Monoclonal antibody combination for detecting apolipoprotein E4 and use thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting apolipoprotein E4 and application thereof. The combination comprises monoclonal antibody 4E10 and monoclonal antibody 3H7, and the complementarity determining region (CDR) sequences thereof are shown as SEQ ID NO. 1 to SEQ ID NO. 12. In a double antibody sandwich ELISA, 4E10 is used as a coating antibody, and 3H7 is used as a labeled antibody, so that APOE4 subtypes can be effectively recognized, high specificity and high sensitivity detection of APOE4 can be realized, the detection limit reaches 100 pg / mL, and the method is suitable for quantitative detection of APOE4 in serum, cerebrospinal fluid and the like. The method provides a reliable immunological tool for early risk assessment of Alzheimer's disease, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for detecting apolipoprotein E4 and its application. Background Technology

[0002] Apolipoprotein E (APOE) is a polymorphic protein with significant biological and clinical diagnostic value. It regulates lipoprotein metabolism and cholesterol transport in the cardiovascular and central nervous systems, and these functions are highly dependent on different isoforms and lipidation states. In humans, APOE consists of 299 amino acids with a molecular weight of 34 kD and has three main isoforms: APOE2, APOE3, and APOE4, encoded by the APO α2, α3, and α4 alleles, respectively. Wild-type APOE3 is the most common (approximately 78%), while APOE2 and APOE4 are considered variants. The differences between APOE2, APOE3, and APOE4 are minimal, differing only in amino acids at positions 112 and 158: APOE2 is 112 Cys, 158 Cys; APOE3 is 112 Cys, 158 Arg; and APOE4 is 112 Arg, 158 Arg. APOE2 has a weak binding affinity to the low-density lipoprotein receptor (LDLR), leading to its association with high plasma cholesterol levels and making it a risk factor for atherosclerosis. APOE4 significantly increases total cholesterol concentration and plasma LDL levels in healthy individuals, playing a crucial role in blood lipid transport and metabolism, and the clearance of β-amyloid protein (Aβ) from the brain. It is considered a major genetic risk factor for early-onset Alzheimer's disease (AD).

[0003] Given that APOE protein is associated with risk factors for various diseases, its significance for disease risk management and prediction has received increasing attention. APOE testing typically involves genotyping, which uses specific molecular biology techniques (such as PCR, genotyping chips, and sequencing) to detect specific loci in the APOE gene, identifying the ε2, ε3, or ε4 alleles, thereby determining an individual's genotype (e.g., ε3 / ε3, ε3 / ε4, etc.). Studies have found that heterozygous (ε2 / ε4, ε3 / ε4) or homozygous ε4 gene (ε4 / ε4) carriers have a significantly higher chance of developing Alzheimer's disease (AD) than non-carriers. Compared with the ApoE ε3 allele, carrying one ApoE ε4 allele (heterozygote) can increase the risk of sporadic Alzheimer's disease by about 3.7 times, while carrying two ApoE ε4 alleles (homozygote) can increase the risk by about 12 times. The APOE ε4 allele is considered to be the strongest known genetic risk factor for sporadic Alzheimer's disease. More than 40% of sporadic Alzheimer's patients are ApoE ε4 positive, and ApoE4 genotyping may be one of the most valuable indicators for early diagnosis of AD.

[0004] Differences in APOE genotypes lead to variations in protein expression. Multiple studies have measured APOE protein concentrations in the cerebrospinal fluid (CSF) of AD patients and cognitively normal controls. Results show that APOE protein levels in the CSF of AD patients are significantly lower than in cognitively normal individuals, and this reduction is particularly pronounced in APOE ε4 allele carriers. Among APOE proteins, APOE4 is considered less functional than APOE2 or APOE3 in areas such as lipid binding, promoting Aβ clearance, and supporting neuronal repair. Therefore, reduced APOE4 concentration and functional impairment increase the risk of AD and may play an important role in the development of AD.

[0005] The ApoE ε4 gene is a well-established risk gene for sporadic Alzheimer's disease and is recommended as an important early warning risk factor for Alzheimer's disease, especially for individuals with a family history of Alzheimer's. Patients carrying the ApoE ε4 gene who experience subjective cognitive decline or mild cognitive impairment are at high risk of dementia progression. Timely disclosure of ApoE ε4 gene testing results is crucial for early diagnosis, assessment, and treatment, and for preventing disease progression. However, current APOE protein detection technologies have limitations. Most commercially available APOE protein testing kits are based on immunoturbidimetric techniques, which can only measure the concentration of total APOE protein in a sample and cannot effectively distinguish between the three subtypes: APOE2, APOE3, and APOE4. Therefore, a tool that can directly and specifically detect APOE4 protein is lacking as an immunological alternative or supplementary method to the ε4 allele detection in APOE genotyping. Summary of the Invention

[0006] In view of the above-mentioned shortcomings and deficiencies in the prior art, the present invention provides a monoclonal antibody combination for detecting apolipoprotein E4 (APOE4) and its application, aiming to solve the technical problems that existing detection methods cannot specifically identify APOE4 protein and are difficult to distinguish APOE subtypes.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, this application provides a monoclonal antibody combination for detecting apolipoprotein E4, the monoclonal antibody combination including monoclonal antibody 4E10 and monoclonal antibody 3H7;

[0009] The heavy chain variable region of monoclonal antibody 4E10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0010] The light chain variable region of monoclonal antibody 4E10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0011] The heavy chain variable region of monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0012] The light chain variable region of monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0013] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.14.

[0014] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.16.

[0015] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO. 18.

[0016] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 20.

[0017] In a further embodiment, apolipoprotein E4 includes recombinant apolipoprotein E4 and natural apolipoprotein E4.

[0018] Secondly, the application of the above-mentioned combination of monoclonal antibodies in the preparation of tools for detecting apolipoprotein E4.

[0019] In a further embodiment, the tools include reagents, kits, test strips, and antibody chips.

[0020] In a further embodiment, the kit includes a double-antibody sandwich ELISA kit.

[0021] In a further embodiment, the ELISA kit uses monoclonal antibody 4E10 as the coating antibody and monoclonal antibody 3H7 as the labeling antibody.

[0022] Beneficial effects:

[0023] This invention provides a monoclonal antibody combination for detecting apolipoprotein E4 (APOE4), comprising monoclonal antibody 4E10 and monoclonal antibody 3H7, with CDR sequences shown in SEQ ID NO. 1-6 and SEQ ID NO. 7-12, respectively. 4E10 specifically recognizes the key epitope of arginine at position 112 of the APOE4 protein, enabling efficient capture of APOE4 in the sample without cross-reactivity with APOE2 or APOE3. 3H7 binds to a conserved region common to the APOE protein, making it suitable for detecting total APOE expression levels. In a double-antibody sandwich ELISA system, 4E10 is used as the coating antibody to capture APOE4, and 3H7 is used as the labeling antibody for signal amplification, achieving highly specific and sensitive detection of APOE4 protein, with a detection limit of up to 100 pg / mL. This antibody combination can effectively distinguish APOE4 from other subtypes (APOE2 / APOE3), that is, effectively identify the APOE4 subtype. It is suitable for detection in various biological samples such as serum and cerebrospinal fluid, and provides a reliable immunological tool for early risk assessment and warning of diseases such as Alzheimer's disease, which has important clinical application value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Image showing the results of SDS-PAGE protein identification;

[0026] Figure 2 The diagram shows the identification results of recombinant proteins APOE2 (A), APOE3 (B), and APOE4 (C).

[0027] Figure 3 For the specificity and sensitivity analysis of double antibody sandwich ELISA;

[0028] Figure 4 The binding reaction of recombinant proteins APOE2 (left), APOE3 (middle), and APOE4 (right) with monoclonal antibodies. Detailed Implementation

[0029] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0030] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0031] Currently, most APOE genotyping methods rely on sequencing, which is costly and complex. While APOE protein detection primarily uses immunoturbidimetry to measure total APOE protein concentration, this method cannot accurately distinguish between different protein subtypes. Furthermore, there are currently no specific monoclonal antibodies or methods for detecting APOE4 protein. Therefore, establishing a method that can specifically detect APOE4 protein as a supplement to individual APOE genotyping remains essential. This method could potentially be more cost-effective, simpler to operate, and directly reflect the presence and levels of functional proteins in the body.

[0032] Recombinant apolipoprotein E4 refers to apolipoprotein E4 that is artificially expressed and produced using gene recombination technology.

[0033] Natural apolipoprotein E4 refers to the APOE4 protein obtained by direct isolation and purification from biological samples such as blood and cerebrospinal fluid of humans or other organisms.

[0034] This invention utilizes hybridoma technology to screen for APOE4 monoclonal antibodies, which have been verified to specifically detect APOE4 protein without cross-contamination with APOE2 and APOE3 proteins. This provides significant reference value for serum APOE protein detection and even early warning diagnosis of Alzheimer's disease. The technical solution of this application, combined with specific APOE4 typing or total APOE protein detection, can comprehensively assess an individual's APOE protein profile, facilitating more accurate Alzheimer's disease risk stratification and early warning, and improving the accuracy of clinical diagnosis.

[0035] Example 1

[0036] 1. Expression of recombinant proteins APOE2, APOE3, and APOE4

[0037] Based on the APOE4 amino acid sequence AAB59397.1, sequence analysis was performed, the N-terminal signal peptide (1-18 aa) was removed, and a start codon ATG was added to the N-terminus. The gene was synthesized by Qingke Biotechnology and cloned into the pET32a vector, and its nucleotide sequence is shown in (SEQ ID NO.21):

[0038] AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTCGTGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0039] The amino acid sequence is (shown in SEQ ID NO.22):

[0040] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0041] APOE2 is created by site-directed mutation of APOE4's 112R and 158R to 112C and 158C, respectively, and then cloned into the pET32a vector.

[0042] The nucleotide sequence is shown in SEQ ID NO.23:

[0043] AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAATGCCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0044] The amino acid sequence is (shown in SEQ ID NO.24):

[0045] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKCLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0046] APOE3 is created by site-directed mutation of APOE4's 112R to 112C and then cloned into the pET32a vector.

[0047] The nucleotide sequence is shown in SEQ ID NO.25:

[0048] AAAGTGGAACAGGCTGTTGAAACCGAACCAGAACCGGAACTTCGTCAGCAGACCGAATGGCAGTCTGGTCAGCGTTGGGAACTGGCTCTGGGTCGTTTCTGGGACTACCTGCGTTGGGTTCAGACCTTGTCTGAACAGGTTCAAGAAGAACTGCTGTCTTCTCAGGTTACTCAAGAACTGCGTGCGTTGATGGACGAAACCATGAAAGAACTGAAAGCGTACAAATCTGAACTGGAAGAACAGCTGACTCCAGTTGCAGAAGAAACTCGTGCTCGTCTGTCTAAAGAACTTCAGGCGGCTCAGGCGCGTCTGGGTGCTGACATGGAAGACGTTTGCGGTCGTCTGGTTCAGTACCGTGGTGAAGTTCAGGCAATGCTGGGTCAGTCTACCGAAGAACTGCGTGTTCGTCTGGCGAGCCATCTGCGTAAACTGCGTAAACGTCTGCTGCGTGATGCTGATGACCTTCAGAAACGTCTGGCTGTTTACCAGGCAGGTGCTCGTGAAGGTGCAGAACGTGGTCTGTCTGCTATCCGTGAACGTTTGGGTCCGCTGGTTGAACAGGGTCGTGTACGTGCAGCTACCGTTGGTTCTCTGGCAGGTCAGCCACTGCAAGAACGTGCTCAGGCATGGGGTGAACGTCTGCGTGCTCGTATGGAAGAAATGGGTTCTCGTACCCGTGATCGTCTGGACGAAGTGAAAGAACAGGTTGCTGAAGTTCGTGCGAAACTGGAAGAACAGGCGCAGCAGATCCGCCTCCAGGCGGAAGCGTTCCAGGCTCGTCTTAAGAGCTGGTTCGAGCCACTGGTTGAAGACATGCAGCGTCAGTGGGCAGGTCTGGTTGAGAAAGTTCAGGCTGCGGTTGGTACTTCCGCAGCTCCGGTTCCGTCTGACAACCAC。

[0049] The amino acid sequence is (shown in SEQ ID NO.26):

[0050] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLR DADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH.

[0051] Recombinant plasmids pET32a-APOE2, pET32a-APOE3, and pET32a-APOE4 were transformed into BL21(DE3) competent cells and induced to express their contents using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium (containing 100 μg / mL ampicillin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until the OD600 reached 0.6-0.9. A final concentration of 0.1 mMIPTG was added, and the cells were induced at 30°C with shaking at 200 rpm for 4 hours before collection.

[0052] In the following text, when APOE2, APOE3 and APOE4 recombinant proteins are mentioned together, they will be collectively referred to as APOE2 / 3 / 4 recombinant proteins.

[0053] 2. Purification of APOE2 / 3 / 4 recombinant protein

[0054] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purification process was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C. Figure 1 In the middle, M represents the protein marker, and numbers 1, 2, and 3 represent the purified APOE2 / 3 / 4 recombinant proteins, respectively. All three target protein bands are located around 55 kDa, which is consistent with the expected molecular weights (APOE2: 53.0 kDa, APOE3: 53.0 kDa, APOE4: 52.4 kDa), indicating successful expression and effective purification of the recombinant proteins. Each protein band is clear, single, and free of obvious impurities, indicating high purity.

[0055] 3. Identification of APOE2 / 3 / 4 recombinant protein

[0056] Purified APOE2 / 3 / 4 recombinant proteins were coated onto microplates, and their reaction with APOE positive antibodies was identified by indirect ELISA. The APOE antibody was a commercially available APOE polyclonal antibody (Wuhan Sanying, 30535-1-AP). First, the recombinant proteins were coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. Then, the plates were washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. Polyclonal antibodies were diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of a microplate coated with the antigen. A negative control was prepared by diluting purified rabbit IgG polyclonal antibody at the same concentration. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-rabbit secondary antibody (Solepro, diluted 5000 times with PBS) was added. The plates were incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to terminate the reaction. The OD450 nm value was measured using a microplate reader. Results are as follows: Figure 2 The APOE2 / 3 / 4 antigens and APOE polyclonal antibodies both showed binding reactions, and the purified APOE2 / 3 / 4 recombinant protein could be used for subsequent experiments.

[0057] 4. Screening of APOE4 monoclonal antibodies

[0058] Based on sequence analysis, the peptide was synthesized using conventional solid-phase peptide synthesis. Peptides with amino acid sequences 110-117aa were selected for design and synthesis. The resulting APOE4-specific peptide, with the amino acid sequence shown in SEQ ID NO.27 (DVRGRLVQ), was named ppAPOE4 and conjugated to KLH (for the immunization phase) and BSA (for the screening and identification phase), respectively. The peptide synthesis was performed by Wuhan Baiyixin. The synthesized peptide was used for mouse immunization and monoclonal antibody screening.

[0059] 4.1 Mouse Immunization

[0060] To more effectively screen for APOE4-specific monoclonal antibodies targeting the differentially expressed locus 110-117aa, mouse immunization was performed using KLH-conjugated ppAPOE4 peptide. Specifically, female BALB / c mice were immunized subcutaneously at multiple sites after 6 weeks of age with a dose of 20 μg / mouse, after mixing the KLH-ppAPOE4 peptide with an equal volume of Freund's complete adjuvant (total volume 200 μL). In week 2, a booster immunization was performed intramuscularly with a dose of 20 μg / mouse mixed with an equal volume of MF59 adjuvant. In weeks 4 and 6, the spleens of mice were directly injected with insulin at a dose of 5 μg / mouse. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization with 20 μg of KLH-ppAPOE4 peptide via intraperitoneal pulse, and the spleens were collected 3 days later for hybridoma cell preparation.

[0061] 4.2 Screening of hybridoma cells

[0062] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones were screened using an indirect ELISA method to obtain those that were positive for both BSA-ppAPOE4 peptide and recombinant APOE4 protein, and negative for both APOE2 and APOE3 recombinant proteins. Since the immunogen was a KLH vector-conjugated peptide, the BSA vector-conjugated peptide was used for detection to screen for specific cell lines targeting APOE4 protein. Positive cells were cloned to a monoclonal state using a limiting dilution method, and then the cell lines were expanded and cryopreserved.

[0063] Indirect ELISA method for screening positive clones:

[0064] Recombinant APOE2, APOE3, and APOE4 proteins, as well as BSA-ppAPOE4 peptide, were coated into microplates using a carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at a concentration of 1 μg / mL. The plates were incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well. The plates were incubated at 37°C for 2 hours, followed by washing once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and blotted dry. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, add 50 μL / well of TMB chromogenic buffer, and incubate at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to terminate the reaction, and measure the OD450nm value using a microplate reader. Positive cell lines that react with both BSA-ppAPOE4 peptide and APOE4 recombinant protein, but not with APOE2 or APOE3 recombinant proteins, were selected for subsequent experiments. The screening results are shown in Table 1.

[0065] Table 1. Screening results of unpurified APOE4 monoclonal antibodies

[0066]

[0067] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing approximately 2.5 × 10⁻⁶ cells) was injected intraperitoneally into female BALB / c mice. 6 (1 cell), about 10 days later, when the mouse abdomen was significantly swollen, ascites fluid was collected using a sterile syringe needle.

[0068] 5. Purification of APOE4 monoclonal antibody

[0069] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0070] 6. Identification of purified APOE4 monoclonal antibody

[0071] The purified antibody was tested for its binding activity with APOE4 using the aforementioned indirect ELISA method. The antibody was coated with BSA-ppAPOE4 peptide and recombinant proteins APOE2, APOE3, and APOE4, respectively, and diluted to 10 μg / ml, 1 μg / ml, and 100 ng / ml for detection. Table 2 shows only the results at 1 μg / ml. Finally, monoclonal antibodies 1E4, 4E10, 8F7, and 8F9 were selected for downstream assays.

[0072] Table 2. Identification results of purified APOE4 monoclonal antibodies

[0073]

[0074] 7. Screening of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4 recombinant proteins

[0075] 7.1 Mouse Immunization

[0076] Since the three recombinant proteins APOE2, APOE3, and APOE4 differ only at a few amino acid sites, to obtain monoclonal antibodies more efficiently, mice were immunized with high-purity APOE3, and monoclonal antibody screening was performed using APOE2, APOE3, APOE4, and a reverse screening antigen. Specifically, purified APOE3 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. In week 2, a booster immunization was performed by intramuscular injection of a mixture of 20 μg / mouse and an equal volume of MF59 adjuvant. In weeks 4 and 6, mice were immunized by direct injection of insulin into the spleen at a dose of 5 μg / mouse. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of APOE4 recombinant protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0077] 7.2 Screening of hybridoma cells

[0078] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for APOE2, APOE3, and APOE4 recombinant proteins were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET32a vector containing Trx and His tags, background components needed to be screened to identify specific cell lines targeting APOE protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0079] Indirect ELISA method for screening positive clones:

[0080] Recombinant proteins APOE2, APOE3, and APOE4, as well as other recombinant proteins of the pET32a vector (pET32a-HPV18 / E7, His tag, preparation method see patent 202510855254.X), were coated in microplates. The coating buffer was carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6. The coating concentration was 1 μg / mL, and the plate was incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well, incubated at 37°C for 2 hours, and then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4). The plates were then blotted dry. 50 μL of cell culture supernatant was added, and the plate was incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to terminate the reaction. Measure the OD450nm value using a microplate reader. Select positive cell lines that react with APOE2, APOE3, and APOE4 recombinant proteins but not with the control recombinant protein for subsequent experiments.

[0081] Table 3. Screening results of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4

[0082]

[0083] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing approximately 2.5 × 10⁻⁶ cells) was injected intraperitoneally into female BALB / c mice. 6 (1 cell), about 10 days later, when the mouse abdomen was significantly swollen, ascites fluid was collected using a sterile syringe needle.

[0084] 8. Purification of monoclonal antibodies that cross-react with APOE2 / APOE3 / APOE4

[0085] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0086] Using the indirect ELISA method described above, the purified antibody was diluted to 1 μg / ml, and the reaction of the antibody with recombinant proteins APOE2, APOE3, and APOE4 was identified. Other recombinant proteins of the pET32a vector (pET32a-HPV18 / E7, His tag) were used as negative antigen controls. The results are shown in Table 4.

[0087] Table 4. Identification results of purified monoclonal antibodies that exhibit cross-reactivity with APOE2 / APOE3 / APOE4

[0088]

[0089] 9. Specific detection of APOE4 using a paired double-antibody sandwich ELISA

[0090] To improve the efficiency of pairing screening, monoclonal antibodies with high binding titers were selected for downstream experiments. Since the monoclonal antibodies used for peptide screening are APOE4-specific, while those used for recombinant protein screening may be APOE2 / 3 / 4 cross-reactive, they were divided into two groups during pairing screening: an APOE4 epitope-specific monoclonal antibody group and an APOE2 / 3 / 4 cross-reactive monoclonal antibody group. These groups were HRP-labeled and coated, with the APOE4 epitope-specific monoclonal antibody group coated with the APOE2 / 3 / 4 cross-reactive monoclonal antibody group for combination screening, and vice versa. This approach aimed to efficiently obtain monoclonal antibody pairings that specifically recognize APOE4. Based on the antibody identification results, monoclonal antibodies with a binding detection value higher than 1.5 with APOE4 protein at a 1ug / ml dilution were selected for pairing in the APOE4 epitope-specific monoclonal antibody group. For the cross-reaction of APOE2 / APOE3 / APOE4, the purified monoclonal antibodies selected were monoclonal antibodies 3G5, 3H7, 3F11 and 4D1 (all of which had high readings with the three antigens).

[0091] HRP-labeled monoclonal antibody

[0092] The selected labeled antibody was diluted to a final concentration of 2 mg / mL using carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). 2 mg of HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 1 mg (0.5 mL) of the diluted antibody solution was added to the matching tube containing HRP, and the mixture was pipetted and incubated at room temperature for 1 hour, with regular mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0093] Screening of paired monoclonal antibodies

[0094] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was patted dry and blocked with 3% sucrose and 2% BSA at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. The test antigen APOE4 and control antigens APOE2 and APOE3 recombinant proteins were diluted to 100 ng / ml with PBS and added to an ELISA plate at 50 μL / well. The plate was incubated at 37°C for 35 min. After washing four times with PBST, the plate was blotted dry. Then, 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS was added and the plate was incubated at 37°C for 35 min. The plate was washed four more times and blotted dry. 50 μL / well of TMB chromogenic buffer was added and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD was measured using an ELISA reader. 450 nm value. The results are shown in Tables 5 and 6. The coated antibody and labeled antibody have the highest P / N value.

[0095] Table 5: Pairing results and P / N ratio analysis of APOE cross-antibody coating and APOE4-specific antibody HRP marker.

[0096]

[0097] Table 6: Pairing results and P / N value analysis of APOE4 specific antibody coating and APOE cross-antibody HRP labeling.

[0098]

[0099] The data in Tables 5 and 6 show that the combination of APOE4-specific antibody 4E10 coating and APOE cross-antibody 3H7-HRP labeling results in the highest P / N value.

[0100] Using the APOE4-specific monoclonal antibody 4E10 as the coating antibody, the APOE4 protein in the sample can be captured efficiently and specifically. The APOE cross-reactive monoclonal antibody 3H7, as the HRP-labeled detection antibody, further enhances the signal intensity. Since APOE2 and APOE3 proteins cannot be effectively recognized by 4E10, they are not captured; even if 3H7 binds to them, no positive signal is generated. This combination achieves highly sensitive and specific detection of APOE4.

[0101] 10. Optimization of the double-antibody sandwich ELISA method

[0102] The purified monoclonal antibody 4E10 was diluted with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose and 2% BSA at 150 μL / well. The plates were incubated at 37°C for 2 h, and the blocking buffer was discarded. The test antigen (recombinant APOE4 protein) and control antigen (mixed APOE2 and APOE3) were diluted with PBS at 100 ng / mL and added to the microplates at 50 μL / well. The plates were incubated at 37°C for 35 min, washed four times with PBST, and then coated with PBS. HRP-labeled monoclonal antibody 3H7 diluted 1000, 2000, and 3000 times, 50 μL / well, was incubated at 37°C for 35 min. After washing the plate four times and patting it dry, 50 μL / well of TMB chromogenic buffer was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction, and the OD was measured using a microplate reader. 450 nm value. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The screening process is shown in Table 7.

[0103] Table 7: Optimization Results of Double Antibody Sandwich ELISA

[0104]

[0105] As shown in Table 7, the optimal conditions for the double-antibody sandwich ELISA method are: monoclonal antibody 4E10 coated with antibody at a concentration of 1 μg / mL and HRP-labeled monoclonal antibody 3H7 diluted 2000 times. This detection method is not used for disease diagnosis and treatment.

[0106] 11. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting APOE4

[0107] After determining the optimal coating concentration of 1 μg / mL and the HRP-labeled monoclonal antibody dilution of 2000-fold, and following the above detection steps, APOE4 recombinant protein was first serially diluted with PBS buffer to concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL. Simultaneously, APOE2 and APOE3 recombinant proteins were added to each well at the same concentration (50 μL) for detection, to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 3 It can be seen that the double-antibody sandwich ELISA composed of this group of paired antibodies still showed a positive reaction when the APOE4 recombinant protein was diluted to 100pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0108] 12. Identification of binding activity of paired antibodies

[0109] Following the aforementioned indirect ELISA method, paired monoclonal antibodies 4E10 and 3H7 were serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Another murine-derived unrelated monoclonal antibody, HPV18 E7 monoclonal antibody (Santa Cruz, F-7), was used as a negative control (Ctrl). The binding activity of the antibodies to the APOE2 / 3 / 4 recombinant protein was measured. Results are as follows... Figure 4 The results showed that 4E10 and 3H7 still reacted positively with APOE4 at a dilution of 1 ng / ml, and 3H7 still reacted positively with APOE2 / 3 at a dilution of 10 ng / ml. 4E10 did not react with APOE2 / 3. Both antibodies had high antibody activity. Figure 4 The results, in order, show the binding reactions of recombinant proteins APOE2 (left), APOE3 (middle), and APOE4 (right) with the two antibodies.

[0110] 13. Variable region gene sequence of monoclonal antibodies

[0111] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0112] Sequence of the coated monoclonal antibody 4E10:

[0113] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.18:

[0114] GACATTTGTGATGTCACAGTCTCCATCCAGTCTGTTTGCATTCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAACTGGTACCAGCAGAAACCAGGAAATATTCCTAAAGTATTGATCTATGAGGCTTCCAACTTGCAC ACAGGGCTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACACTAATCATCAGGAACCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACACGGTCAAAGTTATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0115] The amino acid sequence of the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.14:

[0116] DIVMSQSPSSLFAFLGDTITITCHASQNINVWLNWYQQKPGNIPKVLIYEASNLHTGVPSRFSGSGSGTGFTLIIRNLQPEDIATYYCQHGQSYPRTFGGGTKLEIKRTV.

[0117] Light chain CDR area annotation:

[0118] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.4: HASQNINVWLN;

[0119] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO. 5:

[0120] CDR-L2: EASNLHT;

[0121] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO. 6:

[0122] CDR-L3: QHGQSYPRT.

[0123] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.17:

[0124] GAGTTCCAGCTGCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACAGTTTTACTAACTACTGGATACAGTGGGTAAAACAGAGGCCTGGACAGGGTTCTGGAATGGATTGGGGCTATTTATCCTGGAGATGGTGATACTAGATA CCCTCAGAAGTTCAGGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCTTGACTTCTGAGGACTCTGCGGTCTATTTTTGTTCAAGAGAGGTACGATCTTTTTATTTTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0125] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.13:

[0126] EFQLQQSGAELARPGASVKLSCKASGYSFTNYWIQWVKQRPGQGLEWIGAIYPGDGDTRYPQKFRGKATLTADKSSSTAYMQLSSLTSEDSAVYFCSREVRSFYFLDYWGQGTSVTVSS.

[0127] Heavy chain CDR region annotation:

[0128] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.1:

[0129] CDR-H1: NYWIQ;

[0130] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.2:

[0131] CDR-H2: AIYPGDGDTRYPQKFRG;

[0132] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 4E10 is shown in SEQ ID NO.3:

[0133] CDR-H3: EVRSFYFLDY.

[0134] The sequence of HRP-labeled monoclonal antibody 3H7:

[0135] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.20:

[0136] GATATCCAGATGACACAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCACCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGGTGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCCGGTGTCTACA CTGGACTCTGGAGTCCCTGACAGGTTCGCTGGCAGTGAATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGACTTTATTATTGCTGGCAAGGTACACATTTTCCCACGTGGGCGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0137] The amino acid sequence of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.16:

[0138] DIQMTQTPLTLSVTIGQPASTSCKSSQSLLDSGGKTYLSWLLQRPGQSPKRLIYPVSTLDSGVPDRFAGSESGTDFTLKISRVEAEDLGLYYCWQGTHFPTWAFGGGTKLEIKRTV.

[0139] Light chain CDR area annotation:

[0140] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 10:

[0141] CDR-L1: KSSQSLLDSGGKTYLS;

[0142] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 11:

[0143] CDR-L2: PVSTLDS;

[0144] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 12:

[0145] CDR-L3: WQGTHFPTWA.

[0146] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.19:

[0147] CAGCGTGAGCTGCAGCAGTCTGGTGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCGGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCCGAGAAGAGGCTGGAATGGGTCGCATCTATTGGTAATGGTGGTAGCGCCTTCTATCC AGACAGTGTGAAGGGCCGATTCACCATCTCCAGAAATAATGCCGGGAACACCCTGTTCCTGCAAATGAGCAGTCTGAGGTCTGAAGACACGGCCATGTATTATTGTACAAGAGGCGGGGACTATTATGATTACGACGGGACTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0148] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.15:

[0149] QRELQQSGGGLVKPGGSLKLSCAASGFTFGNYAMSWVRQTPEKRLEWVASIGNGSAFYPDSVKGRFTISRNNAGNTLFLQMSSLRSEDTAMYYCTRGGDYYDYDGTYWGQGTLVTVSS.

[0150] Heavy chain CDR region annotation:

[0151] The amino acid sequence of the complementarity-determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 7: NYAMS;

[0152] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO. 8:

[0153] SIGNGGSAFYPDSVKG;

[0154] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 3H7 is shown in SEQ ID NO.9: GGDYYDYDGTY.

[0155] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0156] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody combination for detecting apolipoprotein E4, characterized in that, The monoclonal antibody combination includes monoclonal antibody 4E10 and monoclonal antibody 3H7; The heavy chain variable region of the monoclonal antibody 4E10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 4E10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 3H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting apolipoprotein E4 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4E10 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 4E10 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting apolipoprotein E4 according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting apolipoprotein E4 according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4E10 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4E10 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting apolipoprotein E4 according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3H7 is shown in SEQ ID NO.

20.

6. The monoclonal antibody combination for detecting apolipoprotein E4 according to claim 1, characterized in that, The apolipoprotein E4 includes recombinant apolipoprotein E4 and natural apolipoprotein E4.

7. The use of the combination of monoclonal antibodies according to claim 1 in the preparation of a tool for detecting apolipoprotein E4.

8. The application according to claim 7, characterized in that, The tools include reagents, kits, test strips, and antibody chips.

9. The application according to claim 8, characterized in that, The kit includes a double-antibody sandwich ELISA kit.

10. The application according to claim 9, characterized in that, The ELISA kit uses monoclonal antibody 4E10 as the coating antibody and monoclonal antibody 3H7 as the labeling antibody.

Images