

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

9. 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 pharmaceutical composition, a detection reagent, or a detection kit; wherein the pharmaceutical composition, detection reagent, or detection kit is used to monitor ApoE levels.

Images