Apolipoprotein e detection reagent and use thereof

Abstract

Provided are an apolipoprotein E detection reagent and the use thereof. The reagent comprises at least one of an ApoE2 protein detection reagent, an ApoE3 protein detection reagent and an ApoE4 protein detection reagent. One of a capture antibody and a detection antibody used for detecting ApoE2 protein or ApoE4 protein is a specific monoclonal antibody, and the capture antibody and the detection antibody used for detecting ApoE3 are both specific monoclonal antibodies. Genotyping of ApoE and quantitative detection of different genotypes of proteins are performed on the basis of an immunological detection method. The detection method has the advantages of being simple, easy to operate, short in time and cheap. A genotype detection result is highly consistent with a fluorescence PCR result. Genotyping of ApoE (6 types) can be performed, which is used to replace the nucleic acid detection and guide medication, and clarify the correlation of the ApoE4 genotype homozygosity / heterozygosity and the protein concentration of ApoE4 with AD.

Description

Apolipoprotein E detection reagent and its application Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an apolipoprotein E detection reagent and application thereof. Background Art

[0002] Apolipoprotein E (ApoE) is a lipid transport protein composed of 299 amino acids with a molecular weight of 34.2 kDa. ApoE comprises three allelic variants (ε2, ε3, and ε4), which encode three isoforms: ApoE2 (mutant), ApoE3 (wild-type), and ApoE4 (mutant). These isoforms differ only in amino acid positions 112 and 158 of their primary structure. ApoE is primarily produced in the liver, but small amounts are also synthesized in the brain, kidneys, bones, adrenal glands, and macrophages. In the brain, it is primarily synthesized and secreted by astrocytes and microglia. The primary physiological function of ApoE is to participate in receptor-mediated lipid transport, storage, utilization, and excretion in the liver. By binding to the low-density lipoprotein receptor and the hepatic ApoE receptor, ApoE promotes the degradation of chylomicrons, low-density lipoproteins, and very-low-density lipoproteins in the blood.

[0003] ApoE is the main risk factor for late-onset Alzheimer's disease (AD) in people over 65 years old. ApoE*ε2 has a protective effect on AD, while ApoE*ε4 increases the risk of AD. Carrying one ApoE*ε4 increases the risk of AD by 3-4 times, while carrying two ApoE*ε4s increases the risk by 9-15 times. A large number of studies have found that ApoE not only regulates the expression levels of β-amyloid plaques, tau protein, and TDP43 protein in the brain of AD patients, but also affects normal brain function (Nature Reviews Neurology, 2019, 15(9): 501-518.).

[0004] ApoE gene is closely related to blood lipid levels. ApoE ε4 gene may be an important etiological factor of hyperlipidemia. In a 2014 study on the correlation between ApoE gene polymorphism and coronary heart disease (CHD) in Chinese people, Zhang, MD et al. analyzed 61 studies totaling approximately 6634 CHD patients and 6393 experimental controls. The results showed that the risk of CHD in Chinese ε4 carriers was 96% higher than that of ε3 / 3 homozygous genotype. In independent analysis and comparison of each genotype, people with genotypes of ε2 / 4, ε3 / 4, and ε4 / 4 were all confirmed to be more likely to suffer from CHD than ε3 / 3 (Journal of the American College of Cardiology, 2014, 9(4):e95463.). A survey of 1,443 European adolescents found that the dynamics of ApoE genotype were strongly correlated with blood lipid / lipoprotein concentrations. ApoEε4 gene carriers had the highest plasma apoB / cholesterol concentrations and an increased risk of AS and other cardiovascular diseases (Clinica Chimica Acta; International Journal of Clinical Chemistry, 1999, 286(1–2):115.).

[0005] Among the existing genotyping detection methods, direct sequencing, fluorescence quantitative PCR (CN 110029159 A A nucleotide sequence group for ApoE genotyping detection and its application), fluorescence chip method and mass spectrometry (Clinica Chimica Acta 519 (2021) 267–275), and microfluidic technology for ApoE protein detection and typing (CN109870567A Microfluidic chip and sorting method for ApoE protein sorting based on immunoassay) all have problems such as high cost, high equipment requirements, or cumbersome procedures, which make it difficult to meet clinical testing needs. Regarding the quantitative detection of ApoE protein, current detection methods mainly focus on the quantitative detection of total ApoE protein, and no quantitative detection of proteins of different genotypes has been found.

[0006] Summary of the Invention

[0007] The present invention uses immunological detection methods to perform ApoE genotyping and quantitative detection of proteins of different genotypes. This detection method is simple, easy to use, time-saving, and inexpensive. The genotype detection results are highly consistent with the fluorescence PCR results. The quantitative detection results of proteins of different genotypes are more correlated with the disease than the total protein detection results. The present invention can perform ApoE typing (six types), which can be used to replace nucleic acid testing and guide medication. It clarifies the correlation between ApoE4 homozygous / heterozygous genotype and ApoE4 protein concentration and AD, indicating Aβ+ risk and suggesting cardiovascular disease risk.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention first provides an ApoE-specific monoclonal antibody, specifically an ApoE2-specific monoclonal antibody, an ApoE3-specific monoclonal antibody, or an ApoE4-specific monoclonal antibody;

[0010] The ApoE2-specific monoclonal antibody is prepared from an ApoE2-specific monoclonal antibody immunogen, and the ApoE2-specific monoclonal antibody immunogen is formed by coupling the polypeptide LLRDADDLQKCLAVYQAGAR with a carrier protein;

[0011] The ApoE3-specific monoclonal antibody is ApoE3-specific monoclonal antibody 1 or ApoE3-specific monoclonal antibody 2, ApoE3-specific monoclonal antibody 1 and ApoE3-specific monoclonal antibody 2 are prepared from ApoE3-specific monoclonal antibody immunogen 1 and ApoE3-specific monoclonal antibody immunogen 2, respectively. The ApoE3-specific monoclonal antibody immunogen 1 is formed by coupling the polypeptide GADMEDVCGRLVQYRGEVQA with a carrier protein; the ApoE3-specific monoclonal antibody immunogen 2 is formed by coupling the polypeptide LLRDADDLQKRLAVYQAGAR with a carrier protein;

[0012] The ApoE4-specific monoclonal antibody is prepared from an ApoE4-specific monoclonal antibody immunogen, and the ApoE4-specific monoclonal antibody immunogen is formed by coupling a polypeptide GADMEDVRGRLVQYRGEVQA with a carrier protein.

[0013] As a further preferred embodiment, the ApoE2-specific monoclonal antibody (G63 antibody) is secreted and prepared by the hybridoma cell line G63. The hybridoma cell line G63 was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on September 7, 2020, with the deposit number CGMCC No. 20296. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the classification name is: Mouse Hybridoma Cell.

[0014] The sequence of the heavy chain variable region of the G63 antibody was shown in SEQ ID NO: 1, specifically:

[0015] The light chain variable region sequence of the G63 antibody is shown in SEQ ID NO: 2, specifically:

[0016] The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 1 is shown in SEQ ID NO: 3, specifically:

[0017] The light chain variable region sequence of ApoE3-specific monoclonal antibody 1 is shown in SEQ ID NO: 4, specifically:

[0018] The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 2 is shown in SEQ ID NO: 5, specifically:

[0019] The light chain variable region sequence of ApoE3-specific monoclonal antibody 2 is shown in SEQ ID NO: 6, specifically:

[0020] The ApoE4-specific monoclonal antibody (M30 antibody) was secreted and prepared by the hybridoma cell line M30. The hybridoma cell line M30 was deposited at the General Microbiology Center of the China Culture Collection Administration (CGMCC) on September 7, 2020, with the deposit number CGMCC No. 20300. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the classification name is: Mouse Hybridoma Cell.

[0021] The sequence of the heavy chain variable region of the M30 antibody was sequenced as shown in SEQ ID NO: 7, specifically:

[0022] The light chain variable region sequence of the M30 antibody is shown in SEQ ID NO: 8, specifically:

[0023] The present invention also provides an ApoE detection reagent, which includes at least one of an ApoE2 protein detection reagent, an ApoE3 protein detection reagent, and an ApoE4 protein detection reagent, and is used to detect any one of ApoE2, ApoE3, and ApoE4, or to detect any two of ApoE2, ApoE3, and ApoE4, or to detect ApoE2, ApoE3, and ApoE4 simultaneously.

[0024] The ApoE2 protein detection reagent can specifically identify ApoE2 protein. The ApoE2 protein detection reagent includes the above-mentioned ApoE2-specific monoclonal antibody and a paired antibody. The ApoE2-specific monoclonal antibody recognizes an epitope near Cys158, and the paired antibody is an ApoE universal antibody.

[0025] The ApoE3 protein detection reagent can specifically identify ApoE3 protein. The ApoE3 protein detection reagent includes an ApoE3-specific monoclonal antibody and a paired antibody. The ApoE3-specific monoclonal antibody is ApoE3-specific monoclonal antibody 1 or ApoE3-specific monoclonal antibody 2. The paired antibody is ApoE3-specific monoclonal antibody 2 or ApoE3-specific monoclonal antibody 1. The epitope recognized by ApoE3-specific monoclonal antibody 1 is near Cys112, and the epitope recognized by ApoE3-specific monoclonal antibody 2 is near Arg158.

[0026] The ApoE4 protein detection reagent can specifically identify the ApoE4 protein. The ApoE4 protein detection reagent includes an ApoE4-specific monoclonal antibody and a paired antibody. The ApoE4-specific monoclonal antibody recognizes an epitope near Arg112, and the paired antibody is an ApoE universal antibody.

[0027] In a further embodiment, the ApoE detection reagent also includes an ApoE protein detection reagent.

[0028] The ApoE protein detection reagent can recognize ApoE protein and can detect different types of ApoE proteins such as ApoE2, ApoE3 and ApoE4. The two paired antibodies used to detect ApoE protein are both universal ApoE antibodies and are not specific for ApoE2, ApoE3 or ApoE4.

[0029] The aforementioned ApoE-specific monoclonal antibodies, the genes encoding the ApoE-specific monoclonal antibodies, the aforementioned hybridoma cell lines, or the aforementioned ApoE detection reagents can be used to prepare apolipoprotein E detection kits or apolipoprotein E detection test strips. Specifically, the apolipoprotein E detection kit is a magnetic particle chemiluminescence detection kit, and the apolipoprotein E detection test strip is an immunochromatographic test strip. Samples for testing can be collected from plasma, saliva, or urine.

[0030] The above-mentioned ApoE-specific monoclonal antibody and the encoding gene of the ApoE-specific monoclonal antibody, the above-mentioned hybridoma cell line or the above-mentioned ApoE detection reagent can also be used to establish a lipid-lowering efficacy evaluation system for statins, which is used to guide the rational use of drugs; or to establish a cardiovascular disease risk system; or to establish an Alzheimer's disease auxiliary diagnosis system; or to establish a cod liver oil supplementation effect evaluation system.

[0031] The present invention further provides an apolipoprotein E magnetic particle chemiluminescence detection kit, which comprises the above-mentioned ApoE detection reagent.

[0032] The present invention further provides an apolipoprotein E immunochromatographic test strip, and the kit comprises the above-mentioned ApoE detection reagent.

[0033] The apolipoprotein E magnetic particle chemiluminescence detection system provided by the present invention includes an apolipoprotein E detection module and an ApoE genotyping module. The apolipoprotein E detection module is used to detect the content of ApoE2, ApoE3 and ApoE4 proteins in a sample, or to detect the content of ApoE2, ApoE4 and ApoE proteins; the apolipoprotein E detection module includes the above-mentioned ApoE detection reagent.

[0034] The ApoE genotyping module can be used in the following operations:

[0035] ① Determine the genotype based on the test results of ApoE2, ApoE3, and ApoE4 proteins:

[0036] (1) ApoE2+ApoE3-ApoE4-, the genotype was determined to be ApoEε2 / ε2;

[0037] (2) ApoE2+ApoE3+ApoE4-, the genotype was determined to be ApoEε2 / ε3;

[0038] (3) ApoE2+ApoE3-ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0039] (4) ApoE2-ApoE3-ApoE4+, the genotype was determined to be ApoEε4 / ε4;

[0040] (5) ApoE2-ApoE3+ApoE4-, the genotype was determined to be ApoEε3 / ε3;

[0041] (6) ApoE2-ApoE3+ApoE4+, the genotype was determined to be ApoEε3 / ε4.

[0042] ② Determine the genotype based on the test results of ApoE2, ApoE4, and ApoE proteins:

[0043] (1) ApoE2+ApoE+ApoE4-, and ApoE2 / ApoE (the ratio of the two detection concentrations)>0.75, the genotype is determined to be ApoEε2 / ε2;

[0044] (2) ApoE2+ApoE+ApoE4-, and ApoE2 / ApoE≤0.75, the genotype was determined to be ApoEε2 / ε3;

[0045] (3) ApoE2+ApoE+ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0046] (4) ApoE2-ApoE+ApoE4+, and ApoE4 / ApoE>0.75, the genotype was determined to be ApoEε4 / ε4;

[0047] (5) ApoE2-ApoE+ApoE4+, and ApoE4 / ApoE≤0.75, the genotype was determined to be ApoEε3 / ε4;

[0048] (6) ApoE2-ApoE+ApoE4-, the genotype was determined to be ApoEε3 / ε3.

[0049] The apolipoprotein E immunochromatographic detection system provided by the present invention includes an apolipoprotein E immunochromatographic detection module and an ApoE genotyping module. The apolipoprotein E immunochromatographic detection module is used to detect the positive and negative of ApoE2, ApoE3 and ApoE4 proteins in a sample. The apolipoprotein E immunochromatographic detection module includes:

[0050] Colloidal gold test strip A: Includes colloidal gold pad A and nitrocellulose membrane A. Colloidal gold pad A is evenly coated with ApoE3-specific monoclonal antibody 1 and colloidal gold marker. Nitrocellulose membrane A is coated with ApoE2-specific monoclonal antibody as the ApoE2 detection line and ApoE3-specific monoclonal antibody 2 as the ApoE3 detection line.

[0051] Colloidal gold test strip B: Includes colloidal gold pad B and nitrocellulose membrane B. ApoE3-specific monoclonal antibody 2-colloidal gold label is evenly applied to the colloidal gold pad B. Nitrocellulose membrane B is coated with ApoE4-specific monoclonal antibody (ApoE4 detection line) and ApoE3-specific monoclonal antibody 1 (ApoE3 detection line).

[0052] The ApoE genotyping module can be used in the following operations:

[0053] Genotype determination is performed based on the test results of different ApoE2, ApoE3, and ApoE4 proteins:

[0054] (1) ApoE2+ApoE3-ApoE4-, the genotype was determined to be ApoEε2 / ε2;

[0055] (2) ApoE2+ApoE3+ApoE4-, the genotype was determined to be ApoEε2 / ε3;

[0056] (3) ApoE2+ApoE3-ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0057] (4) ApoE2-ApoE3-ApoE4+, the genotype was determined to be ApoEε4 / ε4;

[0058] (5) ApoE2-ApoE3+ApoE4-, the genotype was determined to be ApoEε3 / ε3;

[0059] (6) ApoE2-ApoE3+ApoE4+, the genotype was determined to be ApoEε3 / ε4.

[0060] The beneficial effects of the present invention are:

[0061] (1) In the ApoE detection reagent of the present invention, one of the capture antibody and the detection antibody used to detect ApoE2 protein or ApoE4 protein is a specific monoclonal antibody, and the capture antibody and the detection antibody used to detect ApoE3 protein are both specific monoclonal antibodies. By using more specific monoclonal antibodies, the sensitivity and specificity of the kit of the present invention are greatly improved.

[0062] (2) The present invention uses immunological detection methods to perform ApoE genotyping and quantitative detection of different genotype proteins. The detection method is simple, easy to operate, time-saving, and inexpensive; the genotype detection results are highly consistent with the fluorescence PCR results;

[0063] (3) The present invention found that the quantitative detection results of ApoE proteins of different genotypes are more correlated with the disease than the total protein detection results.

[0064] (4) The present invention can perform ApoE typing (6 types), which can be used to replace nucleic acid testing and guide medication, clarify the correlation between ApoE4 homozygous / heterozygous genotype and ApoE4 protein concentration and AD, indicate Aβ+ risk, and suggest cardiovascular disease risk.

[0065] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0067] Figure 1 shows the SDS-PAGE images of ApoE2, ApoE3, and ApoE4 proteins.

[0068] FIG2 shows an ApoE2 calibration curve, wherein the Y-axis represents the logarithmic value of the luminescence value and the X-axis represents the logarithmic value of the concentration of the ApoE2 calibrator.

[0069] FIG3 shows an ApoE4 calibration curve, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic value of the concentration of the ApoE4 calibrator.

[0070] FIG4 shows an ApoE3 calibration curve, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic value of the concentration of the ApoE3 calibrator.

[0071] FIG5 shows an ApoE calibration curve, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic value of the concentration of the ApoE calibrator. DETAILED DESCRIPTION

[0072] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0073] Example 1 Expression and purification of ApoE2, ApoE3, and ApoE4 proteins

[0074] The ApoE2, ApoE3, and ApoE4 genes were inserted into the pcDNA3.1 vector containing a 6×His tag, generating the ApoE2-pcDNA3.1, ApoE3-pcDNA3.1, and ApoE4-pcDNA3.1 vectors. Each of the three vectors was then transformed into DH5α cells. Positive clones were selected and cultured extensively, and the recombinant plasmids were extracted using a high-purity plasmid extraction kit. The recombinant plasmids were transformed into 293T cells, along with the empty pcDNA3.1 vector as a negative control. The cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2 for 72 hours. The supernatants were collected and filtered through a 0.22 μm filter.

[0075] The obtained 500mL filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions, and the equilibrium buffer was 50mM PBS, 10mM imidazole, 150mM NaCl, pH 7.6. After the sample was loaded, 10mL was washed; eluted with 50mM PBS 250mM imidazole, 150mM NaCl, pH 7.6, and the eluate was collected. The protein solution was concentrated using a 3kD ultrafiltration tube, and the protein was stored in pH 7.450mM PBS buffer and stored at -80°C. The purified protein was subjected to SDS-PAGE electrophoresis purity identification. The molecular weight of the three proteins was about 36kD, and the purity reached more than 95%, see Figure 1. The amino acid sequence of the ApoE2 protein is shown in SEQ ID NO:9, specifically:

[0076] The amino acid sequence of ApoE3 protein is shown in SEQ ID NO: 10, specifically:

[0077] The amino acid sequence of ApoE4 protein is shown in SEQ ID NO: 11, specifically:

[0078] Example 2 Preparation of ApoE2, ApoE3, and ApoE4-specific monoclonal antibodies

[0079] Immunogen preparation:

[0080] ApoE2-specific monoclonal antibody immunogen: synthetic peptide LLRDADDLQKCLAVYQAGAR, N-terminally coupled to KLH protein.

[0081] ApoE4-specific monoclonal antibody immunogen: synthetic peptide GADMEDVRGRLVQYRGEVQA, N-terminally coupled to KLH protein.

[0082] Two ApoE3-specific monoclonal antibody immunogens: peptide 1GADMEDVCGRLVQYRGEVQA and peptide 2LLRDADDLQKRLAVYQAGAR were synthesized respectively, and the N-termini of the two peptides were coupled to KLH proteins respectively.

[0083] Mouse immunization: Three batches of BALB / c mice were immunized with the immunogen, three mice each time. Each mouse received 50 μg of antigen. The antigen was mixed with Freund's complete adjuvant in a 1:1 ratio for the first immunization, followed by a 1:1 ratio with Freund's incomplete adjuvant. Immunizations were repeated two weeks apart for a total of three immunizations. Ten days after the third immunization, blood was collected by tail removal and serum titer assayed.

[0084] Hybridoma cell fusion and screening: After the titer of mouse serum reached 1:100,000, mice were boosted intraperitoneally with 30 μg of antigen (without adjuvant). Three days later, spleen cells from the immunized mice were aseptically harvested and mixed with SP2 / 0 mouse myeloma cells at a ratio of approximately 4:1 in a 50 mL centrifuge tube. The cells were washed twice with culture medium, and the supernatant was discarded. The supernatant was then added with 1 mL of prewarmed 50% PEG-1450 for 1 minute. The cells were gently shaken in a water bath for 90 seconds. Immediately, 15 mL of serum-free DMEM prewarmed at 37°C was slowly added dropwise. The cells were incubated at 37°C for 5 minutes, and then serum-free DMEM was added to 40 mL. The cells were centrifuged at 1000 rpm / min for 10 minutes, the supernatant discarded, and 40 mL of prewarmed HAT medium was added. The mixture was gently pipetted to mix thoroughly, and 100 μL was transferred to each well of a 96-well culture plate seeded with feeder cells. The cells were cultured in an incubator. The supernatant was identified, and positive hybridoma clones were screened three times for positive and negative clones.

[0085] ApoE2 hybridoma cell lines were positively screened using ApoE2 protein and negatively screened using ApoE3 and ApoE4 proteins. Hybridoma cell lines with the strongest reaction to ApoE2 protein and no cross-reaction with ApoE3 and ApoE4 proteins were selected to ensure that the screened hybridoma cell lines had high affinity and high specificity. The hybridoma cell line that secretes ApoE2-specific monoclonal antibodies is cloned as G63. Hybridoma cell line G63 was deposited at the General Microbiology Center of the China Culture Collection Administration on September 7, 2020, with the deposit number CGMCC No. 20296.

[0086] The sequence of the heavy chain variable region of the G63 antibody is shown in SEQ ID NO: 1. The sequence of the light chain variable region of the G63 antibody is shown in SEQ ID NO: 2.

[0087] ApoE3 hybridoma cell lines were positively screened using ApoE3 protein and negatively screened using ApoE2 or ApoE4 proteins. Hybridoma cell lines with the strongest reaction to ApoE3 protein and no cross-reaction with ApoE2 or ApoE4 proteins were selected to ensure that the selected hybridoma cell lines had high affinity and high specificity. The results showed that two hybridoma cell lines secreted ApoE3-specific monoclonal antibodies 1 and 2, respectively.

[0088] The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 1 is shown in SEQ ID NO: 3, and the light chain variable region sequence of ApoE3-specific monoclonal antibody 1 is shown in SEQ ID NO: 4.

[0089] The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 2 is shown in SEQ ID NO: 5, and the light chain variable region sequence of ApoE3-specific monoclonal antibody 2 is shown in SEQ ID NO: 6.

[0090] ApoE4 hybridoma cell lines were positively screened using ApoE4 protein and negatively screened using ApoE2 and ApoE3 proteins. Hybridoma cell lines with the strongest reaction to ApoE4 protein and no cross-reaction with ApoE2 and ApoE3 proteins were selected to ensure that the screened hybridoma cell lines have high affinity and high specificity. The hybridoma cell line that secretes ApoE4-specific monoclonal antibodies is cloned as M30. Hybridoma cell line M30 was deposited at the General Microbiology Center of the China General Microbiology Culture Collection Administration (CGMCC) on September 7, 2020, with the deposit number CGMCC No. 20300.

[0091] The sequence of the heavy chain variable region of the M30 antibody was shown in SEQ ID NO: 7, and the sequence of the light chain variable region of the M30 antibody was shown in SEQ ID NO: 8.

[0092] Ascites antibody preparation and purification: After 10-12 week old BALB / c mice were adaptively raised for one week, liquid paraffin was injected intraperitoneally at 0.5 mL / mouse. Seven days later, hybridoma cells with stable antibody secretion and good condition were inoculated into the abdomen at approximately 1×10 6 ~2×10 6 After 7-10 days, the abdomen of the mouse was swollen and the ascites was extracted. The ascites supernatant was collected and purified by Protein A-Sepharose affinity chromatography to obtain antibodies with a purity greater than 90%.

[0093] Monoclonal antibody activity assay: Microplates were coated with 0.1 μg / mL of recombinant ApoE2, ApoE3, and ApoE4 proteins in carbonate buffer (pH 9.5) in a volume of 100 μL at 4°C overnight. Ascites-derived specific monoclonal antibodies (ApoE2-specific monoclonal antibody, ApoE3-specific monoclonal antibody, ApoE4-specific monoclonal antibody) and control antibodies were then diluted in a 1 μg / mL gradient. The control antibody for the ApoE2-specific monoclonal antibody was ApoE2 (E6U9I) antibody from CST (Cell Signaling Technology), and the control antibody for the ApoE4-specific monoclonal antibody was ApoE4 (4E4) antibody from CST. Goat anti-mouse IgG-HRP / goat anti-rabbit IgG-HRP (50 ng / mL) were added to determine the titer of the purified monoclonal antibodies (S / N>2.1). The titers of the ApoE2-specific monoclonal antibody, ApoE3-specific monoclonal antibody, and ApoE4-specific monoclonal antibody were all 1:128,000, which were all greater than the control antibody.

[0094] Monoclonal Antibody Specificity Analysis: Microplates were coated with 100 μL of CBS buffer containing 1 μg / mL of ApoE2, ApoE3, and ApoE4 proteins overnight at 4°C. Serial dilutions of each specific monoclonal antibody (500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, and 0 ng / mL) were added to the microplates. Then, goat anti-mouse IgG-HRP (50 ng / mL) was added. The ApoE2-specific monoclonal antibody was confirmed to specifically recognize ApoE2, and the ApoE4-specific monoclonal antibody to specifically recognize ApoE4. Both antibodies showed no cross-reactivity with other proteins. ApoE3-specific monoclonal antibody 1 can react with ApoE2 and ApoE3 proteins, but has no cross-reaction with ApoE4 protein; ApoE3-specific monoclonal antibody 2 can react with ApoE3 and ApoE4 proteins, but has no cross-reaction with ApoE2 protein.

[0095] Example 3 Establishment of a chemiluminescence detection system for ApoE2, ApoE3, ApoE4 and ApoE magnetic microparticles

[0096] ApoE2 detection system: The methodological model of the present invention is a double antibody sandwich method. 50 μL of biotin-labeled ApoE2-specific monoclonal antibody (G63 antibody) as a capture antibody and 50 μL of sample were added to the instrument in sequence. The reaction was allowed to proceed for 10 minutes. 20 μL of 1 μm streptavidin magnetic beads were added and reacted for 5 minutes. The beads were washed three times with detergent. 100 μL of acridinium ester-labeled ApoE universal antibody (Huamei Bio, catalog number CSB-PA07599A0Rb) was added as a detection antibody. After reacting for 10 minutes, the beads were washed three times. The instrument sent the reaction mixture into a darkroom, and 100 μL of chemiluminescent pre-excitation solution and 100 μL of chemiluminescent excitation solution were added in sequence to conduct a luminescent reaction. Finally, the luminescence intensity was recorded, and the ApoE2 content of the sample was calculated from the calibration curve. The linear range of the calibration curve is 250 to 8000 ng / mL. Figure 2 shows the ApoE2 calibration curve, where the Y-axis represents the logarithmic luminescence value and the X-axis represents the logarithmic concentration of the ApoE2 calibrator. Blood samples were diluted 25-fold before loading, saliva samples were diluted 2-fold, and urine samples were diluted 10-fold.

[0097] ApoE4 detection system: The methodological model of the present invention is a double antibody sandwich method. 50 μL of biotin-labeled ApoE4-specific monoclonal antibody (M30 antibody) as a capture antibody and 50 μL of sample are added to the instrument in sequence. The reaction is allowed to proceed for 10 minutes. 20 μL of 1 μm streptavidin magnetic beads are added and reacted for 5 minutes. The beads are then washed three times with detergent. 100 μL of acridinium ester-labeled ApoE universal antibody (Huamei Bio, catalog number CSB-PA07599A0Rb) is added as a detection antibody. The beads are reacted for 10 minutes and washed three times. The instrument then transfers the reaction mixture to a darkroom. 100 μL of chemiluminescent pre-excitation solution and 100 μL of chemiluminescent excitation solution are added in sequence to initiate a luminescent reaction. Finally, the luminescence intensity is recorded, and the ApoE4 content of the sample is calculated from the calibration curve. The linear range of the calibration curve is 250 to 8000 ng / mL. Figure 3 shows the ApoE4 calibration curve, where the Y-axis represents the logarithmic luminescence value and the X-axis represents the logarithmic concentration of the ApoE4 calibrator. Blood samples were diluted 25-fold before loading, saliva samples were diluted 2-fold, and urine samples were diluted 10-fold.

[0098] ApoE3 detection system: The methodological model of the present invention is a double antibody sandwich method. 50 μL of biotin-labeled ApoE3-specific monoclonal antibody 1 as the capture antibody and 50 μL of the sample are added to the instrument in sequence. The reaction is carried out for 10 minutes. 20 μL of 1 μm streptavidin magnetic beads are added and reacted for 5 minutes. The beads are washed three times with washing solution. 100 μL of acridinium ester-labeled ApoE3-specific monoclonal antibody 2 is added as the detection antibody. The reaction is carried out for 10 minutes and then washed three times. The instrument sends the reaction mixture into a darkroom and adds 100 μL of chemiluminescent pre-excitation solution and 100 μL of chemiluminescent excitation solution in sequence for luminescent reaction. Finally, the luminescence intensity is recorded and the ApoE3 content of the sample being tested is calculated from the calibration curve. The linear range of the calibration curve is 250 to 8000 ng / mL. Figure 4 is the ApoE3 calibration curve, where the Y-axis represents the logarithm of the luminescence value and the X-axis represents the logarithm of the concentration of the ApoE3 calibrator. Blood samples needed to be diluted 25-fold before loading, saliva samples needed to be diluted 2-fold, and urine samples needed to be diluted 10-fold.

[0099] ApoE detection system: The methodological model of the present invention is a double antibody sandwich method, that is, the instrument sequentially adds 50 μL of biotin-labeled ApoE universal capture antibody (Thermo, item number 701241) and 50 μL of sample, reacts for 10 minutes, adds 20 μL of streptavidin magnetic beads with a particle size of 1 μm and reacts for 5 minutes, washes three times with washing solution, adds 100 μL of acridinium ester-labeled ApoE universal detection antibody (Huamei Bio, item number CSB-PA07599A0Rb), reacts for 10 minutes and then washes three times. The instrument sends the reaction mixture into a dark room, sequentially adds 100 μL of chemiluminescent pre-excitation solution and 100 μL of chemiluminescent excitation solution for luminescent reaction, and finally records the luminescent intensity. The ApoE content of the sample being tested is calculated from the calibration curve. The linear range of the calibration curve is 250 to 8000 ng / mL. Figure 5 is the ApoE calibration curve, where the Y axis represents the logarithm of the luminescence value and the X axis represents the logarithm of the concentration of the ApoE calibrator. Blood samples needed to be diluted 25-fold before loading, saliva samples needed to be diluted 2-fold, and urine samples needed to be diluted 10-fold.

[0100] Example 4 Establishment of ApoE2, ApoE3, and ApoE4 Immunochromatographic Detection System

[0101] Preparation of colloidal gold particles: Add 1 mL of 2% chloroauric acid solution to 200 mL of pure water, mix well, and heat to boiling in a 100°C water bath. Under vigorous stirring, add 3 mL of 1% trisodium citrate at one time. When the chloroauric acid aqueous solution turns wine red, continue stirring and react for 15 minutes. After the reaction is completed, transfer to room temperature and slowly stir until cool. Add pure water to make up the solution volume to 200 mL. The preparation of colloidal gold with a particle size of 20 nm is completed. Store at 4°C in the dark.

[0102] Antibody-labeled colloidal gold: Pipette 10 mL of 20 nm gold sol solution into a centrifuge tube, add 42 μL of 0.2 M K₂CO₃ solution and 30 μL (1 mg / mL) ApoE3-specific monoclonal antibody 1, mix thoroughly, and incubate in the dark for 2 h. Add 100 μL of 10% BSA solution to block the mixture and refrigerate at 4°C overnight. Centrifuge the solution at 12,000 rpm for 20 min at 4°C, discard the supernatant, and add 2 mL of reconstitution solution (0.01 M phosphate buffer (pH 8.0) + 1% BSA + 5% sucrose). The resulting ApoE3-specific monoclonal antibody 1-colloidal gold label, referred to as antibody label A, is used for card A preparation. ApoE3-specific monoclonal antibody 2-colloidal gold label, referred to as label B, is prepared using the same method and used for card B preparation.

[0103] The prepared ApoE3-specific monoclonal antibody 1 / 2-colloidal gold label was evenly spread on the glass fiber at 3 μL / cm and dried in an oven at 37° C. for 16 h to prepare colloidal gold pads A or B.

[0104] Preparation of A-card nitrocellulose membrane: Use a microinjector to take ApoE2-specific monoclonal antibody, ApoE3-specific monoclonal antibody 2 and goat anti-mouse IgG antibody at a concentration of 1 mg / mL, respectively. Use a film streaker to paint ApoE2-specific monoclonal antibody as the ApoE2 line, ApoE3-specific monoclonal antibody 2 as the ApoE3 line, and goat anti-rabbit IgG antibody as the C line on the nitrocellulose membrane. After streaking, dry in a 37°C oven for 2 hours and then place in a sealed box with a desiccant for storage.

[0105] Preparation of B-card nitrocellulose membrane: Use a microinjector to take 1 mg / mL of ApoE4-specific monoclonal antibody, ApoE3-specific monoclonal antibody 1 and goat anti-mouse IgG antibody, respectively. Use a film streaker to paint ApoE4-specific monoclonal antibody as the ApoE4 line, ApoE3-specific monoclonal antibody 1 as the ApoE3 line, and goat anti-rabbit IgG antibody as the C line on the nitrocellulose membrane. After streaking, dry in a 37°C oven for 2 hours and then seal and store in a sealed box with a desiccant.

[0106] Sample pad preparation: Soak the glass fiber in Tris-HCl buffer (0.25% Triton X-100, 0.05M Tris-HCl, 0.15M NaCl, pH adjusted to 8.0) for 30 minutes, then place in a 37°C oven to dry and store until ready for use. Use a strip cutter to cut the sample pad into 3 mm strips for later use.

[0107] Assembly of colloidal gold test strip A: stick the sample pad, colloidal gold pad A, nitrocellulose membrane A and absorbent paper on the PVC backboard in sequence, then install the card housing of the colloidal gold test strip, and the test strip preparation and assembly are completed.

[0108] Assembly of colloidal gold test strip B: stick the sample pad, B colloidal gold pad, B card nitrocellulose membrane and absorbent paper on the PVC backboard in sequence, and then install the card housing of the colloidal gold test strip. The test strip preparation and assembly are completed.

[0109] Sample processing: Blood samples need to be diluted 25 times before loading, saliva samples need to be diluted 2 times, and urine samples need to be diluted 10 times.

[0110] Sample detection: Use a dropper to add 3-4 drops of the diluted sample solution (about 100μL) to the sample pad of the test strip and react for 10-15 minutes. If the color development exceeds 30 minutes, it will be invalid.

[0111] Result judgment:

[0112] Negative reaction: T line does not show color, C line shows color;

[0113] Positive reaction: both T line and C line show color;

[0114] Failure reaction: The C line does not show color, but the T line shows color; or both the C line and the T line do not show color, then the test fails or the test strip is invalid.

[0115] Example 5: Determination of ApoE genotype based on the protein detection results of ApoE2, ApoE3, and ApoE4 magnetic microparticle chemiluminescence system

[0116] Using the detection system described in Example 3, ApoE2, ApoE3, and ApoE4 levels (concentrations) were measured in plasma, saliva, and urine samples from 30 healthy individuals (HC) and 30 individuals with Alzheimer's disease (AD). All plasma, saliva, and urine samples were paired. Samples with a concentration ≥250 ng / mL were considered positive for the assay; samples with a concentration <250 ng / mL were considered negative for the assay.

[0117] Genotype determination is performed based on the ApoE2, ApoE3, and ApoE4 protein test results as shown below:

[0118] (1) ApoE2+ApoE3-ApoE4-, the genotype was determined to be ApoEε2 / ε2;

[0119] (2) ApoE2+ApoE3+ApoE4-, the genotype was determined to be ApoEε2 / ε3;

[0120] (3) ApoE2+ApoE3-ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0121] (4) ApoE2-ApoE3-ApoE4+, the genotype was determined to be ApoEε4 / ε4;

[0122] (5) ApoE2-ApoE3+ApoE4-, the genotype was determined to be ApoEε3 / ε3;

[0123] (6) ApoE2-ApoE3+ApoE4+, the genotype was determined to be ApoEε3 / ε4;

[0124] The gold standard is the ApoE genotype sequencing results of the corresponding buccal swab sample. The test results for 60 samples are shown in the table below. As can be seen, the ApoE genotype test results for the 60 samples based on ApoE2, ApoE3, and ApoE4 proteins were used for the ApoE genotype test, and the results were 100% consistent with the sequencing results of the corresponding buccal swab samples.

[0125] Example 6: Determination of ApoE genotype based on the protein detection results of ApoE2, ApoE4, and ApoE magnetic microparticle chemiluminescence system

[0126] The detection system described in Example 3 was used to detect the ApoE2, ApoE4, and ApoE content (concentration) of 30 healthy plasma, saliva, and urine samples (HC) and 30 Alzheimer's plasma, saliva, and urine samples (AD). Plasma, saliva, and urine samples were all paired samples. A sample detection concentration of ≥250 ng / mL was determined to be positive for the detected indicator, and a sample detection concentration of <250 ng / mL was determined to be negative for the detected indicator. Genotype determination was performed based on the detection results of different proteins of ApoE2, ApoE, and ApoE4 as shown below:

[0127] (1) ApoE2+ApoE+ApoE4-, and ApoE2 / ApoE (the ratio of the two detection concentrations)>0.75, the genotype is determined to be ApoEε2 / ε2;

[0128] (2) ApoE2+ApoE+ApoE4-, and ApoE2 / ApoE≤0.75, the genotype was determined to be ApoEε2 / ε3;

[0129] (3) ApoE2+ApoE+ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0130] (4) ApoE2-ApoE+ApoE4+, and ApoE4 / ApoE>0.75, the genotype was determined to be ApoEε4 / ε4;

[0131] (5) ApoE2-ApoE+ApoE4+, and ApoE4 / ApoE≤0.75, the genotype was determined to be ApoEε3 / ε4;

[0132] (6) ApoE2-ApoE+ApoE4-, the genotype was determined to be ApoEε3 / ε3;

[0133] The gold standard is the ApoE genotype sequencing results of the corresponding buccal swab sample. The test results for 60 samples are shown in the table below. As can be seen, the ApoE genotype test results of the 60 samples based on ApoE2, ApoE, and ApoE4 protein detection were used for the ApoE genotype test of the samples, and the results were 100% consistent with the sequencing results of the corresponding buccal swab samples.

[0134] Example 7: Determination of ApoE genotype based on the results of ApoE2, ApoE3, and ApoE4 immunochromatographic protein detection

[0135] ApoE2, ApoE3, and ApoE4 were detected using the detection system described in Example 4 in 30 healthy subjects' plasma, saliva, and urine samples (HC) and 30 subjects with Alzheimer's disease (AD). All plasma, saliva, and urine samples were paired. Genotype determination was performed based on the ApoE2, ApoE3, and ApoE4 protein detection results as shown below:

[0136] (1) ApoE2+ApoE3-ApoE4-, the genotype was determined to be ApoEε2 / ε2;

[0137] (2) ApoE2+ApoE3+ApoE4-, the genotype was determined to be ApoEε2 / ε3;

[0138] (3) ApoE2+ApoE3-ApoE4+, the genotype was determined to be ApoEε2 / ε4;

[0139] (4) ApoE2-ApoE3-ApoE4+, the genotype was determined to be ApoEε4 / ε4;

[0140] (5) ApoE2-ApoE3+ApoE4-, the genotype was determined to be ApoEε3 / ε3;

[0141] (6) ApoE2-ApoE3+ApoE4-, the genotype was determined to be ApoEε3 / ε4;

[0142] The gold standard is the ApoE genotype sequencing results of the corresponding buccal swab sample. The test results for 60 samples are shown in the table below. As can be seen, the ApoE genotype test results for the 60 samples based on ApoE2, ApoE3, and ApoE4 proteins were used for the ApoE genotype test, and the results were 100% consistent with the sequencing results of the corresponding buccal swab samples.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. An ApoE-specific monoclonal antibody, characterized in that, The ApoE-specific monoclonal antibody is an ApoE2-specific monoclonal antibody, an ApoE3-specific monoclonal antibody, or an ApoE4-specific monoclonal antibody; The ApoE2-specific monoclonal antibody is prepared from an ApoE2-specific monoclonal antibody immunogen, and the ApoE2-specific monoclonal antibody immunogen is formed by coupling the polypeptide LLRDADDLQKCLAVYQAGAR with a carrier protein; The ApoE3-specific monoclonal antibody is ApoE3-specific monoclonal antibody 1 or ApoE3-specific monoclonal antibody 2. ApoE3-specific monoclonal antibody 1 and ApoE3-specific monoclonal antibody 2 are respectively prepared from an ApoE3-specific monoclonal antibody immunogen 1 and an ApoE3-specific monoclonal antibody immunogen 2. The ApoE3-specific monoclonal antibody immunogen 1 is formed by coupling the polypeptide GADMEDVCGRLVQYRGEVQA with a carrier protein; the ApoE3-specific monoclonal antibody immunogen 2 is formed by coupling the polypeptide LLRDADDLQKRLAVYQAGAR with a carrier protein; The ApoE4-specific monoclonal antibody is prepared from an ApoE4-specific monoclonal antibody immunogen, and the ApoE4-specific monoclonal antibody immunogen is formed by coupling the polypeptide GADMEDVRGRLVQYRGEVQA with a carrier protein.

2. The ApoE-specific monoclonal antibody according to claim 1, wherein The heavy chain variable region sequence of the ApoE2-specific monoclonal antibody is as shown in SEQ ID NO:1, and the light chain variable region sequence of the ApoE2-specific monoclonal antibody is as shown in SEQ ID NO:

2.

3. The ApoE-specific monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 1 is as shown in SEQ ID NO:3, and the light chain variable region sequence of ApoE3-specific monoclonal antibody 1 is as shown in SEQ ID NO:4; The heavy chain variable region sequence of ApoE3-specific monoclonal antibody 2 is as shown in SEQ ID NO:5, and the light chain variable region sequence of ApoE3-specific monoclonal antibody 2 is as shown in SEQ ID NO:

6.

4. The ApoE-specific monoclonal antibody according to claim 1, wherein The heavy chain variable region sequence of the ApoE4-specific monoclonal antibody is as shown in SEQ ID NO:7, and the light chain variable region sequence of the ApoE4-specific monoclonal antibody is as shown in SEQ ID NO:

8.

5. A hybridoma cell line secreting the ApoE-specific monoclonal antibody according to any one of claims 1-4.

6. The hybridoma cell line according to claim 5, characterized in that, The ApoE2-specific monoclonal antibody is prepared by secreting from the hybridoma cell line G63, and the preservation number of the hybridoma cell line G63 is CGMCC No.20296.

7. The hybridoma cell line according to claim 5, characterized in that, The ApoE4-specific monoclonal antibody is prepared by secreting from the hybridoma cell line M30, and the preservation number of the hybridoma cell line M30 is CGMCC No.20300.

8. The coding gene of the ApoE-specific monoclonal antibody according to any one of claims 1-4.

9. An ApoE detection reagent, characterized in that, The ApoE detection reagent includes at least one of an ApoE2 protein detection reagent, an ApoE3 protein detection reagent, and an ApoE4 protein detection reagent; The described ApoE2 protein detection reagent includes the ApoE2-specific monoclonal antibody and the paired antibody described in claim 1 or 2, and the paired antibody is a universal ApoE antibody; The described ApoE3 protein detection reagent includes the ApoE3-specific monoclonal antibody and the paired antibody described in claim 1 or 3. The ApoE3-specific monoclonal antibody is ApoE3-specific monoclonal antibody 1 or ApoE3-specific monoclonal antibody 2, and the paired antibody is ApoE3-specific monoclonal antibody 2 or ApoE3-specific monoclonal antibody 1; The described ApoE4 protein detection reagent includes the ApoE4-specific monoclonal antibody and the paired antibody described in claim 1 or 4, and the paired antibody is a universal ApoE antibody.

10. An ApoE detection reagent according to claim 9, characterized in that, The ApoE detection reagent also includes an ApoE protein detection reagent, and the described ApoE protein detection reagent includes two paired universal ApoE antibodies.

11. Use of the ApoE-specific monoclonal antibody according to any one of claims 1-4, the hybridoma cell line according to any one of claims 5-7, the coding gene according to claim 8, and the ApoE detection reagent according to claim 9 or claim 10 in any one of the following: (1) Preparation of an apolipoprotein E detection kit or an apolipoprotein E detection test strip; (2) Establishment of a lipid-lowering efficacy evaluation system for statins; (3) Establishment of a cardiovascular disease risk system; (4) Establishment of an auxiliary diagnosis system for Alzheimer's disease; (5) Establishment of a cod liver oil supplementation effect evaluation system.

12. The application according to claim 11, wherein, The described detection kit is a magnetic particle chemiluminescence detection kit, and the described detection test strip is an immunochromatographic test strip.

13. A chemiluminescence detection kit for apolipoprotein E, characterized in that, Includes the ApoE detection reagent according to claim 9 or claim 10.

14. A chemiluminescence detection system for apolipoprotein E, characterized in that, Includes an apolipoprotein E detection module and an ApoE gene typing module, The described apolipoprotein E detection module is used to detect the contents of ApoE2, ApoE3, and ApoE4 proteins in a sample, or to detect the contents of ApoE2, ApoE4, and ApoE proteins in a sample. The apolipoprotein E detection module includes the ApoE detection reagent according to claim 9 or claim 10; The described ApoE gene typing module determines the ApoE genotype according to the detection results of ApoE2, ApoE3, and ApoE4 proteins: (1) ApoE2+ApoE3-ApoE4-, the determined genotype is ApoEε2 / ε2; (2) ApoE2+ApoE3+ApoE4-, the determined genotype is ApoEε2 / ε3; (3) ApoE2+ApoE3-ApoE4+, the determined genotype is ApoEε2 / ε4; (4) ApoE2-ApoE3-ApoE4+, the determined genotype is ApoEε4 / ε4; (5) ApoE2-ApoE3+ApoE4-, the determined genotype is ApoEε3 / ε3; (6) ApoE2-ApoE3+ApoE4+, the determined genotype is ApoEε3 / ε4; Or determine the ApoE genotype according to the detection results of ApoE2, ApoE4, and ApoE proteins: (1) ApoE2 + ApoE + ApoE4-, and the concentration of ApoE2 / the concentration of ApoE > 0.75, the genotype is determined as ApoEε2 / ε2; (2) ApoE2 + ApoE + ApoE4-, and the concentration of ApoE2 / the concentration of ApoE ≤ 0.75, the genotype is determined as ApoEε2 / ε3; (3) ApoE2 + ApoE + ApoE4+, the genotype is determined as ApoEε2 / ε4; (4) ApoE2 - ApoE + ApoE4+, and the concentration of ApoE4 / the concentration of ApoE > 0.75, the genotype is determined as ApoEε4 / ε4; (5) ApoE2 - ApoE + ApoE4+, and the concentration of ApoE4 / the concentration of ApoE ≤ 0.75, the genotype is determined as ApoEε3 / ε4; (6) ApoE2 - ApoE + ApoE4-, the genotype is determined as ApoEε3 / ε3.

15. Apolipoprotein E immunochromatographic test strip, characterized in that, It includes the ApoE detection reagent described in claim 9.

16. An apolipoprotein E immunochromatographic detection system, characterized in that, It includes an apolipoprotein E immunochromatographic detection module and an ApoE genotyping module. The apolipoprotein E immunochromatographic detection module is used to detect the positive and negative of ApoE2, ApoE3, and ApoE4 proteins in a sample. The apolipoprotein E immunochromatographic detection module includes: Colloidal gold test strip A: It includes an A colloidal gold pad and an A card nitrocellulose membrane; an ApoE3-specific monoclonal antibody 1-colloidal gold conjugate is evenly spread on the A colloidal gold pad; an ApoE2-specific monoclonal antibody is coated on the A card nitrocellulose membrane as the ApoE2 test line, and an ApoE3-specific monoclonal antibody 2 is used as the ApoE3 test line; Colloidal gold test strip B: It includes a B colloidal gold pad and a B card nitrocellulose membrane; an ApoE3-specific monoclonal antibody 2-colloidal gold conjugate is evenly spread on the B colloidal gold pad; an ApoE4-specific monoclonal antibody is coated on the B card nitrocellulose membrane as the ApoE4 test line, and an ApoE3-specific monoclonal antibody 1 is used as the ApoE3 test line; The ApoE genotyping module determines the ApoE genotype according to the detection results of ApoE2, ApoE3, and ApoE4 proteins: (1) ApoE2 + ApoE3 - ApoE4-, the genotype is determined as ApoEε2 / ε2; (2) ApoE2 + ApoE3 + ApoE4-, the genotype is determined as ApoEε2 / ε3; (3) ApoE2 + ApoE3 - ApoE4+, the genotype is determined as ApoEε2 / ε4; (4) ApoE2 - ApoE3 - ApoE4+, the genotype is determined as ApoEε4 / ε4; (5) ApoE2 - ApoE3 + ApoE4-, the genotype is determined as ApoEε3 / ε3; (6) ApoE2 - ApoE3 + ApoE4+, the genotype is determined as ApoEε3 / ε4.

17. A method for detecting the content of apolipoprotein E, characterized in that, Detect the contents of ApoE2, ApoE3 and ApoE4 proteins in a sample, or detect the contents of ApoE2, ApoE4 and ApoE proteins in a sample, using the ApoE detection reagent according to claim 9 or claim 10.

18. A genotyping method for apolipoprotein E, characterized in that, Perform apolipoprotein E detection and genotyping on a sample using the apolipoprotein E chemiluminescence detection system according to claim 14; or perform apolipoprotein E detection and genotyping on a sample using the apolipoprotein E immunochromatographic detection system according to claim 16.

19. A method for assisting in the diagnosis of Alzheimer's disease, characterized in that, First, perform genotyping of apolipoprotein E in a sample using the method according to claim 18, and then, based on the genotyping results, perform auxiliary diagnosis of Alzheimer's disease.

20. A method for evaluating the lipid-lowering efficacy of a statin drug, characterized in that, First, perform genotyping of apolipoprotein E in a sample using the method according to claim 18, and then, based on the genotyping results, evaluate the lipid-lowering efficacy of statins.

21. A method for assessing the risk of cardiovascular diseases, characterized in that, First, perform genotyping of apolipoprotein E in a sample using the method according to claim 18, and then, based on the genotyping results, assess the risk of cardiovascular disease.

22. A method for evaluating the supplementary effect of cod liver oil, characterized in that, First, perform genotyping of apolipoprotein E in a sample using the method according to claim 18, and then, based on the genotyping results, evaluate the effect of cod liver oil supplementation.

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